Journal of Analytical Toxicology, Vol. 26, September 2002

A General ScreeningMethod for Acidic, Neutral, and Basic Drugs in Whole Blood usingthe Oasis MCX| Column J. Yawney 1, S. Treacy 2, K.W. Hindmarsh 3, and F.J. Burczynski 1,4,*

7Facultyof Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada;2Royal Canadian Mounted Police Toxicology Laboratory, Winnipeg, Manitoba, Canada;3Facultyof Pharmacy, University of Toronto, Toronto, Ontario, Canada;and 4Departmentof Pharmacologyand Therapeutics,Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada

Abstract Solid-phase extraction (SPE) is becoming a commonly used extraction technique. Most existing SPE methods extract a single drug from a relatively clean biological matrix (e.g., plasma, serum, or urine) using a silica-based column. These methods, however, are generally not satisfactory for forensic applications because the majority of biological samples are not as clean (e.g., whole blood, bile, tissues). Silica-based columns also may have reproducibility and stability problems. Polymer-based columns have been developed to overcome some of these limitations. In this study, sequential extraction of acidic, neutral, and basic drugs from whole blood using a polymer-based column, Oasis MCX, was undertaken. The extraction procedure developed involved a conditioning step using methanol followed by water; a three-step wash sequence using water, 0.1M hydrochloric acid, then water/methanol (95:5); and two elution steps. One elution step was for acidic and neutral drugs utilizing acetone/chloroform (1:1), and a second used ethyl acetate/ammonium hydroxide (98:2) for basic drugs. Of the drugs tested, 75% were extractable from whole blood and detectable at therapeutic concentrations. Good recoveries and clean extracts were achieved for the basic drugs; however, the extracts were not as clean for acidic drugs. Unfortunately, the Oasis MCX procedure was not suitable for extracting all drugs (e.g., benzodiazepines).

Introduction Systematic toxicological analysis (STA) is defined as the logical chemical-analytical search for potentially harmful substances whose presence is uncertain and whose identity is unknown (1). Extractions play a major role in STA by isolating the compounds of interest from difficult to analyze matrices into an environment more suitable for analysis. Once isolated, * Correspondingauthor: F.J.Burczynski, Ph.D., Facultyof Pharmacy,Universityof Manitoba, 50 Sifton Road,Winnipeg, MB, Canada R3T 2N2. E-mail: [email protected].

the compounds of interest may be concentrated to make them easier to detect, identify, and quantitate. In the mid-20th century it was common to use liquid-liquid extractions to extract drugs from poison victims (2). With the use of two immiscible solvents, compounds of interest partition into the solvent in which they are most soluble. Although liquid-liquid extraction is still used today, there are a number of drawbacks that limit its usefulness, including decreased selectivity for compounds of interest, emulsion formation, increased solvent use, and subsequent waste leading to environmental and safety issues. Liquid-liquid extractions are labor intensive, time consuming, difficult to automate, and difficult to reproduce the resulting data (3). These drawbacks have led to the development of other separation methods such as solid-phase extraction (SPE). Unlike liquid-liquid extraction where two liquids are used in the extraction process, SPE involves the use of one liquid phase and one solid phase (sorbent). The SPE mechanism is more selective than liquid-liquid extraction because it is based on interactions between sample components and the sorbent, as well as on solvent solubility. The more selective retention mechanism of SPE also has a number of advantages over liquid-liquid extractions such as cleaner extracts (4), increased selectivity for the compounds of interest, no emulsion formation, larger selection of solvents available for use, and smaller volumes of solvent may be used for each extraction. In general, SPE can be used for smaller sample sizes. This is important when sample volumes are limited (e.g., samples from infants). Compounds have been successfully extracted from as little as 50-100 lJL of plasma. SPE also takes less time, and the procedure can be automated. Because of the short analysis time, volatile compounds also may be analyzed without significant

loss (5). The majority of SPE procedures have been optimized for the extraction of a single drug from a relatively clean biological sample such as plasma, serum, or urine. These methods are generally not satisfactory for forensic applications because the

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:]25

Journal of Analytical Toxicology,Vol. 26, September2002

majority of biological samples submitted for analysis are not as clean (e.g., whole blood, bile, tissues). Depending on the state of decomposition, samples may contain large amounts of breakdown products. The majority of general screening methods for whole blood have used silica based mixed mode columns such as Bond Elut Certify| (6-8). Although successful, silica-based sorbents may have a number of drawbacks. First, for bonded phases, the reaction between the organosilane and the silica is incomplete, leaving unreacted silanol groups that can act as cation-exchange sites and creating a secondary retention mechanism. End-capping reduces the amount of free silanols by converting the hydroxyl group to a methoxy group but it is not 100% effective. Second, the sorbent is not stable at pH extremes. At pH levels below2, there can be breakdown of the silyl ether linkage. At higher pHs (> 7.5), there can be dissolution of the silica in aqueous solutions (9). Third, the sorbent must be wet before the sample is loaded. A dry sorbent will significantly decrease the recovery of the drug because of decreased interaction between the sample components and the sorbent. The polymer-based sorbents were developed to overcome the limitations of silica-based sorbents. The Oasis MCX columns (Waters Corp., Milford, MA) contain a mixed-mode cation-exchange sorbent based on a polymer backbone of poly(divinylbenzene-co-N-vinylpyrrolidone) with the addition of sulfonic acid groups to enable it to retain cations. This water-wettable sorbent is promoted for its ability to retain a wide spectrum of both polar and nonpolar compounds and to remain stable from pHi to 14 (10). Increased recoveries and reproducibility with the Oasis MCX columns compared to silica-based columns have been reported (10). However, to date, we are unaware of any published reports describing the use of Oasis MCXcolumns for drug screening from biological samples. The objective of the present study was to develop an extraction procedure for screening therapeutic concentrations of acidic, neutral, and basic drugs from whole blood. The goal was to sequentially extract all three groups of drugs at an acceptable level of recovery (> 70%) and cleanliness using the Oasis MCX column.

Materials and Methods

Materials and reagents Acetone, chloroform, ethyl acetate, and methanol, all highperformance liquid chromatography (HPLC) grade, were purchased from Caledon Laboratories (Georgetown, ON, Canada). Glacial acetic acid, hydrochloric acid, and ammonium hydroxide were of reagent grade from Caledon Laboratories. Potassium dihydrogen phosphate (KH2PO4),A.C.S. grade, was obtained from Fisher Scientific (Fair Lawn, NJ). Distilled, filtered water was obtained from the Milli-Q UF Plus system. Phosphate buffer (0.1M) pH ~ 4.4 was prepared by dissolving 6.81 g of KH2PO4 into 500 mL deionized water. Ammoniated ethyl acetate was prepared fresh daily by adding 2 mL ammonium hydroxide to 98 mL of ethyl acetate and sonicating for 5 min before use. Acetone/chloroform (1:1, v/v) was prepared 326

by adding equal parts of acetone and chloroform and mixing to ensure homogeneity. Water with 5% methanol was prepared by adding sufficient water to 5 mL of methanol to make 100 mL of solution. The Oasis MCX columns (100-mg sorbent mass, 3-mL column volume) were purchased from Waters Corp. A list of the drugs attempted for isolation is presented in Table I. Drug standards were obtained from the appropriate manufacturer. A stock solution of each drug was prepared in ethanol then further diluted with water or ethanol to a set concentration. An aliquot of this diluted drug standard was then added to porcine blood to provide a concentration within the therapeutic range for that drug (Table II). Ethanolic drug standards were run on a gas chromatograph with a nitrogenphosphorus detector (GC-NPD) to determine their retention times before extractions were performed. GC GC analysis (for all drugs except benzodiazepines) was performed using a Varian Star 3600 CX GC equipped with a Varian 8200 CXAutosampler, an NPD, and a 15-m x 0.32-ram i.d. (11am film thickness) DB1 capillary column (J&W Scientific, Folsom, CA).The operating conditions were as follows:injector temperature, 250~ oven temperature, programmed from 90~ with a l-rain hold, ramped to 320~ at 10~ with a 14-rain Table I. Summary of Drugs Tested on the Oasis MCX Column AIIobarbital Alprazolam Amitripty[ine Amobarbita[ Amoxapine Azacyclonal Azatadine Barbital Benzocaine Benztropine grompheniramine Butabarbital Chlordiazepoxide Chlorpheniramine Chlorpromazine Chlorprothixene Clemastine Clomipramine Clonazepam Cocaine Codeine Desalkylflurazepam Desipramine Dextromethorphan Dextrorphan Diazepam Dicydomine Diethylpropion Diltiazem Diphenhydramine

Diphenoxylate Nortriptyline Ephedrine Orphenadrine Ethosuximide Oxazepam Flunarizine Paroxetine Fluoxetine Pentazocine Haloperidol Pentobarbital Heroin Perphenazine Hydrocodone Phencyclidine Hydroxyzine Pheniramine Imipramine Phenobarbital Ketamine Phenyltoloxamine Lidocaine Prochlorperazine Lorazepam Procyclidine Loxapine Propoxyphene Maprotiline Propranolol Meperidine Pseudoephedrine Methadone Quinidine Methaqualone Secobarbital Methotrimeprazine Sertraline Methylecgonine Strychnine MethylenedioxyamphetamineTemazepam Methylphenidate Thiopental Methylprylon Thioridazine Metoprolol Trazodone Midazolam Triazolam Morphine Trifluoperazine Nefazodone Trimipramine Nitrazepam Triprolidine Nordiazepam Verapamil Nordoxepin Zopiclone

Journal of Analytical Toxicology, Vol. 26, September 2002

Table II. Recoveries of Drugs using the Oasis MCX Column

Drug

Concentration (pg/mL)

Numberof Samples(es;us)*

% Recovery • SD

%RSD

AIIobarbital Amitriptyline Amobarbital Amoxapine Azacyclonal Azatadine Barbital Benzocaine Benztropine Brompheniramine Butabarbital Chlordiazepoxide

5 0.25 5 0.1 0.1t 0.014 5 1 0.12 0.022 5 3

10;10 9;9 10;10 10;10 , * 10;10 8;8 3;10 * 10;10 *

81.2 • 5.1 97.4 • 6.5 90.0 • 3.7 81.8 • 14.3 ND ND 28.0 • 3.2 74.0 • 5.4 109.1 • 8.3 ND 90.4 • 5.9 ND

6.3 6.6 4.1 17.5 11.4 7.3 7.6 6.5

Chlorpheniramine Chlorpromazine Chlorprothixene Clemastine Clomipramine Cocaine Codeine Desipramine Dextromethorphan Dextrorphan Dicyclomine Diethylpropion Diltiazem Diphenhydramine Diphenoxylate Ephedrine Ethosuximide Flunarizine Fluoxetine Haloperidol Heroin Hydrocodone Hydroxyzine Imipramine Ketamine Lidocaine Loxapine Maprotiline Meperidine Methadone Methaqualone Methotrimeprazine Methylecgonine MDA Methylphenidate Methyprylon Metoprolol Midazolam Morphine Nefazodone

0.05 t 0.25 0.25 0.1 t 0.25 0.25 0.1 0.25 0.05 0.25 0.08 0.1 0.25 0.05 0.05 t 0.05 2 0.1 0.25 0.015 0.05 0.02 0.05 0.25 0.1 0.5 0.05 0.25 0.5 0.5 0.5 0.05 0.1 t 0.5 0.05 5 0.25 0.25 0.2 1

10;10 10;10 15;15 3;5 10;10 10;10 10;10 10;10 10;10 10;10 15;15 10;10 10;10 10;10 9;10 5;5 37;37 10;10 10;10 * 8;8 * * 10;10 10;10 8;8 15;15 10;10 8;8 8;8 8;8 10;5 5;10 9;10 10;10 10;10 15;15 10;10 10;10 9;9

121.8 • 7.4 78.1 • 12.4 71.4 • 8.5 68.1 • 3.9 69.8 • 2.3 113.8 • 14.5 86.7 • 7.4 81.0 • 6.3 85.2 • 3.6 86.9 • 4.6 92.8 • 11.4 ND 88.2 • 3.3 87.7 • 4.8 73.6 • 10.4 ND ND 55.9 • 6.6 69.2 • 4.2 ND 92.9 • 8.8 ND ND 79.3 • 4.2 83.1 • 6.4 95.5 • 4.9 88.2 • 5.8 69.9 + 4.8 91.6 • 6.9 90.2 • 4.6 86.6 + 3.2 14.1 • 9.5 75.3 • 8.1 58.2 • 14.8 133.6 • 25.2 85.7 • 4.4 87.2 • 17.5 56.3 • 11.7 86.2 • 41.1 78.4 • 7.7

6.1 15.9 11.8 5.8 3.2 12.7 8.6 7.8 4.2 5.3 12.3 3.8 5.5 14.1 11.8 6.1 9.4 5.3 7.7 5.2 6.5 6.9 7.5 5.1 3.7 8.3 10.7 25.4 18.8 5.1 20.1 20.8 47.7 9.8

-

Comments

% Recovery• SD from BondElutCertify (6)

98.2•

below peak area reject

multiple peaks; parent drug not detected

90.0•

coeluted with ISTD

94.4•

95.0 + 1.1 102.5 + 6.3

* Abbreviations:es = extractedsample size, ISTD= internalstandard,ND = not detected,RSD = relativestandarddeviation, SD = standarddeviation,us = unextractedsamplesize. t Testedabovetherapeuticconcentration. Singlespikedwater sample. wPeakswith areacounts below 2500 were not consideredinsufficientsignal-to-noiseratio.

327

Journal of Analytical Toxicology, Vol. 26, September 2002

Table II (continued). Recoveries of Drugs using the Oasis MCX Column Drug Nordoxepin Nortriptyline Orphenadrine Paroxetine Pentazocine Pentobarbital Perphenazine Phencyclidine Pheniramine Phenobarbital Phenyltoloxamine Prochlorperazine Procyclidine Propoxyphene Propranolol Pseudoephedrine Quinidine Secobarbital Sertraline Strychnine Thiopental Thioridazine Trazodone Trifluoperazine Trimipramine Triprolidine Verapamil Zopiclone

Concentration (pg/mL) 0.1 0.05 0.25 0.05 0.25 5 5 0.07 0.05 5 0.05 t 0.01 0.25 0.25 0.25 0.75 0.25 5 0.05 0.5 5 0.25 1 0.05 0.05 0,04 0.15 0.05

Number of Samples(es;us)* * 5;5 10;10 10;10 9;9 10;10 * 10;10 7;2 10;10 10;10 * 10;10 10;10 10;10 10;10 10;10 10;10 10;10 8;8 10;10 10;10 8;8 10;10 15;15 * 10;10 3;5

% Recovery + SD

~

ND ND 85.2 + 3.8 ND 96.1 + 5.0 89.5 + 4.0 ND 100.4 + 7.6 89.3 + 3.9 92.7 + 5.1 122.6 __16.2 ND 83.9 + 5.3 84.8 + 7.2 76.4 + 9.9 136.4 + 28.0 81.2 + 26.7 86.6 + 4.5 58.2 + 5.6 83.1 + 20.7 78.8 +12.5 83.6 + 8.2 87.0 _+8.1 ND 90.9 + 6.5 ND 121.9 + 9.8 107.3 + 24.0

4.5 5.2 4.5 7.6 4.4 5.5 13.3 6.3 8.5 13.0 20.6 33.2 5.2 9.6 24.5 15.9 9.8 9.0 7.2 8.0 22.4

Comments

% Recovery• SD from BondElutCertify (6)

93.1 + 4.0 coeluted with EtOA artifact 81.2 + 6.6

below peak area rejectw 102.5 + 2.3

* Abbreviations: es = extracted sample size, ISTD = internal standard, ND = not detected, RSD = relative standard deviation, SD = standard deviation, us = unextracted sample size. f Tested above therapeutic concentration. .' Single spiked water sample. Peaks with area counts below 2500 were not considered insufficient signal-to-noise ratio.

hold; and detector temperature, 300~ Analysis of the benzodiazepine extracts was performed on an HP 5890 GC equipped with an HP 7673 Autosampler, an electron capture detector, and a 15-m • 0.32-ram i.d. (0.25-1Jm film thickness) DB17 column (J&W Scientific). Operating conditions were as follows:injector temperature, 250~ oven temperature, programmed from 220~ with a l-rain hold, ramped to 290~ at 8~ with a 6.75-rain hold; detector temperature 300~

Sample pretreatment Aliquots (1.0 mL) of porcine blood (preserved with sodium fluoride and anticoagulated with potassium oxalate) were spiked with the appropriate concentration of the drugs of interest (Table II) along with an internal standard (doxapram 250 ng for basic drugs and tolylbarbita125 ng for acidic drugs), and sonicated for 15 min. Methylnitrazepam (50 ng) was used as the internal standard for the benzodiazepines. Nine milliliters of 0.1M phosphate buffer (pH ~ 4.4) was then added and samples vortex mixed (10 s). Diluted blood samples were centrifuged (15 rain at 3000 rpm) and the supernate loaded onto the conditioned extraction column. 328

Extraction procedure The extraction procedure was performed utilizing an automated system. A Zymark RapidTrace SPE Workstation was linked to a Dell Latitude LM laptop computer programmed with RapidTrace software. The workstation had 2 modules, each with a capability of extracting 10 samples. There were eight solvent reservoirs and a nitrogen gas line connected to the modules. Solvents were drawn from the reservoirs and forced through the column by a syringe providing negative and positive pressure. Columns were dried using a stream of nitrogen. The extraction procedure was a modification of the procedure described by Chen et al. (6) and Waters Corp. (personal communication). Oasis MCX columns were placed in the Zymark RapidTrace SPE Workstation and conditioned initially with methanol (2 mL), followed by water (2 mL), each at a rate of 12 mL/min. The manufacturer's literature suggests conditioning is not required for urine samples. Urine may be applied directly to the columns. Conditioning is required, however, for whole blood samples, which are a major case sample in forensic analyses. Sample supernatants were loaded onto the columns at a rate of 1.2 mL/min. Columns were subsequently washed with water (3 mL), 0.1M hydrochloric acid (3 mL), and finally with

Journal of Analytical Toxicology, Vol. 26, September 2002

5% methanol in water (2 mL), each at a rate of 9 mL/min. Acidic and neutral compounds were eluted with acetone/chloroform (1:1, 4 mL, 1.2 mL/min flow rate). Basic compounds were eluted with ethyl acetate/ammonuim hydroxide (98:2, v/v, 4 mL, 1.2 mL/min flow rate). The elution fractions were evaporated to dryness and reconstituted in ethyl acetate (50 laL) prior to GLC injection (1 laL). Cleanliness of the extracts, visual appearance of the GC chromatogram and the percent recovery were used as evaluation parameters for the extraction process.

Determination of extraction efficiency A 100% extraction efficiency was simulated by extracting blood samples spiked only with the internal standards then adding the drugs of interest to the elution fractions of these samples. These were referred to as unextracted samples. The peak-area ratio (PAR) for each drug in the unextracted sample was calculated (i.e. the peak area of the drug of interest was divided by the peak area of the internal standard). Drugs of interest together with the internal standards were extracted from a second set of blood samples and the PAR determined. These samples were the extracted samples. Percent recovery and relative standard deviation were calculated as follows (11):

lute recoveries, because this mimics procedures used in forensic laboratory determinations of unknowns. Although the use of internal standards may seem inappropriate because it is difficult to mimic the analytes under investigation, they are routinely used in forensic analyses as a quality control measure. Least squares regression analysis of the standard curves for the internal standards phenyltoloxamine, methylphenidate, chlorpheniramine, codeine, morphine, heroin, and diphenoxylate were as follows (in lJg/mL): y = 3.181x- 0.028, r 2 = 0.999; y = 2.735x + 0.043, r 2 = 0.993; y = 2.846x - 0.036, r 2 = 0.999; y = 1.533x + 0.018, r 2 = 0.997; y = 0.435x + 0.027, r 2 = 0.976; y = 1.642x + 0.001, r 2 = 0.998; y = 2.284x + 0.042, r 2 = 0.966, respectively.

Results and Discussion

Acidic, neutral, and basic compounds were retained on the Oasis MCX columns and then differentially eluted. The acidic and neutral drugs eluted together in one fraction and basic drugs eluted into a separate second fraction (see Figures I and 2). Diluting the blood samples with a phosphate buffer (pH 4.4) resulted in most of the basic drugs remaining in an ionized % recovery = ( PAR ~ extracted samples ) x 100 state and being retained by ionic interactions on the Column. PAR of unextracted samples Ethyl acetate/ammonium hydroxide (98:2, v/v) reversed the ionization of the basic drugs resulting in their elution. With the exception of the benzodiazepines, all basic drugs were eluted into the basic fraction. The elution fractions were evaporated to %RSD= S(m~an) x l 0 0 dryness prior to reconstitution with ethyl acetate in order to prevent GC column damage from the ammonium hydroxide. The benzodiazepines, a group of weakly basic drugs, were where RSD and SD are the relative standard deviation and stanfound to elute into both fractions. For example, clonazepam, lodard deviation, respectively. razepam, oxazepam, and temazepam eluted into the acidic/neuA recovery of greater than or equal to 70% with relative stantral elution fraction, and alprazolam, desalkylflurazepam, dard deviations below 10% was taken as an acceptable procediazepam, nordiazepam, and triazolam eluted into the basic dure. fraction. Nitrazepam eluted mainly into the basic fraction with Internal standards were used, rather than determining absoa small percentage being eluted into the acidic fraction. In general, the drugs with PKaS of 1.3 to 1.8 were eluted into the acidic fraction and drugs 10~0. with PKaS of 3.2 to 3.4 were eluted into the basic fraction. Exceptions to this were triazolam (PKa 1.3) and desalkylflurazepam (PKa 1.5) both of which eluted into the basic frac> tion. The appearance of benzodiazepines in the two fractions has been previously reported to be due to partial ionization and affinity for the acidic elution solvents (6). Recoveries of the benzodiazepines were not calculated because of the splitting of methylnitrazepam (internal standard) between the 20 10 ..... elution fractions. Use of an external standard Timo (min) or a standard curve could have been used to Figure 1. Acidic fraction of spiked blood extracted with Oasis MCX method. Peak identification: determine recoveries but the larger variability 1, barbital; 2, allobarbital; 3, butabarbital; 4, arnobarbital; 5, pentobarbital; 6, secobarbital; observed in our laboratory with these methods 7, thiopental; 8, phenobarbital; 9, tolylbarbital (internal standard); and 10 and 11, ethyl acetate would have been unacceptable for forensic artifacts. work. 90000.

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Journal of Analytical Toxicology, Vol. 26, September 2002

Recovery Of the 90 drugs tested, 77 were successfully extracted. The remainder of the drugs were not extracted from either a spiked water or blood sample. It should be noted that because many of the drugs not considered extractable were tested at low concentrations (below 0.1 mg/mL), it is possible that they were extracted but were below the detection limit of the GC. Percent recoveries were calculated for 63 of the 77 drugs extracted. For the remaining 14 drugs, recoveries were not calculated, either because a suitable internal standard was not found, the drug coeluted with another drug or artifact, or the drug degraded into multiple peaks. Of the 63 drugs, more than two-thirds had recoveries of approximately 70% greater with relative standard deviations of less than 10% (see Table II).

Comparison with Bond Elut Certify Recoveries from the Oasis MCX procedure were compared with those obtained from Chen et al. (6) who used Bond Elut Certify columns, a mixed-mode silica-based column (see Table

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II). Silica-based columns have been successfully used for extracting drugs from appropriately prepared whole blood samples (6). In the eight drugs common to both studies, there were no statistically significant differences in percent recoveries for seven of the drugs (p > 0.05). Methaqualone had a higher recovery from the Bond Elut Certifycolumns, although it eluted into both the acidic and basic fractions. No splitting of methaqualone was evident with the Oasis MCXprocedure. It is important to note that larger sample sizes and smaller drug concentrations (except in the case of pentobarbital) were used with this study of the Oasis MCX columns. Method validation Validation of the method was accomplished by the blind analysis of five human blood samples provided by Dr. B. Goldberger (Department of Pathology, Immunology & Laboratory Medicine, University of Florida College of Medicine). The basic fractions of these samples were analyzed. The methodology allowed detection of several of the drugs in Table I and several drugs not previously tested which were contained in the unknown human blood samples. Drugs that were detected included methylecgonine and benzoylecgonine (Sample 1); propoxyphene, propoxyphene metabolites, and acetaminophen (Sample 2); venlafaxine and venlafaxine metabolite (Sample 3); methadone, methadone metabolite, propoxyphene,and promethazine (Sample4); and atropine, diazepam, and oxycodone (Sample 5). There were no additional difficulties in applying the methodology to the analysis of human blood.

311

Time (min)

Figure 2. Basic fraction of spiked blood extracted with Oasis MCX method. Peak identification: 1, benzocaine; 2, methylphenidate; 3, meperidine; 4, lidocaine; 5, phenyltoloxamine; 6, chlorpheniramine; 7, methaqualone; 8, methadone; 9, amitriptyline; 10, pentazocine; 11, codeine; 12, morphine; 13, heroin; 14, doxapram (internal standard); I5, strychnine; 16, trazodone; 17, ethyl acetate artifact; 18, diphenoxylate; 19, ethyl acetate artifact; and 20, nefazodone.

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330

Advantages of the Oasis MCX procedure A major advantage of SPE over liquid-liquid extraction is its adaptability for automation. An automated Oasis MCX procedure greatly increased efficiencyas the analyst can concentrate on other responsibilities while samples are being extracted. A second advantage of the Oasis MCX procedure is that it successfully extracts amphoteric drugs such as morphine. In our laboratory, a time-consuming specializedscreening method was used for extraction of morphine like drugs because they were not extractable by the currently used liquid-liquid extraction procedure. With an automated Oasis MCX procedure amphoteric drugs can be extracted along with acidic, neutral, and basic drugs. The presence of cholesterol in the acidic/neutral elution fraction was identified by GC-MS. Having cholesterol elute into this fraction rather than the basic fraction is an advantage when screening for basic drugs because a greater number of basic drugs were eluted at the same retention time as cholesterol (6). GC-MS chromatograms of the acidic and basic fractions were also cleaner than those obtained

Journal o f Analytical Toxicology, Vol. 26, September 2002

from liquid-liquid extraction (see Figures 3-5). The benzodiazepine elution fractions were very dirty, and a suitable internal standard could not be found. It is possible that a second aliquot of blood could be extracted with a different Oasis MCX method that has been optimized specificallyfor the extraction of that group of drugs. An alternative to developinganother SPE method would be to combine this SPE procedure with another analytical procedure to obtain the most comprehensive drug screen. In forensic analyses, it is desirable to have a screening procedure that will aid in the detection of as many drugs as possible. Combining analytical methods is commonly used for analysis of biologicalsamples containing unknown drugs.

Although many drugs were extractable by this procedure, a few were not. These included some antihistamines (e.g., brompheniramine), certain antidepressants (e.g., paroxetine), some antipsychotics (e.g., haloperidol), and other miscellaneous drugs such as ephedrine. It is important to recognize this limitation. Therefore, the most comprehensive drug screen should combine this SPE procedure with another technology.

Disadvantages of the Oasis MCX procedure One drawback of the SPE method was the dirtiness of the acidic fraction. Despite the use of different wash solvents, the acidic/neutral elution fractions were slightly orange in color. After evaporation, some of the reconstituted fractions contained a fine precipitate. Routine analyses of the acidic elution extracts may lead to increased GC maintenance. Occasionally, the basic fractions would also contain a small amount of precipitate, but the solvent was not as darkly colored as the acidic/neutral extracts.

The Oasis MCXprocedure was capable of the differential elution of acidic, neutral, and basic drugs from whole blood. Of the drugs tested, 75% were extractable from whole blood and detectable at therapeutic concentrations. Good recoveries and clean extracts were achieved. Automation of the procedure greatly reduced the amount of time required to extract a large number of samples. This also reduced the chances of human error in procedural techniques. The successful extraction of morphine eliminated the use of a second, time-consuming extraction procedure. Unfortunately,the Oasis MCXprocedure is not suitable for extracting all drugs (e.g., benzodiazepines). It is suggested that the SPE procedure using the Oasis MCX column be used together with other technologies.

Conclusions

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The authors would like to thank the RCMP Forensic Laboratory, Winnipeg, Manitoba for the use of their equipment and technical assistance, Waters Corporation for technical assistance, and Dr. John Templeton for his editorial comments. We gratefully acknowledge the human blood samples kindly provided by Dr. B. Goldberger,Department of Pathology, Immunology & Laboratory Medicine, University of Florida College of Medicine.

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References

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Figure5. Basicfraction of a blood blank extractedby the OasisMCX method.

,

i

30

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