Download Pobierz plik

Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 74 No. 2 pp. 331ñ346, 2017
ISSN 0001-6837
Polish Pharmaceutical Society
ANALYSIS OF BASIC PSYCHOTROPIC DRUGS IN BIOLOGICAL FLUIDS
AND TISSUES BY REVERSED-PHASE HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
ANNA PETRUCZYNIK and MONIKA WAKSMUNDZKA-HAJNOS*
Department of Inorganic Chemistry, Medical University of Lublin, 20-093 Lublin, Poland
Abstract: The review of the RP HPLC analysis of basic psychotropic drugs is presented. It contains sample
preparation methods with centrifugation, protein precipitation, liquid-liquid extraction (LLE), dispersive liquidñliquid microextraction (DLLME), solid-phase extraction (SPE), solid-phase microextraction (SPME),
microwave-assisted extraction (MAE) and RP-HPLC analysis. Chromatographic behavior of basic drugs in
aqueous media - eluents used in reversed phase systems is discussed. Methods of blocking of residue surface
silanolsí interaction are mentioned. Analytical methods used for the analysis are divided into parts according
with the above methods: the use of low-pH eluents, the use of high-pH eluents, the use of silanol blockers, special stationary phases for basic analytes. Literature connected with the sample preparation methods and analytical systems for the drug analysis are cited in details and presented also in Table 1.
Keywords: basic psychotropic drugs, sample preparation, RP HPLC, blocking of residual silanols, retention
behavior of analytes, biological fluids and tissues
The kind of matrix is crucial in elaboration of
analytical method in case of drug monitoring as well
as in toxicological studies.
Drug-drug interaction and side effects often
presented in case of psychotropic drugs can cause
delayed therapeutic effect and could result in poor
patient susceptibility.
In recent years, the most important is an individual medication profile for each individual patient
to intensify a medicinal care. Especially, it is not
possible to schematically dosage of antidepressants
from younger to older populations. Individual differences of patients in responses on exposure of particular drugs are often reported because of various
pharmacokinetics in different organisms. It is cause
of dependencies of therapy results on enzyme activity, age, hepatic and renal diseases and co-medication by the other drugs as well as food intake. Levels
of psychotropic drugs in plasma are significantly
correlated with their therapeutic effects but simultaneously incidences of side effects related also on
dose at least for some drugs (1). Monitoring of psychotropic drugs plays a crucial role in adaptation of
therapy to patient, taking into account individual
physiological changes to evade unfavorable effects,
Psychotherapeutic drugs are used for treatment
of different psychiatric disorders and include
antipsychotic, antidepressant, mood stabilizing, anxiolytic, psychostimulant and nootropic drugs.
Psychotropic drugs are often determined for a
following purposes: studies of pharmacokinetics,
comparison of pharmacokinetic profiles of new
pharmaceutical formulations, investigation of drug
metabolism and monitoring of a drug level in determination of individual therapies.
In most cases psychotropic drugs are determined in blood, rarely in urine samples. However, in
recent years, analysis of alternative materials has
become a subject of interest in clinical and forensic
toxicology. Oral fluid is noninvasive taken sample
in comparison to blood ones for detection of these
drugs. Quantitation of antidepressants in oral fluid
may be used in clinical settings in routine or immediate analyses and also is a useful way for arrangement of therapy for individual patient. In forensic
cases different matrices are often analyzed. Hair is
one of an unconventional biological specimen,
which enables to retrospective analysis of substances which are accumulated in hair and give a
denotation of exposure.
* Corresponding author: e-mail: [email protected]
331
332
ANNA PETRUCZYNIK and MONIKA WAKSMUNDZKA-HAJNOS
intoxication or other discrepancies. Because of significant concentration differences of psychotropic
drugs in body fluids, drug monitoring should be performed for the effective medication control.
Therapeutic drug monitoring gives additional information for more sensible use of psychiatric drugs.
Monitoring of psychotropic drugsí concentration is
useful to raise their efficacy when there are problems with the patient response to the therapeutic
doses of the drug and in detection of overdosing (2).
Because in a therapy of psychiatric patients
rarely monotherapy is applied, detection of two or
more different psychotropic drugs in body fluids is a
challenge for drug monitoring. Clinically significant
depressive symptoms are detectable in cancer
patients, in victims of myocardial infarction and in
patients with Alzheimerís disease (3). Most patients
are comedicated with various drugs. Currently, psychotropic drugs are prescribed in different combinations, which increases a possibility of drug-drug
interactions. Rapid determination of psychotropic
drugs and their metabolites in body fluids is useful
in forensic medicine and in case of patients with
atypical metabolism to reveal abnormal drug levels.
Usually, psychotropic drugs are metabolized
by enzymes of cytochrome P450, which causes high
pharmacokinetic variability of metabolites due to
different factors including genetic, biological and
environmental ones (4). To keep up drug concentrations in biological fluids in the target level clinicians
have to apply individual dose adaptation by use of
drug monitoring.
Because antipsychotic drugs are often connected with the sudden death, detection of them gives
information of their use and contribution to the
cause of decease (5). Often, the difference between
the therapeutic and toxic levels of psychotropic drug
are insignificant, thus the interpretation of the
results of analysis in case of intoxication is complicated due to a high subject variability of drug concentration.
The concentrations of psychotropic drugs in
biological fluids and tissues are usually low. For
analysis of psychotropic drugs in biological tissues
and fluids the efficient separation methods with sensitive detection techniques have to be applied. For
this purpose the use of various chromatographic
methods is reported. All of the so far published
methods required a sample preparation before transferring the sample onto the analytical system.
Analysis of biological fluids and tissues was
often realized by high performance liquid chromatography (HPLC) coupled with different detection methods such as: UV (DAD), fluorimetry,
mass spectrometry (MS) or (MS/MS) and more
rarely electrochemical and chemiluminescence.
HPLC advantages are: high sensitivity, specificity,
generality. The method gives possibility of simultaneous analysis of several mixture components e.g.,
drugs in complex biological samples. Very valuable
tool is HPLC connected with mass-spectrometry
(LC-MS) especially for identification and quantitation of drugs and poisons in clinical and forensic
toxicology.
Recently, the use of miniaturized systems
(UPLC) in pharmaceutical analysis is often reported. The great advantages of miniaturized separation
techniques are as follows: high separation efficiency
and resolution, rapid analysis and minimal consumption of reagents and samples, low limits of
detection and quantification.
Sample preparation
Sample preparation, one of the most important
steps of analytical process, has to be applied due to
isolation of analytes from the matrix, concentration
of the sample and is essential for every biological
analysis.
Due to matrix interferences and low level of
drugs in biological fluids as well as limit of detection of instruments, chromatographic analysis without sample preparation step is hardly possible.
Proteins and other endogenous components may
negatively influence on the drugs separation,
increase column back pressure and suppress electrospray ionization when LCñMS analysis is
applied. For these reasons, purification and preconcentration processes before a chromatographic run,
effect in accurate, reliable and sensitive results of
analysis (6).
In psychotropic drug analyses in biological
matrices, various sample preparation methods are
applied such as: liquidñliquid extraction (LLE) (7),
solid-phase extraction (SPE), on-line SPE with a
column-switching system and methods of protein
precipitation.
Methods using deuterated compounds of psychotropic drugs as internal standards were also
described. Ansermot et al. applied the stable isotope-labeled standards (SIL-IS) that significantly
simplified sample preparation (4). The stable isotope-labeled internal standards were also used for
analysis of antipsychotic drugs in human plasma (8).
Centrifugation
Rarely, for sample preparation of biological
fluids, only centrifugation before HPLC analysis
was applied. For example, serum samples of patients
Analysis of basic psychotropic drugs in biological fluids and tissues by...
treated with escitalopram were centrifuged directly
after delivery for 10 min at 4000 rpm (9). Samples
were then rapidly analyzed. Blood samples for
determination of lamotrigine and its metabolites
were stored in glass tubes containing EDTA. Then,
all the samples were centrifuged, the supernatants
were transferred to polypropylene tubes and stored
frozen at ñ20OC until the analysis. Samples of
human serum containing aripiprazole and its major
metabolite were only vortexed and centrifuged
before LCñMS/MS analysis (10). Human plasma
sample containing olanzapine and its metabolite Ndesmethylolanzapine was transferred to a microcentrifuge tube and then internal standard (IS) working
solution was added and vortex mixed for 20 s (11).
After centrifugation, the supernatant was injected
into the LC-MS/MS system for analysis.
Protein precipitation (PP)
Protein precipitation is a traditional sample
preparation technique especially for the treatment of
biological fluids. The method of serum sample
preparation consisting of protein precipitation by
addition of acetonitrile and centrifugation steps was
often described (8, 12, 13). PP procedure inexpensive, low time- and low labor ñconsuming method,
does not cause destruction of analytes and their concentration changes in a sample. However, in this
method not all matrix components are eliminated.
The proteins of the plasma samples were often precipitated with acetonitrile (14). The supernatant was
dried in a vacuum concentrator and the dried extract
were reconstituted with ammonium acetate (5
mmol/L)/ammonium hydroxide (5 mmol/L) (1 : 1,
v/v) solution. To prepare samples of rat brain tissue,
containing psychotropic drug phenibut, they were
diluted with 0.1% formic acid solution in acetonitrile before analysis. The samples were then mixed
thoroughly, sonicated and centrifuged. The supernatant was collected, and injected into the LC-MSMS system for analysis.
Another procedure was used for extraction of
aripiprazole and dehydroaripiprazole in human plasma samples (2). The extracting solvent ñ mixture of
heptane and isopropanol were added to 1 mL of
plasma sample. After 20 min and centrifugation at
1800 ◊ g at 4OC, the organic layer was removed and
orthophosphoric acid (0.05 mol/L) was added. After
a further 30 s shaking, the aqueous phase was injected into the chromatographic system.
Liquidñliquid extraction (LLE)
Use of liquidñliquid extraction (LLE), based
on different solubility of analytes in two immiscible
333
solvents, is often reported as a sample preparation
method. The method is useful especially for ionizable analytes e.g., basic drugs, when differences in
solubility of ionized form and neutral one can be utilized due of the changes of the lipophilicity and solubility of both forms. However, some drawbacks
such as formation of emulsion, large scale of the
method, high time- and material-consumption
appear. Despite this, LLE is widely used as a
method of sample preparation for drug analyses in
biological fluids. The choice of a suitable solvent is
most significant point in optimization of LLE of
analyzed drugs for getting of high extraction capability (15, 16).
Psychotropic drugs from serum samples were
often successfully extracted using an alkaline LLE
method. The method relays on the following procedure: a sample of human serum containing basic
psychotropic drug is alkalized by use of buffer of pH
> 7 or sodium hydroxide and as neutral form extracted to an organic phase (appropriate solvent) and
mixed or rotated mechanically. After division of
aqueous and organic phases (usually by centrifugation), more volatile organic phase is completely
evaporated in nitrogen stream. Dry residue is dissolved in known volume of mobile phase and analyzed. Buffers such as borate buffer (pH 9) (6), sodium carbonate/bicarbonate buffer (pH 9.5 or 10) (17,
18), Trizma buffer or sodium hydroxide were
applied for alkalization of aqueous phase, depending
on pKa of extracted drug. Extracting solvents used
in the procedures were: 1-chlorobutane (16), methyl
tert-butyl ether (18, 19), ethyl acetate, or sometimes
solvent mixtures: di-isopropyl ether and isoamyl
alcohol (20), heptaneñisoamyl alcohol, hexaneisoamyl alcohol, heptanesñbutanol, heptaneñchloroform (21) etc. Sometimes, more complex procedures
are described. Some examples are described below.
Jones et al. proposed LLE procedure for
extraction of risperidone and 9-hydroxyrisperidone
from plasma (22). Plasma sample was added to sodium carbonate/bicarbonate buffer (pH 10) containing
methyl-risperidone. Mixture of heptaneñisoamyl
alcohol (98 : 2, v/v) were added and rotated on a
blood mixer. The sample was deep frozen at -80OC
to remove an aqueous phase. Decanted organic
phase was evaporated to dryness in the stream of
nitrogen and dissolved in mobile phase before the
analysis.
A sample of plasma containing sertindole, dehydrosertindole norsertindole and internal standard was
mixed with aqueous NaOH and with the extracting
solvent hexaneñisoamyl alcohol (99 : 1, v/v) (23).
The mixture was shaken for 20 min and centrifuged.
334
ANNA PETRUCZYNIK and MONIKA WAKSMUNDZKA-HAJNOS
The organic phase was transferred to another tube and
the aqueous layer was discarded. Next, the HCl solution was added to the organic phase. The mixture was
shaken for 20 min and centrifuged. The aqueous
phase was transferred to a micro vial in autosampler
and injected to HPLC system.
To the blood sample, Trizma buffer and 1chlorobutane were added and mixed for separation
of antipsychotic drugs from blood. The sample was
mixed for 5 min by use of shaker at 1500 rpm and
centrifuged. Then, the obtained organic layer was
placed in a vial, evaporated to dryness at ambient
temperature and dissolved in mobile phase before
the analysis (16).
Eishafeey et al. described the following procedure for extraction of olanzapine in human plasma
(24). Human plasma samples were placed in glass
tubes, and rosuvastatin solution was added to each
and vortexed. The methyl tert-butyl ether was
added, and samples were vortexed for 2 min. The
tubes were then centrifuged for 10 min. The upper
organic phases were transferred to clean glass tubes
and evaporated to dryness using a centrifugal vacuum concentrator. Dry residues were dissolved in
mobile phase, vortexed for 1 min to reconstitute
residues, and then injected onto chromatographic
system.
For preparation of plasma and urine samples
containing fluvoxamine the internal standard solution and acetonitrile were added. After vortexing,
the samples were centrifuged and the supernatant of
each sample was evaporated to dryness under stream
of nitrogen. To the residue, borate buffer and 1,2naphthoquinone-4-sulfonic acid sodium salt solutions were added. Obtained mixture was mixed by
use of shaker and heated at the 70OC for 30 min in a
water bath. After cooling at ambient temperature, it
was acidified by use of hydrochloric acid and
extracted three times by dichloromethane : nbutanol (4 : 1, v/v) mixture. Organic phase was
evaporated to dryness under stream of nitrogen and
dissolved in mobile phase (25).
Similar procedures are applied also for the
other matrices such as saliva or urine. For preparation of oral fluid, sodium carbonate buffer (pH 9.5)
and diazepam as internal standard were added to the
sample. Extraction of drugs was carried out with
ethyl acetate. After centrifugation, the organic phase
was evaporated to dryness at 45OC under a stream of
nitrogen, and the residue was reconstituted in mixture of methanol and mobile phase (17).
LLE was also applied for preparation of urine
samples. Before HPLC analysis of flunitrazepam,
nimetazepam and nitrazepam in urine, internal stan-
dard and 0.5 mL of 1.5 M carbonate buffer at pH 9.5
were added to the sample and extraction was performed with 3 mL of ethyl acetate (26). After centrifugation for 3 min at 2330 ◊ g, the supernatants
were decanted and dried under nitrogen. The
residues were redissolved in 0.5 mL of mixture of
acetonitrile, water and formic acid.
More complex procedures are used in case of
the other matrices ñ tissues. Procedure of postmortem samplesí preparation in body fluids and tissues was reported by Gronewold et al. for doxepin
related death (27). Homogenized gastric contents,
urine and bile were diluted with water and centrifuged. Tissue specimens were cut up and homogenized with an ultra turrax with water. The samples
were stored at 4OC overnight, then vortexed and centrifuged. After addition of sodium hydroxide and the
internal standard to the fluids or tissue homogenates,
extraction was performed using n-hexane : 1butanol (98 : 2, v/v). The samples were then vortexed, gently shaken, and centrifuged. The supernatant was dried under nitrogen and redissolved in
the mobile phase.
Procedure for preparation of hair and nail samples containing clozapine is described by Chen et al.
(28). The hair or nail were washed independently
with deionized water twice by vigorous shaking for
3 min; then the water residue from the two washes
was combined and placed in vessel before analysis.
Next, ethyl acetate was added to the hair or nail samples in the tube and shaken vigorously for 3 min and
repeated once. After that, all organic residues were
combined and saved in another tube for later analysis. After drying at room temperature, the hair or
nail samples were pulverized. A stainless steel
impactor with an agitation rate at 5 cps for 3 min at
liquid nitrogen temperature was used, followed by a
2 min cool-down time, and then grounding again for
3 min. Hair or nail powder was then sonicated in an
ultrasonic bath for an hour with mobile phase and
clozapine-d4. The mixture was then centrifuged at
12 000 rpm; the supernatant was injected into the
UPLC MS/MS system for analysis.
Dispersive liquidñliquid microextraction
(DLLME)
Currently it is a tendency to miniaturization of
the analytical methods including also miniaturization and automation of sample preparation procedures. Because LLE has a lot of drawbacks such as
high time and solvent consumption and is dangerous
to human health, DLLME is often applied. In
DLLME, extraction of analytes is based on dispersion of the extracting solvent in water. The extrac-
Analysis of basic psychotropic drugs in biological fluids and tissues by...
tion process involves two steps. In the first step, the
mixture of extracting and dispersing solvents is rapidly injected to a water sample. A dispersion is
formed and facilitates fast extraction of analytes
from the water sample. In the second step, the dispersion is removed by centrifugation and the
extracting solvent containing analytes is taken for
analysis with a microsyringe. The dispersing solvent
has to be fully soluble in water. Usually acetone,
acetonitrile and methanol are used for this purpose.
The extracting solvent has to have potential for
extracting analytes. Also, it has to be soluble in the
dispersing solvent while its solubility in water has to
be very low. The density of the extracting solvent
has to be definitely different from the density of
335
water to enable phase separation. Selection of
extracting and dispersing solvents is important to
obtain a high performance of extraction. DLLME is
rapid, cheap and easy for operation and exhibits a lot
of advantages such as: low solvent consumption,
high recovery and high enrichment factor.
DLLME was applied for sample preparation of
urine samples containing such psychotropic drugs
as: amitryptiline, clomipramine and thioridazine
before HPLC-UV analysis (29). In order to optimize
DLLME conditions, kind and volumes of organic
solvents (dichloromethane, chloroform, carbon
tetrachloride, chlorobenzene) were determined and
carbon tetrachloride in volume of 20 µL was selected as the most effective for this purpose. (Fig. 1).
Figure 1. Typical HPLC chromatograms of the target drugs for standard solution (A) without extraction, blank human urine (B), urine 1
(C) and 2 (D) after extraction using DLLME. Peaks 1ñ4 are amitryptiline, clomipramine, thioridazine and carbon tetrachloride, respectively. The concentrations of analytes in standard solution and spiked blank urine sample were 5.000 and 0.060 µg/L, respectively. Urine
1 and 2 were from two patients under treatment with some psychotropic drugs including amitryptiline and clomipramine, respectively, and
the concentrations of amitryptiline and clomipramine spiked in urine 1 and 2 were 0.025 µg/L, respectively. DLLME conditions: sample
volume and its pH values: 5.00 mL, 10.0; disperser solvent and its volume: acetonitrile, 0.50 mL; extraction solvent and its volume: CCl4,
20 µL. Chromatographic conditions are described in the text (29).
336
ANNA PETRUCZYNIK and MONIKA WAKSMUNDZKA-HAJNOS
Solid phase extraction (SPE)
In SPE, isolated analytes are partitioned in a
system solid phase/liquid phase. Due to a lot of
advantages such as high recovery, effective pre-concentration, less organic solvent consumption, ease of
operation and possibility of automation, SPE is the
most often applied as sample preparation method.
SPE columns contain sorbent, usually alkyl
bonded silica, but also inorganic adsorbents such as
silica, alumina, polar bonded phases ñ CN-silica,
Diol as well as ion-exchange resins are applied. In
case of basic drugs there is usually C-18 or other
non-polar sorbent. Bed of RP-sorbent should be
conditioned by use of methanol and water and often
pre-washed with the solvent mixture in which analytes are loaded to the column. In case of basic drugs
the solvent used for such purposes contains water,
organic modifier and basic buffer. In such conditions analyte is in unionized form and has affinity to
non-polar sorbent alkyl groups. The second step
relays on the removing polar ballast substances from
the bed by use of solvent of low eluent strength.
Then, analytes are eluted by the solvent mixture of
higher eluent strength, often acidified, which
increases the affinity of basic drugs, being in ionic
forms, to the aqueous phase. Non-polar ballasts can
be removed from the column by use of pure organic
modifier e.g., methanol. Eluted fraction is evaporated to dryness and usually dissolved in mobile phase
(or organic modifier) used for HPLC analysis.
For preparation of human serum samples, containing donazepil, Oasis HLB cartridges were
washed after samples loading with mixture of 5%
methanol in water followed by 0.025 mol/L ammonium chloride buffer (pH 9)/methanol, 50 : 50, v/v
(30). Elution of donepezil was then performed by a
mixture of 0.025 M ammonium formate buffer pH
2.5/methanol 50 : 50, v/v. The plasma samples containing prazepam and its metabolites were vortex
mixed with 0.05 M NaH2PO4 and applied to the
Figure 2. Matrix effect for spiked human plasma sample (200 ng/mL) on MEPS technique. (A) Water spiked sample; (B) plasma sample
after precipitation with ammonium sulfate 0.1%; (C) plasma sample diluted with phosphate buffer solution pH 4.0, 1:1 (v/v) and (D) centrifuged human plasma sample spiked with antidepressants. 1: citalopram; 2: mirtazapine; 3: fluoxetine; 4: paroxetine and 5: sertraline (41).
Analysis of basic psychotropic drugs in biological fluids and tissues by...
HLB Oasis cartridges. The columns were then
washed with 5% methanol in water and 40%
methanol in water containing 2% ammonium
hydroxide. After drying the bed, the analyte was
eluted with mixture of acetonitrile, tetrahydrofuran,
water and formic acid (80 : 1 : 17 : 2, v/v/v/v).
The first step preparation of urine post-mortem
sample, containing phenazepam, preparation consisted of adding of a mixture of PBS buffer (KH2PO4
2.0 g/L, Na2HPO4 ◊ 2H2O 14.4 g/L, KCl 2.0 g/L,
NaCl 80 g/L in water) and sodium acetate buffer
(pH 4.8), deionized water and an internal standard
solution to test material (31). Urine samples were
hydrolyzed with β-glucuronidase/arylsulfatase at
56OC for 30 min. The SPE cartridges were conditioned with methanol and deionized water. The centrifuged sample supernatants were loaded on the cartridges and drawn through under gravity flow. The
cartridges were then washed with both deionized
water and 5% methanol. The cartridges were dried
for 10 min in order to remove washing solutions.
The analytes were then eluted with methanol containing 2% acetic acid. After vortex mixing, sample
was injected into the LCñMS/MS system.
For RP-SPE extraction procedure of basic
drugs also eluents consisting of water ñ organic
modifier in different proportions were used.
Examples are the following: alprazolam, flunitrazepam, and their main metabolites in hemolyzed
blood (32), tandospirone and fluvoxamine in rat
plasma samples (33) on an Oasis HLB SPE
columns, benzodiazepines and tricyclic antidepressants in biological fluids by use of various SPE cartridges (34), tricyclic antidepressants (TCAs) in
urine by use of Lichrolut RP-18 cartridges (13),
diazepam and its metabolites in plasma and brain
samples by use of SPE C2 cartridges (35).
The other sorbents were more rarely applied in
sample-preparation step of plasma and/or tissue
samples.
De Souza synthesized and applied to preparation of plasma samples containing psychotropic
drugs hybrid silica monoliths functionalized with
aminopropyl or cyanopropyl groups as stationary
phase for microextraction by packed sorbent
(MEPS) (6).
SPE procedure applying cation exchange
resins was used for preparation of plasma samples
containing psychotropic drugs and their metabolites
(36). The SPE 96-well plate Oasis MCX support (10
mg), previously prepared by conditioning using 500
µL MeOH and by 500 µL 1 M aqueous citric acid,
was washed by 1 mL of 1 M citric acid in water and
by 1 mL MeOH. Plasma sample diluted with 500 µL
337
1 M citric acid aqueous solution was introduced in
volume of 1000 µL onto conditioned bed and then
analytes were eluted with 500 µL MeOH + ammonium hydroxide 25% (94 : 6, v/v). The eluted samples
were evaporated to dryness and dissolved in mobile
phase prior to analysis. Sample preparation of whole
blood samples for determination of zolpidem and its
metabolite was performed using an ion-exchange
SPE column (37). The SPE columns were conditioned by methanol and 0.1 M HCl. Each blood and
oral fluid sample was spiked with zolpidem-d6
(internal standard, IS). Each sample was diluted (1 :
2) with acetonitrile and centrifuged. The supernatant
was acidified with 0.1 M HCl, transferred to SPE
column and passed through it. Then, the column was
washed with 0.1 M HCl and methanol. Before elution of analytes the column was dried under a vacuum for 5 min. Analytes were eluted with 10% aqueous NH3 in 90% acetonitrile/methanol (1 : 1, v/v).
The eluate was evaporated to dryness under a nitrogen stream. The residue was reconstituted by adding
of mobile phase.
For preparation of human postmortem brain
tissue containing antipsychotic drugs the hybrid
solid phase extraction-precipitation (Hybrid-SPEPPT) technology was applied. (5). Prior to cleanup, human brain samples were homogenized.
Homogenized brain samples were deproteinized
by use of formic acid in acetonitrile with shaking
for 5 min. Then, samples were centrifuged at
10,000 rpm and 48OC for 5 min and the supernatants were transferred to Hybrid-SPE-precipitation cartridges. The eluates were evaporated to
dryness under a nitrogen stream at room temperature and the residues were reconstituted in the
mobile phase.
Microextraction
Solid-phase microextraction (SPME) is another modern technique of sample preparation used to
the analysis of drugs in biological fluids. For example, the method was applied in sample preparation of
human plasma containing nontricyclic antidepressants (38) and in preparation of saliva samples containing tricyclic antidepressants (39).
The variety of microextraction is microextraction by packed sorbent (MEPS). The technique is
based on the miniaturization of conventional SPE
(40) when 1-2 mg of sorbent material is placed in a
syringe with plastic filters between syringe barrel
and injection needle. This makes possible the use of
MEPS on-line connection to liquid or gas chromatograph equipment without modification of the instrument. Chaves et al. used MEPS syringe containing
338
ANNA PETRUCZYNIK and MONIKA WAKSMUNDZKA-HAJNOS
C8 and strong cationic exchange sorbent for
microextraction of antidepressants from human
plasma (Fig. 2) (41).
SPME in polypyrrole-coated capillary was also
applied. Polypyrrole (PPY) is a stationary phase which
exhibit porous structure and good permeability and
multifunctional properties, which enable intermolecular interactions such as: acidñbase, πñπ, dipoleñdipole,
hydrophobic, hydrogen bonding and ion-exchange. It
makes possible the application of above sorbent to
effective sample preparation of analytes with different
properties (42). Plasma samples containing mirtazapine, citalopram, paroxetine, duloxetine, fluoxetine and
sertraline were prepared by SPME-PPY. Good recovery, high sensitivity, precision, and accuracy were
obtained for investigated drugs by this method. Matrix
effect was also investigated (Fig. 3). The SPME efficiency was the highest when the plasma samples were
diluted with phosphate buffer solution at pH 7.0, in
which the drugs (pKa values from 8.7 to 10.2) were
totally or partially in the ionic form.
A polydimethylsiloxane/polypyrrole mixed
phase coated stir bar was developed for the stir bar
sorptive extraction (SBSE) of antidepressants from
plasma samples (43). Chaves et al. optimized the
SBSE variables, such as time, temperature, pH
matrix, ionic strength, and desorption conditions for
optimal condition of plasma samples containing
antidepressants preparation (3). SBSE was also
applied for human serum containing chlorpromazine
and trifluoperazine samples preparation (44).
Microwave-assisted extraction
Microwave-assisted extraction (MAE) has been
rarely applied for isolation of drugs from confections or
body fluids. For extraction of tricyclic antidepressants
from spiked serum samples 0.6 M NaOH was added
and mixed (45). The different extraction solvents containing: n-hexaneñisoamyl alcohol, ethyl acetateñcyclohexane, n-hexaneñacetone and tolueneñisoamyl alcoholñn-hexane were added, then vessels were closed and
placed into the microwave sample preparation system.
Figure 3. Effect of the matrix pH on the SPME efficiency (44).
Analysis of basic psychotropic drugs in biological fluids and tissues by...
Extraction of benzodiazepines from human hair
was performed by MAE (46). To hair sample borate
buffer and ethyl acetate were added. Then, the closed
vessels were placed in the microwave system, where,
for 10 min at 75OC, microwave-assisted extraction
was performed. Then, the content of each vessel was
transferred to a glass tube and centrifuged. Next, the
organic layer was separated and evaporated to dryness under a stream of nitrogen at 40OC. The residue
was then dissolved in the mobile phase.
RP-HPLC
Most psychotropic drugs in their structure have
one or more ionizable groups. The first choice in
HPLC analysis is usually reversed phase system,
when aqueous mobile phases are applied. In such
systems organic electrolytes occur in two forms: as
ions and as undissociated molecules. It causes problems in their analysis, because of different interaction mechanisms of such two forms. There are
hydrophobic interactions of neutral molecule with
alkyl (phenyl) ligands of Van der Waals nature and
ion-exchange or ion-ion interactions of ionized form
of analytes with silanol residues on the stationary
phase surface. Such situation leads to poor separation efficiency, asymmetric peaks and difficulties in
reproducibility of analysis. To avoid these double
interaction the following methods are used: suppression of analyte ionization, suppression of silanol
ionization by the use of buffer at appropriate pH, the
use of ion-pair reagents as eluent additives (anionic
in case of basic analytes, cationic in case of acidic
analytes), silanol blockers and the use of special stationary phases. It allows to optimize conditions for
the analysis of ionizable drugs in pharmaceutical
preparations and/or biological fluids. The examples
of the application of above mentioned analytical
methods are described below.
Mobile phase containing organic modifier and
water
Mobile phases containing only organic modifier
and water were rarely used for analysis of basic psychotropic drugs. The determination of fluvoxamine in
human plasma and urine was performed in system
containing acetonitrile and water as mobile phase (25).
Mobile phase containing addition of salts
Benzodiazepines and tricyclic antidepressants
were determined in biological fluids on C8 column
with mobile phase containing acetonitrile, methanol
and aqueous solution of ammonium acetate (34).
Addition of ammonium acetate to mobile
phase containing acetonitrile and water was also
339
applied for determination of tricyclic antidepressants in biofluids (13).
Tandospirone and fluvoxamine were quantified in the rat plasma on C18 column with mobile
phase containing methanol and aqueous solution of
ammonium acetate (33).
Mobile phases at acidic pH
Chromatographic systems with mobile phases
at low pH to suppress the ionization of the silanols
were often used for analysis of basic psychotropic
drugs.
Kempf et al. developed a method for determination of 105 psychotropic drugs in human serum by
LC-MS (7). Analysis was performed on C18 column
with mobile phase containing formic acid, ammonium formate and acetonitrile.
The determination of paroxetine, venlafaxine,
clozapine, olanzapine, quetiapine, risperidone, and
their active and nonactive metabolites (Ndesmethylsertraline, norfluoxetine, desmethylcitalopram, didemethylcitalopram, N-desmethylvenlafaxine, O-desmethylvenlafaxine, N-desmethylclozapine, N-desmethylolanzapine, 2-hydroxyolanzapine
and 9-hydroxyrisperidone) in human serum was performed on C18 column with mobile phase containing acetonitrile, ammonium acetate and formic acid
(47). The similar chromatographic system was used
for determination of antipsychotic, antidepressant,
anxiolytic and anticonvulsant drugs in plasma samples from schizophrenic patients (6).
The phosphate buffer at acidic pH as component of mobile phase was also applied for determination of antidepressants in plasma samples (43).
Figure 4 presents the chromatogram of the separation of some psychotropic drugs obtained on C18
column with mobile phase containing buffer at pH
3.8.
The use of ultra-high performance liquid chromatography (UHPLC) allows to increase resolution
and sensitivity while decreasing analysis time and
solvents consumption. UHPLC-MS/MS method was
used for the fast quantification of ten psychotropic
drugs: amisulpride, asenapine, desmethylmirtazapine, iloperidone, mirtazapine, norquetiapine, olanzapine, paliperidone, quetiapine and risperidone and
metabolites in human plasma (4). Separation was
performed on an Acquity UPLC BEH Shield RP18
column, using a gradient elution of 10 mM ammonium formate buffer pH 3.0 and acetonitrile. Similar
chromatographic system with mobile phase containing ammonium formate buffer at pH 4.0 was applied
for quantification of antipsychotics in human plasma
on C18 UPLC column (8).
Human serum
Human plasma
Human serum
Human blood
Human blood
Aripiprazole and its major
metabolite
Olanzapine and its metabolite
N-desmethylolanzapine
Ketamine, Lorazepam,
Midazolam and Sufentanil
30 Antipsychotic drugs
Olanzapine
Mirtazapine, Citalopram,
Paroxetine, Duloxetine,
Fluoxetine, Sertraline
Alprazolam, Flunitrazepam,
and their main metabolites
Clozapine
Flunitrazepam,
Nimetazepam
and Nitrazepam
Human plasma
Hemolyzed blood
Hair and nail
Urine
Oral fluid
Human plasma
Olanzapine
25 Benzodiazepines +
Zolpidem
Human plasma
Sample
Fluoxetine, Paroxetine,
Citalopram, Mirtazapine,
Sertraline, Nortriptyline,
Amitriptyline, Desipramine,
Imipramine Clomipramine
Drugs
C18
C 18
Ultra IBD
C18
C18
C18
C18
C18
C18
C18
C18
C18
Column
Mobile phase
Phosphate buffer solution (0.05 mol/L, pH 3.8), acetonitrile (53 : 47)
Acetate buffer 20 mM pH 5, acetonitrile (67 : 33)
Acetonitrile and ammonium acetate buffer (20 mM ammonium acetate buffer
with 0.1% formic acid, pH 4.0) (70 : 30)
(38)
(32)
(28)
(26)
(17)
Mobile phase A: 2 mM ammonium formate, 0.2% formic acid in water and mobile
phase B: 2 mM ammonium formate, 0.2% formic acid in acetonitrile. The initial
condition was 90% A, decreased to 10% A within 8 min and then kept for 2 min.
Finally, the initial condition was restored within 1 min and held for 4 min to
reequilibrate the system
The mobile phase A: 2 mM ammonium formate/ 0.2% formic acid in water and
mobile phase B: 2 mM ammonium formate/0.2% formic acid in acetonitrile.
The initial condition was 90% A, decreased to 10% A within 8 min and then
kept for 2 min. The initial condition was restored within 1 min to 4 min reequilibration
(24)
(16)
Acetonitrile, 0.02M ammonium acetate buffer (70 : 30) and 0.1% formic acid
(12)
Mobile phase A: 50 mM/L ammonium formate adjusted to pH 3.5 with formic
acid, mobile phase B: acetonitrile + 0.1% formic acid. The flow rate and gradient:
equilibration time (-4.00 to 0.00 min) 10% eluent B, flow rate of 1.4 mL/min;
0.00ñ1.00 min: 10% eluent B, flow rate of 1.4 mL/min; 1.01ñ18.00 min: gradient
increase to 100% eluent B, flow rate increase to 2.2 mL/min; 18.01ñ20.00 min: 100% B
(11)
(10)
(19)
(3)
Reference
Acetonitrile and 0.1% formic acid (60/40)
Methanol - 10 mM ammonium acetate in water contained 0.05% (v/v)
formic acid (pH 3.5)
Mobile phase A: 0.1% aqueous formic acid and mobile phase B:
100% acetonitrile. 0% eluent B for 1 min, 95% eluent B for 3 min,
and afterwards reequilibrated with eluent A for 6 min
Acetonitrile-water (50:50 v/v) with 0.1% formic acid
Acetate buffer solution (0.25 mol/L, pH4.5), acetonitrile, methanol (60 : 37 : 3)
Table 1. Chromatographic systems containing mobile phases at acidic pH used for HPLC of psychotropic drugs.
340
ANNA PETRUCZYNIK and MONIKA WAKSMUNDZKA-HAJNOS
Human plasma
Human plasma
Human plasma
18 Psychotropic drugs
Prazepam and main
metabolites
Clonazepam
Urine
Human plasma
Paroxetine
Amitryptiline,Clomipramine,
Thioridazine
Rat brain tissue
Phenibut
C8
C18
C 18
C8
C18
C18
C8
Doxepin, Nordoxepin
C18
Human plasma
Postmortem samples of
body fluids and tissues
C18
Human plasma
Quetiapine and its active
metabolite Norquetiapine
Acquity
UPLC
HSS
C18
C18
Fusion-RP
C8
C18
Column
Citalopram and its
metabolite
Postmortem blood
sample
Human serum
Nordoxepin, Nortriptyline,
Imipramine, Amitriptyline,
Doxepin, Desipramine
Bromazepam, Diazepam,
Fluoxetine, Haloperidol,
Lorazepam, Nordazepam,
Olanzapine, Paroxetine,
Quetiapine Risperidone
Human plasma
Human blood and
oral fluid
Zolpidem and its
metabolite
Fluoxetine, Duloxetine,
Paroxetine, Citalopram,
Mirtazapine, Sertraline
Rat plasma and brain
tissue
Human plasma
Mirtazapine, Citalopram,
Fluoxetine, Sertraline
Paroxetine
Diazepam and its
metabolites
Sample
Drugs
Table 1. Cont.
(53)
(54)
The mobile phase A acetonitrile/water/formic acid (90 : 9 : 1; v/v/v) and the mobile
phase B water/formic acid (99.954 : 0.046; v/v). The gradients: 0.2ñ1.0 min, 50% of mobile
phase A; 1.0ñ2.2 min, 90% of mobile phase A; and 2.2ñ2.8 min, 50% of mobile phase A
(52)
74% methanol in water containing 0.1% (v/v) formic acid
(29)
Acetonitrile (eluent A) and H2O (eluent B), both added by 0.2% of acetic acid, gradient
scheme: an initial 10% A, increasing up to 46.5% in 3 min and then stepping to 90 held
for 1 min. Injection interval was 5.2 min including the reequilibration time
(51)
(27)
(50)
(49)
(48)
(45)
(43)
(37)
(35)
(41)
Reference
Ammonium acetate (0.03 mol/L, pH 5.5), acetonitrile (60 : 40)
Acetonitrile, phosphate buffer (0.02 M, pH = 4.6), and perchloric acid (60%)
(57.25 : 42.5 : 0.25, v/v/v). (21)
Acetonitrileñformic acid (0.1% in water; 8:92, v/v)
Acetonitrile/methanol/4 mM ammonium acetate, pH 3.2 (28 : 7 : 65, v/v/v)
Ammonium formate (10 mmol/L, pH 3.0) ñ methanol (55 : 45, v/v)
Acetonitrileñwater (30:70, v/v) with 0.25% formic acid
Mobile phase A: acetonitrile and mobile phase B: 0.1% formic
acid in water. Gradient program: initial 75% B, during 1 min,
then gradient elution by changing the mobile phase from 75%
to 40% B 1-6 min and then 75% B. Column was equilibrated
under initial conditions for 1.0 min
Acetonitrile and phosphoric buffer at pH 2.36 (1 : 1)
Phosphate buffer solution (0.05 mol/L, pH 3.8) and acetonitrile (57 : 43)
100 mM ammonium acetate at pH 5.8 and methanol (30 : 70)
Acetonitrile, 30 mM phosphate buffer containing
0.3% triethylamine at pH 2.5 (38 : 62)
Potassium buffer 0.05 mol/L pH 4.5 and methanol (55 : 45)
Mobile phase
Analysis of basic psychotropic drugs in biological fluids and tissues by...
341
342
ANNA PETRUCZYNIK and MONIKA WAKSMUNDZKA-HAJNOS
In past few years monolith columns instead of
traditionally packed ones were often applied in the
area of bioanalysis. The resolution and efficiency of
monolith columns are comparable to columns with
adsorbent particles of 3 µm in diameter.
Haloperidol, olanzapine, clonazepam, mirtazapine, paroxetine, citalopram, sertraline, chlorpromazine, imipramine, clomipramine, quetiapine,
diazepam, fluoxetine, clozapine, carbamazepine,
and lamotrigine were determined at sub-therapeutic
levels in plasma samples from schizophrenic
patients by column-switching LC-MS/MS with
organic-inorganic hybrid cyanopropyl monolithic
column (14). The mobile phase consisted of ammonium acetate 5 mmol/L, 0.1% formic acid and acetonitrile.
Other examples of the application of chromatographic systems containing mobile phases at
acidic pH are presented in Table 1.
Mobile phase at basic pH
The interaction of basic cations and free residual silanols can be reduced by the use of eluents of
basic pH. Then, dissociation of bases is suppressed
and ion-exchange interactions of analytes and dissociated silanol groups is blocked. Choong et al. analyzed seven psychotropic drugs and four metabolites
in human plasma on C18 column with mixture of
acetonitrile and ammonium acetate adjusted to pH
8.1 with 25% ammonium hydroxide (Fig. 5) (36).
The mobile phase used for the chromatographic separation of 17 antipsychotic drugs in human
postmortem brain tissue was composed of acetonitrile and aqueous ammonium formate adjusted to pH
8.2 with ammonia (5). Analysis was performed on
C8 column.
The HPLC separations of aripiprazole and
dehydroaripiprazole in human plasma were carried
out on a C18 column with mobile phase containing
acetonitrile: ammonium buffer at pH 8.35 (2).
The mobile phase containing a mixture of acetonitrile and ammonium buffer at pH 8 was applied
for the determination of sertindole and its main
metabolites in human plasma (23).
Mobile phase with addition of silanol blockers
Good peak symmetry and system efficiency for
analysis of basic compounds was often obtained in
systems containing organic amines as silanol blockers.
Silanol groups of gel matrix acts as ionexchanger for cations of basic compounds. Silanol
blockers (basic reagents), as mobile phase additives,
play different roles: at low concentrations they are
responsible for blocking free residual silanols, at
higher concentrations they suppress ionization of
basic analytes. In the first case retention of basic
analytes decreases, in the second an increase of
retention is usually observed.
Quetiapine and some other antipsychotic drugs
in human blood were analyzed on C18 column with
mobile phase containing a mixture of acetonitrile,
water and addition of tetramethylethylenediamine,
adjusted to pH 6.5 by acetic acid (55).
Addition of diethylamine to mobile phase was
applied for separation of tricyclic antidepressant
drugs; nordoxepin, doxepin, desipramine, nortriptyline, imipramine, and amitriptyline in human oral
fluid on C18 column (39).
The mobile phase consisted of dipotassium
hydrogen orthophosphate and triethylamine adjusted to pH 3.7 with orthophosphoric acid and acetonitrile was applied for analysis of risperidone and 9hydroxyrisperidone in human plasma (22).
Lamotrigine and its metabolites in human plasma were also analyzed in chromatographic system
with addition of silanol blocker (56). The determination was performed on C8 column with mobile
phase containing a mixture of methanol, phosphate
buffer at pH 3.5 and 0.17% triethylamine.
Chlorpromazine and trifluoperazine in human
serum were determined on C18 column with mobile
phase containing methanol, sodium acetate buffer at
pH 4.1 and 0.5% triethylamine (44).
Mobile phases containing 20% methanol, 20%
acetonitrile, 20% acetate buffer at pH 3.5 and 0.025 M
DEA was used for analysis of olanzapine and mirtazapine on Polar RP column and 30% MeCN, 20% acetate
buffer at pH 3.5 and 0.025 M DEA on the phenyl-hexyl
column for analysis of risperidone, oxcarbazepine and
quetiapine in human serum samples (57).
Stationary phases
Octadecylsilica or octylsilica stationary phases
are the most popular column packing materials. The
alternatively used stationary phase is the sorbent with
π-π active aromatic moieties introduced to the common n-alkyl chain RP-sites. As a consequence of the
new functionality it causes different retention mechanism based on the π-π interaction and diversifies stationary phase properties. For the stationary phases
showing the π-π interactions, commercially available
are phases such as: cyanopropyl, phenyl, phenylhexyl, biphenyl, pentafluorophenyl. In contrast to
alkyl silica stationary phases, there have been only
few publication on separation and determination of
psychotropic drugs on the π-π type stationary phases.
Quantification of citalopram and their active
main metabolites desmethyl(es) citalopram in
Analysis of basic psychotropic drugs in biological fluids and tissues by...
343
Figure 4. Chromatogram of the antidepressants (a) blank plasma sample spiked with antidepressants at a concentration of 500 ng/mL ((1)
mirtazapine, (2) citalopram, (3) paroxetine, (4) duloxetine, (5) fluoxetine, (6) sertraline); (b) blank plasma sample. Separation was performed in an RP 18 LichroCARTÆ (125 ◊ 4 mm, 5 µm particle size -Merck, Darmstadt, Germany) column, at room temperature (25∞C),
using phosphate buffer solution 0.05 mol/L, pH 3.8, and acetonitrile (53 : 47 v/v) as the mobile phase, in the isocratic mode, at a flow rate
of 1.0 mL/min (45).
human serum was performed on CN column with
mobile phase containing acetonitrile and phosphate
buffer at pH 6.4 (9).
Phenyl column and mobile phase containing
acetonitrile formic acid and ammonium formate
(pH 3.4) was successfully applied for analysis of
tricyclic antidepressant drugs in human oral fluid
(39).
Determination of benzodiazepines in human
hair was performed on phenyl column with mobile
phase containing ammonium formate buffer at pH
3.4 (46).
Pentafluorophenyl (PFP) column was used for
the separation of different drugs including psychotropic drugs in postmortem samples (58). Mobile
phase containing acetonitrile, water, ammonium formate and formic acid were applied.
Donazepil in human serum was analyzed on
phenyl-hexyl column with a mobile phase contain-
ing acetonitrile and phosphate buffer at pH 2.7 (30).
Analysis of phenazepam in urine post-mortem samples was performed on phenyl-hexyl column with
mixture of acetonitrile, ammonium acetate and
formic acid as mobile phase (31). Some psychotropic drugs in fortified human serum samples were
determined on polar RP or on phenyl-hexyl columns
(57).
Separation of psychotropic drugs enantiomers
HPLC was also often applied for psychotropic
drugs enantiomers separation and quantification.
Many drugs including some psychotropic
drugs are mixtures of enantiomers. Because of their
different binding to proteins, enantiomers may have
different action on organism e.g., metabolite of fluoxetine - norfluoxetine enantiomers exhibit different
pharmacological activity. S-norfluoxetine is about
20-times more potent as serotonin reuptake inhibitor
344
ANNA PETRUCZYNIK and MONIKA WAKSMUNDZKA-HAJNOS
in comparison the its enantiomer R-norfluoxetine,
found both in vitro and in vivo experiments.
Separation of fluoxetine and norfluoxetine enantiomers in human plasma was achieved by Silva et
al. on Chiralcel OD-R column and a mobile phase
consisting of potassium hexafluorophosphate and
sodium phosphate solution at pH 3.0 and acetonitrile
(59).
Donazepil enantiomers in human plasma were
determined on a Chiralpak OD column with the
mixture of n-hexane, isopropanol, triethylamine (87
: 12.9 : 0.1, v/v/v) as mobile phase (60).
Figure 5. Total ion current chromatogram of a plasma extract containing drugs: aripiprazole (ARI), atomoxetine (ATO), duloxetine (DUL),
clozapine (CLO), olanzapine (OLA), sertindole(STN), venlafaxine (VEN) and their active metabolites dehydroaripiprazole (DARI), norclozapine (NCLO), dehydrosertindole (DSTN) and O-desmethylvenlafaxine (OVEN) and 100 ng/mL IS. Extracted ion chromatograms at
5 ng/mL. Note the isotopic contribution peak of DSTN and DARI on STN and ARI, respectively. Separation was carried out on a Xbridge
C18 column (2.1 ◊ 100 mm, 3.5 µm) (Waters, Milford,MA,USA) equipped with a guard cartridge (2.1 ◊ 10 mm; 3.5 µm) containing the
same packing material. A 5 µL sample was injected into the system at a flow rate of 300 µL/min. Ammonium acetate 20 mM adjusted to
pH 8.1 with ammonium hydroxide 25% (A) and ACN (B) was used as the mobile phase with the following gradient program: 16% of B
at 0 min, 33.5% of B at 1.31 min, 60% of B maintained from 7.51 to 10.9 min, followed by a washing step at 85% of B from11 to 13 min
and finally, a 5 min reconditioning step at the initial conditions (36).
Analysis of basic psychotropic drugs in biological fluids and tissues by...
REFERENCES
1. Raggi M.A.: Curr. Med. Chem. 9, 1397 (2002).
2. Lancelina F., Djebrani K., Tabaouti K., Kraoul,
L., Brovedani S. et al.: J. Chromatogr. B 867, 15
(2008).
3. Chaves A.R., Silva S.M., Queiroz R.H.C.,
Lancas F.M. Queiroz M.E.C.: J. Chromatogr. B
850, 295 (2007).
4. Ansermot N., Brawand-Amey M., Kottelat A.,
Eap C.B.: J. Chromatogr. A 1292, 160 (2013).
5. Sampedro M.C., Unceta N., GÛmez-Caballero
A., Callado L.F., Morentin B. et al.: Forensic
Sci. Int. 219, 172 (2012).
6. deSouza I.D., Domingues D.S., Queiroz
M.E.C.: Talanta 140, 166 (2015).
7. Kempf J., Traber J., Auw‰rter V., Huppertz
L.M.: Forensic Sci. Int., 243, 84 (2014).
8. Gradinaru J., Vullioud A., Eapa C.B., Ansermot
N.: J. Pharm. Biomed. Anal. 88, 36 (2014).
9. Greiner C., Hiemke C., Bader W., Haen E.: J.
Chromatogr. B 848, 391 (2007).
10. Caloro M., Lionetto L., Cuomo I., Simonetti A.,
Pucci D. et al.: J. Pharm. Biomed. Anal. 62, 135
(2012).
11. Lou H.G., Ruan Z.R., Jiang B., Chen J.L.:
Biomed. Chromatogr. 29, 671 (2015).
12. Nosseir N.S., Michels G., Binder P., Wiesen
M.H.J., M¸ller C.: J. Chromatogr. B 973, 133
(2014).
13. Samanidou V.F., Nika M.K., Papadoyannis
I.N.: J. Sep. Sci. 30, 2391 (2007).
14. Domingues D.S., de Souza I.D., Queiroz
M.E.C.: J. Chromatogr. B 993, 26 (2015).
15. Patteet L., Cappelle D., Maudens K.E., Crunelle
C.L., Sabbe B., Neels H.: Clin. Chim. Acta 441,
11 (2015).
16. Saar E., Gerostamoulos D., Drummer O.H.,
Beyer J.: J. Mass Spectrom., 45, 915 (2010).
17. Jang M., Chang H., Yang W., Choi H., Kim E.
et al.: J. Pharm. Biomed. Anal. 74, 213 (2013).
18. Cho S.-H., Lee H.-W., Im H.-T., Park W.-S.,
Choi Y.-W. et al.: Rapid Commun. Mass
Spectrom. 20, 1293 (2006).
19. Cavalcanti Bedor N.C., Galindo Bedor D.C.,
Miranda de Sousa C.E., Nunes Bonif·cio F., da
Mota Castelo Branco D. et al.: Clin. Exp.
Pharmacol. Physiol. 42, 305 (2015).
20. Mahatthanatrakul W., Nontaput T., Ridtitid W.,
Wongnawa M., Sunbhanichc M.: J. Clin.
Pharm. Ther. 32, 161 (2007).
21. Yasui-Furukori N., Tsuchimine S., Nakagami
T., Fujii A., Sato Y. et al.: Hum.
Psychopharmacol. Clin. Exp. 26, 194 (2011).
345
22. Jones T., Van Breda K., Charles B., Dean A.J.,
McDermottd B.M., Norris R.: Biomed.
Chromatogr. 23, 929 (2009).
23. Canal-Raffin M., Deridet E., Titiera K., Frakra
E., Molimard M., Moore N.: J. Chromatogr. B
814, 61 (2005).
24. Eishafeey A.H., Eisherbiny M.A., Fathallah
M.M.: Clin. Ther. 31, 600 (2009).
25. Ulu S.T.: J. Pharm. Biomed. Anal. 43, 1444
(2007).
26. Lee H.H., Lee J.F., Lin S.Y., Lin Y.Y., Wu C.F.
et al.: Clin. Chim. Acta 420, 134 (2013).
27. Gronewold A., Dettling A., Haffner H.T.,
Skopp G.: Forensic Sci. Int. 190, 74 (2009).
28. Chen H., Xiang P., Shen M.: J. Forensic Leg.
Med. 22, 62 (2014).
29. Xiong C., Ruan J., Cai Y., Tang Y.: J. Pharm.
Biomed. Anal. 49, 572 (2009).
30. Koeber R., Kluenemann H.-H., Waimer R.,
Koestlbacher A., Wittmann M. et al.: J.
Chromatogr. B 881- 882, 1 (2012).
31. Kriikku P., Wilhelm L., Rintatalo J., Hurme J.,
Kramer J., Ojanper‰ I.: Forensic Sci. Int. 220,
111 (2012).
32. Marchi I., Schappler J., Veuthey J.-L., Rudaz
S.: J. Chromatogr. B 877, 2275 (2009).
33. Nishikawa H., Inoue T., Masui T., Izumi T.,
Nakagawa S., Koyama, T.: Psychiatry Clin.
Neurosci. 62, 591 (2008).
34. Uddin M.N., Samanidou V.F., Papadoyannis
I.N.: J. Sep. Sci. 31, 2358 (2008).
35. Mercolini L., Mandrioli R., Iannello C.,
Matrisciano F., Nicoletti F., Raggi M.A.:
Talanta 80, 279 (2009).
36. Choong E., Rudaz S., Kottelat A., Guillarme D.,
Veuthey J.-L., Eap, C.B.: J. Pharm. Biomed.
Anal. 50, 1000 (2009).
37. Piotrowski P., Bocian S., Sliwka K., Buszewski
B.: Adv. Med. Sci. 60, 167 (2015).
38. Silva B.J.G., Lancas F.M., Queiroz M.E.C.: J.
Chromatogr. B 862, 181 (2008).
39. Woüniakiewicz M., Wietecha-Pos≥uszny R.,
Moos A., Wieczorek M., Knihnicki P., Koúcielniak P.: J. Chromatogr. A 1337, 9 (2014).
40. Altun Z., Abdel-Rehim M.: Anal. Chim. Acta
630, 116 (2008).
41. Chaves A.R., Leandro F.Z., Carris J.A., Queiroz
M.E.C.: J. Chromatogr. B 878, 2123 (2010).
42. Chaves A.R., Chiericato G., Queiroz M.E.C.: J.
Chromatogr. B 877, 587 (2009).
43. Melo L.P., Nogueira A.M., Lancas F.M., Queiroz M.E.C.: Anal. Chim. Acta 633, 57 (2009).
44. Bazhdanzadeh S., Talebpour Z, Adib N.,
Aboul-Enein H.Y.: J. Sep. Sci., 34, 90 (2011).
346
ANNA PETRUCZYNIK and MONIKA WAKSMUNDZKA-HAJNOS
45. Woøniakiewicz M., Wietecha-Pos≥uszny R.,
Garbacik A., Koúcielniak P.: J. Chromatogr. A,
1190, 52 (2008).
46. Wietecha-Pos≥uszny R., Woüniakiewicz M.,
Garbacik A., Chesya P., Koúcielniak P.: J.
Chromatogr. A 1278, 22 (2013).
47. Urinovsk· R., Brozmanov· H., SistÌk P., Silh·n
P., KacÌrov· I. et al.: J. Chromatogr. B 907, 101
(2012).
48. Proenca P., Franco J.M., Mustra C., Monteiro
C., Costa J. et al.: Forensic Sci. Int. 227, 85
(2013).
49. Jiang T., Ronga Z., Penga L., Chen B., Xiea Y.
et al.: J. Chromatogr. B 878, 615 (2010).
50. Xiong X., Yang L., Duan J.: Clin. Chim. Acta
423, 69 (2013).
51. Grinberga S., Zvejniece L., Liepinsh E.,
Dambrova M., Pugovics O.: Biomed.
Chromatogr. A 22, 1321 (2008).
52. Vecchione G., Casetta B., Chiapparino A.,
Bertolino A., Tomaiuolo M. et al.: J. Pharm.
Biomed. Anal. 67-68, 104 (2012).
53. Valavani P., Atta-Politou J., Panderi I.: J. Mass
Spectrom. 40, 516 (2005).
54. Cavedal L.E., Mendes F.D., Domingues C.C.,
Patni A.K., Monif T. et al.: J. Mass Spectrom.
42, 81 (2007).
55. Sachse J., Koller J., Hartte S., Hiemke C.: J.
Chromatogr. B 830, 342 (2006).
56. Saracino M.A., Bugamelli F., Conti M., Amore
M., Raggi M.A.: J. Sep. Sci. 30, 2249 (2007).
57. Petruczynik
A.,
WrÛblewski
K.,
Waksmundzka-Hajnos M.: J. Chromatogr. Sci.
53, 394 (2015).
58. Han E., Kim E., Hong H., Jeong S., Kim J. et
al.: Forensic Sci. Int. 219, 265 (2012).
59. Silva B.J.G., Lancas F.M., Queiroz M.E.C.: J.
Chromatogr. A 1216, 8590 (2009).
60. Lili W., Cheng G., Zhiyong Z., Qi Y., Yan, L.
et al.: Chirality 25, 498 (2013).
Received: 21. 03. 2016