CA2148201A1 - Method for extraction, extraction cartridge and automated extraction processing system - Google Patents

Abstract

An extraction cartridge, for extracting a component from a composition, contains a packing composition including a buffer composition, diatomaceous earth and absorbent. The cartridge is made by heating each component to assure it is free from contaminants. The diatomaceous earth is sifted to obtain a particular particle size. The cartridge also includes frits and filters for holding the packing composition within the cartridge. All of the components of the cartridge are selected or processed to assure that no contaminants are leached into the sample being extracted. A processing system automatically processes a plurality of cartridges. The processing system, which is capable of continuous operation, includes a platform, a rack for holding the cartridges, sample vials and extraction tubes. A walking beam conveyor system moves the rack along the platform and a system of robotic arms automatically transfers the sample to be extracted from the sample vial to the extraction cartridge and adds a predetermined amount of solvent into the extraction cartridge.
The extraction process includes adding a sample into the extraction cartridge, waiting a predetermined time, adding a predetermined amount of solvent and collecting the solvent in a collection tube. The processing system can be controlled by a computer system including a dual microprocessor.

Description

21~8201 P12046.S01 NETHOD FOR EXTRACTION, EXTRACTION CARTRIDGE AND
AUTOMATED EXTRACTION PROCB88ING ~ ~M
BACKGROUND OF THE lNV~N-l'lON
1. Field of the Invention The present invention relates to cartridges for extracting a substance from a composition and a process for making the cartridges. Further, the present invention relates to a method for extracting a substance from a composition.
More specifically, the present invention relates to a method for extracting a drug from a biological sample so that the drug is extracted with good recovery and without extraneous components that would cause interferences during analysis.
Still further, this invention relates to an automatic processing apparatus for extracting substances from a composition.

2. Discussion of Background Information One of the main areas of activity of pharmaceutical companies, contract research laboratories and medical laboratories is the determination of drug levels in samples of human and animal complex biological fluids such as blood, plasma, serum and urine. The drugs are usually present in extremely low concentrations and the most sensitive detection devices are required for their quantification. It is therefore nec~ss~ry that the drug be separated from all other materials present in the complex biological fluids since the other materials could cause interference during analysis.
This separation step is generally referred to as the sample preparation step.
The need to extract only the drug from the other components of the complex biological fluid is becoming increasingly important with the development of increasingly sensitive analytical techniques like gas chromotography/electron capture detector (GC/ECD), gas chromotography/nitrogen phosphorus detector (GC/NPD), gas l 2198201 P12046.S01 chromotography/mass spectroscopy (GC/MS) and liquid chromotography/mass spectroscopy (LC/MS). These methods allow drug detection at the low nanogram/ml and even picogram/ml levels.
Extraction of drugs from biological fluids is presently achieved by one of three procedures: liquid-liquid extraction, solid phase extraction and liquid-liquid extraction on a solid phase support, called Thin-Film Liquid-Liquid Extraction.
Each of these procedures will be briefly described.
(a) Liquid-Liquid Extraction Liquid-liquid extraction involves chAking the biological fluid with a water immiscible organic solvent, at a pH that favors the equilibrium distribution of the drug preferentially into the organic solvent. The organic solvent cont~n~ng the extracted drug is then separated from the aqueous phase, and either evaporated prior to injection into a chromatograph, or subjected to additional cleanup procedures (e.g., extraction into an aqueous phase and back extraction into an organic phase) followed by chromatography.
Liquid-liquid extraction has several disadvantages, such as being very labor intensive, subject to variation in extraction efficiency, and prone to operator errors. Also, emulsion formation can prevent a clean separation of the organic solvent from the aqueous phase, and variable amounts of contaminants are carried through the extraction process, with their p.~ e~c~ usually requiring a chromatography time of 5-60 minutes.
(b) Solid Phase Extraction Solid phase extraction involves use of a short plastic column containing a sorbent, designed to differentially adsorb the drug and contaminants. The column is usually preconditioned with a solvent mixture, the biological fluid is then applied, the drug and other ~ . 2148201 P12046.Sol substances are adsorbed or absorbed on the column, water soluble contaminants are washed off, and finally the drug, along with contaminants, is washed off the column with an organic solvent. As with liquid-liquid extraction, the extract can then be evaporated and chromatographed, or further procesce~ before chromatography.
Solid phase extraction has several advantages over liquid-liquid extraction in that it is often somewhat less labor intensive than liquid-liquid extraction, and provides extracts of apparently comparable cleanliness when using high pressure liquid chromatography (HPLC) as the analytical techn~que. However, solid phase extraction has several disadvantages. Specifically, use of gas chromatography (GC) with solid phase extracts reveals that while the columns used in the extraction remove many contaminants from the biological fluid, they also introduce new contaminants from the column themselves, and this has almost invariably prevented the use of solid phase extraction for sensitive GC procedures.
Additionally, evaporation of eluates from many of these columns leaves behind a powdery residue, derived from the column, that renders GC analysis impossible and shortens HPLC
column life.
c) Thin-Film Liquid-Liquid Extraction Thin-film liquid-liquid extraction involves adding biological fluid to a cartridge cont~ining an essentially inert support, usually diatomaceous earth. An organic solvent is then washed through the cartridge to extract the drug from the film of the biological fluid spread over the ine~t support. This tec-hnique is not commonly used with biological fluids, because the extracts are usually too contaminated with endogenous materials to allow subsequent chromatography, and the percentage extracted is often low.

21~8201 P12046.SOl It is generally recognized that thin-film liquid-liquid extraction is disadvantageous in that the proportion of drug recovered from a thin-film liquid-liquid extraction of a biological fluid is inversely related to the selectivity of the extraction. Thus, virtually complete extraction of a drug can be obtained with a polar solvent, such as diethyl ether, but the extract is often too contaminated to be useful.
Conversely, if a highly non-polar solvent, such as pentane, is used for extraction, the extracts tend to be substantially cleaner, but only between about 0-50% of the drug is extracted and the amount extracted is more variable.
Accordingly, while greater selectivity is achieved with relatively non-polar solvents, sensitivity and precision are sacrificed. The sensitivity of the extraction can be improved by extracting the biological fluid with several aliquots of the organic solvent, but precision tends not to be improved, or it can even be degraded due to the errors incurred in multiple operations.
One method for overcoming the disadvantages of thin-film liquid-liquid extractions has been to develop conditions where multiple extractions of the biological fluid with several aliquots of a relatively non-polar solvent could be performed with minimal variability. This should be achievable using commercially available diatomaceous earth cartridges.
However, while these cartridges appear to be useful for a few drugs, they cannot be generally applied for three reasons:
a) Most drugs are either basic, acidic or amphoteric, and these drugs tend not to be extracted with commercially available cartridges unless the pH of the biological, fluid is first adjusted. At high sensitivity levels, the drug tends to be lost through adhesion to glass surfaces once the pH is changed;

`` 2148201 P12046.S01 b) The non-polar solvent required to obtain relatively clean extracts also dissolves fatty materials from the biological fluid, which often interfere with subsequent chromatography; and c) Extracts from these cartridges suffer from moderate to severe contamination with materials derived from the biological fluid and the cartridge itself.
Despite the many attempts to automate solid phase and liquid-liquid extraction techniques, there is no equipment that allows the rapid, continuous processing of a large number of samples. Most systems handle one sample at thronqhr;lts of 3-5 samples per hour. Others can only perform a specific task but not the whole separation procedure. It would therefore be highly desirable to develop a fully automatic, continuous system for the whole extraction process.
Canadian Laid Open Patent Application 2,034,946, which is hereby incorporated by reference in its entirety, discloses an inert solid phase extraction column. The column includes an elongate barrel made from glass or any other material that does not dissolve or leach contaminants. The column contains a packing material, such as silica, that has been cleaned by co~kin~, serial extraction or fluidized bed flow using a variety of solvents. The packing material is held within the barrel by a pair of meshes positioned above and below the packing material. A stop in the barrel prevents the bottom mesh from moving outside the barrel. A ret~ining ring disposed on the top mesh maintains pressure to keep the packing material confined to a predetermined portion of the glass barrel.
SUMMARY OF THE Ihv~NllON
An object of the present invention is to provide an extraction technique based on an extraction cartridge that produces isolated drug samples that can be analyzed without P12046.S01 any further purification steps. Furthermore, it is an object of the present invention to provide an extraction method that can be readily automated to reduce the considerable cost of manual labor due to the time consuming, repetitive nature of presently available procedures.
Another object of the present invention is to provide a fully automated system that will extract drugs from most biological fluids with greatly increased specificity using the principal of repetitive thin-film liquid-liquid extraction.
Another object of the present invention is to provide a multilayer extraction cartridge filled with materials on which the biological sample is absorbed and/or adsorbed and through which a water immiscible organic solvent or solvent mixture is passed in repetitive aliquots to selectively elute the drug.
Still another object of the present invention is to provide a process for manufacturing a multilayer extraction - cartridge.
Still another object of the present invention is to provide automated equipment ~that allows the continuous processing of biological samples with the organic solvents including a fully automatic liquid handling and dispensing system that employs a w~lk;ng beam to continuously move samples and cartridges through the extractor.
It is accordingly an object of the present invention to provide an extraction cartridge, comprising a barrel comprising an upper opening for feeding of a fluid to be extracted and a lower opening for removal of treated fluid;
and a multilayer packing composition within the barrel, the multilayer packing composition comprising a buffer layer comprising a buffer material for adjusting pH of the fluid to be extracted; and a partitioning layer positioned below the buffer layer, the partitioning 13yer comprising a material for spreading fluid into a thin-film.

2 1 ~ 8 2 0 1 P12046.S01 The barrel can comprise an inert material, preferably selected from the group consisting of glass, ceramic and metal, and more preferably the inert material is glass.
The buffer material can comprise particles, preferably having a particle size of about 100 to 1000 microns.
Moreover, the buffer layer can comprise buffer material and an inert material, preferably particles of inert material, preferably having a particle size of about 200 to 3000 microns. Preferably, the inert material is at least one member selected from the group consisting of crystalline silica, glass beads, metal balls, ceramic beads, crushed-rock, washed sand and diatomaceous earth, more preferably crystalline silica.
Moreover, the buffer material can comprise particles of buffer material, preferably having a particle size of about 100 to 1000 microns.
Further, the buffer layer can comprise buffer material coated on particles of inert material, such as particles having a particle size of about 600 to 850 microns. These insert particles are preferably selected from the group consisting of crystalline silica and diatomaceous earth, more preferably diatomaceous earth.
In each of the embodiments of the buffer layer according to the present invention, the buffer material can comprise at least one member selected from the group consisting of oxalic acid, tartaric acid, citric acid, succinic acid, ammonium dihydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, aluminum sulphate, ammonium oxalate, diammonium hydrogen phosphate, sodium bicarbonate, potassium bicarbonate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, borax, tris(hydroxymethyl)methylamine, sodium carbonate, potassium carbonate, trisodium phosphate and tripotassium phosphate. Further, the buffer layer can comprise Pl2046.Sol about 5-50 wt% buffer material and about 9S-50 wt% inert material, more preferably about 10-30 wt% buffer material and about 90-70 wt% inert material, and even more preferably the buffer layer comprises about 20 wt% buffer material and about 80 wt% inert material.
The material for partitioning a fluid into a thin-film can comprise a particulate material, such as a particulate material having a particle size of about 200 to about 3000 microns. The particulate material can comprise at least one member selected from the group consisting of silica gels, fibrous cellulose, fibrous glass and diatomaceous eart~, and is preferably diatomaceous earth.
The multilayer composition can additionally comprise at least one additional layer, preferably a plurality of layers.
These additional layers can comprise at least one component selected from the group consisting of a component for removing moisture, a component for removing extraneous materials, and a component for derivatizing a drug into a more readily analyzable form.
The component for removing moisture can comprise at least one member selected from the group consisting of anhydrous sodium sulphate, anhydrous magnesium perchlorate, anhydrous calcium sulphate, copper sulphate and alumina, preferably anhyd~ous sodium sulphate.
The component for removing extraneous materials can comprise at least one member selected from the group consisting of alumina, Florisil~, and silica gel. The component for derivatizing a drug into a more readily analyzable form can comprise reactive alkyl halides such as pentafluorobenzyl bromide.
The cartridge can include a filter positioned below the multilayer composition for retaining the multilayer packing composition within the barrel, preferably made from at least ' ' 21g8201 P12046.Sol one material selected from the group consisting of stainless steel mesh, glass mesh, porous polyolefins, porous polytetrafluoroethylene, glass fiber mat and cellulose, more preferably cellulose.
The cartridge can include a filter positioned above the multilayer composition, for maint~;n~n~ the multilayer packing composition within the barrel, preferably comprising at least one material selected from the group consisting of stainless steel mesh, cellulosic material and glass fiber mat, more preferably stainless steel mesh.
A further embodiment of the ~ t invention is a ~ethod for making an extraction cartridge comprising heating an inert support at a temperature of about SOO-C for about 3 hours, mixing the heated inert ~rp~rt with a buffer; heating diatomaceous earth at a temperature of about 500 C for about 3 hours; heating an a~or~nt at a temperature of about 500-C
- for about 3 hours; positioning a frit at the bottom of a cartridge; A~ g the heated A~so-lGI.~ to the cartridge;
ng the heated diatomaceollc earth to the cartridge; ~ ng the mixture of heated inert ~G~ ~ and buffer to the cartridge; and positioning a filter in the cartridge.
A ætill further embodiment of the present invention is an extraction cartridge comprising a barrel comprising an upper o~ni~ for feeAin~ of a fluid to be extracted and a lower 25 o~n~ng for removal of treated fluid; and a multilayer packing composition within the barrel, the multilayer packing composition comprising a buffer layer for adjusting pH
including about 5-50 wt% buffer material and about 95-50 wt%
inert ~"rrort; a partitioning layer comprising a particulate material for spreading a fluid into a thin film; a filter positioned above the multilayer packing composition for retaining the multilayer packing composition within the barrel; and a filter positioned below the multilayer packing ` 2148201 P12046.S01 composition for retaining the multilayer composition within the barrel.
An additional emho~iment of the present invention is a method of extracting a component from a fluid comprising adding a fluid cont~i n i ng a component to be extracted to an extraction cartridge comprising a barrel comprising an upper opening for feeding of a fluid to be extracted and a lower opening for removal of treated fluid; and a multilayer packing composition within the barrel, the multilayer packing composition comprising a buffer layer comprising a buffer material for adjusting pH of the fluid to be extracted;-and a partitioning layer positioned below the buffer layer, the partitioning layer comprising a material for spre~ing fluid into a thin-film; waiting a period of time for the fluid to spread out into a thin film over the partitioning layer;
adding a first organic solvent to the cartridge to extract the component from the fluid into the first organic solvent; and collecting the first organic solvent cont~ining the component.
The method for extraction can also include evaporating the collected solvent and adding a second organic solvent to redissolve the extracted component. The fluid being extracted can be a biological fluid. Preferably the biological fluid is selected from the group consisting of human plasma, human serum, human urine, animal plasma, animal serum and animal urine.
The first organic solvent can be at least one member selected from the group consisting of pentane, hexane, carbon tetrachloride, cyclohexane, iso-propylether, chlorobutane, chloroform, diethylether, toluene and ethyl acetate. The second organic solvent is a polar solvent. Preferably, the polar solvent is at least one member selected from the group consisting of methanol, ethyl acetate and ethanol.

P12046.Sol The method of extracting can also include waiting a period of time for the component being extracted to partition between the fluid and the first solvent; and repeating the steps of adding a first organic solvent to the cartridge to extract the component from the fluid into the first organic solvent; and collecting the first organic solvent containing the component.
A still further embodiment of the present invention is an automatic processing system for extraction of components from a fluid, comprising a main processing platform; indexing means for raising, lowering and incrementally advancing a rack-along the main processing platform; and means for automatically transferring fluids at predetermined positions along the main processing platform. The indexing means can be at least one walking beam positioned at the main processing platform.
The processing system can be structured and arranged for - continuous handling of racks, such as by including extensions for the feeding and removal of racks, or can be structured and arranged to permit handling of racks in a batch mode of operation.
The main processing platform includes a working surface, and a walking beam can include at least one elongated member, with the elongated member being mounted so as to be movably positionable between a lower position wherein an upper surface of the elongated member is below the working surface, and an upper position wherein the upper surface of the walking beam is above the working surface.
The at least one elongated member moves from the lower position to the upper position and returns to the lower position, the at least one elongated member is capable of incrementally moving a rack along the main processing platform. The at least one elongated member can be two elongated members. The at least one elongated member can be .

P12046.S01 eccentrically mounted on at least two pulleys, and each of the at least two pulleys is mounted on a rotatable shaft.
The indexing means further comprises a motor for rotating the rotatable shaft, and means for controlling activation of the motor to enable control of a degree of rotation of the rotatable shaft. The means for controlling activation of the motor comprise elements to stop rotation of the at least two pulleys after one revolution to obtain precise incremental movement. The indexing means further includes at least one locking pin movable from a first position above the working surface to a second position below the working surface as the elongated member moves from the lower position to the upper position, and movable from the second position below the working surface to the first position above the working surface as the elongated member moves from the upper position to the lower position. The at least one locking pin extends to a maximum height of about 0.75 inches above the working surface in the first position.
The processing system further includes a rotatable cam associated with each the at least one locking pin, the at least one locking pin including a lower end contacting the cam. The cam is preferably a pear-shaped cam.
The at least one elongated member moves from the lower position to the upper position and returns to the lower position, the at least one elongated member is capable of incrementally moving a rack along the location, and the at least one elongated member is eccentrically mounted on at least two pulleys, and each of the at least two pulleys and the cam is mounted on a rotatable shaft.
The upper surface of the at least one elongated member includes elements for engaging a lower surface of a rack. The elements for engaging can be protrusions on the upper surface of the at least one elongated member or elements for P12046.S01 increasing frictional engagement along the upper surface of the at least one elongated member.
The working surface of the processing system includes means defining openings corresponding to each of the at least one elongated member and the at least one locking pin to permit passage therethrough. The working surface also can include guide rails.
The means for automatically transferring fluids at predetermined positions includes at least one robotic arm movable along a width, length and height of the location.
The at least one robotic arms can be two independently movable robotic arms, wherein each of the two robotic arms includes at least one probe for transferring fluids. The processing system further includes at least one syringe pump connected by at least one valve with the at least one probe. The at least one probe comprises a plurality of probes, preferably four probes.
The means for controlling activation of the motor to enable control of a degree of rotation of the rotatable shaft comprise at least one proximity sensor. Preferably, the proximity sensor is a photos~ncor.
The processing system further includes a wash rack. The wash rack includes at least one wash station and at least one internal stAn~rd tube. The processing system can include an 2S input extension platform for fee~ing a rack onto the main processing platform. The input extension platform includes at least one proximity sensor and a conveyor belt. The processing system can include output extension platform for receiving a rack from the main processing platform. The output extension platform comprises at least one proximity sensor and a conveyor belt.
The processing system can include a computer system for controlling the operation of the indexing means, and the means 21g8201 P12046.S01 for automatically transferring fluids at predetermined positions. The computer system includes a master system and a slave sub-system. The master system controls operation of the means for automatically transferring fluids. The slave sub-system controls operation of the indexing means. The processing system can include a keyboard with a display, and the slave-subsystem controls the keyboard with a display, The automatic processing system further includes at least one rack, the at least one rack including a lower rack including a back portion, a front portion, and a bottom surface, the back portion including openings for receiving collection vessels, and the front portion including a plurality of openings for receiving sample vessels; an upper rack po~itioned above the back portion of the lower rack, the upper rack including a plurality of openings for receiving cartridges; and the bottom surface includes at least one - element for locking the rack along the main processing platform. The at least one element comprises a plurality of equally spaced openings along the bottom surface. The bottom surface of can be a roughened surface.
BRIEF DESCRIPTION OF THE DRAWINGS
Embodiments of the present invention are shown in the accompanying drawings, in which like reference numerals represent similar parts throughout the several views of the drawings, wherein:
Fig. la is a cross-sectional view of the cartridge according to the present invention.
Fig. lb is a cross-sectional view of another embodiment of the cartridge of the present invention.
Figs. 2 and 3 are chromatograms comparing an extraction from a column according to the present invention and commercially available columns.

21482Ql _ P12046.S01 Figs. 4 and 5 are chromatograms comparing an extraction using a buffer mixture with an extract using a coated buffer.
Figs. 6-17 are chromatograms of materials extracted according to the present invention.
SFigs. 17a, 17b and 17c are chromatograms of materials extracted according to the present invention.
Fig. 18 is an elevated, perspective view of the processor system according to the present invention.
Fig. 19 is an elevated, perspective view of the rack 10according to the present invention.
Fig. l9a is a bottom view of the bottom plate of the rack according to the present invention.
Fig. 20 is a bottom, cutaway view of the main processing platform according to the present invention.
15Fig. 21 is a front, cutaway view of the main processing platform according to the present invention.
Fig. 22 is a side, cutaway view of the main processing platform according to the present invention.
Fig. 23 is a top view of the main processing platform according to the present invention.
Fig. 24 is an elevated, perspe~tive view, with the top partially cut away, of the input extension unit according to the present invention.
Fig. 25a is an elevated, perspective view of the robotic arm according to the present invention.
Fig. 25b is an elevated perspective view of the Z-rack of the robotic arm according to the present invention.
Fig. 26a is a side view of the wash station according to the present invention.
Fig. 26b is a cut away view of a washing reservoir according to the present invention.
Fig. 27 is a block diagram of the computer hardware system according to the present invention.

-P12046.S01 Fig. 28 is a block diagram of the method u~ed in the computer control system according to the present invention.
Fig. 29 is a flow chart of the calibration process according to the present invention.
Fig. 30 is a flow chart of the produc~ion process for a single rack according to the present invention.
Fig. 31 is a flow chart of the production process for multiple racks according to the present invention.
Fig. 32 is a flow chart of the method development process according to the present invention.
DErAT~-~n n~ oN OF THE ~k~ D EMBODIMENTS
As shown in Fig. la, the cartridge 1 according to the present invention includes an elongated barrel 2 having an inlet end 9 and an outlet end 8. Optionally, the cartridge can include a flare 14 at the inlet end 9 and a bottom portion 15 extended by a nipple 10 at the outlet end 8. The elongated barrel 2 can be made from any material that does not dissolve or leach contaminants into the composition being extracted.
The cartridge 1 contains a multilayered packing composition 12 positioned LeL~een the inlet end 9 and the outlet end 8. Th~ multilayered packing composition comprises a number of layers to effect a spreading out of the sample, to co~ ol pH and to permit the selective flow of the eluate through the pac~ing composition. To accomplish these ob;ectives the multilayer composition includes at least a buffer layer 3 and a partition layer 4 for spreading the sample being extracted into a thin film on a solid ~u~po~.
Further, the multilayer packing composition can comprise any number of additional layers, such as a layer to remove moisture, a layer to remove other extraneous materials, and a layer including materials that derivatize the drug being extracted so that it is in a more readily analyzable form.
Further, the additional layers can include components to 21g8201 P12046.S01 provide a combination of functions. For example, one additional layer can have any number of combined function, such as combined moisture removing and derivatizing functions.
As illustrated in Fig. la, in a preferred form of the invention, the uppermost layer 3, positioned toward the inlet of the barrel, disperses the sample evenly over the top of the cartridge and adjusts the pH. The layer 4, beneath the layer 3 includes a composition for spreading the sample being extracted into a thin film. Further, in the illustrated embodiment two layers 5 and 6 are shown below the partition layer 4. Either of these layers can have the function of removing traces o water from the organic solvent or mixture of organic solvents, selectively absorb and/or adsorb non-polar materials, other than the drug, that would otherwise contaminate the extract, as well as derivatize the drug being extracted into a more readily analyzable form. For example, layer 5 can be anhydrous sodium sulphate and layer 6 can be basic, acid or neutral alumina to provide specific interactions with drugs or contaminants. Other possible selective absorbers are Florisil, such as Florisil0 A and Florisil PR, and silica gels. An example of a derivatization agent i8 pentafluorobenzyl bromide which reacts preferentially with phenolic compounds at specific pH.
The packing composition 12 is held within the elongated barrel 2 by filters or frits 7, 11, which will be more specifically described later, placed below and above the packing composition 12, respectively. The filter or frits 7, 11 are constructed of materials that do not introduce contaminants and prevent the packing composition from being washed out of the barrel 2, but do not impede liquid flow.
The cartridge 1 according to the present invention can have any dimension depending upon the extraction being performed and the packing composition used. Specifically, the -P12046.S01 cartridge can be from about 10 and 200 mm in height, measured from the top 16 to the bottom portion 15, with an outer diameter, from about 5 and 40 mm and an inner diameter from about 3 and 36 mm. Preferably, the cartridge can be from about 30 and 120 mm in height with an outer diameter, from about 5 and 25 mm and an inner diameter from about 3 and 23 mm. Even more preferably, the cartridge can be about 67.0 mm in height with an outer diameter, of about 14.5 mm and an inner diameter of about 11.7 mm.
As shown in Fig. lb, an alternative embodiment of the cartridge according to the present invention includes a constriction 13 that reduces the diameter of the barrel 2.
The constriction can be placed at any position sufficient to accommodate the packing composition in the barrel. The constriction 13 can be at a distance from about 1 mm to 100 mm from the top of the cartridge, preferably, from about 1 mm to 40 mm from the top of the cartridge, and even more preferably, about 22 mm from the top of the cartridge. The constriction 13 can reduce the diameter of the cartridge from about 0.5 mm to 10 mm, preferably, from about 0.5 mm to 5 mm, and even more preferably, by about 1.0 mm.
In this embodiment, the top filter is positioned immediately under the constriction. The filter is pushed past the constriction 13 causing slight compression of the packing.
On release, the filter will push up against the constriction.
Thus, the packing is immobilized, preventing mixing of the layers during handling and transporting.
As has been described above, the cartridge according to the present invention includes a barrel, a buffer layer, a partition layer, and optionally at least one additional layer such as a drying agent/absorber layer and frits or filters.
Each of the components of the cartridge will be described in detail below.

P12046.S01 Barrel Any material that does not leach contaminants into the extract can be used to construct the barrel. For example, inert materials such as glass, metals including aluminum, nickel, stainless steel, titanium, or ceramic can be used to make the barrel. Preferably, the material of construction of the barrel is glass. An even more preferred barrel is a glass, syringe-type barrel cartridge with a molded, one piece construction available from Burdick and Jackson (Custom SPE
Cartridge Cat. No. 79.99GB supplied by Canlab, 2390 Argentia Rd., Mississauga, Ont.). Prior to being filled with the packing material, the barrel is preferably washed with hot water, brushed with "Sparkleen" (Fisher Scientific, Nepean, Ont K2E 7L6, Cat. No. 04-320) rinsed several times with deionized water, placed in methanol and sonicated for 15 minutes before being dried at 60C.
As noted above, the barrel is made from a material that does not leach materials into the biological fluids or solvents used in the extraction. All barrels made from plastics have resulted in grossly contaminated extracts, with the cont~.n~nts derived from the plastics. This is illustrated in Figs. 2 and 3, which show that the shromatogram from the glass cartridge has substantially fewer peaks than those of the plastic cartridges. The plastic cartridge used in this comparison was the Merck Extrelut QE, available from EM Science, Cherry Hill, N.J. The glass cartridge used was the cartridge IX, described in Table 2a below. In both cases, 1 ml of distilled water was added to the cartridges and extracted with five 1.5 ml aliquots of chlorobutane. The extract was evaporated and reconstituted to 50 microliters with ethanol before being directly injected into a HP 5890 gas chromatograph equipped with either an electron capture detector (Fig. 3) or a nitrogen/phosphorous detector (Fig. 2) _ P12046.S01 and a DB-5, 15 m x 0.32 mm capillary column with 0.25 micron film thicknes6.
Buffer Layer The buffer layer of the packing composition is positioned toward the inlet end of the cartridge to adjust the pH of the sample being extracted and to disperse the sample over substantially the entire diameter of the cartridge so that the sample spreads out substantially evenly into the partition layer.
The composition of the buffer layer includes buffer material for adjusting pH, and preferably an inert support.
For example, the buffer layer can be composed of intermixed buffer and inert support, inert support coated with buffer or simply particles of buffer material. Any known buffer material can be used dep~n~ing upon the pH desired for a particular extraction. Examples of suitable buffer materials are shown in Table 1. Many other solid organic or inorganic salts, bases and acids that dissolve readily in water but do not dissolve in the water immiscible organic solvent used for drug extraction can be also be suitable.
To achieve intermediate pH levels, the buffer materials can be dry mixed with each other before being incorporated with the inert support. All buffer materials are ACS or ~AT~ grade materials.

P12046.S01 ~u~ pH*
1 Oxalic acid 1.3 2 Tartaric acid 2.0 3 Citric acid 2.1 4 Succinic acid 2.7 Ammonium dihydrogen phosphate 4.0 6 Sodium dihydrogen phosphate 4.5 7 Potassium dihydrogen phosphate 4.5 8 Aluminum sulphate 5.5 9 Ammonium oxalate 6.4 Diammonium hydrogen phosphate 7.9 11 Sodium bicarbonate 8.4 12 Potassium bicarbonate 8.4 13 Disodium hydrogen phosphate 9.1 14 Dipotassium hydrogen phosphate 9.1 Borax 9.2 16 Tris(hydroxymethyl)methylamine 10.5 17 Sodium carbonate 11.5 18 Potassium carbonate 11.5 19 Trisodium phosphate 12.0 Tripotassium phosphate 12.0 approximate pH for an 0.1 N aqueous solution The carrier, or inert support, of the buffer layer can be made up of any solid material that has a size range that allows free flow of the liquids and that does not dissolve or in any way contaminate the aqueous sample or the organic solvent used to elute the drug. Furthermore, the carrier must not react with the buffer material. Carrier materials useful in the buffer layer include crystalline silica, glass beads, metal balls, such as stainless steel ball bearings, ceramic beads or chips, diatomaceous earth, crushed rock or washed sand. Preferably, the carrier material is crystalline silica, which has been heat treated at about 500C for about 3 hours to remove any contaminants.
The particle size of the buffer material and the inert support is selected to ensure that the sample flows freely into the middle layer and that the entire sample is exposed to . 2148201 P12046.S01 the buffer material. The particle size of the buffer material is about 100 microns to 1000 microns, preferably, from about 400 microns to 1000 microns, even more preferably, from about 600 microns to 850 microns.
The particle size of the inert support is about 200 microns to 3000 microns, preferably, from about 400 microns to 850 microns, even more preferably, from about 600 microns to 850 microns. The preferred inert support is obtained by sieving commercially available silica sand to obtain a particle size range of about 600 microns to 850 microns.
The amount of inert support and buffer material in the buffer layer can be varied. Specifically, the amounts of each component can range from about 5-50 wt% of buffer material and 95-50% inert material. Preferably, the range can be about 10-30 wt% buffer and 90-70 wt% inert material. Even more preferably, the buffer layer can be about 20 wt% buffer material and about 80% inert support.
The embodiment of the buffer layer comprising an inert support coated with the buffer material is produced in the following manner. A solution of the buffer material in water, or any other solvent in which the buffer has adequate solubility, is added to the inert support and the mixture, in the form of a slurry, stiff paste or partially wetted inert support, and is evaporated to dryness on a rotary evaporator.
Care must be taken not to overheat the mixture to ensure that there is no decomposition of the buffer salt, e.g., both chemically and physically, loss of C02 from sodium bicarbonate, and that the same form of the buffer material is deposited on the inert support every time.
The following tests demonstrate that comparable results are obtained performing an extraction using a cartridge including a buffer layer that is a mixture of buffer material and inert support and an extraction using a cartridge -P12046.S01 including an inert support coated with buffer material.
Specifically, the drugs diazepam and desmethyldiazepam were extracted from plasma using basically identical cartridges;
however, one cartridge contained a buffer layer comprising a mixture of 20% borax crystals and 80~ silica sand, and the other cartridge contained a buffer layer comprising 20~ borax coated on diatomaceous earth. Any free water was evaporated from the wet diatomaceous earth in a rotary evaporator at less than 30C under vacuum generated by a water aspirator.
10As illustrated in Figs. 4 and 5, respectively, the elution pattern from the cartridge using a buffer layer comprising a mixture of 20% borax crystals and 80% silica sand was comparable to the elution pattern from a cartridge using a buffer layer comprising 20% borax coated on diatomaceous earth. The cartridge used in the elution illustrated in Fig.
4 included 1.19 g of buffer/sand mixture. The cartridge used - in the elution illustrated in Fig. 5 included 0.51 g of buffer coated diatomaceous earth.
The other components of the cartridges were the same, including the glass syringe-type barrel available from Burdick and Jackson described above, 0.75 g of diatomaceo~ls earth positioned below the buffer layer, 0.5 g of anhydrous sodium sulphate positioned below the diatomaceous earth, a cellulose filter positioned below the packaging composition. The cartridge that contained the buffer coated diatomaceous earth also included a 60 mesh stainless steel filter positioned above the packing composition.
The drug extracted in Fig. 4 was diazepam and in Fig. 5 was desmethyldiazepam. The extraction process used was: 1 ml of plasma extracted with 6 additions of 1.5 ml of 5% by volume ethylacetate and 95% by volume chlorobutane.

P12046.Sol Partition Layer Positioned below the buffer layer is a layer of material for spreading the sample, whose pH has been adjusted by the buffer layer, into a thin-film over an inert support. Any material that is inert, highly porous, and includes a large surface area can be used for the partition layer.
Specifically, materials that can be used for the this partition layer include silica gel, fibrous materials made of cellulose or glass, and diatomaceous earth. Preferably, the partition layer is composed of diatomaceous earth with large surface area.
The particle size of the partition layer assures free flow of the sample with a minimum of channelling. The particle size can range from about 100 to 3000 microns, preferably, from about 200 to 1000 microns, even more preferably, from about 600 to 850 microns. A large percentage of particles of less than about 200 microns would lead to reduced flow, necessitating much longer total processing times.
A preferred diatomaceous earth for use in the thin film layer is Hydromatrix available from Varian ~csoçiates, Sunnyvale, Ca, Cat. No. AL-001980-03. This diatomaceous earth is sieved to remove particles outside the range useful in the partition layer. The selected material, with a particle size range greater than 600 microns, is heat treated in a standard muffle furnace, with air atmosphere, for about 3 hours at about 500C to remove all organic materials that could be extracted from the column and cause interference during analysis.
Additional Layer The additional layers of the packing composition can have various functions. For example, the layers can remove any water that remains in the eluate, the layers can remove any 21~8201 P12046.S01 additional extraneous materials, and/or the layers can derivatize the drug that is being extracted. For example, the additional layers can include a layer of drying agent for removing any moisture from the eluate. Any material that absorbs water and does not contaminate the eluate can be used, such as anhydrous sodium sulphate, anhydrous magnesium perchlorate, anhydrous calcium sulphate, copper sulphate and alumina. Preferably, ACS grade anhydrous sodium sulphate heated at about 500-C for about three hours to remove any contaminants is used as the drying agent. The particle size of the drying agent can be about 100 microns to 1000 miCrons, preferably, about 200 microns to 1000 microns, even more preferably about 400 to 8S0 microns.
While the additional layers have been shown in the preferred embodiment to be the lowermost layers, the additional layers can be positioned at various locatiGns in the cartridge dep~nAing upon the extraction. For example, the additional layers can be placed above the buffer layer or between the buffer layer and the partition layer.
Furthermore, the additional layers can be included in more than one location.
Frits/Filters The packing composition is held within the barrel by frits or filters, (hereinafter collectively referred to as "filters") positioned above and below the packing composition.
The filter positioned at the bottom of the packing composition prevents fines from the packing composition from entering the collection tube where they could cause clogging problems when the reconstituted extract is being sampled. The bottom filter can be made from any material that does not add contaminants to the eluate and allows the eluate to flow from the cartridge, such as cellulosic material, stainless steel mesh, glass, porous polyolefins, porous polytetraflouroethylene, . 2148201 -P12046.S01 fine cloth held by a plastic ring or glass fiber mat.
Preferably, the bottom filter is made from cellulose. Even more preferably, the bottom filter is a precut, cellulosic filter, type 740-E, from Schleicher and Schuell, available from Canlab Scientific Products Cat. No. F2755-2, which is preferably washed with ethyl acetate and dried with a stream of nitrogen. Cellulose is the preferred material for making the filter because the undesirable cleanliness, cost and flow/wetting characteristics of the other materials are inferior to those of cellulosic materials. The filter is dimensioned to fit snugly within the inner diameter ~f the barrel.
The filter on top of the packing composition retains the packing during transportation to prevent mixing of the layers.
Additionally, the top filter distributes the sample uniformly over the whole diameter of the cartridge. The top filter can be made from stainless steel mesh, random glass fiber mat or cellulosic filter materials. The preferred material is a stainless steel plain weave mesh, with a mesh opening size of about 0.070 inches. As with the bottom filter, the top filter is dimensioned to fit snugly within the inner diameter of the cartridge. The top filter can be held in place by the constriction in the cartridge wall if it has sufficient stiffness and flexibility that it can be pltChÇ~ past the constriction without permanent distortion. Other ways that the top filter can be positioned are by the insertion of a tightly fitting ring of a flexible material immediately after the filter disc, or an application of a bead of adhesive immediately above the filter which will adhere tightly to the glass. However, the materials must be chosen so as to eliminate the possibility of introducing extraneous materials which would be extracted.

Pl2046.S01 Examples of cartridges that have been used for the isolation of drugs from human plasma samples are defined in Table 2a. Each cartridge is substantially similar except for the buffer layer, which is listed in Table 2a. The cartridges used in the examples each include about 1.0 gram of a buffer layer, about 0.75 grams of a partition layer comprising heat treated, sieved diatomaceous earth, and about 0.5 gram of a drying layer including sodium sulphate. The barrel used was the glass syringe type barrel available from Burdick and Jackson, described above. The bottom filter was the cellulosic filter from Schleicher and Schuell (Type 740-E).
The amount of each layer can be varied depen~ing on the size of the cartridge and the type of extraction.
TABLE 2a Cartridge Approx. Type of Buffer Wt ~
type pH Buffer/crystalline silica IX g Borax 20/80 XI 11 Tris* 20/80 VIII 8 Sodium Bicarbonate 30/70 III 3 C-'ric Acid 20/80 tris(hydroxymethyl)~ethylamine The method of extracting a substance from a biological fluid according to the present invention involves the following steps.
A particular cartridge cont~;n;ng a particular buffer mixture, is selected depending upon the chemical properties of the drug being extracted. More specifically, most drugs have one or more functional ~LoU~s which makes them either acid, basic or amphoteric. If a drug has basic characteristics, it will readily pick up a proton (H+); if it is acidic, it will lose a proton. In both instances, a charged species is formed from an electrically uncharged molecule. In aqueous solution, _ P12046.S01 depending on the pH, the drugs can be present in the charged form which is a highly water soluble state or in the uncharged form which is more soluble in the organic solvent. A
particular cartridge is chosen with the goal of putting the drug molecule in the aqueous sample in the uncharged state by selecting the pH of the cartridge buffer to facilitate extraction with an organic solvent.
Thus, an alkaline drug requires a high pH to prevent charge formation, and Cartridge No. IX or No. XI would be appropriate for extracting alkaline drugs. An acid drug requires a low pH, to prevent its dissociation, since-there are already many protons in solution and, accordingly, Cartridge No. III would be appropriate. Amphoteric drugs can behave both as a base and as an acid; it is therefore neceCc~ry to react one of the functional groups with special reagents, e.g., alkyl sulfonates and tetrabutyl ammonium phosphate as reagents for acidic and basic functional groups, respectively. Such special reagents can be added to the sample and/or can be included in one or more layers of the multilayer packing composition in the cartridge.
The process of extraction begins by adding a measured aliguot of biological fluid containing a drug to be extracted to the cartridge. The fluid flows through the buffer layer and spreads out in a thin film over the partition layer. The fluid does not elute from the cartridge, nor are any materials washed off prior to elution of the drug.
After a period of time, from about 1 minute to 30 minutes, preferably about 5 to about 15 minutes, even more preferably about 10 minutes, a first measured aliquot of organic solvent is added, and the eluate is collected. The solvent addition process is repeated for about 5 or 6 times, at intervals from about 1 minute to 20 minutes, preferably 21482~1 P12046.S01 about 3 to 10 minutes, even more preferably about 5 minutes.
All of the eluates are combined in a collection tube.
Examples of organic solvents that can be used include pentane, hexane, cyclohexane, heptane, iso-propyl ether, chlorobutane, carbon tetrachoride, methylene chloride, chloroform, diethyl ether, toluene or ethylacetate. These solvents can be used on their own or in mixtures of two or more in order to achieve selectivity for the extraction of the drug from a particular matrix. Specifically, it has been found that a small amount of ethylacetate (EtAC) added to any of the solvents listed above can improve the extraction efficiency and selectivity. The amount of EtAC in the solvent mixture can be up to 50% by volume. Preferably, the amount of EtAC in the solvent mixture is about 5-10~ volume.
The selection of a particular solvent or solvent mixture for a particular extraction is made through a series of experiments. Different types of cartridges and internal stA~rds may also be evaluated with each of the different solvents chosen. All extracts collected are then analyzed by the same analytical tec-hnique. In this way, the solvent which gives the cleanest extract with the best recovery of the drug can be selected.
The solvent is evaporated from the collection tube using any known evaporation technique and the non-volatile drug is re-dissolved in a small aliquot of a strong, polar solvent, such as methanol, ethyl acetate or ethanol. Samples from this concentrated drug solution are directly injected into a GC, HPLC or LC/MS or other analytical system.
This method is performed using gravity elution without suction or pressure that is usually applied with solid phase cartridges, and without any pretreatment of the cartridges.
Also, the aqueous phase and substantially all contaminants are ` 21~8201 P12046.S01 left on the column. The column contents can be discarded and the barrel recycled.
There are several important parameters that must be carefully controlled for optimum results. Adequate time must be allowed for the biological fluid to penetrate and spread in a thin film over the partition layer. The porosity/absorbtivity of this layer must be controlled so that the sample spontaneously spreads over about 70% of the layer.
Further, adequate time must be allowed between elutions with the organic solvent for re-equilibration to take place effectively. An equilibrium is established between the drug in the aqueous layer and the organic solvent which depends on the relative solubility of the drug in the two media. A
portion of the solvent from the first addition is retained on the spreading layer. Each time more solvent is added, the "old" solvent into which more drug has solubilized is flushed out and replaced with the fresh solvent that has been added.
In this way, the drug is progressively removed from the aqueous layer. If sufficient time is not allowed between additions of solvent, the solvent retained on the spreading layer will not effectively solubilize the drug, and recovery will be lower and variable.
The process according to the present invention is illustrated in the following non-limiting examples. For each example the following information is provided:
a) A table summarizing details of the analytical conditions and including the recoveries achieved at three concentration levels;
b~ A table including a full set of data to establish the between run precision and accuracy for the analysis of these drugs in human plasma, wherein the nominal value is the actual concentration of drug in a sample; the mean is the mean concentration calculated from n actual measurements of sample;

_ P12046.S01 SD is the st~n~rd deviation of the mean; mean as percent of nominal is mean/nominal x 100; CV% is SD/mean x 100; and n is the number of measurements;
c) A chromatogram obtained from the extraction of blank samples, i.e., no drug is injected, the region of the chromatogram where the drug would appear must not exhibit any interference peaks;
d) A chromatogram of an extract from a sample that includes a low level of the drug to be extracted, which can be as low as 50 picogram per milliliter; and e) A chromatogram of an extract from a sample inc~uding an intermediate level of the drug to be extracted. Standard laboratory methods are used to obtain the statistical data.

Example 1 illustrates the extraction of the drug buspirone using extraction cartridge IX defined above. The extraction conditions, including volume of plasma added, internal stAn~Ard, extraction solvent, volume of extraction solvent, volume of reconstitution solvent, and the chromotography conditions for Examples 1-4 are summarized in ~able 2b.
In addition to the plasma and internal stA~Ard, approximately 0.3% wt. of the anticoagulant ethylene diamine tetraacetic acid (EDTA) is added in each of Examples 1-4. The analytical method is summarized in Table 3. The results are summarized in Table 4. Figs. 6-8 are chromatograms of buspirone samples extracted according to the present invention at 0.00 pg/ml, 50.00pg/ml and 8000.00 pg/ml respectively.

Example 2 illustrates the extraction of the drug danazol, at a variety of concentration levels, using extraction cartridge IX. As noted above, the details of the extraction and the chromotography conditions are summarized in Table 2b.

21~8201 ._ Pl2046.S01 The analytical method is summarized in Table 5. The results are summarized in Table 6. Figs. 9-11 are chromatograms of danazol samples extracted according to the present invention at 0.00 ng/ml, 2.03 ng/ml and 248.25 ng/ml respectively.
S EXAMPhE 3 Example 3 illustrates the extraction of the drug alprazolam, at a variety of concentration levels, using an extraction cartridge IX. The details of the extraction and the chromotography conditions are summarized in Table 2b. The analytical method is summarized in Table 7. The results are summarized in Table 8. Figs. 12-14 are chromatograms of alprazolam samples extracted according to the present invention at 0.00 ng/ml, 0.50 ng/ml and 16.00 ng/ml respectively.
EXAMP~E 4 Example 4 illustrates the extraction of the drugs verapamil and norverapamil, at a variety of concentration levels, using an extraction cartridge IX. The details of the extraction and chromotography conditions are summarized in Table 2b. The analytical method is summarized in Table 9.
The results are summarized in Tables 10 and 11. Figs. 15-17 are chromatograms of verapamil and norverapamil samples extracted according to the present invention at 0.00 ng/ml, 2.00 ng/ml and 201.11 ng/ml verapamil and 209.59 ng/ml norverapamil respectively.
EXAMPhE 5 Example 5 illustrates the extraction of the drug glipizide, at a variety of concentration levels using an extraction cartridge III. The details of the chromatography conditions are summarized in Table 2b. The analytical method is summarized in Table 12. Figs. 17a-17c are chromotograms of glipizide samples extracted according to the present invention at 0.00 mg/ml, 15.0 mg/ml and 800 mg/ml.

-P12046.S01 Table 2b Extraction and Chr, tog,aphic Conditions for Examples 1-5 Buspirone Verapamil Danazol Alprazolam Glipizide Example 1 Ncr~er3, il Example 2 Example 3 Example 5 Example 4 Volume of 1.0 ml 1.0 ml 1.0 ml 1.0 ml 1.0 ml Plasma Internal Loratadine Loratadine Loratadine Triazolam Alprazolam Standard Extraction 5X ethyl 5X ethyl 5% ethyl 5X ethyl 5X ethyl Solvent acetate in acetate in acetate in acetate in acetate in chlorobutane isopropyl hexane chlorobutane methylene ether chloride 1 O Volume of 5 x 1.5 ml 5 x 1.5 ml 5 x 1.5 ml 5 x 1.5 ml 5 x 1.5 ml solvent Volume of 25 microliter 25 microliter 50 mcl of 50 mcl of 50 mcl reconstitution (mcl) of of denatured ethyl acetate ethyl methanol solution methanol alcohol acetate Capillary DB-5, 15m x 0.32mm DB 17, 7.5m DB-5, 15m x column 0.25 micron film thickness x 0.25mm; 0.32mm 0.250.25 micron micron film film thickness thickness Detector Nitrogen/Pho~,orous Electron Nitrogen/
capture Phosphorus Oven temp. 255OC 2600C 2620C 2800C 3000C
Injector 325OC 300OC 300OC 3100C 400OC
temp.
Detector 300OC 275OC 300OC 325OC 3000C
temp.
Type of chromatograph: HP 5890 equipped with split/splitless capillary injection port; automatic liquid sampler; integrator data system 214820i P12046.S01 SUMMARY OF THE BUSPIRONE ANALYTICAL METHOD
Matrix : Plasma Volume Required : 1.0 ML
Anticoagulant : EDTA
Extraction Cartridge : Cartridge IX
Concentration Range : 50.0 - 10009.0 pg/ml Chromatography : High resolution capillary column Detection Mode : NPD
Quantitation Method : Internal St~n~rd Method Quantitation by : Peak height ratio Regression : Linear Goodness of Fit : The correlation coefficients were better than or equal to 0.9950 Recovery : 73.8% at 300.00 pg/mL
: 63.2% at 2500.00 pg/mL
: 64.9% at 8000.00 pg/mL
Stability : Stable at room temperature in plasma for 8 hours.
: Stable in plasma after three freeze-thaw cycles.
: Stable in injection solvent for 6.5 hours P12046.S01 w~N-RUN PRECISION AND ACCURACY FOR
BUSPIRONE IN HUMAN PLASMA
Nominal Mean Value Mean + SD as % of Samples (pg/mL) (pg/mL) Nominal CV% n A 50.051.4 + 4.84 102.8% 9.4% 9 B 300.0270.6 + 26.67 90.2% 9.9% 9 - C 2500.02238.1 i 61.34 89.5% 2.7% 8 D 8000.07701.4 + 500.28 96.3% 6.5% 8 E 50.050.7 + 2.87 101.4% 5.7% 5 F 100.1101.5 + 5.19 101.4% 5.1% 5 G 500.5483.8 + 13.66 96.7% 2.8% 4 H 1501.41497.0 + 96.77 99.7% 6.5% 5 I 5004.64922.4 + 404.60 98.4% 8.2% 5 J 7506.87858.5 + 210.13 104.7% 2.7% 3 K 9008.29171.4 + 680.39 101.8% 7.4% 5 L10009.09732.7 + 366.93 97.2% 3.8% 5 P12046.S01 SUMMARY OF THE DANAZOL ANALYTICAL METHOD
Matrix : Plasma Volume Required : 1.0 ml Anticoagulant : EDTA
Extraction Cartridge : Cartridge IX
Concentration range : 2.03 - 400.40 ng/ml Chromatography : High resolution capillary column Detection Mode : NPD
Quantitation Method : Internal St~n~rd Method Quantitation by : Peak Height Ratio Regression : Log polynomial Goodness of fit : The. correlation coefficients were better than or equal to 0.9990 Recovery : 87.2% at 4.97 ng/mL
: 88.1% at 79.44 ng/mL
: 87.4% at 248.25 ng/mL
Stability : Stable at room temperature in plasma for 8.5 hours : Stable in plasma after three freeze-thaw cycles : Stable in injection solvent for 8 hours ~ ~ 2148201 P12046.S01 w~-RUN PRECISION AND ACCURACY FOR

DANAZOL IN HUMAN PLASMA

Nominal Mean Value Mean + SD as % of Samples (pg/mL) (pg/mL) NominalCV% n A 2.032.05 + 0.233 100.8% 11.4% 17 B 4.974.90 + 0.547 97.5% 11.2% 24 C 79.4477.45 + 7.494 96.5% 9.7~ 25 D 248.25259.76 + 19.063 103.6% 7.3% 23 E 2.002.03 + 0.119 101.6% 5.9% 13 F 4.003.89 ~ 0.305 97.2% 7.8% 10 - G 10.0110.04 ~ 0.527 100.3% 5.3% 13 H 50.0551.38 + 4.164 102.7% 8.1% 11 I 100.1098.73 + 5.836 98.6% 5.9% 12 J 200.20203.65 + 11.877 101.7% 5.8% 11 K 300.30297.66 + 20.658 99.1% 6.9% 13 L 400.40400.72 + 18.352 100.1% 4.6% 11 / 21~8201 P12046.S01 SUMMARY OF THE ALPRAZOLAM ANALYTICAL METHOD
Matrix : Plasma Volume Required : 1.0 ml Anticoagulant : EDTA
Extraction Cartridge : Cartridge IX
Concentration range : 0.500 - 20.000 ng/ml Chromatography : High resolution capillary column Detection Mode : ECD
Quantitation Method : Internal St~n~rd Method Quantitation by : Peak Height Ratio Regression : Ln polynomial Goodness of fit : The correlation coefficients were better than or equal to 0.9920 Recovery : 72.1% at 1.406 ng/mL
: 72.9~ at 8.032 ng/mL
: 74.5% at 16.064 ng/mL
Stability : Stable at room temperature in plasma for 6 hours : Stable in plasma after three freeze-thaw cycles : Stable in injection solvent for 7.5 hours P12046.S01 BETWEEN-RUN PRECISION AND ACCURACY FOR
ALPRAZOL~M IN HUMAN PLASMA

Nominal Mean Value Mean + SD as % of Samples (pg/mL) (pg/mL) Nominal CV% n A 0.500.59 i 0.036 117.7% 6.1% 6 B 1.411.36 i 0.103 96.5% 7.6% 44 C 8.038.32 i 0.390 103.5% 4.7% 46 D 16.0616.12 i 1.017 100.3% 6.3% 46 E 0.500.52 i 0.018 103.6% 3.5% 23 F 1.000.95 i 0.055 94.6% 5.8% 22 G 4.003.98 + 0.164 99.6% 4.1% 23 H 6.006.19 i 0.268 103.2% 4.3% 24 I 10.0010.08 i 0.269 100.8% 2.7% 24 J 14.0014.14 i 0.424 101.0% 3.0% 24 K 18.0017.61 i 0.535 97.8% 3.0% 24 L 20.0Q19.98 i 0.580 99.9% 2.9% 24 21~8201 P12046.S01 SUMMARY OF THE VERAPAMIL/NORVERAPAMIL ANALYTICAL METHOD
Matrix : Plasma Volume Required : 1.0 ml Anticoagulant : EDTA
Extraction Cartridge : Cartridge IX
Concentration range : 2.00 - 500.93 ng/ml for Verapamil : 2.06 - 514.76 ng/ml for Norverapamil Chromatography : High resolution capillary column Detection Mode : NPD
Quantitation Method : Internal St~n~rd Method Quantitation by : Pea~ Height Ratio Regression : Ln polynomial Goodness of fit : The correlation coefficients were better than or equal to 0.9987 for Norverapamil/Verapamil Verapamil Recovery : 79.4% at 5.03 ng/mL
: 82.0% at 201.11 ng/mL
: 80.1% at 402.22 ng/mL
Norverapamil Recovery : 80.6% at 5.24 ng/mL
: 86.5% at 209.59 ng/mL
: 91.0% at 419.19 ng/mL
Stability : Stable at room temperature in plasma for 8 hours : Stable in plasma after three freeze-thaw cycles : Stable in injection solvent for 15.5 hours P12046.S01 BETWEEN-RUN PRECISION AND ACCURACY FOR
NORVERAPAMIL IN HUMAN PLASMA

Nominal Mean Value Mean + SD as % of Samples (pg/mL) (pg/mL) Nominal CV% n A 2.06 2.01 + 0.22397.4%11.1% 19 B 5.24 5.03 + 0.54295.~%10.8~ 20 C 209.59 209.00 + 9.77799.7% 4.7%- 20 D 419.19 417.65 + 20.471 99.6% 4.9% 20 E 2.06 2.34 ~ 0.125113.7%5.3% 5 F 4.12 3.48 + 0.16484.6% 4.7% 5 G 10.30 10.15 + 0.71698.6% 7.0% 4 H 51.48 52.88 + 2.762102.7%5.2% 5 I 102.95 109.01 + 7.033105.9%6.5% 4 J 257.38 276.20 + 29.383 107.3% 10.6% 4 K 463.28 419.40 ~ 13.141 90.5% 3.1% 5 L 514.76 530.49 + 21.214 103.1% 4.0% 5 21g8201 P12046.S01 w~N-RUN PRECISION AND ACCURACY FOR
VERAPAMIL IN HUMAN PLASMA
Nominal Mean Value Mean + SD as % of Samples (pg/mL) (pg/mL) Nominal CV% n A 2.00 1.81 i 0.163 90.7% 9.0% 19 B 5.03 4.89 i 0.479 97.2% 9.8% 20 C 201.11 202.33 i 8.786 100.6% 4.3% 20 D 402.22 399.10 + 16.536 99.2% 4.1% 20 E 2.00 2.11 + 0.157 105.7% 7.4~ 5 F 4.01 3.74 i 0.263 93.2% 7.0% 5 G 10.02 10.24 i 0.885 102.2% 8.6% 4 H 50.09 47.58 i 2.008 95.0% 4.2% 5 I 100.19 105.06 i 5.655 104.9% 5.4% 5 J 250.46 268.83 i 16.403107.3% 6.1% 5 K 450.83 434.13 i 11.390 96.3% 2.6% 5 L 500.93 490.50 i 13.163 97.9% 2.7% 5 `~ 2148201 P12046.S01 SUMMARY OF THE GLIPIZIDE ANALYTICAL METHOD
Matrix : Plasma Volume Required : 1.0 ml Anticoagulant : EDTA
Extraction Cartridge : Cartridge III
Concentration range : 15.0 - 1000.0 ng/ml Chromatography : High resolution capillary column Detection Mode : NPD
Quantitation Method : Internal StAn~Ard Method Quantitation by : Peak Height Ratio Regression : Log Polynomial Goodness of fit : The correlation coefficients were better than or e~ual to 0.9950 Recovery : 82.9% at 800.0 ng/mL
Stability : Stable at room temperature in plasma for 8.5 hours : Stable in plasma after three freeze-thaw cycles : Stable in injection solvent for 8 hours ` ..

P12046.S01 The chromatograms for each example show that the injected extracts are extremely clean, thus allowing relatively short run times for the GC which translates into high efficiency.
The extraction process according to the present invention is accurate at low levels of quantification and has good between run precision. Also, the process allows for fast extraction with resulting short processing times. The process further provides extracts with very low background contamination. Since there is so little background contamination, chromatography run times are drastically reduced from an average of 10-20 min per sample; for conventionally prepared samples, down to 3-5 min/sample. As an example, for the druq danazol, the average run time was reduced from greater than 20 minutes to about 3 minutes.
Additionally, the extraction process, according to the present invention, can be controlled by an experienced analyst when working with small numbers of samples. However, when working with typical bioanalytical batch sizes of 60-120 samples, it is difficult for the analyst to keep track of and adhere to the timing required for each addition of solvent for each cartridge. However, the process is well suited to automation, thereby resolving timing control, while permitting highly precise automated liquid handling and pipetting, and unattended extraction that frees the analyst for performing other work.
Accordingly, another aspect of the present invention is an automated processor system 100. As shown in Fig. 18, the processor system according to the present invention comprises an input extension unit 680, a main processing platform 50, an ou~u~ extension unit 84, a robotic arm sampling system 40, a plurality of pumps 30, a plurality of syringe pumps 31, a wash station and internal standards rack 60, a keyboard 90 with a display 95. The automated processor system is shown in Fig.

._ P12046.S01 18 with two racks 72 which hold the cartridges according to the present invention.
As illustrated in Fig. 19, the rack 72 includes an upper rack 70 and a lower rack 71. The upper rack 70 comprises a plurality of plates, such as two plates 73, 74 which are connected by a plurality of posts 75. Each of plates 73, 74 contains a plurality of openings 76 into which cartridges 12 are placed. The openings are constructed and arranged so that the cartridges are held in the upper rack in a substantially vertical orientation.
The top rack 70 is fitted onto the lower rack 71 so that it is positioned over the back portion 77 of lower rack 71.
Lower rack 71 comprises a plurality of plates. Preferably, the lower rack 71 includes upper plate 78, middle plate 79 and lS lower plate 80. Each of the upper, middle and lower plates includes a plurality of openings 82a and 82b for holding collection tubes 110 and sample tubes 115, respectively. The openings 82a and 82b are constructed and arranged so that the collection and sample tubes are substantially vertically oriented. In particular, the collection tubes 110 are positioned in openings 82a in the back portion 77 of lower rack 71 underneath each of the cartridges 12 positioned in the upper rack 70 to collect the eluate that includes the extracted drug and solvent for a correspon~ing cartridge.
Sample tubes 115, for holding the biological fluid containing the drug to be extracted, are positioned in the openings 82b in the front portion 78 of the lower rack 71.
The lower rack 71 also comprises bottom plate 81, which, as will be described below, engages the top surface 212 of elongated member 208, which is shown in Figs. 20-22, so that the elongated member can lift rack 72, move the rack forward and place the rack back down on the working surface 51. The upper rack 70 can hold any number of cartridges 12 with a P12046.S01 corresponding number of collection tubes 110 placed below the cartridges. Preferably, the rack 72 is capable of holding ninety six of each of cartridges 12, collection tubes 110 and sample tubes 115, with the lower rack 71 having 12 columns and 16 rows of openings, and the upper rack 70 having 6 columns and 16 rows of ope~ings. Preferably, the length of the rack is seventeen inches.
Figs. 20-23 illustrate the main processing platform 50.
As shown in Fig. 20, the main processing platform includes a w~lking beam 250 including a motor 200 that drives a pulley 201 attached to a belt 202, which in turn drives a plurality of shafts 203 using pulleys 205 and belt 206. Preferably, the motor operates at approximately 6 rpm. Positioned at each end 224 of shafts 203 are pulleys 207. Also positioned on shafts 203 are cams 216 onto which are mounted locking pins 215.
Both sides of the elongated member 208 are eccentrically mounted on pulleys 207. As shown in Figs. 20 and 22, preferably two elongated members 208 are mounted on pulleys 207 at each of the ends 224 of shafts 203. As shown in Fig.
22, the elongated member 208 is preferably L Ch~pe~ with substantially horizontal portion 209 being substantially perpendicular to substantially vertical portion 210. The top surface 212 of elongated member 208 engages the bottom surface 84 of bottom plate 81, as shown in Fig l9a, to lift the rack 72, move it forward and set it back down on the working platform 51.
The top surface 212 of elongated member 208 and the bottom surface 84 of the rack 72 can be constructed and arranged in any manner that enables the rack to be lifted and moved forward. In particular, while the top surface 212 of the elongated member 208 and the bottom surface 84 of the rack 72 can be constructed with smooth surfaces, it is preferred that at least one of the surfaces include a construction so as "

P12046.S01 to increase friction between the elongated member and the rack. For example, the top surface 212 can include a plurality of protrusions 211 thereon, positioned so as to contact the bottom surface 84 of the rack. Also, the bottom surface 84 of the rack can include openings 411 or recesses 412 corresponding to the protrusions 211, which are illustrated in Fig. l9a. The protrusions and openings can be reversed, so that the protrusions are located on the bottom surface 84 of the rack; however, it is preferred that the protrusions be on the elongated member.
Further, at least one of the top surface 212 or the bottom surface 84 of the rack can be roughened, such as partially illustrated at 410, or include a high friction surface, such as a rubberized surface, to increase the friction therebetween.
As shown in Fig. l9A, the bottom plate 81 includes a - plurality of locking op~in~s 83. The locking openings 83 are dimensioned to receive pins 215 of elongated member 208, shown in Fig. 20, so as to hold a rack 72 in a set position. In other words, the pins 215 and the locking openings 83 cooperate to maintain the rack in indexed positions, as the rack is moved row-by-row along the working surface 51.
Preferably, the bottom plate 81 includes fifteen locking openings 83 equally spaced one inch apart.
As shown in Fig. 23, each elongated member 208 is positioned under slot 214 in the working surface 51. The walking beam 250 moves the rack 72 a precise, predetermined amount along the working surface 51 by turning one revolution, i.e, the motor 200 and the attached pulleys, belts, shafts and elongated members move in one revolution increments. Each revolution moves the rack 72 a predetermined amount and thereby moves another row of cartridges 12, collection tubes llO and samples tubes 115 toward and underneath the robotic - 2l982ol -P12046.S01 sampling system 40 to position another row of cartridges for receiving a sample to be extracted therein.
The locking pins 215, which are preferably about 0.25 inch in diameter, can include an upper portion 231, are raised above the working surface 51 through apertures 230 to engage the locking openings 83 of Fig. l9a of the rack to lock the rack in position. The locking pin 215 is constructed and arranged to protrude through the working surface 51 to the extent nececc~ry to cooperate with locking op~nings 83 to maintain the rack 72 in a set, indexed position. For example, the locking pin 215 can protrude into the locking opening 83 or can go through the op~ning. In a preferred embodiment, locking pin 215, protrudes 0.75 inches above the working surface.
The locking pin 215 is constructed so that it is capable of setting the position of the rack at the end of one revolution of the walking beam. The upper portion 231 of locking pin 215 protruding above the surface of the working area 51 is constructed and arranged to ensure that the locking pin fits tightly into the locking opening 83, and also smoothly releases the rack before the next forward movement takes place. Furthermore, the upper portion 231 of locking pin 215 should have a preferred profile to be capable of correcting small misalignments of the rack without jarring or jamming the rack. For example, upper portion 231 preferably has a tapered shape to provide ease of insertion and release, with precise positioning of the rack.
The height of locking pin 215 and the shape of cam 216 determines the height of the locking pin with respect to the working surface 51. In this regard, cam 216 can have a pear-sh~p~ structure so that, as shaft 203 rotates, portion 216a and 216b alternatively co~e into contact with lower end 215a of locking pin 215. When portion 216a is in contact with the P12046.S01 lower end 215a, the locking pin is in its raised position and when portion 216b is in contact with the lower end 215a, the locking pin is in its lowered position.
The operation of the walking beam to advance the rack by one increment is described as follows. At the beginning of a revolution, the elongated members 208 are positioned below the working surface 51 and locking pin 215 is positioned above the working surface 51 in a locking op~n~nq 83. As the shafts 203 start to rotate, locking pin 215 moves down, and, at the same time, the elongated members 208 move up above the working surface 51 through slots 214 so that the upper surfade 212 engages the bottom surface 84 of rack 72. As the shafts 203 continue to rotate, the elongated members 208 raise rack 72 and move the rack 72 forward a precise, predetermined amount, i.e, one increment. As the revolution of shafts 203 continues, the elongated members 208 lower rack 72 back down onto working surface 51, returning through slots 214 below working surface 51 and the locking pin 215 moves up to lock the rack in the new position by entering a locking opening 83.
At this time, rotation is stopped, and the revolution for moving one increment is complete.
The dimensions of the elongated members, the pulleys and the location of the elongated members on the pulley determine the distance the rack is moved with each revolution.
Preferably, the elongated members are approximately 41 inches long and 2.5 inches in height. If two elongated members are used they are preferably spaced apart approximately 7.25 inches.
Further, the pulleys 207 preferably include a larger portion 221, approximately 2.25 inches in diameter, onto which the walking beam is mounted and a smaller portion 220 approxima~ely 1.5 inches in diameter. A hole 222, preferably 0.25 inches in diameter is located approximately 1.875 inches ^ - 21~8201 P12046.S01 below the top surface 212 of the elongated member for attaching pulley 207 by attachment means 223. The attachment means can be any attachment means known in the art such as screws, bolt and nut or welding. The specific dimensions cited above will result in a movement of the rack of approximately one inch when the wAlk~ng beam completes one revolution.
These dimensions can be varied d~p~nAing on the amount of movement desired.
Also included in the working surface 51 is a plurality of proximity sensors 217, that sense the position of the rack on the working surface. The proximity sensor can be any position sensor known in the art. For example, the sensor can be a mec~An~cal switch positioned above the working surface 51 that is pushed down by the rack as it reaches the position of the sensor thereby indicating the position of the rack and to thereby control stopping of the revolution. The sensor can - also be a photoelectric sensor that is capable of detecting one revolution of the walking beam and automatically stopping the walking beam. This photoelectric sensor includes a wheel with a 0.25 inch diameter opening which allows a light beam to strike the photocell at a predetermined position in the wAlkin~ beam cycle. A preferred sensor is an electromagnetic proximity detector available from lu~CK Inc., Minneapolis, Minn.
As shown in Fig. 22, the motor 200 as well as the components associated therewith are preferably mounted to the lower surface of the working surface 51 by frame 213 using any attachment means known in the art. For example, the motor can be attached using screw, nuts and bolts, or welding.
The main processing platform can also include a plurality of idlers 218 positioned in the working surface for supporting the rack. Preferably, idlers 218 are approximately 1.5 inches in diameter. The rack 72 is guided on the working surface 51 P12046.Sol of the main processing platform 50 by two parallel angles 219.
The idlers are attached to the platform using any attachment means known in the art.
More specifically, the processor system according to the present invention can also include an input extension unit 680 for automatically feeding a rack 72 onto the main processing platform S0, and an uu~uL extension unit 84 for removing the proc~c~^~ rack from the proceCcor system. As shown in Fig.
24, the input extension unit 680 includes a conveyor belt 681 and a geared down 12 rpm drive motor 682 that drives the belt through pulleys 683. The unit also contains a proXimity sensor 217 to sense the rack position. The power and control signals are connected to the computer the main unit by two separate connectors 85, 86. Similar to the main processing platform 50, the input extension unit 680 can also include a plurality of idlers 218 positioned within the surface 87 over which passes the rubber conveyor belt 681. The rack is placed on the rubber conveyor belt 681, which is preferably a rubber timing belt. The rubber timing belt provides enough friction to drive the rack onto the main processing platform.
Preferably, idlers 218 are approximately 1.5 inches in diameter. The rack 72 is guided on the input extension unit 680 by two parallel angles 219. The input extension unit is positioned against the main processing platform with proximity detector 217 positioned toward the main processing platform.
The ouL~L extension unit 84 is preferably substantially identical to the input extension unit, and so as not to be unduly duplicative, the output extension unit is not illustrated in the drawings. In this regard, any ~i~c~l~sion and/or illustration of the input extension unit can also be considered to be a discussion and/or illustration of the ouL~uL extension unit. The output extension unit is -P12046.S01 positioned with the proximity sensor 225 away from the main processing unit.
The plurality of syringe pumps 30, 31 used in the present invention can be any commercially available liquid pumps known in the art. All actions of the syringe pumps and the movement of the automatic sample processor are controlled by a computer system, which will be described below. The computer system links the two syringe pumps and the movement of the automatic sample processor to allow the complete extraction process to be executed continuously and repeatedly.
As noted above, the ~lO~eSS of extraction involves the transfer of precise volumes of liquid. For example, the extraction process begins with the transfer of an internal stAn~A~d, preferably 0.1 ml, from an internal stAn~Ard reservoir 63 to each cartridge in a row of six. This is followed by the transfer of 1.0 ml of sample from a sample tube to the co~LL_~onding cartridge in that row and finally the addition of 5 or 6 volumes of about 1.5 ml of a solvent or solvent mixture to each cartridge at predetermined time intervals.
The transfer of precise volumes of liquid is accomplished using a number of highly specialized syringe pumps. This type of pump is capable of delivering accurate, reproducible amounts of liquid and it can both aspirate and dispense. It is also readily adaptable to different volume requirements, i.e., 50 microliters to 25 ml per full stroke, by simply changing the size of the syringe. Furthermore, the volume dispensed or aspirated is only little affected by the type or viscosity of the liquid to be pumped. This type of pump consists of an accurate stepper motor which moves the plunger in the calibrated syringe barrel at controlled speeds over defined distances resulting in aspiration or dispensing of an accurate volume of liquid. Examples of commercially available 21~8201 P12046.S01 syringe pumps that are useful in the present invention are manufactured by Cavro (Cavro Scientific Instruments, Inc., Sunnyvale, CA 94089).
Specifically, two Cavro type XL3000 modular, single channel pumps (type 724024) are used to transfer the sample, water for washing and internal stAn~Ard. These two pumps are connected by flexible plastic tubing, preferably PTFE or PFA, to the sample probe 48 held in z-rack 46 of Arm I 41a of the robotic sample processor 40, shown in Fig. 25a. One of the single channel pumps is equipped with a syringe of 1 ml capacity, and performs all aliquoting steps involving samples and internal s~An~Ards. The other single channel pump has a capacity of 10 ml, and provides water for the washing process required between any two dissimilar samples handled lS consecutively by the probe 48.
As shown in Fig. 26a, washing is done in the wash station 62. The rack 61 is mounted on the working platform just next to the wAl~ing beam but is still accessible by the robotic arms. A cut-away view of the wash station is shown in Fig.
26b. During washing the probe 48 is placed in cavity 66 having a preferred depth of about 1 inch, and approximately 5-10 ml of water ml is pumped through probe 48 to wash both the inside and outside of the probe 48. As the wash fluid is pumped into the cavity 66, the wash fluid overflows over the top edge 66a of the cavity 66 into the annular chamber 66b and down through opening 66c into tube 64. In this regard, the probe should be inserted into cavity 66 at least to the depth that the probe is inserted into the sample or solvent to ensure proper washing action of the external surface of the probe. Reservoir 67 contains reagent water, such as deionized water, to be used in the process. The rack includes a plurality of internal stAn~Ard reservoirs 63.

~ 21 1820~
P12046.S01 The elution solvent is handled by a four-channel syringe pump which consists of four syringes driven by the same stepper motor. A commercially available four-channel pump is the Cavro XL3004, type 724510. Each syringe dispenses and aspirates through a three-way solenoid valve. This allows the choice of one of four solvents to be delivered to any cartridge. The four syringes are connected to one of the four stainless steel dispensing probes 49 in the z-rack 46 of ARM
II 41b, which are shown in Fig. 25b, by four independent plastic tubes (preferably PTFE) 44. The probes are held in place in the z-rack 46 by two plastic inserts 500 at ~ither end of the rack. These inserts have four holes through which the probes 49 pass. The probes can be made of Type 304W
stainless steel tubing with an outer diameter (O.D.) of 0.058 inches to 0.120 inches and corresponding internal diameter (I.D.) of 0.042 inches and 0.094 inches; preferably with an O.D. of 0.083 inches and I.D. of 0.063 inches. This arrangement allows consecutive dispensing of different solvents without the need of washing the probe and plastic tubing between operations. This leads to a reduction in processing time, a crucial considera'ion for high speed equipment.
The twin arm robotic sample processor 40, Fig. 25a, is a highly sophisticated handling system with mobility in the X, Y and Z directions. It can be directed through its own computer system to position the probes 48 and 45 at any position within its working span with extremely high accuracy.
Appropriate equipment is supplied by Cavro Scientific Instruments, Inc., Sunnyvale, CA, 94089. In particular their model RSP9652 is used for this invention. This model has an accuracy over travel of + 0.02 mm along the X axis, + 0.015 mm along the Y axis, and + 0.015 mm along the Z axis. This processor consists of two independently movable arms 4la and P12046.S01 41b mounted on the X-frame 42 by X-slide 43. Each arm 41a and 41b carries a Z-rack 46 which moves up and down (Z direction) and backwards and forwards (Y direction). To this is attached a sample probe 48 and an insulation block 47 which ensures that probe 48 is not electrically connected to the Z rack.
This allows the probe to detect the level of a conductive liquid through a capacitance device supplied with the proceCFor 40. If the probe finds either no sample or not enough sample in any sample tube, an error message is generated and no further ~oc~æsing of that sample tube and the co~e_~-o..-l;n~ extraction cartridge takes place. Further, the robotic arm is e~pr~ with a self-diagnostic routine that sets the position of the probes when the apparatus is turned on.
Still another aspect of the present invention is the control of the processing system. The control of the processing system can be performed by a software-controlled computer system. In accordance with a particular embodiment of the present invention, the computer system comprises a dual-microprocessor system. The schematic chart in Fig. 27, illustrates an example of an embodiment of the hardware of the computer system according to the present invention. The system is divided into a master system 303, including master mic~o~LoceCcor 300, which directly controls the operation of the robotic arm sampling system 40 and the liquid handling system 318, which includes a plurality of syringe pumps 30, 31, and a slave sub-system 304, including interface board 310, which controls the operation of the keyboard 90, display 95 and rack mobility system 305. The rack mobility system 305 includes the motor 200, elongated members 208 and proximity sensors 217 of the main processing platform 51, as well as the motor 82, conveyor 81 and proximity sensor 217 of the input and output extension units 680, 84.

P12046.S01 The master system can be implemented using any known personal computer including any known microprocessor, such as an IBM compatible PC. The slave sub-system may be implemented using any microprocessor known in the art, such as an 8-bit microprocessor. As shown in Fig. 27, master system 303 may be implemented with a personal computer having a 80386 class microprocessor 300. The 80386 microprocessor is directed through software to control the operation of the robotic arms 40 and the liquid handling system 30, 31. Commands are transmitted and received between the master system and the robotic arms and liquid handling system using ~tA~rd RS-422 communications protocol; these commands are available from CAVRO Inc.
As show in Fig. 27, the slave sub-system 304 comprises an interface board 310 including a miC~ o~ec~or 311. The illustrated board coLle~onds to a board produced by Chip - Mi~Lv_y~ems (Part No. PCB 1500) and includes a Hitachi 64180 microprocessor, which, as noted above, controls the operation of three devices: a keyboard 90, display 95, and a rack mobility system 305.
The slave sub-system microprocessor 311 may be configured so that is not multi-tasked, but rather is fully dedicated to monitor its serial interface for commands iscllP~ by the master. As each such command is received, sub-system microprocessor parses and executes the command in turn. Once each command is executed, a two character value is returned to the master to indicate the outcome of the execution. The master, in turns, takes an appropriate course of action.
The display 9S can comprise any known display capable of displaying alphanumeric information, e.g., being input by keyboard 90. By way of example, the display can comprise a 4 x 20 LED display with full buffering support. It displays - 21~8201 P12046.Sol system information, and messages, and serves as the keyboard's visual verification unit.
The keyboard 90 may comprise any keyboard capable of inputting the alphanumeric information n~e~ to specify all needed information. By way of example, the keyboard can comprise a 64-key ASCII, non-repetitive unit. The slave sub-system 304 should be configured to provide a buffer 315 with the capacity of 2048 characters or more for the keyboard.
The two microp~ ors communicate with each other using a ~t~nA~rd RS-232 (307) communication protocol, via a suitable channel r~nn~n~ at 9600 Baud or faster.
In addition to the mi~Lo~ æror 311, the interface board 310 also includes a universal asynchronous receiver transmitter or UART 312 to implement the RS-232 protocol.
Further, the interface board includeæ RAM 313 and ROM 314.
The keyboard, screen, and rack mobility system are all memory - mapped. That i8, a region of memory is assigned to each of ~- these devices so data can be acc~~~6~ at these assigned memory .
addresses. The memory mapping iæ implemented in part by input/G~L~ ad~ logic 315. The interface board also includes a screen buffer 316 and a keyboard buffer 317 each of which is reserved within RAM 313, for storing data that is sent to and from the ~creen and keyboard ~~p~ctively.
Fig. 28 is a block diagram of the method used to operate the computer co.~Llol system according to the present invention. m e process 400 that operates on the master mi~vp~J~-C-l-or is written in a High Level Language (HLL) that uses a Pascal-like syntax and encourages stru~Lu~ed programming. The ~LcyLamming environment is available from CAVRO Inc., the manufa~ e~ of a robotic sampling system useful in the pl.-~t invention. The ~Gy-amming environment includes a library 401 comprising about 150 functions divided into lo y ~ that allow the software engineer to issue - 21g8201 P12046.S01 commands to the above mentioned subsystems. The method al80 provides means of communicating with the outside world through its I/0 library functions using the system's serial communication port. It is through this serial channel that the master system establishes a link with the interface board of the slave sub ~ em.
Drivers 402, 403 actuate the arms and pumps, re~e~lively, in ~-p~n^? to commands sent by the master system. The drivers 402, 403 are commercially available driver~ that are provided with commercially available robotic arms and pumps. The preferred drivers are part of the robotic arms and pumps supplied by Cavro Scientific I~ uments, Inc., as previously described.
As noted above, the HLL process that controls the function of the maæter mi~Lo~ r communicates with the interface board by s~an~rd communications protocol, for - example RS-232. As shown in Fig. 28, the interface board control~ the rack mobility system, the keyboard, and the e~l using a string command hA~ protocol 404.
The rack mobility system, the keyboard and the screen all ~-~pQn~ ts commands sent by the slave sub _y~em to their ~e~ tive drivers, 405, 406. An example of the drivers useful for co"L~olling the rack mobility systém, the keyboard and the ~creen are available from Chip Mi~ o~Lems.
The method controls the operation of numerous functions. For example, the method ~u~ ols the exe~Lion of the extraction ~lo~es_, known as the production run, which is capable of processing one rack or multiple racks: it also controls the development of additional extraction methods, known as the method development run. Furthermore, a number of utilities are available which include automatic back-up, reprinting of run reports, network support for remote installation and debugging and calibration of the liquid . 2148201 P12046.Sol handling system. The calibration process, production process and method development proces~ will be described in detail below.
- Calibration ProcesR
Prior to using the automatic processing system, it is preferred that the system be calibrated to assure accurate delivery of samples, internal stAnA~rds and solvents by measuring the amount of liquid that is delivered by each of the syringes in the robotic sampling system. The schematic diagram shown in Fig. 29, illustrates a preferred method of calibrating the automatic ~ e__ing system using the computer col.~,ol system. The calibration process begins, as the user places a balance on the working area~ Ihe balance, which can be any known deviae that ao~ ely measured a quantity of liquid, interfaces with the computer u~ing any well known computer interface, such as a serial interface.
A syringe is selected for calibration and an operator - instructs the system to start the calibration process. A
predetermined amount of fluid is delivered from the chosen syringe onto the balance and the results of the measurement are ,~_o~ded by the process. m is operation is repeated at least ten times. A ~ is ~ ted which analyzes the - results by st~n~-rd statistical methods. If it A~re~rs that the syringe i~ not delivering an a~ e amount of fluid, a troubleshooting guide i6 available for help in determining what steps to take to adjust the amount of fluid being delivered by the syringe. If another syringe is to be calibrated, the process is repeated, if not, the balance is removed and the system is ready for G~ ion.
Production Process For A Sinqle Rack The production process for ~Gc~__ing one rack is illustrated by the flow chart Fig 30. The production proces~
is divided into Arm 1, which describes the ~ovement of the _59 _ P12046.Sol rack and the addition of internal st~n~rd and sample to the extraction cartridge, and Arm 2 which describes the operation of the solvent addition system.
Arm 1 of the production program begins at step 450 wherein a rack 72, is loaded by an operator with extraction cartridges 12 and a corre~po~ding number of sample tubes 115 and collection tubes 110, is placed on either the input extension unit 680 or directly on the main processing platform so -At step 451, the rack is advanced to the start position 80 that the first row of cartridges, samples and extraction tubeæ is ~cce~cible to the robotic sampling system 80 that the addition of in~er~Al st~ rd and samples can begin. This o~ in the following manner. First, if the input extension unit i8 used, the proximity sen-or 217 in the input extension unit 680 ~ends a signal to the computer which ~ignals the - motor 682 to drive the col,~eyor 681 to move the rack 72 onto the working surface 51 of the main p~ ing platform 50.
When a proximity sensor 217 in the main pro~e~sing platform 50 detects the p. -~nce of a rack 72, the motor 682 driving the rack 72 from the input extension unit 680 stops. The proximity rQn~Qr 217 sends a signal to the computer which signals the motor 200 to move the wA~ g beam 250 a - sufficient number of revolutions so that the first row of cartridges 12, collection tubes 110 and sample tubes 115 is positioned underneath the robotic sampling system 40.
The next step in the production ~Icylam 452 is the selection of an internal stAnAArd for the samples that are in the rack. This is done by the operator who inputs the instructions as to which internal st~n~rd to use. Also, the operator inputs which solvent to deliver by the solvent addition system, which is activated automatically by the process.

, ,, 21~8201 -P12046.S01 The addition of internal st~n~rd and sample to the extraction cartridge begins at step 453 with the addition of an internal s~nA~rd to each cartridge in the first row. The first pipette 48 moves to an internal stAn~rd tube 63 which S holds the internal st~n~rd, whose type has been inputted into the computer, and picks up a predetermined volume of internal stAn~srd. The first pipette 48 then moves above the first row of cartridges 12 and deposits the internal standard in each cartridge. The first pipette 48 then moves to the wash station 62 where it is tho~ ly washed.
The next step in the production process 454 is the pickup of the sample from a sample tube and the dispensing of the sample into the cartridge. This is done by the first pipette 48 which moves to the first sample tube in the first row where it picks up the ~ample, moves to the first cartridge in the row and deposits the sample. The ~ e_s of picking up a sample and depositing it in a cartridge is repeated along the first row until each of the samples is deposited in their co}~ o~ ng ca~tridges. The first pipette 48 moves to the wash station 62 and is washed between each sample addition.
After the first row is complete, the process, at step 455 determines if there are more rows of samples to be depoited into cartridges. If the answer is yes, as shown in the flow chart at step 456, a signal is sent to the computer which signal~ the motor 200 to move one revolution and thereby index the rack forward one position so that the next row i8 under the robotic sampling system 40. The next row is ~LGce- -od in the same manner and Arm 1 of the production process continues until all of the rows have been completed. When there are no more rows that need to have internal standard and samples added to the extraction cartridges, Arm 1 of the production process is complete.

21~8201 P12046.Sol Operating concurrently with Arm 1 is the solvent addition system 457. This system tracks the time that has elapsed from -~ when the sample is placed in the cartridge as well as the time between each addition of solvent. As was described above, both of these times are important to obt~i n~ ~g a complete extraction of a particular substance from the sample. Unlike the addition of the internal s~nA~rd and sample, the addition of solvent is not completed on a row by row basis. As will be described below, the process includes a time matrix that tracks the time each sample is added to the cartridge and the time be~ee.. each solvent addition. Accordingly, if a sample in the first row needs its fourth solvent addition, and the next sample that needs a ~olvent addition is in the third row, the computer is capable of tracking both of these times and instructing the solvent system to add solvent accordingly.
For example, as the first sample is added to the - cartridge, t~.e time matrix in Arm 2 is checked at step 458 to determine when a solvent addition is due in the cartridge ;-~, which is illustrated at step 459. If the answer is yes, step 460 adds solvent to the cartridge in the following manner. A
signal is sent from the computer which a-~ivates ~eCQn~
pipette 45, to add a predetermined amount of solvent to the cartridge. The time matrix is updated at step 461 to start counting the next time interval for the next addition of solvent. At step 462, the computer determines if more solvent additions are n~e~, if yes, Arm 2 con~n~es by updating the time matrix and determining the next sample that needs a solvent addition. If no more solvent additions are required, a report is printed at step 472 and the process is completed, ending the production run.
As noted above, the processing of each row, including placing the samples into the cartridges by first pipètte 48 and addition of the solvent by second pipette 45 continues P12046.Sol simultaneously until the entire rack 72 is processed with the addition of sample, internal s~n~rd and fiolvent. When the processing of the rack is completed, the process instructs the - system to remove the rack from the main pLocs-~ing platform, by ~en~i ng a signal to the motor co~ olling the conveyor on the ou~ extension unit 84 to move the rack off the main processing unit so and onto the o~u~ extension unit 84. An operator then removes the rack for further processing of the collection tubes, as well as recycling of the cartridges.
Production Proce~8 for Multi~le Racks Fig. 31 i8 a flow chart of the production process for multiple racks. The production program for multiple racks is substantially similar to the production process for a single rack with the addition of a ~QI~ ent process 500 that determines if another rack is ready for ~ ing. At ~tep 501, a proximity ~^cr sends a fiignal to the computer if another rack has been placed on the input extension unit. If the answer iæ yes, the information about this rack is manually entered into the computer at s~ep 502, and a flag, Rack-Ready, is ~et at step 503 indicating that another rack is ready to be pro~er~^~. At 6tep 504 the computer ask~ if the flag PGM_l_END, which will be described below, has been set, if the answer is yes, than no additional rack is ready for processing and the proce~s ends.
As noted above, the co~ ent process fiets a flag if another rack ifi ready to be ~- 3~_ -e~ . At step 505, the computer afiks if the flag Rack Ready has been set. If the answer i~ yes, then the processing of the next rack starts by advancing the rack to the start position when the internal fit~ rd and sample addition for the last row of the rack has been completed. If the answer is no, the co~puter, at step s06 sets the flag PGM 1 END, which is used during the P12046.Sol concurrent process, as described above, and Arm 1 is completed.
So as not to be unduly duplicative, the remaining steps of the production development process for multiple racks will not be described because they are substantially identical to the steps of the production process for a single rack which are described above.
Method DeveloPment Process Fig. 32 is a flow chart of the method development proce~s. The method development process allows a carefully designed, complex e~ iment to be carried out automatically.
Normally, an analyst must determine the best cartridge, solvent and internal s~n~rd to be used to extract a particular drug, through a pains~king series of trial and error experiments. The method development process can complete this task in a matter of hours.
As is shown in Fig. 32, the method development process is substantially similar to the production process for a single ~- rack illustrated in Fig. 30. The difference LeL~ell the method development process and the production process for a single rack is step 480. At this step, instead of imputing an internal ~t~ rd and solvent that are known to be capable of extracting the drug, the operator inputs a series of combinations of intern~l st~ rds and solvents.
For example, if the apparatu~ is e~lirr~ to deliver four internal stAn~rd and four solvents, it is possible to test sixteen different combinations of solvent and internal st~n~rd on the same drug to determine which combination pr~A-lcec a complete, clean extraction for a particular cartridge. Furthermore, the same combination of solvents and internal st~n~rds can be applied to a number of different cartridges to determine which cartridge is best suitèd for extracting a particular drug.

. .
-P12046.Sol So as not to be unduly duplieative, the remaining steps of the method development process will not be described because they are substantially identical to the steps of the production process whieh are deseribed above.
Any manual input of data to the automatic processing system ean also be aeeomplished automatieally by using well known automatie data entry systems. For example, the data ean be inputted using a bar eode indicating the information cooperating with any automated bar eode reader.
As has been deseribed above, the present invention ineludes among other fea~uue_, an extraetion eartridge, a method of extraeting subst~neefi from fluids, and an automatie p~G~e_-ing system, whieh includes an apparatus for automatieally performing an extraetion ~lGCeSS and a eomputer system for eontrolling the automatie ~Gce_sing.
The y~o~ ring ~ystem aeeording to the present invention has the following advantages. me locking pins ensure absolute positive and ac~ e registration of the sample rack. The finite movements of the beam and rack allow the eomputer to ealeulate the exaet position of eaeh sample tube and provide a~y.v~riate instruetions to the robotie arms.
Careful design of the eam eG..~olling the positioning of the raek ean minimize the possibility of spi-lling sample or extraet. m is is done by graded aeeeleration at the beginning of a raek movement and ~etting it gently baek down despite the eonsiderable weight of a fully loaded raek.
Further advantages of the p w~ssing system aeeording to the present invention are that the apparatus is simple and reliable. The w~lki~g beam allows precise, reprodueible positioning of the sample using locating pins, and ready identifieation of the exaet position of a sample after any number of raek movements. me system is fully automatèd with eomplete synehronization between rack movement, position of 21~8201 P12046.S01 the two robotic arms and the liquid handling system. The processing system allows for the extraction of over 140 samples per hour.
Still further advantages of the processing system according to the present invention include the ability to produce extracts biological fluids that are clean enough for direct injection in HPLCs, GCs and for some drugs directly into mass spectrometers without prior chromatography. The processing system is a single dedicated system with high throughput. The system~ provides a range of preprogrammed methods adequate for the extraction of most drugs a~d the ability for the user to rapidly develop methods for other drugs. Once actuated the system is fully automatic and operates unattended. Additional racks of samples, which can contain different drugs, are continuously processed. The system included built in detection of errors and problems, a calibration routine and a self-diagnostic routine. All working parts are readily accessible for easy service and the apparatus is designed to be resistant to solvent spills.
The automated extraction process according to the present invention has numero--s advantages when compared to solid phase extraction or liquid-liquid extraction. Solid phase extraction may include conditioning of the extraction cartridge, manual measurement and transfer of sample, internal stAnA~rd, buffer and solvent, an organic wash step and vacuum or pressurization. Liquid-liquid also may include manual measurement and transfer of sample, internal st~n~rd, buffer and solvent and an organic wash step as well as shaking and centrifugation of the sample. In contrast, the automated extraction process according to the present invention does not require a conditioning step, provides automatic sample, internal standard and solvent transfer, does not need measurement of buffer, and does not need vacuum, 21~8201 -P12046.S01 pressurization, shaking, or centrifugation. The extracts produced by the present invention are cleaner with a higher percent of drug recovery than extracts produced by liquid-liquid or solid phase extraction.
While the invention has been described with reference to several exemplary embodiments, it is understood that the words which have been used herein are words of description and illustration, rather than words of limitations. Changes can be made, without departing from the scope and spirit of the invention in its aspects. The invention extends to all functionally equivalent structures, methods and uses, such as are within the scope of the appended claims.

Claims (110)

1. An extraction cartridge, comprising:
a barrel comprising an upper opening for feeding of a fluid to be extracted and a lower opening for removal of treated fluid; and a multilayer packing composition within said barrel, the multilayer packing composition comprising:
a buffer layer comprising a buffer material for adjusting pH of the fluid to be extracted; and a partitioning layer positioned below said buffer layer, said partitioning layer comprising a material for spreading a fluid into a thin film.

2. The cartridge according to claim 1, wherein said barrel comprises an inert material.

3. The cartridge according to claim 2, wherein said inert material is selected from the group consisting of glass, ceramic and metal.

4. The cartridge according to claim 3, wherein said inert material is glass.

5. The cartridge according to claim 1, wherein said buffer material comprises particles.

6. The cartridge according to claim 5, wherein said particles comprise a particle size of about 100 to 1000 microns.

7. The cartridge according to claim 6, wherein said buffer material comprises at least one member selected from the group consisting of oxalic acid, tartaric acid, citric acid, succinic acid, ammonium dihydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, aluminum sulphate, ammonium oxalate, diammonium hydrogen phosphate, sodium bicarbonate, potassium bicarbonate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, borax, tris(hydroxymethyl)methylamine, sodium carbonate, potassium carbonate, trisodium phosphate and tripotassium phosphate.

8. The cartridge according to claim 1, wherein said buffer layer comprises buffer material and an inert material.

9. The cartridge according to claim 8, wherein said inert material comprises particles of inert material.

10. The cartridge according to claim 9, wherein said particles comprise a particle size of about 200 to 3000 microns.

11. The cartridge according to claim 10, wherein said inert material is at least one member selected from the group consisting of crystalline silica, glass beads, metal balls, ceramic beads, crushed rock, washed sand and diatomaceous earth.

12. The cartridge according to claim 11, wherein said inert material comprises crystalline silica.

13. The cartridge according to claim 9, wherein said inert material is heated at a temperature of about 500°C for about 3 hours.

14. The cartridge according to claim 9, wherein said buffer material comprises particles of buffer material.

15. The cartridge according to claim 14, wherein said particles of buffer material comprise a particle size of about 100 to 1000 microns.

16. The cartridge according to claim 15, wherein said buffer material comprises at least one member selected from the group consisting of oxalic acid, tartaric acid, citric acid, succinic acid, ammonium dihydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, aluminum sulphate, ammonium oxalate, diammonium hydrogen phosphate, sodium bicarbonate, potassium bicarbonate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, borax, tris(hydroxymethyl)methylamine, sodium carbonate, potassium carbonate, trisodium phosphate and tripotassium phosphate.

17. The cartridge according to claim 14, wherein said buffer layer comprises about 5-50 wt% buffer material and about 95-50 wt% inert material.

18. The cartridge according to claim 17, wherein said buffer layer comprises about 10-30 wt% buffer material and about 90-70 wt% inert material.

19. The cartridge according to claim 18, wherein said buffer layer comprises about 20 wt% buffer material and about 80 wt% inert material.

20. The cartridge according to claim 9, wherein said buffer layer comprises buffer material coated on said particles of inert material.

21. The cartridge according to claim 20, wherein said particles comprise a particle size of about 600 to 850 microns.

22. The cartridge according to claim 21, wherein said inert material is selected from the group consisting of crystalline silica and diatomaceous earth.

23. The cartridge according to claim 22, wherein said inert material comprises diatomaceous earth.

24. The cartridge according to claim 20, wherein said buffer material comprises at least one member selected from the group consisting of oxalic acid, tartaric acid, citric acid, succinic acid, ammonium dihydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, aluminum sulphate, ammonium oxalate, diammonium hydrogen phosphate, sodium bicarbonate, potassium bicarbonate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, borax, tris(hydroxymethyl)methylamine, sodium carbonate, potassium carbonate, trisodium phosphate and tripotassium phosphate.

25. The cartridge according to claim 20, wherein said buffer layer comprises about 5-50 wt% buffer material and about 95-50 wt% inert material.

26. The cartridge according to claim 25, wherein said buffer layer comprises about 10-30 wt% buffer material and about 90-70 wt% inert material.

27. The cartridge according to claim 26, wherein said buffer layer comprises about 20 wt% buffer material and about 80 wt% inert material.

28. The cartridge according to claim 1, wherein said material for spreading a fluid into a thin film comprises a particulate material.

29. The cartridge according to claim 28, wherein said particulate material comprises a particle size of about 200 to about 3000 microns.

30. The cartridge according to claim 29, wherein said particulate material comprises at least one member selected from the group consisting of silica gels, fibrous cellulose, fibrous glass and diatomaceous earth.

31. The cartridge according to claim 30, wherein said particulate material comprises diatomaceous earth.

32. The cartridge according to claim 28, wherein said particulate material is heated to a temperature of about 500°C
for about 3 hours.

33. The cartridge according to claim 1, wherein said multilayer composition further comprises at least one additional layer.

34. The cartridge according to claim 33, wherein said at least one additional layer comprises a plurality of layers.

35. The cartridge according to claim 33, wherein said at least one additional layer comprises at least one component selected from the group consisting of a component for removing moisture, a component for removing extraneous materials, and a component for derivatizing a drug into a more readily analyzable form.

36. The cartridge according to claim 35, wherein said component for removing moisture comprises at least one member selected from the group consisting of anhydrous sodium sulphate, anhydrous magnesium perchlorate, anhydrous calcium sulphate, anhydrous copper sulphate and alumina.

37. The cartridge according to claim 36, wherein said component for removing water comprises anhydrous sodium sulphate.

38. The cartridge according to claim 35, wherein said component for removing extraneous materials comprises at least one member selected from the group consisting of alumina, Florisil and silica gel.

39. The cartridge according to claim 35, wherein said component for derivatizing a drug into a more readily analyzable form comprises pentafluorobenzyl bromide.

40. The cartridge according to claim 35, wherein said component is heated to a temperature of about 500°C for about 3 hours.

41. The cartridge according to claim 1, further comprising a filter positioned below said multilayer composition for retaining said multilayer packing composition within said barrel.

42. The cartridge according to claim 41, wherein said filter positioned below said multilayer composition comprises at least one material selected from the group consisting of stainless steel mesh, glass mesh, porous polyolefins, porous polytetrafluoroethylene, glass fiber mat and cellulose.

43. The cartridge according to claim 42, wherein said filter positioned below said multilayer composition comprises cellulose

44. The cartridge according to claim 1, further comprising a filter positioned above said multilayer composition, for maintaining said multilayer packing composition within said barrel.

45. The cartridge according to claim 41, further comprising a filter positioned above said multilayer composition, for maintaining said multilayer packing composition within said barrel.

46. The cartridge according to claim 44, wherein said filter positioned above said multilayer composition comprises at least one material selected from the group consisting of stainless steel mesh, cellulosic material and glass fiber mat.

47. The cartridge according to claim 46, wherein said filter positioned above said multilayer composition comprises stainless steel mesh.

48. A method for making an extraction cartridge comprising:
heating an inert support at a temperature of about 500°C
for about 3 hours;
mixing the heated inert support with a buffer;
heating diatomaceous earth at a temperature of about 500°C for about 3 hours;
heating an adsorbent at a temperature of about 500°C for about 3 hours;
positioning a first filter at the bottom of a cartridge;
adding the heated adsorbent to the cartridge;
adding the heated diatomaceous earth to the cartridge;
adding the mixture of heated support and buffer to the cartridge; and positioning a second filter in the cartridge.

49. An extraction cartridge, for extracting essentially completely a component from a composition with extraneous components, comprising:

a barrel comprising an upper opening for feeding of a fluid to be extracted and a lower opening for removal of treated fluid; and a multilayer packing composition within said barrel, said multilayer packing composition comprising:
a buffer layer for adjusting pH including about 5-

50 wt% buffer material and about 95-50 wt% inert support;
a partitioning layer comprising a particulate material for spreading a fluid into a thin film;
a filter positioned above said multilayer packing composition for retaining said multilayer packing composition within said barrel; and a filter positioned below said multilayer packing composition for retaining said multilayer composition within said barrel.
50. The cartridge according to claim 49, wherein said buffer material and said inert material comprise particles having a particle size of about 600 to 850 microns, and said particulate material in said partitioning layer has a particle size of about 600 to 850 microns.

51. The cartridge according to claim 50, wherein said inert material comprises crystalline silica and said particulate material comprises diatomaceous earth.

52. The cartridge according to claim 49, wherein said filter positioned below said multilayer packing composition comprises cellulose, and said filter positioned above said multilayer packing composition comprises stainless steel mesh.

53. The cartridge according to claim 49, wherein said barrel is composed of glass.

54. A method of extracting a component from a fluid comprising:
a) adding a fluid containing a component to be extracted to the cartridge as claimed in claim 1;

b) waiting a period of time for the fluid to spread out into a thin film over the partitioning layer;
c) adding a first organic solvent to the cartridge to extract the component from the fluid into the first organic solvent; and d) collecting the first organic solvent containing the component.

55. The method according to claim 54, further comprising evaporating the collected solvent.

56. The method according to claim 55, further comprising adding a second organic solvent to redissolve the extracted component.

57. The method according to claim 54, wherein the fluid is a biological fluid.

58. The method according to claim 57, wherein the biological fluid is selected from the group consisting of human plasma, human serum, human urine, animal plasma, animal serum and animal urine.

59. The method according to claim 54, wherein the first organic solvent comprises at least one member selected from the group consisting of pentane, hexane, carbon tetrachloride, cyclohexane, iso-propylether, chlorobutane, chloroform, diethylether, toluene and ethyl acetate.

60. The method according to claim 56, wherein the second organic solvent comprises a polar solvent.

61. The method according to claim 60, wherein the polar solvent is at least one member selected from the group consisting of methanol, ethyl acetate and ethanol.

62. The method according to claim 54, further comprising:
waiting a period of time for the component being extracted to partition between the fluid and the first solvent; and repeating steps c and d.

63. An automatic processing system for extraction of components from a fluid, comprising:
a main processing platform;
indexing means for raising, lowering and incrementally advancing a rack along said main processing platform; and means for automatically transferring fluids at predetermined positions along said main processing platform.

64. The processing system according to claim 63, wherein said indexing means comprise a walking beam positioned at said main processing platform.

65. The processing system according to claim 64, wherein said main processing platform comprises a working surface, said walking beam comprises at least one elongated member, said at least one elongated member being mounted so as to be movably positionable between a lower position wherein an upper surface of said elongated member is below said working surface, and an upper position wherein said upper surface of said walking beam is above said working surface.

66. The processing system according to claim 65, wherein, said at least one elongated member moves from said lower position to said upper position and returns to said lower position, said at least one elongated member is capable of incrementally moving a rack along said main processing platform.

67. The processing system according to claim 66, wherein said at least one elongated member comprises two elongated members.

68. The processing system according to claim 66, wherein said at least one elongated member is eccentrically mounted on at least two pulleys, and each of said at least two pulleys is mounted on a rotatable shaft.

69. The processing system according to claim 68, wherein said indexing means further comprise a motor for rotating said rotatable shaft, and means for controlling activation of said motor to enable control of a degree of rotation of said rotatable shaft.

70. The processing system according to claim 69, wherein said means for controlling activation of said motor comprise elements to stop rotation of said at least two pulleys after one revolution to obtain precise incremental movement.

71. The processing system according to claim 65, wherein said indexing means further comprise at least one locking pin movable from a first position above said working surface to a second position below said working surface as said elongated member moves from said lower position to said upper position, and movable from said second position below said working surface to said first position above said working surface as said elongated member moves from said upper position to said lower position.

72. The processing system according to claim 71, wherein said at least one locking pin extends to a maximum height of about 0.75 inches above said working surface in said first position.

73. The processing system according to claim 71, further including a rotatable cam associated with each said at least one locking pin, said at least one locking pin including a lower end contacting said cam.

74. The processing system according to claim 73, wherein said cam comprises a pear-shaped cam.

75. The processing system according to claim 71, wherein, as said at least one elongated member moves from said lower position to said upper position and returns to said lower position, said at least one elongated member is capable of incrementally moving a rack along said location, and said at least one elongated member is eccentrically mounted on at least two pulleys, and each of said at least two pulleys and said cam is mounted on a rotatable shaft.

76. The processing system according to claim 65, wherein said upper surface of said at least one elongated member includes elements for engaging a lower surface of a rack.

77. The processing system according to claim 76, wherein said elements for engaging comprise protrusions on said upper surface of said at least one elongated member.

78. The processing system according to claim 76, wherein said elements for engaging comprise elements for increasing frictional engagement along said upper surface of said at least one elongated member.

79. The processing system according to claim 71, wherein said working surface includes means defining openings corresponding to each of said at least one elongated member and said at least one locking pin to permit passage therethrough.

80. The processing system according to claim 79, including guide rails on said working surface.

81 The processing system according to claim 63, wherein said means for automatically transferring fluids at predetermined positions comprises at least one robotic arm movable along a width, length and height of said location.

82. The processing system according to claim 81, wherein said at least one robotic arms comprises two independently movable robotic arms.

83. The processing system according to claim 82, wherein each of said two robotic arms includes at least one probe for transferring fluids.

84. The processing system according to claim 83, further including at least one syringe pump connected by at least one valve with said at least one probe.

85. The processing system according to claim 84, wherein said at least one probe comprises a plurality of probes.

86. The processing system according to claim 85, wherein said plurality of probes comprises four probes.

87. The processing system according to claim 69, wherein said means for controlling activation of said motor to enable control of a degree of rotation of said rotatable shaft comprise at least one proximity sensor.

88. The processing system according to claim 87, wherein said proximity sensor comprises a photosensor.

89. The processing system according to claim 63, further comprising a wash rack.

90. The processing system according to claim 89, wherein said wash rack comprises at least one wash station and at least one internal standard tube.

91. The processing system according to claim 63, further comprising an input extension platform for feeding a rack onto said main processing platform.

92. The processing system according to claim 91, wherein said input extension platform comprises at least one proximity sensor and a conveyor belt.

93. The processing system according to claim 91, further comprising an output extension platform for receiving a rack from said main processing platform.

94. The processing system according to claim 93, wherein said output extension platform comprises at least one proximity sensor and a conveyor belt.

95. The processing system according to claim 63, comprising a computer system for controlling the operation of said indexing means, and said means for automatically transferring fluids at predetermined positions.

96. The processing system according to claim 95, wherein said computer system comprises a master system and a slave sub-system.

97. The processing system according to claim 96, wherein said master system controls operation of said means for automatically transferring fluids.

98. The processing system according to claim 97, wherein said slave sub-system controls operation of said indexing means.

99. The processing system according to claim 98, further comprising a keyboard with a display, and said slave-subsystem controls said keyboard with a display,

100. The processing system according to claim 63, further comprising at least one rack, said at least one rack comprising:
a lower rack including a back portion, a front portion, and a bottom surface, said back portion including openings for receiving collection vessels, and said front portion including a plurality of openings for receiving sample vessels;
an upper rack positioned above said back portion of said lower rack, said upper rack including a plurality of openings for receiving cartridges; and said bottom surface includes at least one element for locking said rack along said main processing platform.

101. The processing system according to claim 100, wherein said at least one element comprises a plurality of equally spaced openings along said bottom surface.

102. A rack for holding a plurality of extraction cartridges, collection vessels and sample vessels, comprising:
a lower rack including a back portion, a front portion, and a bottom surface, said back portion including openings for receiving collection vessels, and said front portion including a plurality of openings for receiving sample vessels;

an upper rack positioned above said back portion of said lower rack, said upper rack including a plurality of openings for receiving cartridges; and said bottom surface comprising at least one element for locking said rack in position.

103. The rack according to claim 102, wherein said at least one element comprises a plurality of equally spaced openings along said bottom surface.

104. The rack according to claim 102, wherein said bottom surface comprises a roughened surface.

105. An automatic processing system for extraction of components from a fluid, comprising:
a main processing platform;
indexing means for raising, lowering and incrementally advancing a rack along said main processing platform, said indexing means comprising a walking beam positioned at said main processing platform;
means for automatically transferring fluids at predetermined positions along said main processing platform;
said main processing platform comprises a working surface, said walking beam comprises at least one elongated member, said at least one elongated member being eccentrically mounted on a rotatable shaft so as to be movably positionable between a lower position wherein an upper surface of said elongated member is below said working surface, and an upper position wherein said upper surface of said walking beam is above said working surface, and as said at least one elongated member moves from said lower position to said upper position and returns to said lower position, said at least one elongated member is capable of incrementally moving a rack along said main processing platform; and means for controlling activation of a motor to enable control of a degree of rotation of said rotatable shaft.

106. The processing system according to claim 105, wherein said indexing means further comprise at least one locking pin movable from a first position above said working surface to a second position below said working surface as said at least one elongated member moves from said lower position to said upper position, and movable from said second position below said working surface to said first position above said working surface as said elongated member moves from said upper position to said lower position.

107. The processing system according to claim 105, wherein said at least one elongated member comprises two elongated members.

108. A method of extracting a substance from a plurality of samples comprising:
feeding a rack, comprising a plurality of rows holding a plurality of extraction cartridges, a plurality of sample tubes and a plurality of collection tubes positioned below said plurality of extraction cartridges, onto a processing system comprising a main processing platform;
transferring fluids and samples into the plurality of extraction cartridges along one row of cartridges;
indexing the rack by raising, lowering and incrementally advancing the rack along the main processing platform;
repeating transferring fluids and samples into the plurality of extraction cartridges along another row of cartridges; and adding to each of the extraction cartridges a water-immiscible solvent, a plurality of times, to collect an extract in a corresponding one of the plurality of collection tubes.

109. The process according to claim 108, wherein said plurality of cartridges comprises:

a barrel comprising an upper opening for feeding of a fluid to be extracted and a lower opening for removal of treated fluid; and a multilayer packing composition within said barrel, the multilayer packing composition comprising:
a buffer layer comprising a buffer material for adjusting pH of the fluid to be extracted; and a partitioning layer positioned below said buffer layer, said partitioning layer comprising a material for spreading a fluid into a thin film.

110. A method of extracting a component from a fluid comprising:
a) adding a fluid containing a component to be extracted to a cartridge contained in a rack in an automatic processing system as recited in claim 65, the cartridge comprising a barrel comprising an upper opening for feeding of a fluid to be extracted and a lower opening for removal of treated fluid;
and a multilayer packing composition within said barrel, the multilayer packing composition comprising:
a buffer layer comprising a buffer material for adjusting pH of the fluid to be extracted; and a partitioning layer positioned below said buffer layer, said partitioning layer comprising a material for spreading a fluid into a thin film;
b) waiting a period of time for the fluid to spread out into a thin film over the partitioning layer;
c) adding a first organic solvent to the cartridge to extract the component from the fluid into the first organic solvent; and d) collecting the first organic solvent containing the component.