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.     

LC-ESI-MS Method for the Determination of Mizolastine in Human Plasma

A sensitive and rapid liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method has been developed and validated for the determination of mizolastine in human plasma using dipyridamole as the internal standard (I.S.). Plasma samples were simply pretreated with methanol for deproteinization. Chromatographic separation was performed on an Agilent Zorbax C₁₈ column with a mobile phase of 10 mM ammonium acetate buffer containing 0.1% formic acid–methanol (20:80, v/v) at a flow rate of 1 mL min⁻¹. The electrospray ionization (ESI) interface was employed in a single quadrupole mass spectrometer. The analytes were protonated in the positive ESI interface and detected in single ion monitoring (SIM) mode. Chromatographic separation was achieved in less than 3.5 min. The linearity was established over the range of 0.5–600 ng mL⁻¹. The lower limited of quantification (LLOQ) of the method was 0.5 ng mL⁻¹. The intra- and inter-run standard deviations were both less than 11.2%. The method was applied to study the pharmacokinetics of the mizolastine sustained-release tablets in healthy volunteers.     

RP-HPLC Determination of Linarin in Beagle Dog Plasma After Administration of Yejuhua Injection

A sensitive, simple, and accurate reversed-phase column liquid chromatographic (RP-LC) method with daidzein as an internal standard and UV detection at 348 nm has been developed for determination of linarin in beagle dog plasma. Plasma protein was precipitated by addition of acetonitrile and the remaining solution was evaporated to dryness. The resulting residue was then reconstituted in methanol and injected on to a 250 mm × 4.6 mm i.d., 5 μm particle, ODS analytical column. The mobile phase was a gradient prepared from acetonitrile and an aqueous solution (0.4%) of phosphoric acid; the flow rate was 1.0 mL min⁻¹. Response was a linear function of concentration over the range 3.4–1,373.3 ng mL⁻¹; the correlation coefficient (R ²) was 0.993. Mean recovery was 74.2%. Within-day and between-day precision were better than 8.8% relative standard deviation (RSD). The limit of quantification was 3.4 ng mL⁻¹. This RP-LC method was used successfully in pharmacokinetic studies of linarin in beagle plasma after administration of Yejuhua injection.     

Rapid Determination of Montelukast in Human Plasma by LC-ESI-MS/MS and Its Application to a Bioequivalence Study

Abstract

A rapid liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) method was developed for the determination of montelukast in human plasma. The extraction of montelukast from plasma (300 µL) involved protein precipitation. Quantitation was performed using LC-ESI-MS/MS, operating in the positive ion and selective reaction monitoring (SRM) mode. The total chromatographic run time for the analysis was 1.5 min. A linear dynamic range was established from 5 to 800 ng mL^?1 for montelukast. The method was fully validated especially with regard to real subject sample analysis. It was successfully applied to a bioequivalence study in 18 healthy human subjects under fed condition (human subjects were allowed to eat as per the prescribed diet for bioequivalence study).     

LC-ESI-MS Determination of Bilobalide and Ginkgolides in Canine Plasma

A sensitive and selective method using liquid chromatography with electrospray ionization mass spectrometric detection was developed for the quantification of bilobalide and ginkgolides in canine plasma. The analytes were extracted with diethyl ether-dichloromethane-isopropanol (6:3:1, v/v) after spiking the samples with daidzein (internal standard). The lower limit of quantification (LLOQ) of the method was 2.5 μg L⁻¹ for ginkgolide B and 10.0 μg L⁻¹ for bilabolide, ginkgolide A and ginkgolide C. The accuracy of the method was within 15% of the actual values over a wide range of plasma concentrations. The intra-day and inter-day precision was better than 15% (R.S.D.). Finally, the LC-ESI-MS method was successfully applied to study the pharmacokinetics of ginkgolides and bilabolide after administration of Ginkgo biloba extracts to dogs.     

Simultaneous Determination of Four Active Components in a Compound Formulation by Liquid Chromatography

Summary A rapid and accurate LC method is described for simultaneous determination of pseudoephedrine hydrochloride (PSE), acetaminophen (AMP), dextromethorphen hydrobromide (DEX), and diphenhydramine hydrochloride (DPH) in a compound formulation. Chromatographic separation of the four drugs was achieved on a Hypersil CN column (150 mm × 4.6 mm, 5 m particle) by use of a mobile phase comprising a mixture of 3 mM ion-pairing solution, 2% aqueous triethylamine solution, and 2 M phosphoric acid, 68:48:88 (v/v), pH 3.0, delivered at 1.0 mL min^–1. Compounds were detected at 215 nm and the run time was less than 10 min. The linearity, accuracy, and precision of the method were found to be acceptable over the concentration ranges 6.1–36.4 g mL^–1 for PSE, 65.0–390.0 g mL^–1 for AMP, 3.1–18.6 g mL^–1 for DEX, and 5.0–30.0 g mL^–1 for DPH.     

Simultaneous Determination of Metformin Hydrochloride, Cyanoguanidine and Melamine in Tablets by Mixed-Mode HILIC

A simple and specific hydrophilic interaction liquid chromatography (HILIC) procedure for the quantification of metformin hydrochloride (MFH) and its impurities in bulk pharmaceuticals and finished dosage forms has been developed. The method is based on hydrophilic interaction of the analytes with silica. The influence of the weaker solvent, acetonitrile, pH and the nature and ionic strength of the buffer was studied. Linearity range and percent recoveries for MFH were 100–400 μg mL⁻¹ and 100.62%, respectively. Good validation data were obtained for all compounds. The method separates impurities cyanoguanidine (CGD), melamine (MLN) and other degradation products with a run time of less than 13 min. Degradation studies involved thermal stress, hydrolysis at various pHs and chemical and photolytic oxidation.     

HPLC Analysis and Pharmacokinetics of Picroside I in Dog Plasma

A sensitive and selective HPLC–UV method established for determination of picroside I in dog plasma has been used to study the pharmacokinetics of the drug after intravenous administration of three different doses. Sample pretreatment consists in deproteination by addition of acetonitrile; l-ascorbic acid was used to improve the stability of picroside I. The lower limit of quantification of picroside I was 0.05 μg mL⁻¹. The recovery of the method was up to 90%. After intravenous administration to dogs picroside I was mainly distributed in the central compartment and was rapidly eliminated from the plasma; the mean elimination half-life was 30.54 ± 4.34, 30.20 ± 3.78, and 34.02 ± 1.88 min for doses of 2.5, 5, and 15 mg kg⁻¹, respectively, and the respective values of AUC _0–∞ were 81.04 ± 19.95, 198.50 ± 27.77, and 586.44 ± 103.08 μg min mL⁻¹. The different doses had no significant effect on the main pharmacokinetic data and the kinetics seemed to be linear in dosage range 2.5–15 mg kg⁻¹.     

HPLC Determination and Pharmacokinetic Study of Valdecoxib in Human Plasma

A sensitive, simple, and accurate high-performance liquid chromatographic method has been developed for determination of valdecoxib and the internal standard rofecoxib in human plasma. Protein was precipitated from plasma samples by addition of perchloric acid (HClO₄); the drug was then extracted with diethyl ether. Separation was performed on a Cosmosil C₁₈ column (150 mm × 4.6 mm i.d., 5 m particles) with ammonium acetate buffer-acetonitrile, 60:40 (v/v), containing 0.1% TEA, pH 6.5, as mobile phase. Detection and quantification were performed by UV-visible detection at 239 nm. Detection and quantification limits were 3 and 5 ng mL^–1, respectively. The linear concentration range for valdecoxib was 5–400 ng mL^–1. The validated RP HPLC method was used for determination of the pharmacokinetic data for the drug in humans.     

Validation of an HPLC Method for Determination of Cefepime (a Fourth-Generation Cephalosporin). Determination in Human Serum, Cerebrospinal Fluid, and Urine. Pharmacokinetic Profiles

A high-performance liquid-chromatographic method with detection at 256 nm has been developed and validated for analysis of cefepime in several biological matrices. Serum samples were deproteinized with acetonitrile and extracted once with dichloromethane. For urine and cerebrospinal fluid samples, only a microfiltration step was necessary. The method was validated in accordance with the recommendations of the International Conference on Harmonization (ICH), the Food and Drug Administration (FDA), and the Center for Drug Evaluation and Research (CDER). The method was used to determine levels of the drug in the serum, cerebrospinal fluid, and urine of twelve patients treated with Maxipime. The results obtained were compared with those from previously published HPLC methods.     

Simultaneous Determination of Tramadol and Acetaminophen in Human Plasma by LC–ESI–MS

This study presents a high-performance liquid chromatography–electrospray ionization–mass spectrometric (LC–ESI–MS) method for the simultaneous determination of tramadol and acetaminophen in human plasma using phenacetinum as the internal standard. After alkalization with saturated sodium bicarbonate, both compounds were extracted from human plasma with ethyl acetate and were separated by HPLC on a Hanbon LiChrospher CN column with a mobile phase of 10 mM ammonium acetate buffer containing 0.5% formic acid–methanol (40:60, v/v) at a flow rate of 1 mL min⁻¹. Analytes were determined using electrospray ionization in a single quadrupole mass spectrometer. LC–ESI–MS was performed in the positive selected-ion monitoring (SIM) mode using target ions at [M+H]⁺ m/z 264.3 for tramadol, [M+H]⁺ m/z 152.2 for acetaminophen and [M+H]⁺ m/z 180.2 for phenacetinum. Calibration curves were linear over the range of 5–600 ng mL⁻¹ for tramadol and 0.03–16 μg mL⁻¹ for acetaminophen. The inter-run relative standard deviations were less than 14.4% for tramadol and 12.3% for acetaminophen. The intra-run relative standard deviations were less than 9.3% for tramadol and 7.9% for acetaminophen. The mean plasma extraction recovery for tramadol and acetaminophen were in the ranges of 82.7–85.9 and 83.6–85.3%. The method was applied to study the pharmacokinetics of a new formulation of tramadol/acetaminophen tablet in healthy Chinese volunteers.     

Simultaneous Determination of Olanzapine and Fluoxetine by HPLC

A rapid, specific reversed phase HPLC method has been developed for simultaneous determination of olanzapine and fluoxetine in their formulations. Chromatographic separation of these two pharmaceuticals was carried out on an Inertsil C₁₈ reversed phase column (150 mm × 4.6 mm, 5 μm) with a 40:30:30 (v/v/v) mixture of 9.5 mM sodium dihydrogen phosphate (pH adjusted to 6.8 ± 0.1 with triethylamine), acetonitrile and methanol as mobile phase. The flow rate 1.2 mL min⁻¹ and the analytes are monitored at 225 nm. Paroxetine was used as internal standard. The assay results were linear from 25 to 75 μg mL⁻¹ for olanzapine (r ² ≥ 0.995) and 100–300 μg mL⁻¹ for fluoxetine (r ² ≥ 0.995), showed intra- and inter-day precision less than 1.0%, and accuracy of 97.7–99.1% and 97.9–99.0%. LOQ was 0.005 and 0.001 μg mL⁻¹ for olanzapine and fluoxetine, respectively. Separation was complete in less than 10 min. Validation of the method showed it to be robust, precise, accurate and linear over the range of analysis.     

High-Performance Liquid Chromatographic Determination of Isofraxidin in Rat Plasma

For the first time a high-performance liquid chromatographic (HPLC) method, with liquid-liquid extraction and ultraviolet (UV) absorbance detection, has been developed for quantification of isofraxidin in rat plasma. The analysis was performed on a Diamonsil C₁₈ column (200 mm × 4.6 mm i.d., 5 μm particle size) with acetonitrile–0.05% phosphoric acid, 26:74 (v/v), as isocratic mobile phase. The linear range was 0.05–8.0 μg mL⁻¹ and the lower limit of quantification was 0.05 μg mL⁻¹. The intra and inter-day relative standard deviation (RSD) for measurement of 0.25, 2.0, and 6.0 μg mL⁻¹ quality-control (QC) samples ranged from 5.7 to 6.4% and from 6.3 to 7.9%, respectively. Accuracy, as relative error (RE), was from ±5.8% to ±7.3%. The method was validated for specificity, accuracy, and precision and was successfully used in a pharmacokinetic study of isofraxidin in rat plasma after administration of Ciwujia extract.     

HPLC Determination of DCJW in Rat Plasma and Its Application to Pharmacokinetics Studies

A high-performance liquid chromatography–UV method for determining DCJW concentration in rat plasma was developed. The method described was applied to a pharmacokinetics study of intramuscular injection in rats. The plasma samples were deproteinized with acetonitrile in a one-step extraction. The HPLC assay was carried out using a VP-ODS column and the mobile phase consisting of acetonitrile–water (80:20, v/v) was used at a flow rate of 1.0 mL min⁻¹ for the effective eluting DCJW. The detection of the analyte peak area was achieved by setting a UV detector at 314 nm with no interfering plasma peak. The method was fully validated with the following validation parameters: linearity range 0.06–10 μg mL⁻¹ (r > 0.999); absolute recoveries of DCJW were 97.44–103.46% from rat plasma; limit of quantification, 0.06 μg mL⁻¹ and limit of detection, 0.02 μg mL⁻¹. The method was further used to determine the concentration–time profiles of DCJW in the rat plasma following intramuscular injection of DCJW solution at a dose of 1.2 mg kg⁻¹. Maximum plasma concentration (C _max) and area under the plasma concentration–time curve (AUC) for DCJW were 140.20 ng mL⁻¹ and 2405.28 ng h mL⁻¹.     

用反相高效液相色谱法测定人体血浆中盐酸氟桂利嗪含量

采用反相高效液相色谱法以桂利嗪为内标测定人体血浆中盐酸氟桂利嗪的含量.以V甲醇:V乙腈:V缓冲液=58:20:22的体系为流动相,用hypersilBDSC8不锈钢色谱柱、紫外检测器(检测波长254nm),血浆样品经乙酸乙酯萃取处理后进样测定.血浆中盐酸氟桂利嗪的含量在5～300μg/L范围内与盐酸氟桂利嗪的峰面积和桂利嗪的峰面积之比呈线性关系(r=0.9997),方法对盐酸氟桂利嗪的平均回收率为83.3%～85.0%,检出限为3μg/L(S/N=3).用该法测定了12名单次口服30mg西比灵(盐酸氟桂利嗪)胶囊的健康志愿者血浆中盐酸氟桂利嗪含量.结果表明,该药在血浆中含量平均达峰时间为(2.67±0.91)h,平均峰质量浓度为(154.9±66.0)μg/L.该法适用于盐酸氟桂利嗪药代动力学的临床监测和研究.     

Determination of the Enantiomers of Tamsulosin Hydrochloride and its Synthetic Intermediates by Chiral Liquid Chromatography

A chiral liquid chromatographic method is described for the determination of the enantiomers of tamsulosin hydrochloride and its synthetic intermediates. Enantioseparation was achieved on a Chiralcel OD-R column (250 mm × 4.6 mm, 10 m) using a mobile phase consisting of a mixture of 0.5 mol L^–1 sodium perchlorate and acetonitrile (80:20, v/v, pH 4.0). The flow rate was 0.4 mL min^–1 and detection was at 223 nm. Excellent enantiomer separations were achieved for tamsulosin hydrochloride and its synthetic intermediates. No other methods are available for the separation of these enantiomers. The method developed in this study has been successfully applied for purity control.     

Simultaneous Determination of Pioglitazone and Glimepiride by High-Performance Liquid Chromatography

A rapid and accurate HPLC method has been developed for simultaneous determination of pioglitazone and glimepiride. Chromatographic separation of the two pharmaceuticals was performed on a Cosmosil C₁₈ column (150 mm × 4.6 mm, 5 m) with a 45:35:20 (v/v) mixture of 0.01 m triammonium citrate (pH adjusted to 6.95 with orthophosphoric acid), acetonitrile, and methanol as mobile phase, at a flow rate of 1.0 mL min^–1, and detection at 228 nm. Separation was complete in less than 10 min. The method was validated for linearity, accuracy, precision, limit of quantitation, and robustness [1, 2]. Linearity, accuracy, and precision were found to be acceptable over the ranges 2.50–30.00 g mL^–1 for pioglitazone and 0.10–10.00 g mL^–1 for glimepiride.     

Development and Validation of a Liquid Chromatographic Method for Determination of Efavirenz in Human Plasma

A simple, rapid, and sensitive high-performance liquid chromatographic method for estimation of efavirenz in human plasma has been developed and validated. Chromatography was performed with C₁₈ analytical column and 50:50 acetonitrile–phosphate buffer (pH 3.5) as mobile phase. Compounds were monitored by UV detection at 247 nm. The retention time for efavirenz was 6.45 min and that for the internal standard, nelfinavir, was 2.042 min. Response was a linear over the concentration range of 0.1 μg–10 μg mL⁻¹ in human plasma. The method was simple, specific, precise and accurate and was useful for bioequivalence and pharmacokinetic studies of efavirenz.     

Simple High-Performance Liquid Chromatographic Assay, with Post-Column Derivatization, for Simultaneous Determination of Mycophenolic Acid and its Glucuronide Metabolite in Human Plasma and Urine

Two simple high-performance liquid chromatographic (HPLC) methods have been established for simultaneous determination of mycophenolic acid (MPA) and its glucuronide metabolite (MPAG) in human urine, and of their total and unbound forms in human plasma. For total MPA and MPAG analysis sample preparation entailed precipitation of protein with acetonitrile and isolation of the free analytes from the plasma by ultrafiltration. For urine samples, fivefold dilution with water was used. MPAG was determined by UV detection whereas MPA was quantified by fluorescence detection after post-column derivatization with 0.2 M sodium hydroxide solution. For plasma, response was found to be linearly dependent on concentration over the ranges 0.1–40 μg mL^-1 and 0.01–1 μg mL^-1 for total and free MPA, respectively, and 10–200 μg mL^-1 and 2.5–100 μg mL^-1 for total and free MPAG, respectively. For urine, linearity was observed from 0.1 to 50 μg mL^-1 for MPA and 10 to 500 μg mL^-1 MPAG in the urine before dilution. The methods reported were found to be accurate and reproducible for quantifying the level of MPA and MPAG and can thus be used for clinical pharmacokinetic studies and for therapeutic drug monitoring. Contributed equally to this work An erratum to this article is available at .     

High-Throughput Analysis of Sofalcone in Human Plasma by Use of Automated 96-Well Protein Precipitation and LC-MS-MS

A sensitive and selective method for the determination of sofalcone in human plasma was established by use of protein precipitation and liquid chromatography-tandem mass spectrometry. Plasma samples were transferred into 96-well plate using an automated sample handling system and spiked with 10 L of 2 g mL^–1 internal standard solution (d₃-sofalcone). 0.5 mL of acetonitrile was added to the 96-well plate and the plasma samples were then vortexed for 30 sec. After centrifugation, the supernatant was transferred into another 96-well plate and completely evaporated at 40 °C under a stream of nitrogen. The dry residue was reconstituted with mobile phase. All sample transfer and protein precipitation was automated through the application of both the PerkinElmer MultiPROBE II HT and TOMTEC Quadra 96 workstation. The limit of quantitation of sofalcone was 2 ng mL^–1 using a sample volume of 0.2 mL for the analysis. The reproducibility of the method was evaluated by analyzing five samples at nine quality control (QC) levels over the nominal concentration range from 2 ng mL^–1 to 1000 ng mL^–1. Validation of the method showed that the assay has good precision and accuracy. Sofalcone and internal standard produced a protonated precursor ion ([M+H]⁺) at m/z 451 and 454, and both gave a corresponding product ion at m/z 315. The high sample throughput of the method has been successfully applied to a pharmacokinetic study of sofalcone in human plasma.