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
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
:]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.
8
5
"tOO00-
11
|
~000,
~0-
40000.
3~00-
,
S
T
. . . .
j
9
.
.
lg
329
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
,
i
?
10
15
20
25
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.
ADC18, ADCICHANNELB (D:UY'AUG24011,O)
11oo~
'
1ooooo
"v l
N
r~
20000 o
. . . .
'
. . . .
~ "
'
Time (min)
Fig.re3. Elutionfraction
fractionof
of a blood
blood bl~
blank extracted by liquid-liquid extraction.
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
ADC1 B, ADCI C H A N N E L B (C:~'IPCH EM~2~DATAUY~MAR01027.1D)
Z1
8o|
Acknowledgments
I
?oooo-.]1
r,oooo~ I
5oooo-I I
4ooo0-{ I
~-II
.
. . . .
0
. L~l
i
8
. . . .
.....
i
10
~
. . . .
i
15
,
. . . .
,
20
. . . .
i
25
9
.
,
3o
Time (min)
Figure4. Acidic fraction of a blood blank extractedby the OasisMCX method.
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.
A D C I B, ADC1 CHANNEL B (G:~HPCHEM~DATAUY~MAR01023.D)
1100oo
References
'Z
.
.
.
.
9
.
A
9
o
5
lO
15
20
25
T i m e (min)
Figure5. Basicfraction of a blood blank extractedby the OasisMCX method.
,
i
30
1. R.A. de Zeeuw. Drug screening in biological
fluids; the need for a systematic approach.
J. Chromatogr. B 689" 71-79 (1997).
2. R.D. McDowall, J.C. Pearce, and G.S. Murkitt.
Liquid-solid sample preparation in drug analysis. J. Pharm. Biomed. Anal 4(1): 3-21(1986).
3. R.D. McDowall. Sample preparation for
biomedical analysis. J. Chromatogr. 492:3-58
(1989).
4. H.D. Rood. Comparison of liquid-liquid and
solid-phase extraction techniques for drugs in
urine and effects on the lifetime and performance of fused-silica capillary columns. Anal
Chim. Acta 236:115-120 (1990).
331
Journal of Analytical Toxicology,Vol. 26, September2002
5. T.R. Krishnan and I. Ibraham. Solid-phase extraction technique for
the analysis of biological samples. J. Pharm. Biomed. Anal. 12(3):
287-294 (1994).
6. X.H. Chen, J.P. Franke, J. Wijsbeek, and R.A. de Zeeuw. Isolation
of acidic, neutral, and basic drugs from whole blood using a single
mixed-mode solid-phase extraction column. J. Anal ToxicoL 16"
351-355 (1992).
7. X.H. Chen, J. Wijsbeek, J.P. Franke and R.A. de Zeeuw. A singlecolumn procedure on Bond Elut Certify for systematic toxicological analysis of drugs in plasma and urine. J. Forensic ScL 37(1):
61-71 (1992).
8. Z. Huang, X.H. Chen, J. Wijsbeek, J.P.Franke, and R.A. de Zeeuw.
An enzymatic digestion and solid-phase extraction procedure for
the screening for acidic, neutral and basic drugs in liver. The Joint
TIASFT/SOFTInternational Meeting, Tampa, FL, 1994, pp 102-112.
332
9. N. Simpson and K.C. Van Home, Eds. Varian sorbent extraction
technology handbook. Varian Associates, Inc., Harbor City, CA,
1993.
10. Oasis~ Sample Extraction Applications Notebook. Waters Corporation, Milford, MA, 1999.
11. Understanding Biostatistics, T.H. Hassard, Ed. Mosby-Year Book,
St. Louis, MO, 1991, p 7.
12. M.E. Sharp. Evaluation of a screening procedure for basic and
neutral drugs: n-butyl chloride extraction and megabore capillary
gas chromatography. Can. Soc. Forensic Sci. J. 19(2)" 83-101
(1986).
Manuscript received May 18, 2001 ;
revision received May 28, 2002.