Validated HPLC method for determination of carboxylic acid metabolite of clopidogrel in human plasma and its application to a pharmacokinetic study

A new, simple, and reproducible method for determination of carboxylic acid metabolite of clopidogrel in human plasma has been developed. After liquid-liquid extraction in acidic medium with chloroform, samples were quantified on a Nova-pak C(8), 5 microm column using a mixture of 30 mM K(2)HPO(4)-THF-acetonitrile (pH = 3, 79:2:19, v/v/v) as mobile phase with UV detection at 220 nm. The flow rate was set at 0.9 mL/min. Ticlopidine was used as internal standard and the total run time of analysis was about 12 min. The method was linear over the range of 0.2-10 microg/mL of clopidogrel metabolite in plasma (r(2) > 0.999). The within-day and between-day precision values were in the range 1.0-4.8%. The limit of quantification of the method was 0.2 microg/mL. The method was successfully used to study the pharmacokinetics of clopidogrel in healthy volunteers.     

Liquid chromatography-mass spectrometry method for the determination of venlafaxine in human plasma and application to a pharmacokinetic study

A sensitive and selective liquid chromatographic-mass spectrometric (LC-MS) method for the determination of venlafaxine in human plasma has been developed. Samples were prepared using liquid-liquid extraction and analyzed on a C(18) column interfaced with a triple quadrupole mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase was methanol-water containing 10 mmol/L ammonium acetate, pH 7.9 adjusted with aqueous ammonia (80:20, v/v) at the flow rate of 1.0 mL/min. The analyte and internal standard clozapine were both detected by use of selected ion monitoring mode. The method was linear in the concentration range of 1.0-200.0 ng/mL. The lower limit of quantification (LLOQ) was 1.0 ng/mL. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 10.1%. The accuracy determined at three concentrations (5.0, 50.0 and 150.0 ng/mL for venlafaxine) was within +/-10.0% in terms of relative error (RE). The method was successfully applied for the evaluation of pharmacokinetic profiles of venlafaxine capsule in 20 healthy volunteers. The results show AUC, T(max), C(max) and T(1/2) between the testing formulation and reference formulation have no significant difference (p > 0.05). Relative bioavailability was 103.4 +/- 14.1%.     

An HPLC method for the determination of bisoprolol in human plasma and its application to a pharmacokinetic study

A new high-performance liquid chromatographic method is described for the determination of bisoprolol in human plasma. The proposed method was based on the derivatization of bisoprolol with 4-chloro-7-nitro-2,1,3-benzoxadiazole in borate buffer at pH 9.5 to yield a fluorescent product. Chromatographic separation of bisoprolol was achieved by using isocratic elution at a flow rate of 1.2 mL/min on a C18 reversed-phase column (Inertsil, 4 μm, 150 4.6 mm) at 40°C. The mobile phase used for the analysis was methanol-water (70:30, % v/v). Fluorescence detector was used at the excitation and emission wavelengths of 458 and 525 nm, respectively. The method was validated for linearity, limit of detection, limit of quantification, precision, accuracy, recovery and system suitability. The assay was linear over the concentration range of 10-2000 ng/mL. This method was applied in pharmacokinetic studies of bisoprolol preparations in healthy volunteers.     

Validated LC-MS-MS method for the determination of quetiapine in human plasma: application to a pharmacokinetic study

A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the determination of quetiapine was developed and validated over the linearity range 1-1500 ng/mL with 0.1 mL of plasma using clozapine as the internal standard. Detection was performed on a triple-quadrupole tandem mass spectrometer using positive electrospray ionization and quantification was performed by selected reaction monitoring mode. The MS-MS ion transitions monitored were m/z 384.1 → 253.1 and 327.0 → 270.0 for quetiapine and clozapine, respectively. The between- and within-run precision was less than 7.44% and accuracy was less than 10.2%. The lower limit of quantification was 1 ng/mL. The extraction recoveries of quetiapine were over 90%. The method is proved to be accurate and specific, and was applied to the pharmacokinetic study in healthy Chinese volunteers.     

HPLC method for determination of diclofenac in human plasma and its application to a pharmacokinetic study in Turkey

A simple high-performance liquid chromatography (HPLC) method has been developed for determination of diclofenac in human plasma. The method was validated on Ace C(18) column using UV detection. The mobile phase consisted of 20 mM phosphate buffer (pH 7) containing 0.1% trifluoroacetic acid-acetonitrile (65:35, v/v). Calibration curve was linear between the concentration range of 75-4000 ng/mL. Intra- and inter-day precision values for diclofenac in plasma were less than 3.6, and accuracy (relative error) was better than 5.3%. The limits of detection and quantification of diclofenac were 25 and 75 ng/mL, respectively. Also, this assay was applied to determine the pharmacokinetic parameters of diclofenac in healthy Turkish volunteers who had been given 50 mg diclofenac.     

High-performance liquid chromatographic method for determination of leucovorin in plasma: validation and application to a pharmacokinetic study in healthy volunteers

A sensitive and reliable high-performance liquid chromatographic (HPLC) method, using a solid-phase extraction (SPE), was developed and validated for determination of leucovorin (LV) in human plasma. Plasma sample was extracted by using a Sep-Pak cartridge which could be renewable. The sample was analyzed by HPLC with UV detection at 286 nm. The method was shown to perform selectively and sensitively for LV. The main metabolite of LV, 5-methyltetrahydrofolic acid, and endogenous substances in plasma did not show any interference in the analysis. The limit of detection was 10 ng/mL for LV in plasma and the linear range was 50-1500 ng/mL in plasma. The relative standard deviation (RSD) of intra-day and inter-day assays was 2.8-6.1% and 2.4-5.3%, respectively. The extraction recoveries of LV in plasma were over 90%. The method was proved to be applicable to the pharmacokinetic study of LV in healthy volunteers after a single oral administration (75 mg). The pharmacokinetic parameters and relative bioavailability were investigated for domestic LV tablet and capsule vs an imported tablet.     

Development and validation of a HPLC-MS/MS method for the determination of venlafaxine enantiomers and application to a pharmacokinetic study in healthy Chinese volunteers

An HPLC‐MS/MS method has been developed and validated for the determination of venlafaxine enantiomers in human plasma and applied to a pharmacokinetic study in healthy Chinese volunteers. The method was carried out on a vancomycin chiral column (5 µm, 250 × 4.6 mm) maintained at 25°C. The mobile phase was methanol–water containing 30 mmol/L ammonium acetate, pH 3.3 adjusted with aqueous ammonia (8:92, v/v) at the flow rate 1.0 mL/min. A tandem mass spectrometer with an electrospray interface was operated in the multiple reaction monitoring mode to detect the selected ions pair at m/z 278.0 → 120.8 for venlafaxine enantiomers and m/z 294.8 → 266.7 for estazolanm (internal standard). The method was linear in the concentration range of 0.28–423.0 ng/mL. The lower limit of quantification was 0.28 ng/mL. The intra‐and inter‐day relative standard deviations were less than 9.7%. The method was successfully applied for the evaluation of pharmacokinetic profiles of venlafaxine enantiomers in 18 healthy volnteers. Validation parameters such as the specificity, linearity, precision, accuracy and stability were evaluated, giving results within the acceptable range. Pharmacokinetic parameters of the venlafaxine enantiomers were measured in the 18 healthy Chinese volunteers who received a single regimen with venlafaxine hydrochloride capsules. The results show that AUC_(0–∞), C_max and t_1/2 between S‐venlafaxine and R‐venlafaxine are significantly different (p < 0.05). Copyright © 2010 John Wiley & Sons, Ltd.     

A high performance liquid chromatography-tandem mass spectrometric method for the determination of mefenamic acid in human plasma: application to pharmacokinetic study

In the present study, the development and validation of an LC‐MS/MS method for quantifying mefenamic acid in human plasma is described. The method involves liquid–liquid extraction using diclofenac as an internal standard (IS). Chromatographic separation was achieved on a Thermo Hypurity C₁₈, 50 × 4.6 mm, 5 µm column with a mobile phase consisting of 2 m m ammonium acetate buffer and methanol (pH 4.5 adjusted with glacial acetic acid; 15:85, v/v) at a flow‐rate of 0.75 mL/min and the total run time was 1.75 min. Analyte was introduced to the LC‐MS/MS using an atmospheric pressure ionization source. Both the drug and IS were detected in negative‐ion mode using multiple reaction monitoring m/z 240.0 → 196.3 and m/z 294.0 → 250.2, respectively, with a dwell time of 200 ms for each of the transitions. The standard curve was linear from 20 to 6000 ng/mL. This assay allows quantification of mefenamic acid at a concentration as low as 20 ng/mL in human plasma. The observed mean recovery was 73% for the drug. The applicability of this method for pharmacokinetic studies has been established after successful application during a 12‐subject bioavailibity study. Copyright © 2012 John Wiley & Sons, Ltd.     

A sensitive and specific liquid chromatography/tandem mass spectrometry method for determination of pinaverium bromide in human plasma: application to a pharmacokinetic study in healthy volunteers

A sensitive and specific method using liquid chromatography–electrospray tandem mass spectrometry (LC‐MS/MS) for the determination of pinaverium bromide in human plasma was developed and validated. Pinaverium bromide and an internal standard (paclitaxel) were isolated from plasma samples by precipitating plasma, and determined by LC‐MS/MS in multiple‐reaction monitoring mode. The main metabolite of pinaverium bromide and endogenous substances in plasma did not show any interference. The calibration curve was linear over the plasma concentration range of 10.0–10000.0 pg/mL with a correlation coefficient of 0.9979. The relative standard derivations intra‐ and inter‐day at 30.0, 300.0 and 8000.0 pg/mL in plasma were less than 15%. The absolute recoveries of pinaverium bromide and the internal standard were 99.7–111.7 and 106.2%, respectively. The lower limit of quantitation was 10 pg/mL. The analytical method was successfully applied to study the pharmacokinetics of pinaverium bromide tablets in healthy Chinese volunteers. Copyright © 2011 John Wiley & Sons, Ltd.     

A simplified and reliable LC–tandem mass spectrometry method for determination of ulipristal acetate in human plasma and its application to a pharmacokinetic study in healthy Chinese volunteers

In this study, a simplified, sensitive and reliable LC–tandem mass spectrometry method was established and validated for the quantification of ulipristal acetate (UPA) in human plasma and for the investigation of pharmacokinetic profile of UPA following a single oral administration of ella (UPA 30-mg tablet) in healthy Chinese volunteers. Plasma samples were analyzed after being processed by protein precipitation with methanol. Chromatographic separation was performed on a Kinetex EVO C₁₈ column (2.1 × 50 mm, 2.6 μm) using gradient elution with a mobile phase composed of methanol and water containing 2 mm ammonium acetate and 0.3% formic acid at a flow rate of 0.3 mL/min. The chromatographic running time was 4.0 min per sample. The MS detection was performed via an LC system with the positive ion electrospray ionization interface in multiple reaction monitoring mode using the transition of m/z 476.2 → 134.1 for UPA and m/z 479.3 → 416.2 for UPA-d3 [internal standard (IS)], respectively. UPA and IS were monitored without severe interference from the biological matrices. The method was linear over the wide concentration range of 0.300–300 ng/mL. The intra- and inter-day precision and accuracy were well within the limits required for bioanalytical assays. The method was first used to describe the pharmacokinetic characteristic of UPA after a single oral administration of ella in healthy Chinese volunteers. Based on a between-study comparison, there were statistically significant differences (p < .05) between Chinese and Caucasian volunteers for the systemic exposure of UPA, suggesting that race seems to significantly impact the systemic exposure of UPA.     

Absorption, concentration and determination of trace amounts of air pollutants by flow injection method coupled with a chromatomembrane cell system: application to nitrogen dioxide determination

The concentration distribution of an analyte in a chromatomembrane cell (CMC) was examined by using various air samples of different air pollutant (NO₂) concentrations and volumes, and the results obtained could be explained by a proposed principle of the concentration distribution of the analyte in the CMC. This principle was for the first time proved experimentally in the present study. On-line preconcentration and continuous determination of the air pollutant (NO₂) in air samples were realized by coupling a three-hole CMC with a flow injection analysis (FIA) system, where a triethanolamine (TEA) aqueous solution (2 g l⁻¹) was used as an absorbing solution for NO₂ in the air samples. A calibration method with standard nitrite aqueous solutions was developed for the determination of NO₂ in the air samples. Concentrations of NO₂ in indoor air and its diluted air samples were determined by the proposed CMC/FIA method. The volume of air sample necessary for the measurement was decreased to only 5 ml. The measuring time for one sample was about 5–6 min even when a 20 ml air sample was used.     

Liquid chromatography-tandem mass spectroscopic method for the determination of zerumbone in human plasma and its application to pharmacokinetics

A rapid, sensitive, specific and selective LC-MS/MS method for the determination of zerumbone (ZER) in human plasma using 2,4-diamino-6-(4-methoxyphenyl)-1,3,5-triazine (DMTZ) as an internal standard (IS) has been developed and validated. ZER was chromatographed on C8 column using a mobile phase of acetonitrile/water (80:20, v/v) at a flow rate of 0.25 ml min(-1) . Quantitation was achieved using ESI+ interface, employing multiple reaction monitoring (MRM) mode at m/z 219 > 81 and 218 > 134 for ZER and IS, respectively. The calibration standards were linear over a range of 5-3000 ng ml(-1) (r(2)=0.9994) with an LLOQ of 5 ng ml(-1) (RSD %; 11.4% and bias%; 9.5%). Intra- and inter-day precision of ZER assay ranged from 0.18 to 3.56% with accuracy (bias) that varied between -5.09 and 4.3%, demonstrating good precision and accuracy. Recoveries of ZER and the IS from human plasma were above 85%. The developed method was validated for the determination of ZER in rat plasma. Linearity, stability of ZER and the ME on rat plasma were discussed. The applicability of the developed method was demonstrated by measuring ZER in rat plasma samples following intravenous and intraperitoneal administration of ZER prepared in hydroxypropyl-β-cyclodextrin (HPβCD) and sodium carboxymethyl cellulose (CMC), respectively, in 20 mg kg(-1) and this study indicated a clear significant difference (p<0.05) in pharmacokinetic parameters of ZER in ZER/HPβCD complex compared with ZER in CMC preparation.     

Validation of a HPLC‐ESI MS/MS method for the determination of clonidine in human plasma and its application in a bioequivalence study in Chinese healthy volunteers

A rapid and sensitive liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) method to determine clonidine in human plasma was developed and fully validated. Sample preparation was involved an one‐step extraction with diethyl ether. Donepezil was employed as the internal standard (IS). Chromatographic separation was performed on a Hypersil BDS C₁₈ column (i.d. 2.1 × 50 mm, particle size 3μm) with a mobile phase of methanol–water (containing 0.1% formic acid; 60:40, v/v) at a flow rate of 200 μL/min. The peaks were detected by mass spectrometry using the electrospray ion source in selected reaction monitoring mode. The extraction recovery was 72.53–85.25%. The method was found to be linear in a concentration range of 0.02–6.00 ng/mL and the lower limit of quantification was 0.02 ng/mL. The within‐ and between‐batch precisions at three concentrations were 4.33–16.47 and 7.24–17.24% with accuracies of ?2.47–10.91 and 1.86–10.19%, respectively. This validated method was successfully used for a bioequivalence study of two clonidine transdermal patches on healthy volunteers. The results suggested that the test formulation of clonidine patch met the regulatory criterion for bioequivalence to the reference formulation, but a larger sample size should be needed for the estimation of bioequivalence. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of a liquid chromatographic method for the determination of hydroxymethylfurfural and alpha-ketoglutaric acid in human plasma

Hydroxymethylfurfural (HMF) and alpha-ketoglutaric acid (KG) have been recently investigated as potential cancer cell damaging agents. We herein report for the first time a validated quantitative assay for their simultaneous determination in human plasma which is amenable to be applied in the future screening of the target compounds in human probands in order to properly design a targeted chemotherapeutic regimen for certain types of malignant tumors.A simple liquid chromatographic method in conjunction to derivatization after a two-step optimized solid phase clean-up procedure is described. The method is based on the reaction of HMF and KG with 2-nitrophenylhydrazine or 2,4-dinitrophenylhydrazine in an aqueous environment. Reaction conditions were studied with respect to pH, reagent volume, reaction temperature and time. Exact testing of such parameters beside careful selection of the mobile phase composition rendered feasible the quantification of the chemically significantly differing analytes along a single chromatographic run. The formed derivatives could be separated isocratically by reversed-phase LC on a C₈-column. Detection in the UV and in the visible range is possible. Results showed good recovery and reproducibility with detection limits (S/N = 3) down to 2 picomoles analyte on column. Resolution of the syn and anti geometric isomers of the HMF and KG derivatives is possible. The isomeric ratio in relation to the reaction pH is discussed.     

Validation of an LC/MS method for the determination of gemfibrozil in human plasma and its application to a pharmacokinetic study

Gemfibrozil, a fibric acid hypolipidemic agent, is increasingly being used in clinical drug–drug interaction studies as an inhibitor of drug metabolizing enzymes and drug transporters. The validation of a fast, accurate and precise LC/MS method is described for the quantitative determination of gemfibrozil in an EDTA‐anticoagulated human plasma matrix. Briefly, gemfibrozil was extracted from human plasma by an acetonitrile protein precipitation method. The assay was reproducible with intra‐assay precision between 1.6 and 10.7%, and inter‐assay precision ranging from 4.4 to 7.8%. The assay also showed good accuracy, with intra‐assay concentrations within 85.6–108.7% of the expected value, and inter‐assay concentrations within 89.4–104.0% of the expected value. The linear concentration range was between 0.5 and 50 µg/mL with a lower limit of quantitation of 0.5 µg/mL when 125 µL of plasma were extracted. This LC/MS method yielded a quick, simple and reliable protocol for determining gemfibrozil concentrations in plasma and is applicable to clinical pharmacokinetic studies. Copyright © 2010 John Wiley & Sons, Ltd.     

LC‐MS/MS method for the determination of agomelatine in human plasma and its application to a pharmacokinetic study

A sensitive and selective LC‐MS/MS method for the determination of agomelatine in human plasma was developed and validated. After simple liquid–liquid extraction, the analytes were separated on a Zorbax SB‐C₁₈ column (150 × 2.1 mm i.d., 5 µm) with an isocratic mobile phase consisting of 5 mm ammonium acetate solution (containing 0.1% formic acid) and methanol (30:70, v/v) at a flow‐rate of 0.3 mL/min. The MS acquisition was performed in multiple reaction monitoring mode with a positive electrospray ionization source. The mass transitions monitored were m/z 244.1 → 185.3 and m/z 285.2 → 193.2 for agomelatine and internal standard, respectively. The methods were validated for selectivity, carry‐over, matrix effects, calibration curves, accuracy and precision, extraction recoveries, dilution integrity and stability. The validated method was successfully applied to a pharmacokinetic study of agomelatine in Chinese volunteers following a single oral dose of 25 mg agomelatine tablet. Copyright © 2013 John Wiley & Sons, Ltd.     

Simple and sensitive HPLC method for determination of amrubicin and amrubicinol in human plasma: application to a clinical pharmacokinetic study

A simple and sensitive high‐performance liquid chromatographic (HPLC) method was developed for determination of amrubicin and its metabolite amrubicinol in human plasma. After protein precipitation with methanol without evaporation procedure, large volume samples were injected and separated by two monolithic columns with a guard column. The mobile phase consisted of tetrahydrofuran–dioxane–water (containing 2.3 mM acetic acid and 4 mM sodium 1‐octanesulfonate; 2:6:15, v/v/v). Wavelengths of fluorescence detection were set at 480 nm for excitation and 550 nm for detection. Under these conditions, linearity was confirmed in the 2.5–5000 ng/mL concentration range of both compounds. The intra‐ and inter‐day precision and intra‐ and inter‐day accuracy for both compounds were less than 10%. The method was successfully applied to a clinical pharmacokinetic study of amrubicin and amrubicinol in cancer patients. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of a CE method for the determination of mycophenolic acid in human plasma: a comparison with HPLC

Mycophenolate mofetil (MMF) is a widely used drug for the maintenance of immunosuppressive therapy in renal-transplant recipients. MMF is rapidly metabolized in vivo to mycophenolic acid (MPA), a reversible, noncompetitive inhibitor of inosine monophosphate dehydrogenase, which represents a limiting enzyme in lymphocyte proliferation. MPA shows large interindividual pharmacokinetic variability: its monitoring is therefore of primary importance to achieve adequate immunosuppression with minimized risk of graft rejection or toxicity. We developed a CE method for the determination of total MPA (tMPA) in plasma, based on easy sample preparation; CE evaluation of tMPA was performed in 30 mmol/L sodium-borate with 10 mmol/L SDS (pH 10.00) at 25 degrees C using a 60 cm (54.5 to window) uncoated capillary with UV detection at 254 nm wavelength. MPA was readily detectable in plasma; the CE method was linear in the range of 0.7-120 microg/mL (r >0.992). Intra- and interassay imprecision was <7% except for the lowest spiked MPA concentration, which had an intra-assay RSD% of 14.7 compared to 18.3 interassay. Data by CE were compared with results obtained by a validated HPLC method. CE assay of tMPA exhibited a very good correlation (r(2) >0.988) with respect to HPLC; Bland-Altman difference versus average showed a mean of -0.18 microg/mL +/- 1.14 SD. CE determination of tMPA is a robust, sensitive and reproducible method with the advantage over HPLC of being fast, simple and unexpensive, also enabling quick assessment of MPA for pharmacokinetic studies.     

An improved LC-MS/MS method for quantitative determination of metolazone in human plasma and its application to a pharmacokinetic study

A simple, rapid and accurate liquid chromatography-tandem mass spectrometry method has been developed. After a liquid-liquid extraction procedure, samples were chromatographed on an Agilent TC-C(18) (150 × 4.6 mm, 5 μm) column using an isocratic elution mobile phase composed of methanol and distilled water (70:30, v/v) at a flow rate of 0.5 mL/min. After single-dose administration of 0.5, 1 and 2 mg metolazone, the t(1/2) values were 6.6 ± 2.8, 7.9 ± 1.2 and 7.6 ± 1.9 h, respectively. The pharmacokinetic parameters of multiple doses (1 mg metolazone) were as follows: t(1/2) was 8.9 ± 1.0 h; C(max) was 22.4 ± 5.0 ng/mL; and AUC(0-48) was 156.8 ± 31.6 ng h/mL.     

Validation and pharmacokinetic application of a method for determination of doxazosin in human plasma by high-performance liquid chromatography

A simple high-performance liquid chromatographic method for the determination of doxazosin in human plasma was developed and validated. Prazosin was used as internal standard. After extraction twice with ethyl acetate, chromatographic separation of doxazosin in human plasma was carried out using a reversed-phase Apollo C18 column (250 x 4.6 mm, 5 microm) with mobile phase of methanol-acetonitrile-0.04 m disodium hydrogen orthophosphate (22:22:56, v/v/v) adjusted to pH 4.9 with 0.9 m phosphoric acid and quantified by fluorescence detection operated with an excitation wavelength of 246 nm and an emission wavelength of 389 nm. The lower limit of quantification (LLOQ) of this assay was 1 ng/mL using 500 microL human plasma. Linearity was established over the range 1-25 ng/mL (r2 > 0.9994). The intra- and inter-day accuracy ranged from 90.5 to 104.4% and the coefficient of variation were not more than 8.6% for both intra- and inter-day precision, over the range of the calibration curve. The absolute recoveries of doxazosin and prazosin from human plasma were more than 91%. Doxazosin demonstrated acceptable short-term, long-term and freeze-thaw stability in human plasma. The assay has been successfully applied to plasma sample ana-lysis for pharmacokinetic study.