Determination of Pitavastatin in Human Plasma by LC–MS–MS

A simple and rapid LC–MS–MS assay was developed and validated for the quantitative determination of pitavastatin in human plasma. Sample pretreatment involved simple protein precipitation by addition of acetonitrile. Separation was on an Agilent 1.8 μm Zorbax SB-C18 column (150 mm × 4.6 mm) at 25 °C using isocratic elution with methanol–0.1% formic acid in water (85:15, v/v) at a flow rate of 0.4 mL min^?1. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the ion transitions m/z 422.0 → 290.1 for pitavastatin, and m/z 330.1 → 192.1 for paroxetine (IS). LC–MS–MS was found to improve the quantitation of pitavastatin in plasma and was successfully applied in pharmacokinetic studies.     

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%.     

Simultaneous Determination of Tamsulosin and Dutasteride in Human Plasma by LC–MS–MS

A sensitive and selective liquid chromatographic tandem mass spectrometric (LC–MS–MS) method was developed for simultaneous identification and quantification of tamsulosin and dutasteride in human plasma, which was well applied to clinical study. The method was based on liquid–liquid extraction, followed by an LC procedure with a Gemini C-18, 50 mm × 2.0 mm (3 μm) column and using methanol:ammonium formate (97:3, v/v) as the mobile phase. Protonated ions formed by a turbo ionspray in positive mode were used to detect analytes and internal standard. MS–MS detection was by monitoring the fragmentation of 409.1 → 228.1 (m/z) for tamsulosin, 529.3 → 461.3 (m/z) for dutasteride and 373.2 → 305.3 (m/z) for finasteride (IS) on a triple quadrupole mass spectrometer. The lower limit of quantification for both tamsulosin and dutasteride was 1 ng mL^?1. The proposed method enables the unambiguous identification and quantification of tamsulosin and dutasteride for clinical drug monitoring.     

Determination of Fenofibric Acid in Human Plasma by LC–MS/MS and LC–UV

A sensitive, high-throughput and economic liquid chromatographic method for determination of fenofibric acid in human plasma was developed and validated by ultraviolet detection and tandem mass spectrometry, then applied in pharmacokinetic study to investigate Lipanthyl™ 200 mg MC bioavailability under food and fasting conditions. Fenofibric acid with 2-chloro fenofibric acid-d6 (internal standard) was extracted from 100 µL of human plasma by acetonitrile in a single extraction step. 25 and 2 µL from supernatant were injected onto ACE column, 50 mm, 5 micron with 4.6 mm inner diameter for LC–UV and 2.1 mm for LC–MS/MS, and both systems were eluted isocratically by water:methanol:formic acid (35:65:0.1, v/v/v), with a constant flow rate of 1 mL min⁻¹. The established calibration curve was linear between 0.05–20 µg mL⁻¹, and the within- and between-day precisions were all below 13 % in both LC–MS/MS and LC–UV systems during validation, and accuracies ranged between 91 and 112 %. Twenty-eight healthy adult subjects participated in this clinical study, and the pharmacokinetic parameters including coefficient of variation were calculated and discussed. A dramatic decrease in C _max and AUC0-72 (3.63- and 1.85-fold, respectively) were observed for Lipanthyl™ MC under fasting conditions with more variable inter subject measurements comparing to the fed state.

     

Simultaneous Determination of Atenolol and Chlorthalidone by LC–MS–MS in Human Plasma

A simple, sensitive, selective and rapid liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous separation and quantitation of atenolol and chlorthalidone in human plasma using metoprolol and hydrochlorothiazide as internal standard. Following solid phase extraction, the analytes were separated by an isocratic mobile phase on a reversed-phase C₁₈ column and analyzed by MS in the multiple reaction-monitoring mode (atenolol in positive and chlorthalidone in the negative ion mode). The limit of quantitation for this method was 10 and 15 ng mL^?1 and the linear dynamic range was generally 10–2,050 ng mL^?1 and 15–3,035 ng mL^?1 for atenolol and chlorthalidone, respectively.     

LC–ESI–MS Determination of Flupentixol in Human Plasma

Flupentixol and an internal standard, loperamide were extracted from human plasma by liquid–liquid extraction and analyzed on a Thermo Hypersil HyPURITY C18 column, with 10 mM ammonium acetate–acetonitrile–methanol (26:62:12, v/v/v) as mobile phase, coupled with electrospray ionization mass spectrometry (ESI–MS). The protonated analyte was quantified by selected-ion monitoring (SIM) with a quadrupole mass spectrometer in a positive-ion mode. The calibration curve was linear (r = 0.9990) over the concentration range: 0.039–2.5 ng mL^?1. Intra-day and inter-day precision (RSD%) were less than 13.05%. The established method was successfully applied for the determination of pharmacokinetics of flupentixol in human plasma.     

LC–ESI–MS Determination of Lovastatin in Human Plasma

A convenient, selective and sensitive liquid chromatographic-electrospary ionization mass spectrometry (LC–ESI–MS) method was developed and validated to determine lovastatin in human plasma. The analyte was extracted from human plasma samples by typical liquid–liquid extraction, separated on a C₁₈ column by using the mobile phase consisting of water–methanol (13:87, v/v). Simvastatin was used as the internal standard (IS). The method was linear within the range of 0.1–20 ng mL⁻¹. The lower limit of quantification (LLOQ) was 0.1 ng mL⁻¹. The intra- and inter-run precision, calculated from quality control (QC) samples was less than 10.2%. The accuracy as determined from QC samples was in the range of 99.3–102.9% for the analyte. The mean recoveries for lovastatin and IS were 84.8 and 88.0%, respectively. The method was successfully applied for evaluation of the pharmacokinetic of lovastatin in healthy volunteers.     

LC�CMS�CMS Quantitative Determination of Gefitinib in Human Serum and Cerebrospinal Fluid

A specific, sensitive, and rapid method based on high-performance liquid chromatography coupled to tandem mass spectrometry (LC?CMS?CMS) was developed for determination of gefitinib in human serum and cerebrospinal fluid (CSF). The analyte was detected by tandem mass spectrometry operating in positive electrospray ionization mode with multiple reaction monitoring (MRM). Gefitinib was extracted from serum or CSF samples with ethyl acetate using icotinib as internal standard. The method was validated over the concentration range of 1.00?C1,000 ng mL^?1 in human serum and 0.05?C50.0 ng mL^?1 in CSF. For both matrices, inter- and intraday precision (CV%) were less than 15% and accuracy was within 85?C115%. Average extraction recoveries were 78.9 and 61.8% in human serum and CSF, respectively. Linearity, recovery, matrix effects, and stability were validated in the two matrices. The method was successfully used for analysis of clinical samples from lung cancer patients with brain metastases treated with gefitinib in the dosage range of 250?C500 mg day^?1.     

Determination of Drospirenone in Human Plasma by LC–Tandem-MS

A bioanalytical method has been developed and validated for determination of drospirenone in human plasma. The analytical method consists in the extraction of plasma sample with dichloromethane, followed by determination of drospirenone by LC–MS–MS using levonorgestrel as internal standard. Separation was achieved on a Peerless cyano column with an isocratic mobile phase consisting of methanol and ammonium formate buffer. Protonated ions formed by a Turboionspray in the positive mode were used to detect analyte and IS. The MS–MS detection was by monitoring the fragmentation for drospirenone and for levonorgestrel on a triple quadrupole mass spectrometer. The assay was calibrated over the range 5–100 ng mL^?1 with a correlation coefficient of 0.9998. Validation data showed intra-batch (n = 6) CV% ≤ 5.58 and RE (%) between ?3.34 and +6.27 and inter-batch (n = 18) CV% < 6.08 and RE (%) between ?1.84 and +6.73. Mean extraction recovery were 83.31–92.58% for three QC samples and 89.32% for IS. Plasma samples were stable for three freeze-thaw cycles, or 24 h ambient storage, or 1 and 3 months storage at ?20 °C. Processed samples (ready for injection) were stable up to 72 h at autosampler (4 °C). This method has been used for analyzing plasma samples from a bioequivalence study with 12 volunteers.     

Simultaneous Determination of Paracetamol and Caffeine in Human Plasma by LC–ESI–MS

A rapid, selective and convenient liquid chromatography–mass spectrometric method for the simultaneous determination of paracetamol and caffeine in human plasma was developed and validated. Analytes and theophylline [internal standard (I.S.)] were extracted from plasma samples with diethyl ether-dichloromethane (3:2, v/v) and separated on a C₁₈ column (150 × 4.6 mm ID, 5 μm particle size, 100 Å pore size). The mobile phase consisted of 0.2% formic acid–methanol (60:40, v/v). The assay was linear in the concentration range between 0.05 and 25 μg mL^?1 for paracetamol and 10–5,000 ng mL^?1 for caffeine, with the lower limit of quantification of 0.05 μg mL^?1 and 10 ng mL^?1, respectively. The intra- and inter-day precision for both drugs was less than 8.1%, and the accuracy was within ±6.5%. The single chromatographic analysis of plasma samples was achieved within 4.5 min. This validated method was successfully applied to study the pharmacokinetics of paracetamol and caffeine in human plasma.     

Simultaneous Determination of Levodopa, Benserazide and 3-O-Methyldopa in Human Serum by LC–MS–MS

For the first time a sensitive, specific and rapid LC–MS–MS assay is presented for the simultaneous determination of levodopa (L-DP), 3-O-methyldopa (3-OMD) and benserazide (BSZ) in human serum. The three compounds were extracted from human serum by protein precipitation followed by dilution of the supernatant with aqueous formic acid. In serum, linearity was observed between 50 and 1,000 ng mL^?1 of L-DP, 3-OMD and BSZ, respectively. Intra-day and inter-day RSD values were below 10.56 and 6.22% at concentrations of 120, 360 and 720 ng mL^?1. The presented method showed excellent specificity and sensitivity compared with other methods reported. It was applied to a pharmacokinetic study and demonstrated its applicability to pre-clinical and clinical pharmacological research.     

Simultaneous Determination of Ramipril and Its Active Metabolite Ramiprilat in Human Plasma by LC–MS–MS

A selective, rapid and sensitive liquid chromatography tandem mass spectrometry method has been developed for the simultaneous determination of ramipril and ramiprilat in human plasma using enalapril as the internal standard via one-step extraction with ethyl acetate under acidic condition. The analysis was carried out on a Diamonsil C₁₈ column (150 mm × 4.6 mm i.d., 5 μm) with a mobile phase consisting of 1% formic acid-acetonitrile (25:75, v/v) at a constant flow rate of 0.5 mL min^?1. The detection was performed on a triple-quadruple tandem mass spectrometer by selective reaction monitoring mode via electrospray ionization. Linear calibration curves of ramipril and ramiprilat were obtained in the concentration range of 0.107–107.0 and 0.262–105.0 ng mL^?1, respectively. The intra- and inter-day precision (RSD) values were below 8.2 and 4.8% for ramipril, 10.4 and 12.3% for ramiprilat, and accuracy (RE) were within ±5.5 and ±3.2%, respectively at all QC levels. The method was utilized to support clinical pharmacokinetic studies in healthy volunteers following oral administration of ramipril tablets.     

Determination of Eleutherosides in Dietary Supplements by LC–MS/MS

A simple and specific method for the simultaneous determination of eleutherosides B and E in powdered rhizomes of Eleutherococcus senticosus extract and in solid and liquid dietary supplements was developed and validated. E. senticosus extracts, often mixed with other plants or herbal extracts, are widely used in food supplements because of the tonic and adaptogenic activities referred to the eleutherosides B and E. In this study, samples were analyzed by a liquid chromatography-electrospray tandem mass spectrometry (LC–ESI-MS/MS) method operated in single reaction monitoring (SRM). Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values were fixed at 3 μg L^?1 and 10 μg L^?1, respectively, whereas linearity was established within 10–1,000 μg L^?1 range for both compounds. Good precision was obtained for both eleutherosides in terms of intra-day precision (RSD % lower than 4 %) and inter-day precision (RSD % lower than 6 %). Good percentage recoveries were obtained for both eleutherosides (91.5–103.6 %). Finally, the developed method was successfully applied to analyze a number of solid and liquid commercial dietary supplements containing E. senticosus extracts, also mixed with other herbal extracts.     

Determination of Memantine in Human Plasma by LC–MS–MS: Application to a Pharmacokinetic Study

A sensitive and selective liquid chromatography–tandem mass spectrometry method for the determination of memantine was developed and validated over the linearity range 0.1–25 ng mL^?1 with 0.5 mL of plasma using procainamide as the internal standard. This analysis was carried out on a Cosmosil 5C₁₈-MS column and the mobile phase was composed of methanol: 0.5% formic acid (50:50, v/v). Detection was performed on a triple–quadrupole tandem mass spectrometer using positive ion mode electrospray ionization and quantification was performed by multiple reaction monitoring mode. The MS–MS ion transitions monitored were m/z 180 → 107 and 236 → 163 for memantine and procainamide, respectively. The between- and within-day precision was less than 10.9% and accuracy was less than 2.5%. The lower limit of quantification (LLOQ) was 0.1 ng mL^?1. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of memantine in healthy Chinese volunteers.     

Determination of adrenolytic drugs by SPME–LC–MS

Five adrenolytic drugs have been analyzed by liquid chromatography–mass spectrometry (LC–MS). Samples were prepared by solid-phase microextraction (SPME) using polypyrrole fibers coated on stainless steel support as an adsorbent for the drugs. Adsorption efficiencies were 95% and were close for all the drugs investigated. Relative standard deviations (RSD), calculated for samples prepared in standard solutions, were in the range 2.5–13%, however RSD values for the drugs in human plasma were 2.5–4.5%. Using LC–MS the limit of detection (LOD) and the limit of quantification (LOQ) were in the ranges 0.11–0.18 and 0.39–0.54 ng mL⁻¹, respectively, for the five drugs.     

LC–MS–MS Determination of Rotenone, Deguelin, and Rotenolone in Human Serum

For the first time we report a rapid and sensitive LC–MS–MS method for quantification of rotenone, deguelin, and rotenolone in human serum. The analytical procedure involves extraction with ethyl acetate without further clean-up. The active ingredients were separated on a C₈ reversed-phase column by isocratic elution. Eleven simultaneous transitions of precursor ions were monitored. Excellent selectivity and sensitivity enables quantification and identification of low levels of rotenoids (LOD 2 ng mL^?1, LOQ 5 ng mL^?1) in human serum.     

LC–MS Simultaneous Determination of Itopride Hydrochloride and Domperidone in Human Plasma

A rapid, simple, sensitive and specific liquid chromatography–tandem mass spectrometry method was developed and validated for simultaneous quantification of itopride hydrochloride and domperidone in human plasma. Both drugs were extracted by liquid–liquid extraction with ethyl acetate and saturated borax solution. The chromatographic separation was performed on a reversed-phase C18 column with a mobile phase of water–methanol (2:98, v/v) containing 0.5% formic acid. The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The assay exhibited linearity over the concentration range of 3.33–500 ng mL^?1 for itopride hydrochloride and 3.33–100 ng mL^?1 for domperidone in human plasma. The precursor to product ion transitions of m/z 359.1–72.3 and 426.0–147.2 were used to measure itopride hydrochloride and domperidone respectively. The method was found suitable for the analysis of plasma samples collected during phase 1 pharmacokinetics study of itopride HCl 50 mg and domperidone 20 mg in 12 healthy volunteers after single oral doses of the combination drug.     

Determination and Pharmacokinetic Study of 10-O-Dimethylaminoethylginkgolide B in Rat Plasma by LC–MS

To evaluate the pharmacokinetics of a novel analogue of ginkgolide B, 10-O-dimethylaminoethylginkgolide B (XQ-1) in rat plasma in pre-clinical studies, a sensitive and specific liquid chromatographic method with electrospray ionization mass spectrometry detection (LC–ESI–MS) was developed and validated. After a simple extraction with ethyl acetate, XQ-1 was analyzed on a Shim-pack C18 column with a mobile phase of a mixture of 1 μmol L⁻¹ ammonium acetate containing 0.02% formic acid and methanol (55:45, v/v) at a flowrate of 0.3 mL min⁻¹. Detection was performed in selected ion monitoring (SIM) mode using target ions at [M + H]⁺ m/z 496.05 for XQ-1 and m/z 432.10 for the internal standard (lafutidine). Linearity was established for the concentration range from 2 to 1,000 ng mL⁻¹ . The extraction recoveries ranged from 86.0 to 89.9% in plasma at concentrations of 5, 50, and 500 ng mL⁻¹. The lower limit of quantification was 2 ng mL⁻¹ with 100 μL plasma. The validated method was successfully applied to a pharmacokinetic study after intragastic administration of XQ-1 mesylate in rats at a dose of 20 mg kg⁻¹.     

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.