Determination of several psychiatric drugs in whole blood using
capillary gas–liquid chromatography with nitrogen phosphorus
detection: comparison of two solid phase extraction procedures
Carolina Sanchez de la Torre, Marıa A. Martınez*, Elena Almarza
National Institute of Toxicology and Forensic Sciences, Ministry of Justice, C/Luis Cabrera 9, 28002 Madrid, Spain
Received 23 August 2004; received in revised form 4 November 2004; accepted 3 December 2004
Available online 26 January 2005
Abstract
A simple and reliable gas chromatographic method with nitrogen-phosphorus detection without derivatization was developed
for the detection of several psychiatric drugs in whole blood as part of systematic toxicological analyses (STA). Drugs included
mirtazapine, chlorpromazine, methotrimeprazine (levomepromazine), clothiapine, olanzapine, clozapine, haloperidol, and
thioridazine. All drugs were studied at concentrations of 100–2000 mg/L, except haloperidol that was studied at concentrations
of 400–8000 mg/L. In order to select the best blood purification procedure and therefore increase the signal to noise ratio we have
compared two solid-phase extraction (SPE) columns, Chem Elut and Bond Elut Certify, for their recovery, precision, sensitivity
and matrix purification efficiency. Recoveries for these drugs using Chem Elut columns at 500 and 2000 mg/L (2000 and
8000 mg/L for haloperidol) were in the range 21–65%, with intra-assay and inter-assay precisions of less than 17% and 19%,
respectively. Limits of detection (LODs) and limits of quantitation (LOQs) for mirtazapine, chlorpromazine, methotrimepra-
zine, clothiapine, olanzapine, clozapine, and thioridazine ranged from 62 to 161 mg/L and from 205 to 531 mg/L, respectively.
LOD and LOQ for haloperidol were 442 and 1458 mg/L, respectively. Recoveries of these compounds using Bond Elut Certify
columns at 500 and 2000 mg/L (2000 and 8000 mg/L for haloperidol) were in the range 44–97%, with intra-assay and inter-assay
precisions of less than 7% and 14%, respectively. LODs and LOQs for mirtazapine, chlorpromazine, methotrimeprazine,
clothiapine, olanzapine, clozapine, and thioridazine ranged from 37 to 66 mg/L and from 122 to 218 mg/L, respectively. LOD
and LOQ for haloperidol were 156 and 515 mg/L, respectively. Linearity was observed in the studied range for all compounds
with r2 values of >0.999. The use of the mixed-mode bonded-silica Bond Elut Certify columns showed advantages comparing
with Chem Elut columns for the screening of these psychotropic agents such as higher recoveries, cleaner extracts, better
sensitivity, better precision and less solvent consumption and subsequent disposal.
# 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Systematic toxicological analysis; Psychiatric drugs; Solid phase extraction; GC; Screening
www.elsevier.com/locate/forsciint
Forensic Science International 155 (2005) 193–204
1. Introduction
Psychoses are among the most severe psychiatric dis-
orders, in which there is not only marked impairment of
* Corresponding author.
E-mail address: [email protected] (M.A. Martınez).
0379-0738/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights r
doi:10.1016/j.forsciint.2004.12.007
behavior but also a serious inability to think coherently, to
comprehend reality, or even to gain insight into the presence
of these abnormalities. These common disorders (affecting
perhaps 0.5–1.0% of the population at some age) typically
include symptoms of false beliefs (delusions) and abnormal
sensations (hallucinations). Representative syndromes in
this category include schizophrenia, brief psychoses, and
eserved.
C. Sanchez de la Torre et al. / Forensic Science International 155 (2005) 193–204194
delusional disorders, although psychotic features also are
uncommon in the major mood disorders, particularly mania
and severe melancholic depression. Antipsychotic agents are
used primarily in the management of patients with psychotic
or other serious psychiatric illnesses marked by agitation and
impaired reasoning.
Depression is also one of the most common and most
debilitating mental disorders. It is characterized by feelings
of intense sadness and despair, mental slowing and loss of
concentration, pessimism, lack of pleasure, self-deprecation,
and variable agitation. Antidepressant agents, which act
blocking neuronal uptake of biogenic amines, represent
the first choice in the treatment of moderate to severe
depressive illness [1].
Pharmacological treatment is essential for an adequate
management of these psychiatric disorders. Toxicity to
antipsychotics and antidepressants can worsen patient status
and may be associated with elevated blood levels. Drug
interactions can be recognized more readily if routine blood
level monitoring of patients is the rule rather than the
exception. Deliberate and accidental cases of self-poisoning
with these therapeutic drugs have become a major medical
problem because of the widespread use of these therapeutic
drugs and the severity of their toxic actions. This fact has led
to the development of reliable analytical methods for their
analysis.
In systematic toxicological analysis (STA) one of the
main purposes is drug screening. Antipsychotics and anti-
depressants are some of the most frequently encountered
drugs in forensic and clinical cases. In STA the substances
present are not known at the start of the analysis. In such
cases the extraction procedure cannot be directed to a given
substance but must be a general procedure where a com-
promise must be reached so that the substances of interest are
capable of being detected with high recovery, efficiency, and
minimal presence of potential interfering substances. Tra-
ditionally, liquid–liquid extraction (LLE) has been routinely
used in most forensic laboratories. As an extraction method,
solid phase extraction (SPE) is growing in use as a very
effective procedure for sample pre-treatment and clean-up of
biological samples in all types of drug testing laboratories,
including those which carry out forensic toxicology. SPE
offers several advantages over traditional LLE, including
better specificity, the ability to obtain cleaner extracts, good
reproducibility, avoidance of emulsion formation, the ability
to automate the extraction procedure, and substantial reduc-
tion in the volume of solvent required [2].
There are numerous publications on SPE for relatively
clean biological samples such as plasma, serum or urine
[3–12]. Until now, only a few publications have described
SPE methods for whole blood [13–23]. However, hemolyzed
whole blood is in practice the matrix most frequently
encountered in forensic toxicology. It is worth mentioning
that, post-mortem whole blood is not as fluid as ante-mortem
serum or plasma and therefore generally requires more
workup.
The Chem Elut column, a diatomaceous earth support, is a
SPE procedure closely related to conventional LLE. The
aqueous sample is added to the top of the column, adsorbed,
and distributed into a film over the diatomaceous earth, which
is an hydrophilic packing porous material which acts as
support for the aqueous phase. The high surface area of the
inert diatomaceous earth ensures that the organic eluents
remain immiscible with the aqueous matrix, eliminating
emulsion problems, and facilitating efficient interaction
between the sample and the organic solvent [24,25]. However,
for STA purposes where acidic, neutral, and basic substances
may be present, this type of SPE must be carried out with at
least two columns: one for the acidic and neutral substances
and one for basic and neutral substances and often large
volumes of hazardous organic solvents are required [2].
The Bond Elut Certify column, a mixed-mode bonded
silica SPE extraction support containing hydrophobic chains
(octylsilane, n-C8) and strong cation-exchange moieties (ben-
zenesulphonylpropylsilane), has the ability to retain acidic,
neutral, basic and amphoteric drugs on one column. This is a
great advantage in the field of toxicology where blood speci-
mens available for analysis are sometimes very small and a
search of a very wide variety of drugs is required [20,22,23].
The use of capillary gas–liquid chromatography using
fused-silica capillary columns and nitrogen-phosphorus
detection (GC-NPD) has proved to be a powerful tool in
the area of underivatized drug analysis in biological extracts.
Since many drugs are nitrogen-containing compounds,
traces of them can be selectively detected in the screening
toxicological analysis with excellent sensitivity and minimal
interference from other non-nitrogenous compounds, both
endogenous and exogenous [26]. This is crucial for whole
blood drug analysis considering that it is not a clean matrix
and the concentrations of the drugs or metabolites are much
smaller compared with urine.
This paper presents a rapid and sensitive validated
method which allows the simultaneous determination of
whole blood therapeutic and/or toxic concentrations of
the antidepressant mirtazapine and the antipsychotics: chlor-
promazine, methotrimeprazine (levomepromazine), clothia-
pine, olanzapine, clozapine, haloperidol, and thioridazine,
using GC-NPD analysis without derivatization (see Fig. 1).
Mepivacaine and prazepam were used to control the whole
analytical procedure. Two SPE analytical procedures were
compared, Chem Elut and Bond Elut Certify columns, for
their recovery, precision, sensitivity, and matrix purification
efficiency.
2. Experimental
2.1. Materials
Mirtazapine was donated by Organon (Roseland, NJ,
USA), clothiapine was donated by Novartis Pharma AG
(Basel, Switzerland), and olanzapine was donated by Eli
C. Sanchez de la Torre et al. / Forensic Science International 155 (2005) 193–204 195
Fig. 1. Chemical structures of the eight psychotropic studied in this work and the compounds used to control the analytical procedure.
Lilly (Dunderrow, Kinsale Co., Cork, Ireland). Chlorproma-
zine hydrochloride, methotrimeprazine maleate, clozapine,
haloperidol, thioridazine hydrochloride, mepivacaine hydro-
chloride, and prazepam were purchased by Sigma–Aldrich
(Buch, Switzerland).
Individual stock solutions were prepared by dissolving
10 mg of each pure compound in 10 mL of methanol. These
stock solutions were stored in glass tubes and maintained at
�27 8C. Appropriate dilution with methanol yielded the
working solutions containing all compounds involved in
the study. The extraction standard solution was prepared
by diluting the stock solution of mepivacaine with deionized
water to 16 mg/L. The chromatographic standard solution
was prepared by diluting the stock solution of prazepam with
methanol to 5 mg/L.
All chemicals (Merck, Darmstadt, Germany) and sol-
vents (Scharlau, Barcelona, Spain) were of analytical grade.
Borate buffer (saturated solution, pH 9.0) and dichloro-
methane–isopropanol (85:15) were used for the Chem Elut
procedure. Phosphate buffer (0.1 M, pH 6.0), 0.01 M acetic
acid, acetone–dichloromethane (1:1), and dichloromethane–
isopropanol–ammonia (78:14:8) were used for the Bond Elut
Certify procedure.
Chem Elut CE 1010 columns (10 mL of column reservoir
volume), Bond Elut Certify columns (130 mg of sorbent
mass, 3 mL of column reservoir volume) and a VAC-ELUT
SPS 24 vacuum manifold system for the manual mixed-
mode bonded silica SPE were purchased from Varian Sam-
ple Preparation Products (Harbor City, CA).
A P-Selecta sonication bath and a P-Selecta Centronic S
centrifuge were both purchased from Selecta (Barcelona,
Spain).
A pool of citrated human whole blood samples was
provided by Comunidad de Madrid Blood Bank (Madrid,
Spain) and verified to be drug free. No interferences were
found for the studied compounds and the samples were kept
frozen at �27 8C until used.
2.2. Instrumentation
The analysis of the extracts was performed on a Hewlett-
Packard (HP) (Avondale, PA) model 5890 Series II gas
chromatograph equipped with a nitrogen-phosphorus detec-
tor and linked to a HP 3396A integrator. The column was a
25 m � 0.20 mm i.d. fused silica capillary column coated
with cross-linked methylsilicone (0.11 mm film thickness)
C. Sanchez de la Torre et al. / Forensic Science International 155 (2005) 193–204196
(HP Co., PA). The following temperature program was used
with a total run time of 18 min, including 2 min of equili-
bration time. The initial column temperature was set to
180 8C for 1 min, and then increased to 300 8C at 10 8C/
min. This final temperature was held for 3 min. The carrier
gas was helium (Air Liquid, Madrid, Spain) delivered at a
column head pressure of 195 kPa. Injection port and detector
temperatures were 280 and 300 8C, respectively. The NPD
was operated at a baseline of 30–35 pA. The splitting ratio
was 1:20.
Deactivation of insert liners were performed according to
the following procedure traditionally used in our laboratory.
Insert liners were silanized with dimethyldichlorosilane:to-
luene (5:100) after standing in contact with the solution for
30 min. Excess dimethyldichlorosilane was rinsed away
with toluene:methanol (90:100) after soaking for 30 min
and the process was completed with an acetone rinse of
30 min. After removing the excess of solvent, they were
packed with Supelco silanized glass wool (from Supelco
Park, Bellefonte, PA). In order to minimize a reduction in the
performance of the chromatographic system, it is essential to
ensure that insert liners are kept clean. Routinely, they were
replaced before each batch of injections (approximately 100
injections).
2.3. Preparation of spiked whole blood samples
Human whole blood (100 mL) was spiked with mirta-
zapine, chlorpromazine, methotrimeprazine, clothiapine,
olanzapine, clozapine, and thioridazine at three concentra-
tion levels 0.1, 0.5 and 2 mg/L, respectively, while the
respective concentrations for haloperidol were 0.4, 2 and
8 mg/L. The spiked blood was sonicated for 15 min at room
temperature and then it was submitted in parallel to SPE
using Chem Elut and Bond Elut Certify columns.
2.4. Chem Elut extraction procedure
The extractions were performed using a Chem Elut
extraction procedure described in our previous work [20].
Briefly, each aliquot (2 mL), was treated with 7.0 mL of
borate buffer (pH 9.0) and 100 mL of a mepivacaine
aqueous solution of 16 mg/L as extraction standard, vortex
mixed for 5 min, and loaded onto the Chem Elut columns.
After 5 min, elution of the sample by gravity was carried
out adding two aliquots of 10 mL of dichloromethane–
isopropanol (85:15). Eluates were evaporated under a
nitrogen stream. The extraction residues of blood were
reconstituted with 200 mL of methanolic solution of chro-
matographic standard (5 mg/L) and 2 mL were injected for
GC analysis.
2.5. Bond Elut Certify extraction procedure
The extraction procedure with Bond Elut Certify col-
umns was also described in our previous work [20]. It was
performed on a VAC-ELUT SPS 24 vacuum manifold
system. To each 2.5 mL aliquot, 7.5 mL of phosphate buffer
(pH 6.0) and 125 mL of a mepivacaine aqueous solution of
16 mg/L as extraction standard were added, sonicated for
5 min and centrifuged at 4000 rpm for 10 min, then 8 mL of
the supernatant, equivalent to 2 mL of whole blood, were
used for further extraction. The columns were precondi-
tioned with 1 mL methanol, followed by 1 mL 0.1 M phos-
phate buffer (pH 6.0) under light vacuum (approximately
2 in. Hg) to avoid the columns becoming dry before the
application of the sample. The whole blood was applied onto
the columns and drawn through completely at a flow rate of
approximately 1.5 mL/min. The columns were washed with
2 mL deionized water. The columns were acidified by
passing through 0.5 mL of 0.01 M acetic acid. Then the
columns were dried under full vacuum (15 in. Hg) for 4 min.
Methanol (60 mL) was added, and the columns were dried
under full vacuum for 1 min.
After the column outlets were wiped with tissue, the
labelled evaporation tubes recently rinsed with methanol,
in order to avoid retention of polar drugs in the walls of the
glass tubes, were placed into the manifold basin. To each
column, first 3.5 mL of acetone–dichloromethane (1:1)
was added, and after 3 mL of dichloromethane–isopropa-
nol–ammonia (78:14:8). The eluents were pulled through
completely at a flow rate of 0.8 and 0.5 mL/min, respec-
tively and the combined eluates were evaporated under a
nitrogen stream. The extraction residues of blood were
reconstituted with 200 mL of methanolic solution of chro-
matographic standard (5 mg/L) and 2 mL were injected for
GC analysis.
2.6. Validation of the methods
The spiked whole blood samples, prepared as described
before in Section 2.3, were sonicated for 15 min at room
temperature and then submitted in parallel to SPE with
Chem Elut and Bond Elut Certify columns.
Calibration curves were prepared with five methanolic
standard solutions of these therapeutic drugs. The concen-
trations for mirtazapine, chlorpromazine, methotrimepra-
zine, clothiapine, olanzapine, clozapine, and thioridazine
were 0.5, 1, 5, 15 and 30 mg/L, while the concentrations
for haloperidol were 2, 4, 20, 60 and 120 mg/L, respec-
tively. The concentration of prazepam (internal standard)
was fixed at 5 mg/L. Drugs to prazepam area ratios were
measured, and the calibration curves were generated from
least-squares linear regression. The regression lines were
used to calculate the absolute recoveries (n = 6) of indi-
vidual drugs from spiked blood at three concentration
levels.
The intra-assay precision was assessed at three concen-
trations, 0.1, 0.5 and 2.0 mg/L for all the compounds, except
haloperidol, that was studied at 0.4, 2.0 and 8.0 mg/L. They
were extracted and analyzed on the same day (n = 6). The
inter-assay precision was assessed by analyzing a set of 10
C. Sanchez de la Torre et al. / Forensic Science International 155 (2005) 193–204 197
spiked blood samples at the three concentrations on different
days, by different operators, using fresh methanolic calibra-
tion curves.
The limits of detection (LODs) and quantitation (LOQs)
were determined as the lowest concentration giving a
response of three times and 10 times, respectively, the
average of the base line noise defined from six control
samples.
The linearity of the method for mirtazapine, chlorpro-
mazine, methotrimeprazine, clothiapine, olanzapine, cloza-
pine, and thioridazine, was checked by preparing six
replicates of the blood calibration curves at three different
concentrations, ranging from 100 to 2000 mg/L (from 400 to
8000 mg/L for haloperidol), by addition of known amounts
of each drug to human whole blood.
3. Results and discussion
3.1. Chromatography
The Bond Elut Certify columns provided cleaner extracts
without interference of endogenous substances compared to
the Chem Elut columns. Figs. 2 and 3 show representative
GC-NPD chromatograms of extracted human whole blood
for each extraction method. Good chromatographic results
with reliable separations of these eight psychiatric drugs and
the chromatographic standard were achieved under the
chromatographic conditions described in relatively short
chromatographic times (16 min). Excellent chromato-
graphic behavior with sharp and symmetrical peaks without
derivatization was also obtained.
The selectivity of the method was studied for possible
matrix influence and for the possible interference of other
substances that can be present in combination with the
studied drugs. The influence of the matrix was studied by
analyzing a series of different pools of human whole blood
(n = 6) and checking for the presence of any interfering
substances at the retention time of the compounds of interest.
The absence of interferences from the matrix was further
confirmed by the daily use of the method with many different
samples. The potential interference of other drugs was
investigated by checking the retention time of the studied
drugs and other drugs, potentially detectable under the
described chromatographic conditions (Table 1). Mirtaza-
pine were found to co-elute with desipramine and elute close
to doxepin, causing peaks not to be completely resolved. In
real cases in which these compounds were present simulta-
neously, quantification can be accomplished using other
technique, such as GC–MS mode SIM or HPLC-DAD.
It is also interesting to report that both extraction pro-
cedures provided chromatograms without interferences in
the areas corresponding to the retention time of the studied
compounds and the internal standards. Caffeine, a common
beverage used in the population, did not interfere with the
studied drugs.
Mepivacaine was added as extraction internal standard to
control the extraction procedures. Mepivacaine, an uncom-
mon prescribed drug, presents good extraction recoveries
(100% with a relative standard deviation of 5%) using both
SPE procedures. Furthermore, this compound does not
interfere with a large variety of abused and therapeutic
drugs. Prazepam was chosen as chromatographic internal
standard because of its similar molecular structure and
physicochemical behavior. It is not widely used or pre-
scribed. The chromatographic internal standard was added
after the extraction, but prior to the introduction of the
extract into the gas chromatograph, in order to correct for
differences in the final volume of the concentrated extract
and injection volumes and to check chromatographic beha-
vior. The use of mepivacaine as extraction internal standard
(surrogate) and prazepam as chromatographic standard
allowed us to control the whole analytical procedure during
the routine performance of STA.
The nitrogen-containing structures of mirtazapine chlor-
promazine, methotrimeprazine, clothiapine, olanzapine, clo-
zapine, haloperidol, and thioridazine together with the use of
SPE columns are responsible for the high sensitivity with
minimal interference obtained using a nitrogen phosphorus
detector.
3.2. Comparison of the efficiencies of the two
SPE procedures
According to our experience in the routine practice in our
laboratory, both SPE methods under these chromatographic
conditions allow the determination of a wide variety of drugs
in the performance of STA. Evaluating their efficiencies is
one of the main reasons we have conducted this comparative
validation study.
A summary of the most representative analytical para-
meters studied are shown in Table 2. The accuracy of the
methods was expressed as percent recovery. Recoveries for
these therapeutic drugs using Chem Elut columns at 500 and
2000 mg/L (2000 and 8000 mg/L for haloperidol) were in the
range 21–65% with intra-assay and inter-assay precisions of
less than 17% and 19%, respectively. LODs and LOQs for
mirtazapine, chlorpromazine, methotrimeprazine, clothiapine,
olanzapine, clozapine, and thioridazine ranged from 62 to
161 mg/L and from 205 to 531 mg/L, respectively. LOD and
LOQ for haloperidol were 442 and 1458 mg/L, respectively.
Recoveries of these compounds using Bond Elut Certify
columns at 500 and 2000 mg/L (2000 and 8000 mg/L for
haloperidol) were in the range 44–97% with intra-assay
and inter-assay precisions of less than 7% and 14%, respec-
tively. LODs and LOQs for mirtazapine, chlorpromazine,
methotrimeprazine, clothiapine, olanzapine, clozapine, and
thioridazine ranged from 37 to 66 mg/L and from 122 to
218 mg/L, respectively. LOD and LOQ for haloperidol were
156 and 515 mg/L, respectively. Excellent linearity was
observed in the studied range for all tested therapeutic drugs,
r2 > 0.999 (n = 6).
C.
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Fig. 2. Representative GC-NPD chromatograms of human whole blood after extraction using Chem Elut columns. (A) Blank whole blood, I = caffeine. (B) Blood spiked with 0.5 mg/L of each
therapeutic drug, I = caffeine, 1 = mepivacaine (extraction standard), 2 = mirtazapine 3 = chlorpromazine, 4 = methotrimeprazine, 5 = prazepam (chromatographic standard), 6 = clothiapine,
7 = olanzapine, 8 = clozapine, 9 = haloperidol, and 10 = thioridazine.
C.
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Fig. 3. Representative GC-NPD chromatograms of human whole blood after extraction using Bond Elut Certify columns. (A) Blank whole blood, I = caffeine. (B) Blood spiked with 0.5 mg/L of
each therapeutic drug, I = caffeine, 1 = mepivacaine (extraction standard), 2 = mirtazapine, 3 = chlorpromazine, 4 = methotrimeprazine, 5 = prazepam (chromatographic standard), 6 = clothia-
pine, 7 = olanzapine, 8 = clozapine, 9 = haloperidol, and 10 = thioridazine.
C. Sanchez de la Torre et al. / Forensic Science International 155 (2005) 193–204200
Table 1
Retention times (absolute and relative to diazepam) of drugs detect-
able under the described chromatographic conditions
Compound RTabsolute (min) RRT
Fenfluramine 1.47 0.05
Chlomethiazole 1.48 0.05
Ephedrine 1.66 0.08
Piracetam 1.82 0.10
Amphepramone 1.95 0.12
Pyrithyldione 1.95 0.12
Fenproporex 2.18 0.16
Metronidazole 2.23 0.17
Acetaminophen 2.37 0.19
Methylphenidate 2.78 0.25
Meperidine 2.89 0.27
Caffeine 3.13 0.30
Theobromine 3.21 0.31
Phenazone 3.39 0.34
Viloxazine 3.45 0.35
Thiopental 3.46 0.35
Fluoxetine 3.50 0.36
Diphenhydramine 3.54 0.37
Lidocaine 3.60 0.37
Fluvoxamine 3.62 0.38
Oxprenolol 3.77 0.40
Aminopyrine 3.78 0.40
Doxylamine 3.85 0.41
Theophylline 3.88 0.42
Propyphenazone 3.99 0.43
Phenobarbital 4.01 0.44
Tramadol 4.07 0.45
Trimetazidine 4.32 0.48
Chlorpheniramine 4.48 0.51
Cyclizine 4.64 0.53
Metoprolol 4.64 0.53
Clenbuterol 4.80 0.56
Mepivacaine 4.92 0.58
Fluconazole 5.02 0.59
Venlafaxine 5.04 0.59
Ticlopidine 5.15 0.61
Brompheniramine 5.20 0.62
Dextromethorphan 5.48 0.66
Propranolol 5.53 0.67
Methadone 5.57 0.68
Primidone 5.78 0.71
Atropine 5.85 0.72
Etamiphyllin 5.88 0.72
Propoxyphene 5.92 0.73
Mianserin 5.93 0.73
Amitriptyline 5.94 0.73
Nortriptyline 6.04 0.75
Levorphanol 6.05 0.75
Pirlindole 6.07 0.75
Moclobemide 6.10 0.76
Imipramine 6.14 0.76
Trimipramine 6.14 0.76
Medazepam 6.17 0.77
Pyrilamine 6.18 0.77
Doxepin 6.18 0.77
Desipramine 6.25 0.78
Table 1 (Continued )
Compound RTabsolute (min) RRT
Mirtazapine 6.25 0.78
Cyclobenzaprine 6.26 0.78
Trihexyphenidyl 6.37 0.80
Halazepam 6.46 0.81
Pentazocine 6.56 0.83
Biperiden 6.59 0.83
Timolol 6.60 0.83
Bupivacaine 6.63 0.84
Melitracen 6.63 0.84
Phenytoin 6.75 0.86
Alimemazine 6.76 0.86
Oxazepam 6.85 0.87
Bisoprolol 7.01 0.90
Maprotiline 7.01 0.90
Cyproheptadine 7.18 0.92
Phenylbutazone 7.20 0.92
Sertraline 7.23 0.93
Codeine 7.25 0.93
Bromhexine 7.33 0.94
Buflomedil 7.33 0.94
Dothiepine 7.37 0.95
Pentoxyphilline 7.40 0.96
Lorazepam 7.47 0.97
Citalopram 7.51 0.97
Lamotrigine 7.57 0.98
Tetrazepam 7.62 0.99
Cloperastine 7.63 0.99
Clomipramine 7.64 0.99
Diazepam 7.69 1.00
Nordiazepam 8.10 1.06
Chlordiazepoxide 8.21 1.08
Chlorpromazine 8.25 1.09
Flumazenil 8.25 1.09
Methotrimeprazine 8.52 1.13
Clobazam 8.55 1.13
Trimethoprim 8.64 1.14
Paroxetine 8.80 1.17
Midazolam 8.90 1.18
Bromazepam 8.93 1.19
Loxapine 8.95 1.19
Flunitrazepam 8.97 1.20
Metoclopramida 9.12 1.22
Ketotifen 9.15 1.22
Chloroquine 9.18 1.23
Amoxapine 9.21 1.23
Ambroxol 9.25 1.24
Prazepam 9.34 1.25
Laudanosine 9.52 1.28
Trifluoperazine 9.66 1.30
Clothiapine 9.71 1.31
Olanzapine 9.90 1.34
Zolpidem 10.36 1.41
Flurazepam 10.37 1.41
Quinidine 10.43 1.42
Quinine 10.44 1.42
Tiapride 10.47 1.42
Clozapine 10.94 1.50
Hydroxyzine 11.05 1.51
Alprazolam 11.41 1.57
C. Sanchez de la Torre et al. / Forensic Science International 155 (2005) 193–204 201
Table 1 (Continued )
Compound RTabsolute (min) RRT
Haloperidol 11.48 1.58
Diltiazem 11.53 1.59
Cinnarizine 12.45 1.73
Etoperidone 12.57 1.74
Sulpiride 12.75 1.77
Thioridazine 12.80 1.78
Verapamil 13.13 1.83
Trifluoperazine 13.68 1.91
Periciazine 13.74 1.92
Trazodone 14.06 1.97
Lofepramine 14.09 1.98
Sulforidazine 14.85 2.09
Clebopride 15.46 2.19
Based on the obtained data, the use of the Bond Elut
Certify columns resulted in more advantages besides the
detection of therapeutic drugs, such as better and more easily
reproducible recoveries, cleaner extracts, better sensitivity,
and less solvent consumption and disposal.
Table 2
Extraction recovery (%), intra-assay and inter-assay precision (R.S.D. %), li
eight psychotropic drugs in whole blood using Chem Elut and Bond Elu
Compound Recovery (%)
mean (n = 6)
Intra-assay
precision
R.S.D. (%) (n = 6)
In
pr
R.
0.5 mg/L 2.0 mg/L 0.5 mg/L 2.0 mg/L 0.
Mirtazapine
Chem Elut 65 64 10 9 12
Bond Elut Certify 90 92 2 5 4
Chlorpromazine
Chem Elut 21 22 12 10 15
Bond Elut Certify 56 76 6 5 12
Methotrimeprazine
Chem Elut 21 23 12 6 15
Bond Elut Certify 44 55 6 5 14
Clothiapine
Chem Elut 33 34 17 11 19
Bond Elut Certify 79 82 2 4 5
Olanzapine
Chem Elut 42 38 11 9 15
Bond Elut Certify 80 97 5 7 10
Clozapine
Chem Elut 46 47 14 9 18
Bond Elut Certify 70 79 3 4 6
Haloperidola
Chem Elut 36 29 11 10 13
Bond Elut Certify 83 83 2 4 5
Thioridazine
Chem Elut 21 23 13 9 19
Bond Elut Certify 48 58 5 6 11
a Two milligram per liter for haloperidol.
3.3. Considerations regarding strategy in the
performance of STA
It is crucial to have validated analytical methods in order
to get accreditation in a forensic or clinical laboratory.
Toxicologists must have absolute confidence in their proce-
dures in order to uphold toxicological results in courts of
law. Furthermore, STA always requires well-characterized
and validated methods in order to produce reliable results
that can be interpreted satisfactorily. Moreover, STA drug
concentrations are low and biological matrix are usually not
clean.
The Chem Elut extraction procedure, with at least two
columns: one for the acidic and neutral substances and other
for basic and neutral substances, has been routinely used in
our laboratory for sample pre-treatment and clean-up of
biological samples in the last recent years. The use of the
Bond Elut extraction procedure, using just one column, as
regular part of drug testing has been introduced in our
laboratory, after developing several series of comparative
nearity (r2), limits of detection (LOD) and quantitation (LOQ) for the
t Certify columns and analyzed by GC-NPD
ter-assay
ecision
S.D. (%) (n = 10)
Linearity r2
(n = 6)
Limit of
detection
LOD (mg/L)
(n = 6)
Limit of
quantitation
LOQ
(mg/L) (n = 6)5 mg/L 2.0 mg/L
11 0.999 63 208
7 0.999 45 148
11 0.999 139 459
10 0.999 46 152
11 0.999 122 403
10 0.999 52 172
13 0.999 65 214
5 0.999 46 152
11 0.999 123 406
10 0.999 66 218
10 0.999 62 205
5 0.999 37 122
15 0.999 442 1458
5 0.999 156 515
12 0.999 161 531
10 0.999 62 205
C. Sanchez de la Torre et al. / Forensic Science International 155 (2005) 193–204202
Table 3
List of therapeutic, toxic, and fatal levels for the studied psychiatric drugs published in the literature
Compound Therapeutic level (mg/L) Toxic level (mg/L) Fatal level (mg/L) Reference
Mirtazapine (serum) 0.02–05 N.A.a N.A. [27]
Chlorpromazine (blood) 0.1–2 0.8–3 >1 (mean = 5) [28]
Methotrimeprazine (serum) 0.03–0.15 0.5 N.A. [27]
Clothiapine N.A. N.A. N.A. –
Olanzapine (serum) 0.01–0.05 (0.1) 0.2 N.A. [27]
Clozapine (serum) 0.1–0.6 0.8–1.3 3 [27]
Haloperidol (blood) 0.0005–0.0009 0.05–0.5 N.A. [28]
Thioridazine (blood) 0.05–5 1–5 >2 (mean = 5) [28]
a Not available.
Fig. 4. GC-NPD chromatogram of whole blood obtained from an
attempted suicide after extraction using a Bond Elut Certify column.
Detected are, 1 = caffeine, 2 = mepivacaine (extraction standard),
3 = methotrimeprazine, 4 = midazolam, 5 = prazepam (chromato-
graphic standard), 6 = thioridazine, 7 = mesoridazine, and 8 = sul-
foridazine.
studies with different types of drugs regarding these two SPE
procedures [20,22,23].
The combination of the chromatographic techniques is
highly recommended to achieve the most comprehensive
drug screen. Knowing that some drugs cannot be detected in
the GC-NPD screening, or show poor chromatographic
properties or display thermal instability during injection,
it is more convenient to have a single sample extract to
complete the screening or confirmation. This was the reason
we used the Bond Elut Certify columns since both acidic-
neutral and basic eluates were obtained.
3.4. Utility of the method
The utility of the methods is demonstrated comparing the
literature levels with the LODs obtained in this study.
According to therapeutic and toxic situations published in
the literature [27,28] and shown in Table 3, our assay using
the described Bond Elut Certify method, of choice, allows
detection and quantitation of mirtazapine, chlorpromazine,
methotrimeprazine, clothiapine, clozapine, and thioridazine
at therapeutic levels. Olanzapine can only be detected at high
therapeutic and toxic concentrations. It is important to
considerate that the olanzapine data published in the litera-
ture refers to serum instead whole blood. Haloperidol can
only be detected at toxic concentrations. Not all of the drugs
levels are available in Table 3 since some of the studied
drugs have been recently introduced in the market with
subsequent lack of data.
3.5. Application to a case history
The SPE method of choice, the Bond Elut Certify
procedure, was applied to the analysis of the blood sample
collected in ICU from a 20-year-old woman who tried to
commit suicide by ingestion of Meleril1 (thioridazine). The
woman, who suffered schizophrenia and had a previous
history of drug abuse, manifested a Glasgow coma score
of 7 on admission to hospital. According to our compre-
hensive toxicological screening protocol the blood sample,
was previously tested for abuse drugs on a Hitachi 902
Automatic Analyzer using Cedia1 reagents, and for ethanol
and other volatiles on a headspace GC-FID equipment. The
results were negative for all abuse drugs including ethanol.
Fig. 4 shows the chromatogram of the Bond Elut Certify
blood extract, demonstrating the good performance of the
method. After screening with the method described here,
methotrimeprazine, thioridazine, mesoridazine, sulforida-
zine (both psychoactive thioridazine metabolites), and mid-
azolam, a sedative agent administrated in the hospital, were
detected and quantified using GC-NPD and confirmed using
GC–MS mode TIC (total ion chromatogram), under the
same chromatographic conditions. The toxicological find-
ings are shown below in Table 4.
C. Sanchez de la Torre et al. / Forensic Science International 155 (2005) 193–204 203
Table 4
Toxicological findings in case history
Bond Elut Certify procedure Blood concentrations
(mg/L)
Methotrimeprazine <LOQ
Thioridazine 1.6
Mesoridazine 0.4
Sulforidazine 0.3
4. Conclusions
In conclusion, the use of Bond Elut Certify mixed-phase
columns resulted in numerous advantages for the detection
of eight psychiatric drugs including improved and more
reproducible recoveries, cleaner extracts, better sensitivity
and reduced solvent consumption, and represents an excel-
lent alternative to traditional Chem Elut columns.
Consequently, it is concluded that the combination of
Bond Elut Certify columns for extraction of blood samples
and GC-NPD for detection provides a sensitive and repro-
ducible method of choice which allows the determination of
therapeutic and toxic concentrations of mirtazepine, chlor-
promazine, methotrimeprazine, clothiapine, olanzapine, clo-
zapine, and thioridazine, and toxic concentrations of
haloperidol in blood. These results are in accordance with
our previous studies and complete the analytical data for the
detection of psychotropic drugs [20,22,23].
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