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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. 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