Simultaneous Quantification of Oroxylin A and Its Metabolite Oroxylin A-7-O-Glucuronide: Application to a Pharmacokinetic Study in Rat

A sensitive liquid chromatography?Celectrospray ionization?Ctandem mass spectrometry (LC?CESI?CMS?CMS) method was developed and validated for the quantification of cepharanthine (CEP) in beagle dog plasma. The chromatographic separation was performed on an Agilent-C18 column and the mobile phase was composed of methanol:water with 10 mM ammonium acetate (20:80, v/v). Detection was operated in the positive ion mode and the tandem mass spectrometer was tuned in the multiple reactions monitoring mode (MRM) to monitor m/z transitions 607 ?? 365 for CEP and 285 ?? 193 for the internal standard (IS) diazepam. This method exhibited a linear range of 5?C2,500 ng mL^?1. The precision (RSD%) and accuracy (RME%) of the assay were <8.7 and 2.4%, respectively. The limit of quantification was 5 ng mL^?1 and no significant matrix effect was observed. The validated method has been successfully applied to pharmacokinetic study of CEP in beagle dog.     

Determination of Doxapram Hydrochloride in Rabbit Plasma by LC-MS-MS and Its Application

A sensitive and selective liquid chromatography tandem mass spectrometry (LC-MS-MS) method for determination of doxapram hydrochloride in rabbit plasma was developed. After addition of urapidil hydrochloride as internal standard (IS), protein precipitation by 10% trichloroacetic acid in methanol (w/v) was used as sample preparation. Chromatographic separation was achieved on a Zorbax SB-C18 (2.1 mm × 50 mm, 3.5 μm) column with acetonitrile–water as mobile phase with gradient elution. Electrospray ionization (ESI) source was applied and operated in positive ion mode; multiple reaction monitoring (MRM) mode was used for quantification using target fragment ions m/z 378.9 → 291.8 for doxapram hydrochloride and m/z 387.9 → 204.6 for the IS. Calibration plots were linear over the range of 2–1000 ng mL^?1 for doxapram hydrochloride in plasma. Lower limit of quantitation (LLOQ) for doxapram hydrochloride was 2 ng mL^?1. Mean recovery of doxapram hydrochloride from plasma was in the range 83.7–91.5%. RSD of intra-day and inter-day precision were less than 9%, respectively. This method is simple and sensitive enough to be used in pharmacokinetic research for determination of doxapram hydrochloride in rabbit plasma.     

LC–MS/MS Method for Detection of a Highly Selective KCa3.1 Blocker,TRAM-34, in Rat Plasma

1-[(2-Chlorophenyl)diphenylmethyl]pyrazole (TRAM-34) is a highly selective K_Ca3.1 channel blocker. TRAM-34 was commonly used to study the role of K_Ca3.1 in the pathogenesis of disease in vivo, but there was no validated analytical method. Here, we describe the first validated LC–MS/MS analytical method for TRAM-34. Solid-phase extraction (SPE) was performed to extract TRAM-34 from the rat plasma. Chromatographic separation was achieved on the phenyl column. A triple quadrupole mass spectrometer was operated in positive-mode electrospray ionization. There were two multiple-reaction monitoring (MRM) transitions for TRAM-34: m/z 277.2 → 165.1 (for quantification) and m/z 277.2 → 241.2 (for qualification). Bifonazole was used as an internal standard. The lower limit of quantification (LLOQ) achieved was 1 ng mL^?1 and the run time was 7.5 min. The linear range was from 1 to 1,000 ng mL^?1. The pharmacokinetics profile was acquired for rats following an intraperitoneal injection of TRAM-34, with the following pharmacokinetics parameters found: C _max 17.03 ± 1.34 ng mL^?1; T _max 8.67 ± 3.06 h; and T _1/2 13.45 ± 2.72 h. In addition, a suspected metabolite of TRAM-34 was found using this LC–MS/MS method. Given the results of the detailed validation process and its application to TRAM-34 pharmacokinetics, it is clear that a fast, selective, precise, and reproducible TRAM-34 LC–MS/MS analytical method was successfully established.     

Simultaneous Estimation of Vinpocetine and Its Metabolite, Apovincaminic Acid, in Beagle Plasma by LC-MS-MS

A method was developed to determine vinpocetine and its metabolite, apovincaminic acid, in beagle plasma by LC-MS-MS. After protein precipitation with methanol, the supernatant of the sample was concentrated and injected into an Agilent Zorbax XDB-C₁₈ column. The sample was separated by a mobile phase consisting of acetonitrile and 0.2% formic acid solution, and the reading was determined on an Agilent 6410 Triple Quad Tandem mass spectrometer in multiple reaction monitoring mode with the following transitions: m/z 351.5 ?? 280.2/266.3 for vinpocetine, 323.2 ?? 236.1/280.2 for apovincaminic acid, and 411.2 ?? 191.1 for the internal standard. The intra- and inter-day variances were less than 15% (RSD%), and average recoveries were higher than 80%. The linearity ranges (LR) between 0.1 and 20.0 ng mL^?1 for vinpocetine (r ² = 0.9980) and between 1.0 and 200.0 ng mL^?1 for apovincaminic acid (r ² = 0.9995) were established. In summary, this method is sensitive, specific, and appropriate for in vivo study of various dosage forms of vinpocetine.     

LC–MS/MS Determination of Antihypertension Drugs in Rat Plasma and Urine: Applications to Pharmacokinetics

A sensitive, rapid and reproducible LC–MS/MS method for the determination of olmesartan (OLM), amlodipine (ALM) and hydrochlorothiazide (HCZ) in rat plasma and urine has been developed and validated. Irbesartan (IRB) was used as an internal standard. The analytes were separated on a Waters XTerra-C₁₈ column using gradient elution with acetonitrile and 10 mM ammonium acetate buffer (pH 3.5, adjusted with acetic acid) at a flow rate of 1.0 mL min^?1. The three analytes were ionized by positive ion electrospray using multiple-reaction monitoring (MRM) mode to monitor precursor?→?product ion transitions m/z 447.31?→?234.97 for OLM, 408.87?→?238.18 for AML and 290.1?→?204.85 for HCZ. The specificity, matrix effect, recovery, sensitivity, linearity, accuracy, precision, and stabilities were all validated over the concentration range 0.4–100 ng mL^?1 for AML, 0.2–100 ng mL^?1 for OLM, 0.1–100 ng mL^?1 for HCZ. The mean concentrations (C_max) are 10.32, 587, and 3.4 for OLM, ALM, and HCZ, respectively, by the oral administration of 15 mg kg^?1 of each analyte.     

LC-MS-MS Determination and Pharmacokinetic Study of Clozapine in Human Plasma

A sensitive and selective liquid chromatographic method coupled with tandem mass spectrometry was established and validated for the determination and pharmacokinetic study of clozapine in human plasma. Ethyl acetate extraction was used for plasma sample preparation with mirtazapine as internal standard. Chromatographic separation was achieved on a Hanbon Kromasil C₁₈ (250 mm × 4.6 mm, 5 μm) column by isocratic elution with a mixture of 70 volumes of methanol and 30 volumes of water containing 0.2% ammonium acetate and 0.1% formic acid as mobile phase delivered at 1.0 mL min^?1. The MS-MS detection was carried out on a tandem mass spectrometer using positive electrospray ionization and multiple reaction monitoring with argon for collision-induced dissociation. The ion transitions were monitored as follows: m/z 327 to m/z 270 for clozapine and m/z 266 to m/z 195 for the internal standard (mirtazapine), respectively. Calibration curves were generated over the concentration range from 0.10 to 200 ng mL^?1 with the lower limit of quantification of 0.10 ng mL^?1, and two segments of linear calibration curves were established by regressing in the way of least-square in the range from 0.10 to 5.0 and 5.0 to 200 ng mL^?1, respectively. The intra- and inter-day precision and accuracy were determined at three different concentration levels, 0.20, 10.0 and 100 ng mL^?1, and were all better than 15% (n = 5). This specific and sensitive liquid chromatography coupled with tandem mass spectrometry has been successfully applied to a pharmacokinetic study of clozapine after a single oral dose of 25 mg in healthy Chinese volunteers.     

Simultaneous Determination of Pethidine and Atropine in Rabbit Plasma by LC�CMS�CMS and Its Application to a Pharmacokinetic Study after Intramuscular Administration

A sensitive and selective liquid chromatography?Ctandem mass spectrometry method for the determination of pethidine and atropine in rabbit plasma was developed and validated. The analytes and internal standard (IS) are extracted from plasma by liquid?Cliquid extraction using ethyl acetate, and separated on a Zorbax SB-Aq column (2.1 × 150 mm, 3.5 ??m) using acetonitrile?C0.1% formic acid as mobile phase with gradient elution. Electrospray ionization source was applied and operated in positive ion mode, and multiple reaction monitoring mode was used for quantification using target fragment ions m/z 247.8 ?? 219.7 for pethidine, m/z 289.9 ?? 123.8 for atropine and m/z 295.0 ?? 266.8 for IS, respectively. The assay is linear over the range of 5?C1,000 ng mL^?1 for pethidine and atropine, with a lower limit of quantification of 3 ng mL^?1 for pethidine and 5 ng mL^?1 for atropine. Intra-day and inter-day precision are less than 11% and the accuracy are in the range of 90.4?C106.3%. Furthermore, the newly developed method is successfully used for the determination of pethidine and atropine in rabbit plasma for pharmacokinetic study.     

A Sensitive LC–ESI–MS–MS Method for the Determination of Huperzine A in Human Plasma: Method and Clinical Applications

A sensitive and specific liquid chromatography–electrospray ionization–tandem mass spectrometry method has been developed and validated for the quantification of huperzine A in human plasma. After the addition of trimetazidine, the internal standard (IS) and sodium hydroxide, plasma samples were extracted using 5 mL ethyl acetate. The compounds were separated on an Agilent Zorbax SB C₁₈ column (100 mm × 2.1 mm ID, dp 3.5 μm) using an elution system of 10 mM ammonium acetate solution–methanol–formic acid (18:82:0.1, v/v) as the mobile phase. The quantification of target compounds was obtained by using multiple reaction monitoring (MRM) transitions: m/z 243.1, 210.1 and 267.2, 166.0 were measured in positive mode for huperzine A and IS. Linearity was established for the range of concentrations 0.01–4.0 ng mL^?1 with a coefficient of correlation (r) of 0.9991. The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.01 ng mL^?1. The method has been successfully applied to study the pharmacokinetics of huperzine A in healthy male Chinese volunteers.     

Immunoaffinity Column Clean-Up and Liquid Chromatography with Post-Column Derivatization for Analysis of Aflatoxins in Traditional Chinese Medicine

A rapid tandem mass spectrometric (MS-MS) method for the quantification of gabapentin (GBP) in human plasma using 4-phenyl-4-aminobutanoic acid as an internal standard (IS) has been developed and validated. The drug and the internal standard were analyzed, by flow injection analysis without chromatographic separation, using a mobile phase of acetonitrile-water-formic acid (50:50:0.025, v/v/v) at a flow rate of 0.1 mL min^?1. The run-cycle time was <3 min injection-to injection. Quantitation was achieved using multiple reaction monitoring (MRM) scan at MRM transitions m/z 172 > 154 and m/z 180 > 117 for GBP and the IS, respectively. Ion suppression study indicated practically no suppressive effect of plasma constituents on the mass ions detection of GBP and IS, when measured in MRM scanning mode. Calibration curves were linear over the concentration range of 0.1–10 μg mL^?1 (r > 0.999) with a limit of quantification of 0.1 μg mL^?1 (RSD%; 7.6 and % DEVs; ?3.0 to +17.0%). Validation data showed that the RSD% values were in the range of 1.85 to 13.06%, whereas, the % DEVs values ranged from ?1.4 to +10.0% indicating good precision and accuracy. Analytical recoveries of GBP from spiked human plasma were in the range of 98.9 to 101.3%. On the other hand, recoveries of GBP from stored human plasma samples were in the range of 100.0 to 107.5% indicating that GBP was stable in plasma, with no appreciable degradation, when stored at ?20 °C. The developed method was applied for GBP monitoring in plasma samples of patients treated with GBP.     

Determination of Dothiepin in Human Plasma by LC–ESI–MS and its Application to Bioequivalence Studies

An LC–MS method for the determination of dothiepin in human plasma was developed and validated. Sample preparation involved extraction with n-hexane:2-propanol (95:5). Separation was on an Ultimate XB C18 column (2.1 × 150 mm, 5 μm). A single-quadrupole mass spectrometer with an electrospray interface was operated in the selected-ion monitoring mode to detect the [M+H]⁺ ions at m/z 296 for dothiepin and at m/z 278 for the internal standard (amitriptylene). The method demonstrated good linearity from 0.78 ng mL^?1 (the LOQ) to100 ng mL^?1. The mean extraction recovery was 82.4% for dothiepin and and 84.2% for the internal standard. The intra-day and inter-day precision ranged from 8.5 to 11.4% and 9.7 to 12.1% (RSD), respectively. The method was successfully applied to bioequivalence studies of dothiepin hydrochloride tablets to obtain the pharmacokinetic parameters.     

LC–MS–MS Determination of Nikethamide in Human Plasma

A sensitive LC–MS–MS method with electrospray ionization has been developed for determination of nikethamide in human plasma. After addition of atropine as internal standard, liquid–liquid extraction was used to produce a protein-free extract. Chromatographic separation was achieved on a 150 mm × 2.1 mm, 5 μm particle, Agilent Zorbax SB-C₁₈ column, with 45:55 (v/v) methanol–water containing 0.1% formic acid as mobile phase. LC–MS–MS was performed in multiple reaction monitoring mode using target fragment ions m/z 178.8 → 107.8 for nikethamide and m/z 289.9 → 123.8 for the internal standard. Calibration plots were linear over the range of 20.0–2,000 ng mL^?1. The lower limit of quantification was 20.0 ng mL^?1. Intra-day and inter-day precisions were better than 4.2 and 6.1%, respectively. Mean recovery of nikethamide from human plasma was in the range 65.3–71.1%.     

UPLC-MS-MS Method for the Determination of N-(2,6-Dimethoxypyridine-3-yl)-9-methylcarbazole-3-sulfonamide in Rat Plasma and Its Application to a Pharmacokinetic Study

The purpose of the study is first to develop a sensitive and rapid ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method for the determination of a new synthesized tubulin ligand, N-(2,6-dimethoxypyridine-3-yl)-9-methylcarbazole-3-sulfonamide (IG-105), in rat plasma. The analyte and internal standard (carbamazepine) were extracted by liquid/liquid extraction with petroleum ether/diethyl ether (2:1, v/v). The chromatographic separation was performed on an Acquity UPLC BEH C₁₈ column with a mobile phase gradient consisting of methanol and water. The mass spectrometric detection was performed by triple-quadrupole mass spectrometry with multiple reaction monitoring (MRM) via an ESI source operating in positive ionization mode. The mass transitions m/z 398??153 and m/z 237??194 were used to assay the analyte and IS, respectively. The method demonstrated good linearity over a concentration range of 0.67?C333.33 ng mL^?1, and the lower limit of quantitation (LLOQ) of IG-105 was 0.67 ng mL^?1. The intra- and inter-day precision (relative standard deviation) values were <6%, and the accuracy (relative error) was <5% at three quality control levels. The extraction recovery of IG-105 and IS was 84.45 and 88.5%, respectively. Finally, the validated method was successfully applied to a pharmacokinetic study of IG-105 in rat plasma.     

LC–MS Determination and Pharmacokinetic Study of Salidroside in Rat Plasma after Oral Administration of Traditional Chinese Medicinal Preparation Rhodiola crenulata Extract

A simple, rapid and sensitive liquid chromatography–mass spectrometry (LC–MS) method was developed for the quantification of salidroside in rat plasma and the study of its pharmacokinetics after oral administration of 15 g kg^?1 Rhodiola crenulata extract to Wistar rats. A 200 μL plasma sample was extracted by acetonitrile and performed on Kromasil C₁₈ column (150 mm × 4.6 mm, 5 μm) with the mobile phase of acetonitrile–water (11:89) within a run time of 8 min. The analyte was monitored with electrospray ionization (ESI) by selected ion monitoring (SIM) mode. The target ions were m/z 299.20 for salidroside and m/z 150.00 for internal standard (IS) paracetamol. A good linear relationship was obtained over the range of 100–20,000 ng mL^?1 and the lower limit of quantification was 100 ng mL^?1. The validated method was successfully applied for the pharmacokinetic study of salidroside in rat. After oral administration of Rhodiola crenulata extract, the main pharmacokinetic parameters T _max, T _1/2, C _max, AUC _0?t and AUC _0?∞ were 0.56 ± 0.21 h, 7.91 ± 4.42 h, 3,386 ± 2,138 ng mL^?1, 16,146 ± 6,558 ng h mL^?1 and 18,599 ± 6,529 ng h mL^?1, respectively.     

LC–MS Method for the Quantification of Clonazepam in Rat Plasma

A sensitive and specific high-performance liquid chromatography–tandem mass spectrometry method has been developed and validated for the determination of clonazepam in rat plasma. Clonazepam and internal standard diazepam were extracted from plasma samples by a single-step protein precipitation. The chromatographic separation was performed on a Dikma ODS-C18 reversed-phase column at 40 °C. The mobile phase composed of a premix of solvent A (0.1% formic acid–4 mM ammonium acetate–water)–solvent B (acetonitrile) (13:87, v/v) at a flow-rate of 0.7 mL min^?1. Positive electrospray ionization was utilized as the ionization source. Clonazepam and the internal standard were determined using multiple reaction monitoring of precursor → product ion transitions at m/z 316.0 → 270.0 and m/z 285.1 → 193.2, respectively. The lower limit of quantification was 0.25 ng mL^?1 using 50 μL plasma samples and the linear calibration range was from 0.25 to 128 ng mL^?1. The within- and between-batch RSDs were lower than 15% and the relative recoveries of clonazepam ranged from 97.4 to 104.7%. The mean extraction recoveries of clonazepam and IS were 79.7 and 77.6%, respectively. The method has been successfully applied to the pharmacokinetic studies in rat after oral administration of clonazepam.     

Simultaneous Quantitation of Paracetamol, Pseudoephedrine and Chlorpheniramine in Dog Plasma by LC-MS-MS

A simple, rapid and sensitive liquid chromatography/electrospray tandem mass spectrometry quantitative detection method, using amantadine as internal standard, was developed for the simultaneous analysis of paracetamol, pseudoephedrine and chlorpheniramine concentrations. Analytes were extracted from plasma samples by liquid–liquid extraction with n-hexane–dichloromethane–2-propanol (2:1:0.1, v/v), separated on a C18 reversed-phase column with 0.1% formic acid–methanol (40:60, v/v) and detected by electrospray ionization mass spectrometry in positive multiple reaction monitoring mode. Calibration curves for plasma were linear over the concentration range 10–10,000 ng mL^?1 of paracetamol, 2–2,000 ng mL^?1 of pseudoephedrine and 0.2–200 ng mL^?1 of chlorpheniramine. The method has a lower limit of quantitation of 10 ng mL^?1 for paracetamol, 2.0 ng mL^?1 for pseudoephedrine and 0.2 ng mL^?1 for chlorpheniramine. Recoveries, precision and accuracy results indicate that the method was reliable within the analytical range, and the use of the internal standard was very effective for reproducibility by LC-MS-MS. This method is feasible for the evaluation of pharmacokinetic profiles of a novel multicomponent sustained release formulation containing 325 mg of paracetamol, 30 mg of pseudoephedrine hydrochloride and 2 mg of chlorpheniramine maleate. It is the first time the pharmacokinetic evaluation of a novel sustained-action formulation containing paracetamol, pseudoephedrine and chlorpheniramine has been elucidated in vivo using LC-MS-MS.     

HPLC–MS Analysis of Isoniazid in Dog Plasma

A simple, rapid, and sensitive liquid chromatography–mass spectrometric (LC–MS) method was developed and validated for the determination of isoniazid in dog plasma. Plasma samples were deproteined with methanol and separated on a C₁₈ column interfaced with a single quadrupole mass spectrometer, using 0.1% formic acid–acetonitrile (91:9 v/v) as mobile phase. Detection was performed by positive electrospray ionization with selected ion monitoring at m/z 138 for isoniazid and 152 for entecavir maleate internal standard. Linearity was obtained over the range of 25–5,000 ng mL^?1, with a lower limit of quantification of 25 ng mL^?1. The intra- and inter-day precision was less than 2.7% in terms of relative standard deviation. Accuracy, expressed as relative error, ranged from ?2.0 to 8.0%. Plasma samples were analysed within 5 min. The method was successfully applied to the evaluation of the pharmacokinetics of isoniazid in dog plasma.     

Determination of Rizatriptan in Human Plasma by Liquid Chromatography Stable Isotope Dilution Electrospray MS�CMS for Application in Bioequivalence Study

A simple, sensitive, selective, rapid, rugged, reproducible and specific liquid chromatography?Ctandem mass spectrometry (LC?CMS/MS) method was used for quantitative estimation of rizatriptan (RZ) in human plasma using rizatriptan-d ₆ (RZD6) as internal standard (IS). Chromatographic separation was performed on Ascentis Express RP Amide C18, 50 × 4.6 mm, 2.7 ??m column with isocratic mobile phase composed of 10 mM ammonium formate:acetonitrile (20:80 v/v) at flow rate of 0.5 mL min^?1. RZ and RZD6 were detected with proton adducts at m/z (amu) 270.2 ?? 201.2 and 276.1 ?? 207.1, respectively, in multiple reaction monitoring (MRM) positive mode. Liquid?Cliquid extraction was used and validated over a linear concentration range of 0.1?C100.0 ng mL^?1 with correlation coefficient r ² ?? 0.9981. The limit of quantification (LOQ) and limit of detection (LOD) were found to be 0.1 ng mL^?1 and 12.5 fg, respectively. Intra- and inter-day precision were within 1.7?C3.1% and 2.8?C3.7%, and accuracy within 96.0?C101.7% and 99.7?C101.4% for RZ. Drug was found to be stable throughout three freeze?Cthaw cycles. The method was successfully employed for analysis of plasma samples following oral administration of RZ (10 mg) in 25 healthy Indian male human volunteers under fasting conditions.     

Rapid LC-APCI-MS-MS Method for Simultaneous Determination of Phenacetin and Its Metabolite Paracetamol in Rabbit Plasma

A highly sensitive liquid chromatographic-atmospheric pressure chemical ionization-tandem mass spectrometric method is developed to quantitate phenacetin and its metabolite paracetamol in rabbit plasma. The analytes and internal standard oxazepam are extracted from plasma by liquid–liquid extraction using ethyl acetate, and separated on a Zorbax SB-C₁₈ column (2.1 mm × 150 mm, 5 μm) using acetonitrile–0.1% formic acid in water (40:60 v/v) at a flow of 0.4 mL min^?1. Detection is carried out by multiple reaction monitoring on a ion-trap LC-MS-MS system with an atmospheric pressure chemical ionization interface. The assay is linear over the range 4–1,600 ng mL^?1 for phenacetin and 3–2,000 ng mL^?1 for paracetamol, with a lower limit of quantitation of 4 ng mL^?1 for phenacetin and 3 ng mL^?1 for paracetamol. Intra- and inter-day precision are less than 7.1% and the accuracy are in the range 97.3–103.5%. The validated method is successfully used to analyze the drug in samples of rabbit plasma for pharmacokinetic study.     

Determination of Asiaticoside in Rat Plasma by LC–MS–MS and Its Application to Pharmacokinetics Studies

A rapid and sensitive LC–MS–MS method was developed and validated for the determination of asiaticoside in rat plasma. Asiaticoside was extracted by protein precipitation with acetonitrile, and separated on a C₁₈ column. The total analytical time was relatively short (4 min), and the limit of quantification was 38 ng mL^?1 using 100 μL of rat plasma. Asiaticoside and the internal standard (felodipine) were monitored in the multi-reaction-monitoring mode as follows: m/z 957.4 → 469.3 and m/z 382.2 → 145.1, respectively. Calibration was linear over a concentration range from 38 to 7,600 ng mL^?1, and the correlation coefficient was greater than 0.998. The recoveries of asiaticoside from plasma were better than 85%, and RSDs of inter-day and intra-day assays were below 10.1%. The method is sensitive and specific, and suitable for pharmacokinetic studies of asiaticoside in rats.     

Sensitive and Selective LC-MS-MS Assay for the Quantification of Palonosetron in Human Plasma and Its Application to a Pharmacokinetic Study

A highly sensitive and selective liquid chromatography-tandem mass spectrometry method was developed for the determination of palonosetron in human plasma samples. Chromatographic conditions and mass spectral parameters were optimized in order to achieve a limit of quantification of approximately 0.03 ng mL^?1. Palonosetron and citalopram (internal standard) were extracted by liquid–liquid extraction under alkaline conditions using saturated sodium bicarbonate. Separation was achieved with a Hanbon Lichrospher C₁₈ column and detection was carried out by tandem mass spectrometry using positive electrospray ionization in selected reaction monitoring mode. The target ions of palonosetron and citalopram were to m/z 297.00 → 297.00 and 325.00 → 325.00 respectively. Calibration curves were linear over the range of approximately 0.03–10 ng mL^?1. Precision and accuracy of this method was acceptable. The method was successfully applied to a pharmacokinetic study with healthy Chinese volunteers after intravenous administration of a single dose of 0.125, 0.25 or 0.5 mg palonosetron hydrochloride.