An improved HPLC-ED method for monitoring plasma levels of clozapine and its active metabolites in schizophrenic patients

Summary An HPLC method with electrochemical detection has been developed for the determination of clozapine and its main metabolites, desmethylclozapine and clozapine N-oxide, in human plasma. An accurate pretreatment of the biological samples was implemented by means of solid phase extraction (SPE) on HLB cartridges. This improved pretreatment, together with a new mobile phase, allows for the accurate determination of clozapine N-oxide, which could not be quantitated by a previous method. The method uses only 100 μL of plasma for one complete analysis and shows good recovery values for all three analytes. The eluates from the SPE procedure were chromatographed in a reversed phase C18 column using a mobile phase composed of phosphate buffer, acetonitrile and methanol. Clozapine, desmethylclozapine and clozapine N-oxide were eluted in less than 10 minutes, without any interference from the biological matrix. Linearity was observed over the 2.50–150 ng mL⁻¹ (clozapine and desmethylclozapine) or 1.25–75 ng mL⁻¹ clozapine N-oxide) range for the three analytes, with satisfactory repeatability values. The limit of detection was 0.3 ng mL⁻¹ for clozapine and desmethylclozapine, samples of patients treated with Leponex gave good results. No interference from other common central nervous system drugs was found. This method seems to be a useful tool for pharmacokinetic studies and for clinical monitoring, because of its need for small plasma samples and its high sensitivity and selectivity.     

Separation and HPLC analysis of 15 benzodiazepines in human plasma

Benzodiazepines (BZDs) are often prescribed to schizophrenic or depressed patients, as a part of polypharmacy regimens. An HPLC method has been developed for the simultaneous determination of 15 BZDs in human plasma. Separation was obtained by using a C8 RP column and a mobile phase composed of 65% aqueous phosphate buffer at pH 3.0 and 35% ACN. The UV detector was set at 220 nm and clomipramine was used as the internal standard. A careful pretreatment procedure of plasma samples was developed, using SPE with C1 cartridges, which gives high extraction yields (> 97%). The LOQs were always lower than 7.6 ng/mL and the LODs always lower than 2.6 ng/mL for all analytes. The method was successfully applied to plasma samples from depressed and schizophrenic patients undergoing polypharmacy with one or more BZDs. Precision data, as well as accuracy results, were satisfactory and no interference from other drugs was found. Hence, the method seems to be suitable for the therapeutic drug monitoring (TDM) of patients undergoing therapy with one or more BZDs.     

Liquid chromatography/tandem mass spectrometry method for the simultaneous determination of olanzapine, risperidone, 9-hydroxyrisperidone, clozapine, haloperidol and ziprasidone in rat plasma

A simple, sensitive and rapid liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method was developed and validated for simultaneous quantification of olanzapine, clozapine, ziprasidone, haloperidol, risperidone, and its active metabolite 9-hydroxyrisperidone, in rat plasma using midazolam as internal standard (IS). The analytes were extracted from rat plasma using a single step liquid-liquid extraction technique. The compounds were separated on a Waters Atlantis dC-18 (30 mm x 2.1 mm i.d., 3 microm) column using a mobile phase of acetonitrile/5 mM ammonium formate (pH 6.1 adjusted with formic acid) with gradient elution. All of the analytes were detected in positive ion mode using multiple reaction monitoring (MRM). The method was validated and the specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, recoveries and stability were determined. LLOQ was 0.1 ng/mL and correlation coefficient (R(2)) values for the linear range of 0.1-100 ng/mL were 0.997 or greater for all the analytes. The intra-day and inter-day precision and accuracy were better than 8.05%. The relative and absolute recovery was above 77% and matrix effects were low for all the analytes except for ziprasidone. This validated method has been successfully used to quantify the plasma concentration of the analytes after chronic treatment with antipsychotic drugs.     

Simultaneous determination of clozapine, clozapine N-oxide, N-desmethylclozapine, risperidone, and 9-hydroxyrisperidone in plasma by high performance liquid chromatography with ultraviolet detection

A simple high performance liquid chromatography techniques with ultraviolet detection (HPLC–UV) method is described for the simultaneous determination of clozapine (CZP), clozapine N-oxide (CNO), N-desmethylclozapine (NCZ), risperidone (RSP) and 9-hydroxyrisperidone (9-OHRSP) in human plasma. After extraction process, the analytes were separated on a C₁₈ column (150 mm×3.9 mm i.d.) by the mobile phase (methanol–water–dimethylamine, 60:40:0.04 (v:v:v)). Relative recoveries of five analytes were quantitative. The precision and accuracy of intra- and interday assays were all below 8.2% for R.S.D. and 5.6% for RE, respectively. Based on 1 ml of plasma, the limits of detection were 2.0 ng/ml for CZP, 0.2 ng/ml for CZP N-oxide, 1.0 ng/ml for NCZ, 1.0 ng/ml for RSP, and 0.5 ng/ml for 9-OHRSP (S/N=3). The calibration curves were linear (r≥0.988). This method was applied to therapeutic drug monitoring of schizophrenia patients receiving CZP or RSP therapy.     

Urine polyamines determination using dansyl chloride derivatization in solid-phase extraction cartridges and HPLC

The derivatization of biogenic amines such as putrescine, cadaverine, spermidine and spermine with dansyl chloride in solid phase extraction cartridges is described. Different types of filling materials were tested in order to have the highest retention of the different analytes. The best results were obtained by using C18 cartridges. The optimal conditions were: amine solution buffered at pH 12, 2 mM dansyl chloride (acetone-bicarbonate solution 20 mM (pH 9-9.5), 2 + 3 v/v) as reagent concentration, room temperature and 30 min reaction time. The developed procedure was applied to the determination of these polyamines in urine samples from healthy controls and cancer patients using HPLC with 1,7-diaminoheptane as internal standard. The concentrations ranged from 0.5 to 5 micrograms mL-1 and the detection limits were 10 ng mL-1 for all polyamines. By concentrating the urine extracts, the detection limits were improved down to 2 ng mL-1. The accuracy and the precision of the method were tested. The proposed dansylation method is advantageous with respect to solution dansylation. It improves the total analysis time, avoids high temperatures that can affect the thermal stability of the derivatives and could make possible the automation of the procedure.     

Confirmatory analysis of acetylgestagens in plasma using liquid chromatography-tandem mass spectrometry

A confirmatory method has been developed and validated for the determination of chlormadinone acetate (CMA), megestrol acetate (MGA), melengestrol acetate (MLA) and medroxyprogesterone acetate (MPA) in bovine and porcine plasma. Analytes are extracted from plasma samples using matrix-assisted liquid-liquid extraction (LLE) on Extrelut NT columns followed by C18 solid-phase extraction (SPE). Analytes were analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and quantification was performed using matrix-matched calibration standards in combination with deuterated internal standards. In accordance with Commission Decision 2002/657/EC, two ion transitions were monitored for each analyte. Decision limits (CCalpha) were estimated by analysing 20 blank plasma samples and ranged from 0.1 to 0.2 ng mL(-1). Detection capabilities (CCbeta) were estimated using 20 plasma samples fortified at 0.5 ng mL(-1) and were <0.5 ng mL(-1). In the range 0.5-2 ng mL(-1), the mean intra-laboratory reproducibility of the analytes ranged from 6 to 18% (%R.S.D.). Analytes were shown to be stable in fortified plasma samples for >8 months when stored at -20 degrees C.     

Sensitive quantification of clozapine and its main metabolites norclozapine and clozapine-<Emphasis Type="Italic">N</Emphasis>-oxide in serum and urine using LC-MS/MS after simple liquid–liquid extraction work-up

An LC-MS/MS method for the determination of the atypic neuroleptic clozapine and its two main metabolites norclozapine and clozapine-N-oxide has been developed and validated for serum and urine. After addition of d4-clozapine as deuterated internal standard a fast single-step liquid–liquid extraction under alkaline conditions and with ethyl acetate as organic solvent followed. The analytes were chromatographically separated on a Synergi Polar RP column using gradient elution with 1 mM ammonium formate and methanol. Data acquisition was performed on a QTrap 2000 tandem mass spectrometer in multiple reaction monitoring mode with positive electrospray ionization. Two transitions were monitored for each analyte in order to fulfill the established identification criteria. The validation included the determination of the limits of quantification (1.0 ng/mL for all analytes in serum and 2.0 ng/mL for all analytes in urine), assessment of matrix effects (77% to 92% in serum, 21 to 78% in urine) and the determination of extraction efficiencies (52% to 85% for serum, 59% to 88% for urine) and accuracy data. Imprecision was <10%, only the quantification of norclozapine in urine yielded higher relative standard deviations (11.2% and 15.7%). Bias values were below ±10%. Dilution of samples had no impact on the correctness for clozapine and norclozapine in both matrices and for clozapine-N-oxide in serum. For quantification of clozapine-N-oxide in urine a calibration with diluted calibrators has to be used. Calibration curves were measured from the LOQ up to 2,000 ng/mL and proved to be linear over the whole range with regression coefficients higher than 0.98. The method was finally applied to several clinical serum and urine samples and a cerebro-spinal fluid sample of an intoxicated 13-month-old girl.     

HPLC analysis and pharmacokinetic study of quercitrin and isoquercitrin in rat plasma after administration of Hypericum japonicum thunb. extract

A simple HPLC method was developed for determination of quercitrin and isoquercitrin in rat plasma. Reversed-phase HPLC was employed for the quantitative analysis using kaempferol-3-O-beta-D-glucopyranoside-7-O-alpha-L-rhamnoside as an internal standard. Following extraction from the plasma samples with ethyl acetate-isopropanol (95:5, v/v), these two compounds were successfully separated on a Luna C(18) column (250 x 4.6 mm, 5 microm) with isocratic elution of acetonitrile-0.5% aqueous acetic acid (17:83, v/v) as the mobile phase. The flow-rate was set at 1 mL/min and the eluent was detected at 350 nm for both quercitrin and isoquercitrin. The method was linear over the studied ranges of 50-6000 and 50-5000 ng/mL for quercitrin and isoquercitrin, respectively. The intra- and inter-day precisions of the analysis were better than 13.1 and 13.2%, respectively. The lower limits of quantitation for quercitrin and isoquercitrin in plasma were both of 50 ng/mL. The mean extraction recoveries were 73 and 61% for quercitrin and isoquercitrin, respectively. The validated method was successfully applied to pharmacokinetic studies of the two analytes in rat plasma after the oral administration of Hypericum japonicum thunb. ethanol extract.     

Simultaneous HPLC determination of 14 tricyclic antidepressants and metabolites in human plasma

A HPLC method has been developed for the simultaneous determination of seven tricyclic antidepressants (TCAs) and seven metabolites in human plasma. The analyte separation was obtained using a C8 reversed phase column and a mobile phase composed of 68% aqueous phosphate buffer at pH 3.0 and 32% ACN. The UV detector was set at 220 nm and loxapine was used as the internal standard. A careful pre‐treatment procedure for plasma samples was developed, using SPE on C2 cartridges, which gives satisfactory extraction yields (>80%) and good sample purification. The LOQs were always lower than 9.1 ng/mL and the LODs always lower than 3.1 ng/mL for all analytes. The method was successfully applied to plasma samples from depressed patients undergoing therapy with one or more TCA drugs. Precision data (RSD <8.1%), as well as accuracy results (recovery >80%), were satisfactory and no interference from other drugs was found. Hence the method seems to be suitable for the therapeutic drug monitoring of patients treated with TCAs under monotherapy or polypharmacy regimens.     

Quantitative determination of pravastatin and its metabolite 3α‐hydroxy pravastatin in plasma and urine of pregnant patients by LC‐MS/MS

This report describes the development and validation of a chromatography/tandem mass spectrometry method for the quantitative determination of pravastatin and its metabolite (3α‐hydroxy pravastatin) in plasma and urine of pregnant patients under treatment with pravastatin, as part of a clinical trial. The method includes a one‐step sample preparation by liquid–liquid extraction. The extraction recovery of the analytes ranged between 93.8 and 99.5% in plasma. The lower limits of quantitation of the analytes in plasma samples were 0.106 ng/mL for pravastatin and 0.105 ng/mL for 3α‐hydroxy pravastatin, while in urine samples they were 19.7 ng/mL for pravastatin and 2.00 ng/mL for 3α‐hydroxy pravastatin. The relative deviation of this method was <10% for intra‐ and interday assays in plasma and urine samples, and the accuracy ranged between 97.2 and 106% in plasma, and between 98.2 and 105% in urine. The method described in this report was successfully utilized for determining the pharmacokinetics of pravastatin in pregnant patients enrolled in a pilot clinical trial for prevention of preeclampsia. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous determination of morphine‐6‐d‐glucuronide,morphine‐3‐d‐glucuronide and morphine in human plasma and urine by ultra‐performance liquid chromatography–tandem mass spectrometry: Application to M6G injection pharmacokinetic study

A robust ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for the determination of morphine‐6‐d ‐glucuronide (M6G), morphine‐3‐d ‐glucuronide (M3G) and morphine (MOR) in human plasma and urine has been developed and validated. The analytes of interest were extracted from plasma by protein precipitation. The urine sample was prepared by dilution. Both plasma and urine samples were chromatographed on an Acquity UPLC HSS T₃ column using gradient elution. Detection was performed on a Xevo TQ‐S tandem mass spectrometer in multiple reaction monitoring mode using positive electrospray ionization. Matrix interferences were not observed at the retention time of the analytes and internal standard, naloxone‐D5. The lower limits of quantitation of plasma and urine were 2/0.5/0.5 and 20/4/2 ng/mL for M6G/M3G/MOR, respectively. Calibration curves were linear over the concentration ranges of 2–2000/0.5–500/0.5–500 and 20–20,000/4–4000/2–2000 ng/mL for M6G/M3G/MOR in plasma and urine samples, respectively. The precision was <7.14% and the accuracy was within 85–115%. Furthermore, stability of the analytes at various conditions, dilution integrity, extraction recovery and matrix effect were assessed. Finally, this quantitative method was successfully applied to the pharmacokinetic study of M6G injection in Chinese noncancer pain patients.     

Automatic on‐line solid‐phase extraction with ultra‐high performance liquid chromatography and tandem mass spectrometry for the determination of ten antipsychotics in human plasma

An automatic on‐line solid‐phase extraction with ultra‐high performance liquid chromatography and tandem mass spectrometry method was developed for the simultaneous determination of ten antipsychotics in human plasma. The plasma sample after filtration was injected directly into the system without any pretreatment. A Shim‐pack MAYI‐C₈ (G) column was used as a solid‐phase extraction column, and all the analytes were separated on a Shim‐pack XR‐ODS III column with a mobile phase consisting of 0.1% v/v formic acid in water with 5 mM ammonium acetate and acetonitrile. The method features were systematically investigated, including extraction conditions, desorption conditions, the equilibration solution, the valve switching time, and the dilution for column‐head stacking. Under the optimized conditions, the whole analysis procedure took only 10 min. The limits of quantitation were in the range of 0.00321–2.75 μg/L and the recoveries ranged from 75.9 to 122%. Compared with the off‐line ultra‐high performance liquid chromatography and the reported methods, this validated on‐line method showed significant advantages such as minimal pretreatment, shortest analysis time, and highest sensitivity. The results indicated that this automatic on‐line method was rapid, sensitive, and reliable for the determination of antipsychotics in plasma and could be extended to other target analytes in biological samples.     

Determination of seven selected antipsychotic drugs in human plasma using microextraction in packed sorbent and gas chromatography–tandem mass spectrometry

A method using microextraction by packed sorbent (MEPS) and gas chromatography–tandem mass spectrometry (GC-MS/MS) is described for the determination of seven antipsychotic drugs in human plasma. The studied compounds were chlorpromazine (CPZ), haloperidol (HAL), cyamemazine, quetiapine, clozapine, olanzapine (OLZ), and levomepromazine; promazine, protriptyline, and deuterated CPZ were used as internal standards. The validation parameters included selectivity, linearity and limits of detection and quantitation, intra- and interday precision and trueness, recovery, and stability and were studied according to internationally accepted guidelines. The method was found to be linear between the lower limit of quantitation and 1000 ng/mL, except for OLZ and HAL (200 ng/mL), with determination coefficients higher than 0.99 for all analytes, and extraction efficiencies ranged from 62 to 92 %. Intra- and interday precision ranged from 0.24 to 10.67 %, while trueness was within a ±15 % interval from the nominal concentration for all analytes at all studied levels. MEPS has shown to be a rapid procedure for the determination of the selected antipsychotic drugs in human plasma, allowing reducing the handling time and the costs of analysis. Furthermore, GC-MS/MS has demonstrated to be a powerful tool for the simultaneous quantitation of the studied compounds, enabling obtaining adequate selectivity and sensitivity using a sample volume of as low as 0.25 mL.     

A sensitive and selective liquid chromatographic/electrospray ionization tandem mass spectrometric assay for the simultaneous quantification of alpha-,beta-arteether and its metabolite dihydroartemisinin in plasma,useful for pharmacokinetic studies

A sensitive, selective, specific and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay method was developed and validated for the simultaneous quantitation of alpha-,beta-arteether (alpha-,beta-AE) and its metabolite alpha-dihydroartemisinin (DHA) in monkey plasma using the propyl ether analogue of beta-arteether (PE) as an internal standard. The method involves a simple two-step liquid-liquid extraction with hexane. The analytes were chromatographed on a C(18) reversed-phase chromatographic column by isocratic elution with methanol-ammonium acetate buffer (pH 4) (92 : 8, v/v) and analysed by mass spectrometry in the multiple reaction monitoring mode. The chromatographic run time was 7 min and the weighted (1/x(2)) calibration curves were linear over the range 0.78-200 ng ml(-1). The method was validated in terms of accuracy, precision, absolute recovery, freeze-thaw stability, bench-top stability and re-injection reproducibility. The limit of detection and lower limit of quantification in monkey plasma were 0.39 and 0.78 ng ml(-1) respectively for all the analytes. The intra- and inter-batch precision and accuracy were found to be well within acceptable limits (<15%). All three analytes were stable even after three freeze-thaw cycles (deviation < 15%). The average absolute recoveries of alpha-,beta-AE, DHA and PE, used as an internal standard, from spiked plasma samples were 85.85 +/- 6.56, 70.10 +/- 7.06, 54.37 +/- 3.39 and 93.90 +/- 6.9%, respectively. The assay method described here could be applied to study the pharmacokinetics of alpha-,beta-AE and DHA in rhesus monkeys.     

Analysis of 21 progestagens in various matrices by ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) with diverse sample pretreatment

In this study, a highly sensitive and robust method using an ultra-high-performance liquid chromatography-tandem mass spectrometry combined with solid-phase extraction and ultrasonic extraction for pretreatment and silica gel purification steps has been developed for determination of 21 natural and synthetic progestagens in river surface water and sediments, and influents, effluents, and sludge from municipal wastewater treatment plants, and flush water and feces from swine farms. For the various matrices considered, the optimized method showed satisfactory performance with recoveries of 70–129 % (except AD, 5α-DHP, DPT, HPC), the limits of quantification below 2.30 ng/L for liquid samples and 2.59 ng/g for solid samples (except AD), and good linearity and reproducibility. This developed method was successfully applied in the analysis of progestagens in environmental samples from Liuxi Reservoir, Xintang municipal wastewater treatment plant, and Shunfeng swine farm in South China. Six analytes were detected at trace levels in surface water, effluent, and sediment samples. Seven analytes (0.7 (HPA)–35.1 ng/L (DGT)) were found in the influent samples and three analytes (5.6 (DGT)–11.8 ng/g (5α-DHP)) in the dewatered sludge samples. Moreover, 13 analytes were detected in swine farm, with high concentrations ranging from 23.8 ng/L (ET) to 5,024 ng/L (P) in flush water, and from 20.0 ng/g (MPA) to 1952 ng/g (P) in feces.     

Determination of perfluorinated chemicals in food and drinking water using high-flow solid-phase extraction and ultra-high performance liquid chromatography/tandem mass spectrometry

For this study, we developed methods of determining ten perfluorinated chemicals in drinking water, milk, fish, beef, and pig liver using high-flow automated solid-phase extraction (SPE) and ultra-high performance liquid chromatography/tandem mass spectrometry. The analytes were separated on a core-shell Kinetex C18 column. The mobile phase was composed of methanol and 10-mM N-methylmorpholine. Milk was digested with 0.5 N potassium hydroxide in Milli-Q water, and was extracted with an Atlantic HLB disk to perform automated SPE at a flow rate ranged from 70 to 86 mL/min. Drinking water was directly extracted by the SPE. Solid food samples were digested in alkaline methanol and their supernatants were diluted and also processed by SPE. The disks were washed with 40% methanol/60% water and then eluted with 0.1% ammonium hydroxide in methanol. Suppression of signal intensity of most analytes by matrixes was lower than 50%; it was generally lower in fish and drinking water but higher in liver. Most quantitative biases and relative standard deviations were lower than 15%. The limits of detection for most analytes were sub-nanograms per liter for drinking water and sub-nanograms per gram for solid food samples. This method greatly shortened the time and labor needed for digestion, SPE, and liquid chromatography. This method has been applied to analyze 14 types of food samples. Perfluorooctanoic acid was found to be the highest among the analytes (median at 3.2-64 ng/g wet weight), followed by perfluorodecanoic acid (0.7-25 ng/g) and perfluorododecanoic acid (0.6-15 ng/g).     

Enantioselective determination of chloroquine and its n-dealkylated metabolites in plasma using liquid-phase microextraction and LC-MS

A selective method using three-phase liquid-phase microextraction (LPME) in conjunction with LC-MS-MS was devised for the enantioselective determination of chloroquine and its n-dealkylated metabolites in plasma samples. After alkalinization of the samples, the analytes were extracted into n-octanol immobilized in the pores of a polypropylene hollow fiber membrane and back extracted into the acidic acceptor phase (0.1 M TFA) filled into the lumen of the hollow fiber. Following LPME, the analytes were resolved on a Chirobiotic V column using methanol/ACN/glacial acetic acid/diethylamine (90:10:0.5:0.5 by volume) as the mobile phase. The MS detection was carried out using multiple reaction monitoring with ESI in the positive ion mode. The optimized LPME method yielded extraction recoveries ranging from 28 to 66%. The method was linear over 5-500 ng/mL and precision (RSD) and accuracy (relative error) values were below 15% for all analytes. The developed method was applied to the determination of the analytes in rat plasma samples after oral administration of the racemic drug.     

Analysis of phenoxyalkanoic acid herbicides and their phenolic conversion products in soil by microwave assisted solvent extraction and subsequent analysis of extracts by on-line solid-phase extraction-liquid chromatography

A multiresidue method for the determination of phenoxyalkanoic acid herbicides and their phenolic conversion products in soil was developed. The method was based on microwave-assisted solvent extraction (MASE) of soil samples by an aqueous methanolic mixture and subsequent analysis of extracts by automated solid-phase extraction followed by on-line high-performance liquid chromatography and diode array detection. MASE parameters (extraction temperature and time, composition of the extraction mixture and extraction volume) were optimized with respect to analyte recoveries. The method was validated with two types of soils containing 1.5 and 3.5% organic matter, respectively, both types containing fresh and aged residues of sought analytes. Under the selected analytical conditions when soils with fresh residues were analyzed all target analytes were recovered above 80% from the soil containing 1.5% organic matter, while limits of identification at the level of 20-40 ng/g were achieved. From the soil containing 3.5% organic matter the least polar phenolic analytes exhibited slightly reduced recoveries, while identification limits of 30-50 ng/g were achieved. Samples with aged residues exhibited reduced recoveries for some analytes, the reduction amounting up to 6-12% within 1 month of aging period depending on soil organic matter.     

Dispersive solid-phase extraction of risperidone from plasma samples using graphene oxide aerogels and determination with liquid chromatography

A graphene oxide-based aerogel was synthesized and applied to the extraction and the determinations with the high-performance liquid chromatography-ultraviolet detector. After the characterization of the produced graphene-aerogel, it was utilized as a dispersive solid-phase extraction sorbent for risperidone extraction from plasma samples. Aerogels are materials with a large surface area-to-mass ratio and plenty of core with functional groups which can easily attach to the analytes to extract them to the second phase. The suggested method determined risperidone in plasma samples in the wide dynamic range from 20 ng/ml to 3 μg/ml. The limits of detection and quantification of the developed method were calculated as 2.4 and 8.2 ng/ml, respectively. As a novel feature, the developed method has no need to precipitate plasma proteins, improving the analytical performance of the analysis. Also, for the first time, the produced materials were utilized for the extraction of risperidone from the plasma samples. The obtained results revealed that the developed approach could be employed as an accurate method for the quantification of risperidone in real plasma samples.     

Stereoselective determination of demethyl- and didemethyl-citalopram in rat plasma and brain tissue by liquid chromatography with fluorescence detection using precolumn derivatization

A rapid and sensitive HPLC enantioselective method with fluorescence detection was developed to determine (-)-(R) and (+)-(S) enantiomers of the metabolites of citalopram, demethyl- and didemethyl-citalopram in plasma and brain tissue. This assay involves pre-column chiral derivatization with (-)-(R)-1-(1-naphthyl)ethyl isocyanate followed by separation on a normal-phase silica column. The developed liquid-liquid extraction procedure permits quantitative determination of analytes with recoveries ranged between 81 and 88% with intra- and inter-day relative standard deviations less than 10.5%. Linearity was obtained over the concentration range 5-1000 ng/mL and 100-10,000 ng/g for spiked drug-free plasma and brain tissue, respectively, with detection limits lower than 2.1 ng/mL and 42.8 ng/g.