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 Clopidogrel and Its Carboxylic Acid Metabolite (SR26334) in Human Plasma by LC–ESI–MS–MS: Application to the Therapeutic Drug Monitoring of Clopidogrel

A sensitive and specific liquid chromatography-tandem-mass spectrometry method was developed and validated for the simultaneous determination of clopidogrel and its carboxylic acid metabolite (SR26334) in human plasma using nateglinide and pioglitazone as internal standards. Analytes were extracted from 0.50 mL of plasma using diethyl ether–n-hexane (4:1, v/v). Chromatographic separation was performed on a Teknokroma C₁₈ column with a mobile phase of methanol–water (containing 0.1% formic acid) (80:20, v/v) at a flow rate of 0.20 mL min^?1 within 5.6 min. Linearity was established over the concentration range of 0.005–5 ng mL^?1 for clopidogrel and 20–2,500 ng mL^?1 for SR26334. Intra- and inter-batch standard deviations were less than 9.2% and the accuracy of this assay was found to fall within an acceptable range ≤10.0%. The method was successfully applied to the therapeutic drug monitoring of clopidogrel.     

Estimation of Perospirone in Human Plasma by LC–MS–MS and Its Application to Pharmacokinetics Study

A sensitive liquid chromatography-tandem-mass spectrometry method was developed and validated for the determination of perospirone in human plasma, using quetiapine as internal standard. Plasma samples were extracted from 1 mL of plasma using n-hexane. Chromatographic separation was performed on an Agilent Zorbax SB C₁₈ column with a mobile phase of 5 mM ammonium acetate solution-methanol (12:88, v/v, adjusted to pH 3.8 with glacial acetic acid) at a flow rate of 0.2 mL min^?1. The chromatographic separation was achieved in less than 4.6 min. The linearity was established over the concentration range of 0.05–20 ng mL^?1. Both of the intra- and inter-batch standard deviation was less than 9.8%. The method was successfully applied to study the pharmacokinetic parameters of perospirone hydrochloride tablets in healthy Chinese volunteers.     

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.     

LC-MS-MS Method for Simultaneous Determination of Icariin and Its Active Metabolite Icariside II in Human Plasma

An LC-MS-MS method was revised and validated for simultaneous determination of icariin and its active metabolite icariside II in human plasma. The analytes and daidzein (IS) were extracted by liquid–liquid extraction and analyzed by LC-MS-MS. The separation was performed by a Zorbax SB-C₁₈ column (3.5 μm, 2.1 × 100 mm) with an isocratic mobile phase consisting of methanol–water–formic acid (65:35:0.035, v/v/v) at a flow rate of 0.25 mL min^?1. Detection was performed on a triple quadrupole tandem mass spectrum by multiple reaction monitoring mode using the electrospray ionization technique in positive mode. The method had lower limits of quantitation 0.2 and 0.1 ng mL^?1 for icariin and icariside II, respectively, using 500 μL plasma sample. The linear calibration curves were obtained in the concentration range of 0.2–100 ng mL^?1 for icariin and 0.1–100 ng mL^?1 for icariside II. The RSD values of intra- and inter-day precision calculated from quality control (QC) samples were less than 7.2% for icariin and less than 6.5% for icariside II. The accuracy as determined from QC samples was within 3.8% for each analyte. The method has been applied to determine and evaluate the pharmacokinetic of icariin and its metabolite icariside II in volunteers following oral administration of icariin and extract of Epimedium, respectively.     

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.     

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.     

RP-LC Quantification and Pharmacokinetic Study of Iriflophenone 2-O-α-Rhamnopyranoside in Rat Plasma

Iriflophenone 2-O-α-rhamnopyranoside (IP2R) is one of the main bioactive constituents of the leaves of Aquilaria sinensis (Lour.) Gilg, used in traditional Chinese medicines. A simple, rapid, and sensitive reversed-phase high-performance liquid chromatographic method has been developed for analysis of IP2R in rat plasma after intravenous administration. The analyte was extracted from plasma samples with methanol as deproteinization agent. Analysis was performed on an 250 mm × 4.6 mm i.d., 5-μm particle, C₁₈ column with a 8 mm × 4.6 mm i.d., 5-μm particle, RP-18 guard column; the mobile phase was acetonitrile–H₂O–acetic acid 22:78:0.01 (v/v) at a flow-rate of 1.0 mL min^?1. UV detection was at 289 nm. The calibration plot was linear in the range 0.01–33.33 μg mL^?1 (r = 0.9997, n = 5) in rat plasma. The lower limits of detection and quantification were 0.004 and 0.01 μg mL^?1. Intra-day and inter-day precision were 1.18–3.96 and 1.29–2.81%, respectively. Average extraction recovery from plasma was more than 95%. This assay method was successfully used to study the pharmacokinetics of IP2R in rats after a single dose of 25 mg kg^?1 by intravenous administration; the plasma concentration–time curve of IP2R conformed to a two-compartment open model.     

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.     

A Simple and Sensitive LC Method for Quantification of Cefteram in Human Plasma

A simple and sensitive liquid chromatographic method was developed for quantification of cefteram in human plasma. Amoxicillin was used as an internal standard. The present method used protein precipitation for extraction of cefteram from human plasma. Separation was carried out on a reversed-phase C₁₈ column. The column effluent was monitored by UV detection at 262 nm. The mobile phase was a mixture of methanol and water containing 0.3% v/v triethylamine and 0.6% v/v glacial acetic acid (35:65:0.3:0.6 v/v) at a flow rate of 0.30 mL min^?1. The column temperature was 20 °C. This method was linear over the range of 47.5–4,750.0 ng mL^?1 with determination coefficient greater than 0.99. The mean extraction recovery of cefteram and IS was ≥76.82 and ≥76.49%, respectively, and the method was found to be precise, accurate, and specific during the study. The method was successfully applied for a pharmacokinetic study of cefteram in human.     

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.     

Simultaneous Determination of Acetaminophen, Caffeine, and Chlorphenamine Maleate in Paracetamol and Chlorphenamine Maleate Granules

A simple and rapid HPLC method using phenacetin (PHN) as internal standard has been developed for simultaneous determination of acetaminophen, caffeine, and chlorphenamine maleate in the product compound paracetamol and chlorphenamine maleate granules. Separation and quantitation were achieved on a 250 mm × 4.6 mm, 5 μm particle, C₁₈ column. The mobile phase was methanol 0.05 mol L^?1 aqueous KH₂PO₄ solution, 45:55 (v/v), containing 0.1% triethylamine and adjusted to pH 3.6 by addition of phosphoric acid; the flow rate was 1.0 mL min^?1. Detection of all compounds was by UV absorbance at 260 nm and elution of the analytes was achieved in less than 12 min. The linearity, accuracy, and precision of the method were acceptable to good over the concentration ranges 6.4–153.6 μg mL^?1 for acetaminophen, 5.0–120.0 μg mL^?1 for caffeine, and 9.6–230.4 μg mL^?1 for chlorphenamine maleate.     

LC–MS Determination of p-(1-Dimethylamino ethylimino)aniline: a Metabolite of Tribendimidine in Human Plasma

A sensitive and selective liquid chromatography–mass spectrometric method was developed and validated for the determination of p-(1-dimethylamino ethylimino)aniline (dADT), a metabolite of tribendimidine, in human plasma. The analyte was separated on a Hypersil C₁₈ column (250 × 4.6 mm ID, 5 μm) by isocratic elution with 10 mM ammonium acetate (containing 0.1% triethylamine)-acetonitrile (20:80, v/v) at a flow rate of 1.0 mL min^?1 and measured by electrospray ionization source in positive selective ion monitoring mode at m/z 178. The weighted (1/x ²) calibration curve was linear within a concentration range of 0.5–1,000 ng mL^?1 and displayed a correlation coefficient (r) of 0.9996. The lower limit of quantification was determined to be at 0.5 ng mL^?1. The inter and intra-day precisions (%RSD) were less than 8% and the extraction recoveries ranged from 84.21 to 85.20%. The developed method was successfully applied to the determination of dADT in human plasma as part of a clinical pharmacokinetic study.     

Rapid and Sensitive RP-LC Method for the Quantification and Pharmacokinetic Study of p-Hydroxyphenethyl Anisate in Rat Plasma

A rapid, sensitive and specific reversed-phase liquid chromatographic method was developed and validated for the quantification of p-hydroxyphenethyl anisate (HPA), which is one of the main constituents of Notopterygium Radix (underground parts of Notopterygium incisum and N. forbesii), in rat plasma, and study its pharmacokinetics after the intravenous administration of 40 mg kg^?1 HPA to rats. The method involves a plasma clear-up step using liquid–liquid extraction by ethyl acetate, followed by RP-LC separation and detection. Separation of HPA was performed on an analytical Diamonsil ODS C₁₈ column equipped with a Dikma ODS C₁₈ EasyGuard column using a mobile phase consisting of MeOH–H₂O (75:25, v/v) at a flow-rate of 1.0 mL min^?1. The UV detection was performed at a wavelength of 256 nm. The linear calibration curves were obtained in the concentration range of 0.05–5.0 μg mL^?1 (r = 0.9992, n = 5) in rat plasma with the lower limit of detection of 0.01 μg mL^?1 and the lower limit of quantification of 0.04 μg mL^?1, and the extraction recovery of HPA was calculated to be the range of 82.01–86.66%. The intra- and inter-day precisions in terms of % relative standard deviation were lower than 2.33 and 3.99% in rat plasma, respectively, with accuracies ranging from 91.22 to 110.5%. The developed method was suitable for the determination and pharmacokinetic study of HPA in rat plasma.     

Simultaneous LC–MS Analysis and of Wogonin and Oroxylin A in Rat Plasma, and Pharmacokinetic Studies After Administration of the Active Fraction from Xiao-Xu-Ming Decoction

A rapid and specific high-performance liquid chromatographic method coupled with electrospray ionization mass spectrometric detection has been developed and validated for identification and quantification of wogonin and oroxylin A in rat plasma. Wogonin, oroxylin A, and diazepam (internal standard) were extracted from plasma samples by liquid–liquid extraction with ethyl acetate. Chromatographic separation was achieved on a C₁₈ column with acetonitrile–0.6% aqueous formic acid 35:65 (v/v) as mobile phase at a flow rate of 0.2 mL min^?1. Detection was performed with a single-quadrupole mass spectrometer in selected-ion-monitoring (SIM) mode. Linearity was good within the concentration range 14.4–360 ng mL^?1 for wogonin and 10.8–271 ng mL^?1 for oroxylin A; the correlation coefficients (r ²) were 0.9999. The intra-day and inter-day precision, as RSD, was below 12.4%, and accuracy ranged from 81.1 to 111.9%. The lower limit of quantification was 14.4 ng mL^?1 for wogonin and 10.8 ng mL^?1 for oroxylin A. This method was successfully used in the first pharmacokinetic study of wogonin and oroxylin A in rat plasma after oral administration of the active fraction from Xiao-xu-ming decoction.     

LC Method for Determination and Pharmacokinetic Study of 5,6,7,8,3′,4′-Hexamethoxy-3-sulfonyl Flavone in Rat Plasma

A sensitive, specific and rapid high-performance liquid chromatography method was developed for determination of 5,6,7,8,3′,4′-hexamethoxy-3-sulfonyl flavone in rat plasma. A simple methanol-induced protein precipitation was applied to extract 5,6,7,8,3′,4′-hexamethoxy-3-sulfonyl flavone and Picroside II (the internal standard) from rat plasma. Chromatographic separation was achieved on a Hypersil ODS₂ analytical column (200 mm × 4.6 mm, 5 μm) with acetonitrile–0.04% triethylamine solution (adjusted to pH 5.8 using phosphoric acid) (24:76, v/v) as mobile phase. The calibration curves were linear over the range of 0.2–40 μg mL^?1. Absolute recoveries of 5,6,7,8,3′,4′-hexamethoxy-3-sulfonyl flavone were 82.7–95.9% from rat plasma. The intra- and inter-day relative standard deviation precisions were less than 5 and 9%, respectively. The method was successfully applied to the pharmacokinetic study of 5,6,7,8,3′,4′-hexamethoxy-3-sulfonyl flavone in rats after intravenous administration.     

LC–ESI-MS Determination of Hydroxycamptothecin in Rat Plasma

A rapid and sensitive method using liquid chromatography with electrospray ionization mass spectrometric detection (LC–ESI-MS) was developed and validated for the determination of hydroxycamptothecin in rat plasma. Plasma samples were extracted with ether and separated on a C₁₈ column interfaced with a single quadrupole mass spectrometer, with mobile phase consisting of 0.1% formic acid–methanol (45:55, v/v). Detection was carried out by positive electrospray ionization (ESI) in selected ion recording (SIR) mode at m/z 321 and 305 for hydroxycamptothecin and camptothecin (internal standard), respectively. The linearity was obtained ranged from 2.5 to 1,000 ng mL^?1 and the lower limit of quantification (LLOQ) was determined to be at 2.5 ng mL^?1. The intra- and inter-day precision (%RSD) was less than 5.5% and accuracy (% RE) ranged from 3.8 to 5.3%. This method was applied successfully to a preliminary pharmacokinetic study following the intravenous administration of hydroxycamptothecin to rats.     

LC Determination of Isosorbide-5-Mononitrate in Human Plasma

A simple, sensitive, selective and cost effective LC–UV method was developed for determination of isosorbide mononitrate in human plasma using guaifenesin as an internal standard. Isosorbide mononitrate in plasma was extracted by a single step liquid extraction using tert-butyl methyl ether and chromatographed on a C18 column using water and acetonitrile (80:20 v/v) as mobile phase. The method was validated and exhibited a linear range from 51.6 to 2064.4 ng mL^?1. The inter- and intra-assay accuracy ranged from 97.2–102.7 to 94.2–105.5%, respectively, with precision less than 10% in both the cases. The LLQ was 51.6 ng mL^?1. The validated method was applied to the quantitation of isosorbide mononitrate from plasma samples in a pharmacokinetic study.     

Determination of Tiopronin in Human Plasma and Pharmacokinetics by LC-ESI-MS

A rapid, sensitive and specific method based on high performance liquid chromatography-electrospray ionization-mass spectrometry was developed for the determination of tiopronin in human plasma. In this study, vitamin C and dithiothreitol were used as the reducer and to release and stabilize tiopronin from dimeric mixed forms with endogenous thiols encountered during pretreatment of plasma samples. The separation was successfully achieved on an Agilent SB-Aq column packed with 5 μm C₁₈ silica, using an aqueous formic acid solution (pH 4.5–4.7), 0.5 mM tris(hydroxymethyl)aminomethane and methanol (95:5, v/v) as the mobile phase. Mass spectra were acquired in selective ion monitoring mode, using the [M ? H] ^? ions of tiopronin at m/z 162.0 and the [M ? H]^? of the internal standard sodium cyclamate at m/z 178.0, respectively. This quantitative assay was fully validated with respect to precision, repeatability and accuracy. The correlation coefficients were >0.9995 in the range 0.025–8.15 μg mL^?1 in human plasma. The mean recoveries were above 85%. The limit of quantitation was 0.012 μg mL^?1 with a relative standard deviation of inter-day and intra-day accuracy of less than 15%. This LC-ESI-MS method was also successfully applied to a pharmacokinetic study after oral administration of formulated tiopronin to healthy volunteers. The elimination half-life (T _1/2) was 21.5 ± 11.1 h.     

RP-LC Determination and Pharmacokinetic Study of Ferulic Acid and Isoferulic Acid in Rat Plasma After Taking Traditional Chinese Medicinal-Preparation: Guanxinning Lyophilizer

A sensitive, simple, and accurate method for determination and pharmacokinetic study of ferulic acid and isoferulic acid in rat plasma was developed using a reversed-phase column liquid chromatographic (RP-LC) method with UV detection. Sample preparations were carried out by protein precipitation with the addition of methanol, followed by evaporation to dryness. The resultant residue was then reconstituted in mobile phase and injected into a Kromasil C₁₈ column (250 × 4.6 mm i.d. with 5 μm particle size). The mobile phase was methanol-1% formic acid (33:67, v/v). The calibration plots were linear over the range 5.780–5780 ng·mL^?1 for ferulic acid and 1.740–348.0 ng·mL^?1 for isoferulic acid. Mean recoveries were 85.1% and 91.1%, respectively. The relative standard deviations (RSDs) of within-day and between-day precision were not above 15% for both of the analytes. The limits of quantification were 5.780 ng·mL^?1 for ferulic acid and 1.740 ng·mL^?1 for isoferulic acid. This RP-LC method was used successfully in pharmacokinetic studies of ferulic acid and isoferulic acid in rat plasma after intravenous injection of Guanxinning Lyophilizer.