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.     

Liquid Chromatography‐Electrospray Ionization Mass Spectrometric Method for Determination of Gliclazide in Human Plasma

Abstract

A rapid, sensitive, and specific liquid chromatography‐electrospray ionization mass spectrometric (LC‐ESI‐MS) method has been developed for quantification of gliclazide in human plasma. The analyte and tolbutamide (internal standard, I.S.) were extracted from plasma samples with n‐hexane–dichloromethane (1:1, v/v) and analyzed on a C₁₈ column. The chromatographic separation was achieved within 4.0 min by using methanol–0.5% formic acid (80:20, v/v) as mobile phase and the flow rate was 1.0 mL/min. Ion signals m/z 324.0 and 271.0 for gliclazide and internal standard were measured in the positive mode, respectively. The method was linear within the range of 2.5–2000 ng/mL. The lower limit of quantification (LLOQ) was 2.5 ng/mL. The intra‐ and inter‐day precisions were lower than 2.8% in terms of relative standard deviation (RSD). The inter‐day relative error (RE) as determined from quality control samples (QCs) ranged from ?1.93% to 1.85%. This validated method was successfully applied for the evaluation of pharmacokinetic profiles of gliclazide modified‐release tablets in 20 healthy volunteers.     

GC Determination of Prilocaine HCl in Human Plasma: Analytical Application to Real Samples

A gas chromatographic (GC) method with a rapid and simple sample preparation was developed and validated for determination of prilocaine in human plasma. The validation parameters of linearity, precision, accuracy, recovery, specificity, limit of detection and limit of quantification were studied. The range of quantification for the GC method was 50–300 ng mL^?1 in plasma. Intra- and inter-day precision, expressed as the relative standard deviation (RSD) were less than 4.5%, and accuracy (relative error) was better than 8.0% (n = 6). The analytical recovery of prilocaine HCl from plasma has averaged 96.5% and the recovery of internal standard (lidocaine HCl) reached 96.8%. The limit of quantification (LOQ) and the limit of detection (LOD) of the method for plasma were 50 and 40 ng mL^?1, respectively. Also the developed and validated method was applied to three healthy volunteers to whom a local anaesthesia with citanest was administered.     

Determination of Lorazepam in Rabbit Plasma After Intranasal Administration by LC-MS

An LC-MS method was developed and validated to determine lorazepam in rabbit plasma. Chromatographic separation was performed on a C₁₈ column using methanol-150 nM sodium acetate (62.5:37.5, v/v) as the mobile phase at the flow rate of 0.2 mL min^?1. The retention times for lorazepam and diazepam (internal standard) were 6 and 10 min, respectively. Quantitative analysis was operated in selected ion monitoring (SIM) and positive ion mode using target ions at [M + H]⁺ m/z 284.9 for diazepam and [M + Na]⁺ m/z 342.9 for lorazepam, respectively. The lower limit of quantification (LLOQ) was 1.2 ng mL^?1 and a linear range of 1.2–150 ng mL^?1 with correlation coefficients (r ²) of 0.9968. The intra- and inter-day relative standard deviation was <5 and < 10%, respectively. The accuracy values were higher than 95%. The method is simple, sensitive and repeatable, and has been successfully applied to pharmacokinetics studies of lorazepam-loaded mocroemulsions after intranasal administration in rabbit.     

Simultaneous Determination of Berberine and Palmatine in Rabbit Plasma by LC-MS–MS and Its Application in Pharmacokinetic Study After Oral Administration of Coptidis and Coptidis–Gardeniae Couple Extract

A valid and sensitive LC-MS–MS method is adopted for pharmacokinetics study of berberine and palmatine in rabbit plasma. After mixing with internal standard tetrahydroberberine, plasma samples were pretreated with 1.5 mL acetonitrile. Chromatographic separation was on a C₁₈ column using a mixture of water (containing 10 mmol L^?1 ammonium acetate, pH 3.5) and acetonitrile (50∶50, v/v) as mobile phase. The detection was performed by selected ion monitoring mode via electrospray ionization source operating in the positive ionization mode. The method was linear over the concentration range of 2.0–200.0 ng mL^?1 for berberine and 1.0–100.0 ng mL^?1 for palmatine. The lowest limits of quantitation (LLOQ) were 2.0 ng mL^?1 for berberine and 1.0 ng mL^?1 for palmatine. The intra- and inter-day precision values were less than 14.3% and the deviations were within ±11.0%. The fully validated LC-MS–MS method has been successfully applied to a pharmacokinetic study of berberine, palmatine in rabbit plasma after oral administration of Coptidis and coptidis–gardeniae couple extract. The results indicated that the plasma profiles of the two compounds in rabbit confirmed to one-compartment open model and the combinational utilization with Gardeniae could increase the bioavailability of berberine and palmatine, the two major active components of Coptidis.     

Quantitative Determination of Lobeline Hydrochloride in Rabbit Plasma by LC–MS–MS and Its Application

A sensitive and selective liquid chromatography tandem mass spectrometry method for quantitative determination of lobeline hydrochloride in rabbit plasma was developed and validated. After addition of triazolam as internal standard, protein precipitation by acetonitrile was used as sample preparation. Chromatographic separation was achieved on a Zorbax SB-C18 column with acetonitrile-0.1% formic acid as mobile phase with gradient elution. Electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring mode was used for quantification using target fragment ions m/z 338.1 → 315.8 for lobeline hydrochloride and m/z 342.9 → 308.0 for the IS. Calibration plots were linear over the range of 2–500 ng mL^?1 for lobeline hydrochloride in plasma. Lower limit of quantitation for lobeline hydrochloride was 2 ng mL^?1. Mean recovery of lobeline hydrochloride from plasma was in the range 97.5–102.3%. RSD of intra-day and inter-day precision were both <9%. This developed method is successfully used in pharmacokinetic study of lobeline hydrochloride in rabbit.     

Rapid and Selective LC�CMS�CMS Method for the Determination of cyclo-trans-4-l-Hydroxyprolyl-l-serine (JBP485) in Rat Plasma

A rapid and selective liquid chromatographic/tandem mass spectrometric method for the determination of JBP485 was developed and validated. Following protein precipitation, the analyte and internal standard (JBP923) were separated from human plasma using an isocratic mobile phase on an Elite Kromasil C18 column. An API 3200 tandem mass spectrometer equipped with a Turbo ionSpray ionization source was used as the detector and operated in the positive ion mode. Multiple reaction monitoring using the precursor to product ion combinations of m/z 201.2 ?? 86.2 and m/z 219.2 ?? 86.2 was performed to quantify JBP485 and JBP923, respectively. The method was linear in the concentration range of 0.10?C50.00 ??g mL^?1 using 100 ??L of plasma. The lower limit of quantification was 0.10 ??g mL^?1. The intra- and inter-day relative standard deviations over the entire concentration range were less than 6.65%. Accuracy determined at three concentrations (0.25, 4.00 and 25.00 ??g mL^?1 for JBP485) ranged from ?0.78 to 2.74% in terms of relative error. Each plasma sample was chromatographed within 2.0 min. The method was successfully applied to characterize the pharmacokinetic profiles of JBP485 in rats after an intravenous injection of 6.25 mg kg^?1 JBP485.     

GC–MS Determination of Atenolol Plasma Concentration after Derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide

An analytical procedure was developed and validated for the determination of atenolol in human plasma. Atenolol and metoprolol (internal standard) were extracted from human plasma with a mixture of chloroform and butanol at basic pH. The extracts were derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide and analyzed by GC–MS. Calibration curves were linear over the concentration range 15–250 ng mL^?1. Intra- and inter-day precision values for atenolol in human plasma were less than 7.4, and accuracy (relative error) was better than 6.4%. Recovery of atenolol from human plasma averaged 90.46%. The limits of detection (LOD) and quantitation (LOQ) of atenolol were 5.0 and 15 ng mL^?1. This method was successfully applied to six patients with hypertension who had been given an oral tablet of 50 mg atenolol.     

Simultaneous Determination of Chloroquine and Its Metabolite Desethyl Chloroquine in Human Plasma Using Liquid Chromatography Tandem Mass Spectrometry

This study presents a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of chloroquine (CQ) and its metabolite desethyl chloroquine (DCQ) in human plasma. The method generally involved methanol protein precipitation and LC-MS/MS detection. The mass spectrometer was operated with positive electrospray ionization (ESI) source and the data acquisition was carried out in multiple reaction monitoring (MRM) mode. Quantification was performed by labeled isotope dilution method with matrix-matched calibration curves. Inter-batch accuracies were ranged from 90.2% to 109.8%. Both intra-batch and inter-batch precisions (relative standard deviation, RSD, %) of the analytes were no more than 15.0%. The recoveries were from 73.7% to 79.0%, and the limit of quantification (LOQ) was 1.0 and 0.5 ng/mL for CQ and DCQ, respectively. The linear ranges of CQ/DCQ were 1.0/0.5–500.0/250.0 ng/mL with the linear correlation coefficient R² > 0.999. No significant matrix effect was observed in this study. The carryover effect was significantly minimized by the addition of ethylene glycol in the reconstitution solution and the utilization of basic washing solution and repetitive fast washing gradient program. After optimization, the carryover effect was less than 20%. This method is in possession of sufficient resolution, high sensitivity as well as selectivity and convenient to be applied to the trace determination of CQ and DCQ in human plasma.     

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.     

Sensitive Analysis of Wilforidine in Human Plasma by LC-APCI-MS/MS

Wilforidine is a potentially efficient medicine to cure autoimmune diseases. In this paper, a sensitive and selective liquid chromatographic method coupled with atmospheric -pressure chemical ionization mass spectrometry (LC–APCI–MS/MS) has been developed for quantification of wilforidine in human plasma. Samples were deproteinized with acetonitrile and cleaned by solid-phase extraction. The chromatographic separation was performed on an analytical RRHD C₁₈ column (50 × 2.1 mm) using ammonium acetate solution (10.0 mmol L^?1)/acetonitrile (30/70, v/v) as the mobile phase at a flow rate of 0.7 mL min^?1. Detection was carried out by the positive multiple reaction monitoring mode with transitions of m/z 780 → 684 for wilforidine, and 646 → 586 for aconitine (internal standard), respectively. The calibration curve was linear (r = 0.9991) in the concentration range of 0.5–100.0 μg L^?1 with a lower limit of quantification of 0.5 μg L^?1 in plasma. Intra- and inter-day relative standard deviations were less than 6.8 and 13.1 %, respectively, and the recoveries were between 88.0 and 96.0 %. This accurate and highly specific assay provides a useful method for evaluating the pharmacokinetics of wilforidine in human plasma.     

Sensitive Analysis of Wilforidine in Human Plasma by LC-APCI-MS/MS

Wilforidine is a potentially efficient medicine to cure autoimmune diseases. In this paper, a sensitive and selective liquid chromatographic method coupled with atmospheric -pressure chemical ionization mass spectrometry (LC–APCI–MS/MS) has been developed for quantification of wilforidine in human plasma. Samples were deproteinized with acetonitrile and cleaned by solid-phase extraction. The chromatographic separation was performed on an analytical RRHD C₁₈ column (50 × 2.1 mm) using ammonium acetate solution (10.0 mmol L⁻¹)/acetonitrile (30/70, v/v) as the mobile phase at a flow rate of 0.7 mL min⁻¹. Detection was carried out by the positive multiple reaction monitoring mode with transitions of m/z 780 → 684 for wilforidine, and 646 → 586 for aconitine (internal standard), respectively. The calibration curve was linear (r = 0.9991) in the concentration range of 0.5–100.0 μg L⁻¹ with a lower limit of quantification of 0.5 μg L⁻¹ in plasma. Intra- and inter-day relative standard deviations were less than 6.8 and 13.1 %, respectively, and the recoveries were between 88.0 and 96.0 %. This accurate and highly specific assay provides a useful method for evaluating the pharmacokinetics of wilforidine in human plasma.

     

Rapid and Sensitive UPLC-MS–MS for the Determination of Trimetazidine in Human Plasma

A specific and sensitive UPLC-MS–MS was developed for the determination of trimetazidine in human plasma. The sample preparation was based on a single-step liquid–liquid extraction with acetic ether. The chromatographic separation was on a C₁₈ analytical column (50 mm × 2.1 mm, 1.7 μm) with acetonitrile and 10 mM ammonium acetate (30:70, v/v) as the mobile phase, and a triple-quadrupole mass spectrometer equipped with an electrospray ionization source (ESI) applied for detection. The method was linear over the concentration ranges of 0.25–100.00 ng mL⁻¹ for trimetazidine, and the lower limit of quantification (LLOQ) was 0.25 ng mL⁻¹. The intra- and inter-day relative standard deviation (RSD) were less than 15% and the relative error (RE) were all within 15%. Finally, this method has been successfully applied to analyze plasma samples from a bioequivalence study with 18 volunteers.

     

Rapid and Sensitive UPLC-MS–MS for the Determination of Trimetazidine in Human Plasma

A specific and sensitive UPLC-MS–MS was developed for the determination of trimetazidine in human plasma. The sample preparation was based on a single-step liquid–liquid extraction with acetic ether. The chromatographic separation was on a C₁₈ analytical column (50 mm × 2.1 mm, 1.7 μm) with acetonitrile and 10 mM ammonium acetate (30:70, v/v) as the mobile phase, and a triple-quadrupole mass spectrometer equipped with an electrospray ionization source (ESI) applied for detection. The method was linear over the concentration ranges of 0.25–100.00 ng mL^?1 for trimetazidine, and the lower limit of quantification (LLOQ) was 0.25 ng mL^?1. The intra- and inter-day relative standard deviation (RSD) were less than 15% and the relative error (RE) were all within 15%. Finally, this method has been successfully applied to analyze plasma samples from a bioequivalence study with 18 volunteers.     

LC–ESI–MS Determination of Imperatorin in Rat Plasma After Oral Administration and Total Furocoumarins of Radix Angelica dahuricae and its Application to a Pharmacokinetic Study

A simple, rapid, sensitive and reliable liquid chromatography–electrospray ionization mass spectrometry method for the quantification of imperatorin in rat plasma after oral administration and total furocoumarins of Radix Angelica dahuricae has been established. The plasma samples were deproteinized by adding internal standard (IS) osthole solution, which was prepared by acetonitrile. The analysis was performed on a Shim-pack C₁₈ column (150 × 2.0 mm i.d., 5 μm) using acetonitrile and 0.5% formic acid solution (70:30, v/v) as a mobile phase. The detection was performed on a quadrupole mass spectrometer detector with an ESI interface operated in the selected ion monitoring mode. The linear quantification range of the method was 2–4000 ng mL^?1 in rat plasma with a correlation coefficient greater than 0.99, the limit of detection (LOD) was 0.5 ng mL^?1 and the lower limit of quantification (LLOQ) 2 ng mL^?1. The intra- and inter-day relative standard deviations (RSD) were less than 2.5 and 3.5%, respectively. The recoveries were above 90%. The validated method was successfully applied to a pharmacokinetic study of imperatorin in rats after oral administration and total furocoumarins of Radix Angelica dahuricae.     

A simple high-performance liquid chromatographic determination of mexiletine in human plasma at therapeutic levels

Summary A method for the quantitative determination of mexiletine in human plasma by high-performance liquid chromatography has been described. The plasma samples are buffered to pH 12 and extracted on Clin-Elut columns with diethylether-ethylacetate (1:1), after addition of the internal standard, the 2,4,6 methyl analogue of mexiletine. The minimum detectable amount of mexiletine is 50 ng in 0.5 ml plasma. Recovery is between 96–114% and the relative standard deviation at 1.5 ml^–1 level of mexiletine is 2.1% Accurate determinations of human plasma levels were performed after oral or intravenous treatment.Part of the work was presented at the 29. Kongreß der Deutschen Gesellschaft für Laboratoriumsmedizin, Hamburg 1985.     

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.     

Development and Validation of a Sensitive LC–Tandem-MS Method for the Quantitative Determination of Picroside II in Rat Plasma

A rapid and sensitive method for the quantitative determination of picroside II in rat plasma was developed and validated using liquid chromatographic separation with tandem mass spectrometric detection. The analytes of interest were extracted from rat plasma samples by ethyl acetate after acidification with 1.0% acetic acid solution. Chromatographic separation was achieved on a Hypersil GOLD column (50 × 2.1 mm I.D., 5 μm) using a mobile phase consisting of acetonitrile–0.1% formic acid solution (30:70, v/v) at a flow rate of 0.2 mL min^?1. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via electrospray ionization (ESI). The calibration curve was linear in the concentration range of 1.00–400 ng mL^?1 in rat plasma, with a 1.00 ng mL^?1 lower limit of quantification (LLOQ). Satisfactory results were achieved for intraday repeatability [relative standard deviation (RSD) = 6.4–12.4%] and inter-day precision (RSD = 6.8–14.7%). The accuracy in terms of relative error ranged from ?2.1 to 10.0%. The extraction recoveries of picroside II and icariin (internal standard) were 80.0 and 89.3%, respectively. The developed method was successfully employed to determine picroside II plasma concentrations after oral administration to Wistar rats.     

Simultaneous Determination of Isoniazid and Acetylisoniazid in Human Plasma by Hydrophilic Interaction Liquid Chromatography Tandem Mass Spectrometry and Its Application to a Pharmacokinetics Related to N-Acetyltransferase2 Genetic Polymorphism

A rapid, sensitive, and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with hydrophilic interaction chromatography has been developed and validated for the simultaneous determination of isoniazid and acetylisoniazidin human plasma. Following precipitation of the protein, the analytes were extracted from human plasma, with high extraction recovery (>70%) for both Isoniazid and acetylisoniazid. The analytes were then separated using a hydrophilic interaction chromatography (HILIC) column and detected by electrospray ionization (ESI) mass spectrometry performed with a triple-quadrupole mass spectrometry. The quantification of the analytes was realized by low-energy collision dissociation tandem mass spectrometry (CID-MS/MS) using the multiple reaction monitoring (MRM) mode at m/z of 138.1→121.1 for isoniazid and m/z 180.1 → 138.1 for acetylisoniazid, respectively. The method was linear over the concentration range of 5–50,000 ng/mL for both. The intra-day and inter-day relative standard deviations (RSD) were less than 15% and the relative errors (RE) were all within 15%. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic studies of isoniazid related to NAT2 genetic polymorphism in healthy Chinese subjects. The results showed that there were significant differences in the pharmacokinetic parameters of isoniazid and acetylisoniazid between subjects with and without mutations in the NAT2 gene.     

Development and Validation of a Sensitive LC–Tandem-MS Method for the Quantitative Determination of Picroside II in Rat Plasma

A rapid and sensitive method for the quantitative determination of picroside II in rat plasma was developed and validated using liquid chromatographic separation with tandem mass spectrometric detection. The analytes of interest were extracted from rat plasma samples by ethyl acetate after acidification with 1.0% acetic acid solution. Chromatographic separation was achieved on a Hypersil GOLD column (50 × 2.1 mm I.D., 5 μm) using a mobile phase consisting of acetonitrile–0.1% formic acid solution (30:70, v/v) at a flow rate of 0.2 mL min⁻¹. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via electrospray ionization (ESI). The calibration curve was linear in the concentration range of 1.00–400 ng mL⁻¹ in rat plasma, with a 1.00 ng mL⁻¹ lower limit of quantification (LLOQ). Satisfactory results were achieved for intraday repeatability [relative standard deviation (RSD) = 6.4–12.4%] and inter-day precision (RSD = 6.8–14.7%). The accuracy in terms of relative error ranged from −2.1 to 10.0%. The extraction recoveries of picroside II and icariin (internal standard) were 80.0 and 89.3%, respectively. The developed method was successfully employed to determine picroside II plasma concentrations after oral administration to Wistar rats.