Sensitive Determination of Felodipine in Human and Dog Plasma by Use of Liquid–Liquid Extraction and LC–ESI–MS–MS

Summary A sensitive and selective liquid chromatographic method coupled with electrospray ionization tandem mass spectrometry (LC–ESI–MS–MS) has been developed for quantification of felodipine in human and dog plasma. Compounds were separated on a 2.0 mm × 150 mm, 5.0 m particle, C₈ column with 1 m m ammonium acetate–acetonitrile, 20:80, pH 6.0, as mobile phase at a flow rate of 200 L min^–1. Nifedipine was used as internal standard. Plasma samples were extracted with diethyl ether, the centrifuged upper layer was evaporated, the residue was reconstituted with mobile phase, and the reconstituted samples were injected. The analytical column lasted for at least 1000 injections. By use of multiple reaction monitoring (MRM) mode in MS–MS felodipine and nifedipine were detected without severe interference from the human or dog plasma matrix. Felodipine produced a protonated precursor ion ([M + H]⁺) at m/z 384 and a corresponding product ion at m/z 338. And internal standard (nifedipine) produced a protonated precursor ion ([M + H]⁺) at m/z 347 and a corresponding product ion at m/z 315. Detection of felodipine in human and dog plasma was accurate and precise, with a limit of quantification of 0.05 ng mL^–1. The method has been successfully applied to preliminary pharmacokinetic study of felodipine in human and dog plasma.     

Quantitative estimation of riluzole in human plasma by LC-ESI-MS/MS and its application to a bioequivalence study

A novel simple, sensitive, selective, and rapid high-performance liquid chromatography coupled with tandem mass spectrometry method was developed and validated for quantification of riluzole in human plasma. The chromatography was performed by using a Zorbax-SB-C18 (4.6 × 75 mm, 3.5 μm) column , isocratic mobile phase 0.1% formic acid/acetonitrile (10:90 v/v), and an isotope-labeled internal standard (IS), [¹³C,¹⁵N₂]riluzole. The extraction of drug and internal standard was performed by liquid–liquid extraction and analyzed by MS in the multiple reaction monitoring (MRM) mode using the respective [M+H]⁺ ions, m/z 235.0/165.9 for riluzole and m/z 238.1/169.0 for the IS. The calibration curve was linear over the concentration range 0.5–500.0 ng/ml for riluzole in human plasma. The limit of quantification (LOQ) was demonstrated at 0.5 ng/ml. The within-batch and between-batch precision were 0.6–2.3% and 1.4–5.7%, and accuracy was 97.1–101.1% and 98.8–101.2% for riluzole respectively. Drug and IS were eluted within 3.0 min. The validated method was successfully applied in a bioequivalence study of riluzole in human plasma.     

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 Glimepiride in Human Plasma by LC-MS-MS and Comparison of Sample Preparation Methods for Glimepiride

A sensitive and selective method for quantitation of glimepiride in human plasma was established using liquid chromatography-electrospray ionization tandem mass spectrometry. Three different methods for the sample preparation of glimepiride and an internal standard were investigated (liquid-liquid extraction, solid-phase extraction and protein precipitation). Glipizide was used as an internal standard. Compounds were separated on a C₁₈ column with 80% acetonitrile and 20% deionized water (adjusted to pH 3.5 with acetic acid), as mobile phase at a flow rate of 200 L min^–1. By use of multiple reaction monitoring mode in MS-MS with liquid-liquid extraction and solid-phase extraction, glimepiride and glipizide were detected without severe interference from the human plasma matrix. Glimepiride produced a protonated precursor ion ([M+H]⁺) at m/z 491 and a corresponding product ion at m/z 352, and the internal standard produced a protonated precursor ion ([M+H]⁺) at m/z 446 and a corresponding product ion at m/z 321. The limit of quantitation was 0.1 ng mL^–1, 0.5 ng mL^–1 and 1.0 ng mL^–1 when using liquid-liquid extraction, solid-phase extraction and protein precipitation, respectively. The validation, reproducibility, stability, and recovery of the different sample preparation methods were comparable and all the methods gave reliable results. The method has been successfully applied to pharmacokinetic study of glimepiride in human plasma.     

A liquid chromatography–mass spectrometric method for the quantification of azithromycin in human plasma

A liquid chromatographic mass spectrometric assay for the quantification of azithromycin in human plasma was developed. Azithromycin and imipramine (as internal standard, IS) were extracted from 0.5 mL human plasma using extraction with diethyl ether under alkaline conditions. Chromatographic separation of drug and IS was performed using a C₁₈ column at room temperature. A mobile phase consisting of methanol, water, ammonium hydroxide and ammonium acetate was pumped at 0.2 mL/min. The mass spectrometer was operated in positive ion mode and selected ion recording acquisition mode. The ions utilized for quantification of azithromycin and IS were m/z 749.6 (M + H) ⁺ and m/z 591.4 (fragment) for azithromycin, and 281.1 m/z for internal standard; retention times were 6.9 and 3.4 min, respectively. The calibration curves were linear (r² > 0.999) in the concentration ranges of 10–1000 ng/mL. The mean absolute recoveries for 50 and 500 ng/mL azithromycin and 1 µg/ mL IS were >75%. The percentage coefficient of variation and mean error were <11%. Based on validation data, the lower limit of quantification was 10 ng/mL. The present method was successfully applied to determine azithromycin pharmacokinetic parameters in two obese volunteers. The assay had applicability for use in pharmacokinetic studies. Copyright © 2013 John Wiley & Sons, Ltd.     

A sensitive and selective method for the quantitative analysis of miglitol in rat plasma using unique solid‐phase extraction coupled with liquid chromatography–tandem mass spectrometry

A sensitive, selective and robust liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed for the quantification of miglitol in rat plasma. The sample preparation procedures involved protein precipitation and unique solid‐phase extraction, which efficiently removed sources of ion suppression and column degradation interference present in the plasma. Chromatographic separation was achieved on an amide column using 10 mmol/L CH₃COONH₄ and CH₃CN:CH₃OH (90:10, v/v) as the mobile phase under gradient conditions. Detection was performed using tandem mass spectrometry equipped with an electrospray ionization interface in positive ion mode.The selected reaction monitoring transitions for miglitol and a stable isotope‐labeled internal standard were m/z 208 → m/z 146 and m/z 212 → m/z 176, respectively. The correlation coefficients of the calibration curves ranged from 0.9984 to 0.9993 over a concentration range of 0.5–100 ng/mL plasma. The quantification limit of the proposed method was more than 10 times lower than those of previously reported LC‐MS/MS methods. The novel method was successfully validated and applied to a pharmacokinetic study in rats. Copyright © 2014 John Wiley & Sons, Ltd.     

A new HPLC–MS/MS method for the simultaneous quantification of SGLT2 inhibitors and metformin in plasma and its application to a pharmacokinetic study in healthy volunteers

Monitoring the plasma concentrations of metformin and sodium‐glucose cotransporter‐2 inhibitors (canagliflozin, dapagliflozin and empagliflozin) is essential for pharmacokinetic and bioequivalence studies and therapeutic monitoring. The present work therefore aimed to develop and validate a high‐performance liquid chromatography coupled to tandem mass spectrometry (HPLC–MS/MS) method for the simultaneous quantification of these drugs in human plasma. The analyses were performed using an Agilent 1200 HPLC system coupled to an Applied Biosystems API 3200 triple quadrupole MS/MS with electrospray ionization in positive ion mode. After one‐step protein precipitation of plasma with acetonitrile containing 0.1% formic acid, chromatographic separation was achieved on an Xbridge C₁₈ column, with a mobile phase consisting of a gradient of water and acetonitrile, both containing 1 mm ammonium formate and 0.1% formic acid. Quantification was performed in multiple reaction monitoring mode using m/z 130.1 → 71.1 for metformin, m/z 462.0 → 191.2 for canagliflozin, m/z 426.1 → 167.1 for dapagliflozin and m/z 468.0 → 354.9 for empagliflozin. The proposed method was validated and demonstrated to be adequate for the quantification of metformin, canagliflozin, dapagliflozin and empagliflozin for clinical monitoring, pharmacokinetics and bioequivalence studies.     

A simple,selective and rapid validated method for estimation of anastrazole in human plasma by liquid chromatography–tandem mass spectrometry and its application to bioequivalence study

A rapid, simple and specific method for estimation of anastrazole in human plasma was validated using letrozole as internal standard. The analyte and internal standard were extracted from plasma using simple solid‐phase extraction. The compound were separated on a reverse‐phase column with an isocratic mobile phase consisting of 0.1% formic acid in water and acetonitrile (12 : 88, v/v) and detected by tandem mass spectrometry in positive ion mode. The ion transitions recorded in multiple reaction monitoring mode were m/z 294.1 → 225.1 for anastrazole and m/z 286.1 → 217.1 for internal standard. Linearity in plasma was observed over the concentration range 0.3–30 ng/mL for anastrazole. The mean recovery for anastrazole was 83.7% with a lower limit of quantification of 0.3 ng/mL. The coefficient of variation of the assay was less than 6.8% and the accuracy was 96.1–102.2%. The validated method was applied to a bioequivalence study of 1 mg anastrazole tablet in healthy human volunteers. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous quantification of cilostazol and its primary metabolite 3,4-dehydrocilostazol in human plasma by rapid liquid chromatography/tandem mass spectrometry

A simple, rapid, sensitive and selective liquid chromatography/electrospray tandem mass spectrometry method was developed and validated for the simultaneous quantification of cilostazol and its primary metabolite 3,4-dehydrocilostazol in human plasma using mosapride as an internal standard. The method involves a simple one-step liquid-liquid extraction with a diethyl ether and dichloromethane mixture (7:3). The analytes were chromatographed using an isocratic mobile phase on a reversed-phase C₁₈ column and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective [M+H]⁺ ions, m/z 370/288 for cilostazol, m/z 368/286 for 3,4-dehydrocilostazol and m/z 422/198 for the internal standard. The assay exhibited a linear dynamic range of 5–2,000 ng/mL for cilostazol and 5–400 ng/mL for 3,4-dehydrocilostazol in human plasma. The lower limit of quantitation was 5 ng/mL for both cilostazol and its metabolite. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetics, bioavailability or bioequivalence studies.      

Pharmacokinetic analysis of plasma bicyclol by liquid chromatography–tandem mass spectrometry

Bicyclol is a synthetic drug widely used to treat chronic hepatitis B. This study aimed to develop a selective, sensitive and high‐throughput liquid chromatography–tandem mass spectrometric method for the detection of bicyclol in human plasma. Bicyclol was detected using a multiple reaction monitoring mode, with ammonium adduct ions (m/z 408.2) as the precursor ion and the [M‐CH₃]⁺ ion (m/z 373.1) subjected to demethylation as the product ion. Chromatographic separation was achieved using a Zobax Eclipse XDB‐C₁₈ column with a gradient elution and a mobile phase of 2 mm ammonium formate and acetonitrile. Bicyclol was extracted from plasma matrix by precipitation. A linear detection response was obtained for bicyclol ranging from 0.500 to 240 ng/mL, and the lower limit of quantification was 0.500 ng/mL. The intra‐ and inter‐day precisions were all ≤7.4%, and the accuracies were within ±6.0%. The extraction recovery was >95.9%, and the matrix effects were between 96.0% and 108%. Bicyclol was found to be unstable in human plasma at room temperature, but the degradation was minimized by conducting sample collection and preparation in an ice bath. The validated method was successfully applied to investigate the pharmacokinetics of bicyclol tablets in six healthy Chinese volunteers.     

Determination of roxythromycin in blood serum by liquid chromatography with mass spectrometric detection

A procedure has been developed for the determination of a macrolide antibiotic roxythromycin (RX) in blood serum using HPLC with mass spectrometric detection using clarithromycin (CL) as the internal standard. RX and CL have been extracted from the samples by solid-phase extraction in a cartridge filled with a polar adsorbent, cyanopropylsilyl silica gel. The absolute recoveries of RX and CL are 89.6 and 92.5%, respectively. Chromatographic separation has been performed on a Nucleodur C₁₈ Isis column with the mobile phase composed as follows: water-methanol-acetonitrile-formic aid (499: 250: 250: 1 by volume). Registration has been performed in the mode of selected ion monitoring with m/z 837.7 (RX) and m/z 748.7 (CL). The analytical range for RX is 0.097–14.81 μg/mL, the quantification limit is 0.097 μg/mL, the detection limit is 0.03 μg/mL, and the intraday and interday relative standard deviation are 2–6 and 4–8% respectively. The procedure has been applied to the pharmacokinetic studies of the Rulid pharmaceutical preparation.     

Exploring dried blood spot sampling technique for simultaneous quantification of antiretrovirals: lamivudine, stavudine and nevirapine in a rodent pharmacokinetic study

A high‐performance liquid chromatography/positive ion electrospray tandem mass spectrometry method for the simultaneous quantification of lamivudine, stavudine and nevirapine was developed and validated in dried blood spot (DBS) cards. The analytes were separated using an isocratic mobile phase on a reverse phase column and analyzed by MS/MS in the MRM mode using the respective [M + H]⁺ ions, m/z 230–112 for lamivudine, m/z 225–127 for stavudine, m/z 267–226 for nevirapine, m/z 383–337 for zidovudine (IS). The lower limit of quantification was 1 ng/mL for both lamivudine and stavudine and 10 ng/mL for nevirapine. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The method was successfully applied to quantify them in a rat pharmacokinetic study in whole blood, plasma and DBS cards after a single oral co‐administration at the dose of 10, 2 and 13 mg/kg for lamivudine, stavudine and nevirapine, respectively, to male Wistar rats. Following oral administration the pharmacokinetic results in all the matrices are in close agreement. Thus accomplishment of this method would facilitate the ease of collection of clinical samples on DBS cards for lamivudine, stavudine and nevirapine during human clinical trials and therapeutic drug monitoring. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of 5-n-Butyl-4-{4-[2-(1H-tetrazole-5-yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6-dichloridephenyl)-3H-1,2,4-triazol-3-one, a New Angiotensin Type 1 Receptor Antagonist in Rat Plasma by LC-ESI-MS: Application to Pharmacokinetic Studies

A simple and sensitive reversed-phase LC-ESI-MS method to identify and quantitate 5-n-butyl-4-{4-[2-(1H-tetrazole-5-yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6-dichloridephenyl)-3H-1,2,4-triazol-3-one (1b), a new Angiotensin II type 1 receptor antagonist in rat plasma has been developed and validated. Sample preparation used a simple liquid–liquid extraction with ethyl acetate. Separation was achieved by gradient elution on a C₁₈ column. The mobile phase consisted of acetonitrile and water (0.05% triethylamine and 0.05% acetic acid) at a flow rate of 0.2 mL min⁻¹. The detection utilized selected ion monitoring (SIM) in the negative mode at m/z 507.1 and m/z 407.2 for the deprotonated molecular ions of 1b and the internal standard irbesartan, respectively. The lower limit of quantification was reproducible at 5 ng mL⁻¹ with 100 μL of plasma and the good linear was observed in the 5–500 ng mL⁻¹ range. This concentration range corresponded well with the plasma concentrations of 1b in pharmacokinetic studies. Recoveries of 1b in rat plasma were 76.1, 74.6 and 79.0% at 5, 50 and 500 ng mL⁻¹. The RSD of intra-assay and inter-assay variations were all less than 5%. This validated LC-ESI-MS assay is an economic, quick, precise and reliable method for the analysis of 1b in pharmacokinetic studies.     

Determination of 6-Benzyladenine in Bean Sprout by LC–ESI-IT-MS–MS

A rapid and sensitive method has been developed and validated to determine 6-benzyladenine (6-BA) residues in bean sprout using high-performance liquid chromatography coupled with electrospray ionization ion trap tandem mass spectrometry. Liquid–liquid extraction, using acidified methanol, was used to isolate 6-BA from the sample matrix. The separation was carried out on a Zorbax SB C₁₈ column (150 mm × 4.6 mm i.d., 5 μm) with 0.1% acetic acid/methanol (25:75 v/v) as mobile phase in isocratic mode. The quantitation of 6-BA was based on fragmentation of the molecular ion at m/z 226.1 to a product ion at m/z 91.1. In addition, the recoveries of three extraction solvents by ultrasound extraction and conventional extraction method were compared, and it was found that acidified methanol as the extraction solvent was the best. The calibration curve was linear (r ² > 0.997) in the concentration range of 0.04–10.0 ng mL⁻¹ with a lower limit of quantification of 0.5 ng g⁻¹ for 6-BA in bean sprout. Intra-day and inter-day relative standard deviations (RSDs) were less than 7.1 and 9.8%. Recoveries of 6-BA were between 85.0 and 88.5%. This assay has been successfully applied to the determination of trace 6-BA residues in bean sprout and monitoring the market in Ningbo, China.     

Quantitative determination of ginsenoside Rh2 in rat biosamples by liquid chromatography electrospray ionization mass spectrometry

Ginsenoside Rh₂ is a “hot” natural compound with great potential as a new anti-cancer drug based on abundant pharmacological experiments. However, no systemic pharmacokinetic study of Rh₂ was reported because current analysis methods could not fully meet the requirements. Thus, we developed a simple LC/MS method with highly improved sensitivities for the determination of Rh₂ in rat plasma, bile, urine, feces and most tissues. The tissues and feces were firstly homogenized mechanically using buffer and methanol as the media, respectively. Plasma, bile, urine and tissue homogenates were extracted with diethyl ether for sample preparation. Feces homogenates were directly deproteinized with acetonitrile. The subsequent analysis procedures were performed on a Shimadzu LCMS2010A system (electrospray ionization single quadrupole mass analyzer), with an ODS column (150 mm × 2.0-mm i.d., 5 μm) plus a C18 guard column for separation and ammonium chloride (500 μmol) as mobile phase additive. The proportions of mobile phase were changed timely according to gradient programs. Chlorinated adducts of molecular ions [M + Cl]⁻ of Rh₂ at m/z 657.35 and internal standard digitoxin at m/z 799.55 were monitored in selective ion monitoring mode of negative ions. The method was validated to be accurate, precise and rugged with good linearity in all matrices, according to the FDA guidelines. The lower limits of quantitation in rat plasma, urine and feces were 0.2, 0.2 and 20 ng/mL respectively. Stability studies were also performed, indicating that there were no stability-related problems in the analytical procedure of Rh₂. The proposed method was successfully applied to the preclinical pharmacokinetic research of Rh₂ in rats, including plasma kinetics, tissue distribution and excretion studies.     

Quantification of methyllycaconitine,selective α7 nicotinic receptor antagonist,in rodent plasma and brain tissue by liquid chromatography tandem mass spectrometry – application to neuropharmacokinetics of methyllycaconitine in rats

A sensitive high‐performance liquid chromatography–positive ion electrospray tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the quantification of methyllycaconitine (MLA) in rat plasma and brain tissue. Following acetonitrile protein precipitation, the analyte was separated using a gradient mobile phase on a reversed‐phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H]⁺ ions, m/z 683–216 for MLA and m/z 260–116 for the internal standard. The assay exhibited a linear dynamic range of 0.5–250 ng/mL for MLA in rat plasma and brain tissue. The lower limit of quantification was 0.5 ng/mL. Acceptable precision (<12%) and accuracy (100 ± 6%) were obtained for concentrations over the standard curve range. The method was successfully applied to quantify MLA concentrations in a rodent pharmacokinetic and brain penetration study. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of imidacloprid in paddy water and soil by liquid chromatography electrospray ionization-tandem mass spectrometry

A method for the determination of imidacloprid in paddy water and soil was developed using liquid chromatography electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS). Separation of imidacloprid was carried out on a Shimadzu C18 column (150 mm × 4.6 mm, 4.6 μm) with an acetonitrile-water (50: 50, v/v) mobile phase containing 0.1% of acetic acid. The flow rate was 0.3 mL/min in isocratic mode. The product ion at 209 m/z was selected for quantification in multiple-reaction monitoring scan mode. Imidacloprid residues in soil were extracted by a solid-liquid extraction method with acetonitrile. Water samples were filtered and directly injected for analysis without extraction. Detection limits of 0.5 μg/kg and 0.3 μg/L were achieved for soil and water samples, respectively. The method had recoveries of 90 ± 2% (n = 4) for soil samples and 100 ± 2% (n = 4) for water samples. A linear relationship was observed throughout the investigated range of concentrations (1–200 μg/L), with the correlation coefficients ranging from 0.999 to 1.000.     

Quantitative determination of sauchinone in rat plasma by liquid chromatography-mass spectrometry

A rapid, sensitive and specific method using liquid chromatography with tandem mass spectrometric detection (LC‐MS) was developed for the analysis of sauchinone in rat plasma. Di‐O‐methyltetrahydrofuriguaiacin B was used as internal standard (IS). Analytes were extracted from rat plasma by liquid–liquid extraction using ethyl acetate. A 2.1 mm i.d. × 150 mm, 5 µm, Agilent Zorbax SB‐C₁₈ column was used to perform the chromatographic analysis. The mobile phase was methanol–deionized water (80:20, v/v). The chromatographic run time was 7 min per injection and the flow‐rate was 0.2 mL/min. The tandem mass spectrometric detection mode was achieved with electrospray ionization interface in positive‐ion mode (ESI⁺). The m/z ratios [M + Na]⁺, m/z 379.4 for sauchinone and m/z 395.4 for IS were recorded simultaneously. Calibration curve were linear over the range of 0.01–5 µg/mL. The lowest limit of quantification was 0.01 µg/mL. The intra‐day and inter‐day precision and accuracy of the quality control samples were 2.94–9.42% and 95.79–108.05%, respectively. The matrix effect was 64.20–67.34% and the extraction recovery was 93.28–95.98%. This method was simple and sensitive enough to be used in pharmacokinetic research for determination of sauchinone in rat plasma. Copyright © 2011 John Wiley & Sons, Ltd.     

Development and Validation of LC–MS Method for the Determination of Hydroxyzine Hydrochloride in Human Plasma and Subsequent Application in a Bioequivalence Study

A rapid, simple and specific liquid chromatography-electrospray ionization mass spectrometry method has been developed and validated for the determination of hydroxyzine hydrochloride in human plasma. Samples were separated using a Thermo Hypersil-HyPURITYC18 reversed-phase column (150 mm × 2.1 mm i.d., 5 μm). The mobile phase consisted of 50 mM ammonium acetate (pH 4.0)–methanol–acetonitrile (45:36:19, v/v). Hydroxyzine and its internal standard were measured by electrospray ion source in positive selective ion monitoring mode. The method was validated with a linear range of 1.56–200.0 ng mL⁻¹ and the lowest limit of quantification was 1.56 ng mL⁻¹ for hydroxyzine hydrochloride (r ²= 0.9991). The extraction efficiencies were about 70% and recoveries of the method were in the range of 93.5–104.4%. The intra-day relative standard deviation (RSD) was less than 8.0% and inter-day RSD was within 7.4%. QC samples were stable when kept at ambient temperature for 12 h at −20 °C for 30 days and after four freeze–thaw cycles. The method has been successfully applied to the evaluation of pharmacokinetics and bioequivalence of two hydroxyzine hydrochloride formulations in 12 healthy Chinese volunteers after an oral dose of 25 mg.     

UPLC-MS/MS determination of doxazosine in human plasma

A sensitive, selective and rapid method for the analysis of doxazosine (DOX) in human plasma based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) is described. DOX and tamsulosin, used as an internal standard (IS), were extracted by liquid-liquid extraction, and the chromatography was performed on a C18 UPLC column packed with 1.7 μm particles. The total run time was 2 min. Detection was achieved by the multiple reaction monitoring of the following transitions: m/z 452→344 and m/z 409→228 for DOX and IS, respectively. Transitions of m/z 452→247 and m/z 409→271 were also collected for confirmation purposes. The calibration curve based on peak area ratio was linear up to at least 100 ng ml⁻¹, with a detection limit of 0.02 ng ml⁻¹ (a signal-to-noise ratio of 3). The method showed satisfactory reproducibility, and the short-term stability of the analyte was assessed. The method was successfully applied to the analysis of DOX in human plasma.