A conventional LC-MS method developed for the determination of plasma raltegravir concentrations

Raltegravir belongs to a new class of antiretrovirals acting for a human immunodeficiency virus (HIV)-1 integrase inhibition. Clinical trials of this drug have demonstrated potent antiviral activity in both therapy na?ve and experienced patients. Thus, raltegravir has become an important component of combination treatment regimens used to treat patients with multidrug-resistant HIV-1. The quantification of raltegravir in human plasma is important to support clinical studies and determine pharmacokinetic parameters of raltegravir in HIV-1 infected patients. Recently, the LC-MS/MS superfine system was developed to determine plasma concentration of raltegravir; however, the system needs to be delicately set and the equipment is very expensive. Therefore, we developed a conventional LC-MS method to overcome these difficulties. Subsequently the method was validated by estimating the precision and accuracy for inter- and intraday analysis in the concentration range of 0.010-7.680 microg/ml. The calibration curve was linear in this range. Average accuracy ranged from 97.2 to 103.4%. Relative standard deviations of both inter- and intraday assays were less than 10.4%. Recovery of raltegravir was more than 80.6%. This novel LC-MS method is accurate and precise enough to determine raltegravir levels in human plasma samples.     

The validation of plasma darunavir concentrations determined by the HPLC method for protease inhibitors

Darunavir (DRV) is a new protease inhibitor used to treat human immunodeficiency virus (HIV) type-1. The aim of this study was to validate the determination of plasma DRV concentrations using the HPLC method, a simple procedure for simultaneous determination of seven HIV protease inhibitors and efavirenz. The calibration curve was linear (range of 0.13 to 10.36 microg/ml). The average accuracy ranged from 100.7 to 105.6%. Both the interday and intraday coefficients of variation were less than 6.7%, which was similar to or much lower than previously reported values by the LC/MS/MS method. It is concluded that HPLC can be used to determine plasma DRV concentrations and routinely in the clinical setting; thus, this HPLC method enables further study of DRV pharmacokinetics in conventional hospital laboratories.     

UPLC-MS法测定艾滋合并结核患者血浆中替诺福韦浓度

目的:建立血浆中替诺福韦(tenofovir,TFV)含量测定的方法,测定艾滋合并结核患者给药后血浆中替诺福韦的浓度.方法:血浆采用甲醇沉淀蛋白,色谱柱为Kinetex Phenyl-Hexyl(50 mm×3.0 mm,2.6μm),流动相为5 mmol·L-1甲酸铵溶液(A)-甲醇溶液(B),每个样品分析时间为3....     

LC-MS/MS法测定人血浆中丙酚替诺福韦和替诺福韦的质量浓度

目的 建立LC-MS/MS法测定人血浆中丙酚替诺福韦和其代谢物替诺福韦的质量浓度并比较富马酸丙酚替诺福韦片与原研制剂韦立得的人体生物等效性.方法 采用简单的蛋白沉淀法处理样品,丙酚替诺福韦内标为丙酚替诺福韦-2H5,色谱柱为月旭Ultimate XB C18柱(50 mm×2.1mm,5μm),替诺福韦内标为替诺福韦-...     

RP-HPLC法测定人血浆中盐酸表柔比星的浓度

目的:建立以反相高效液相色谱法测定人血浆中盐酸表柔比星浓度的方法。方法:色谱柱为YWGC18,流动相为水-乙腈(12∶7,用85%磷酸调节体系pH为3.90±0.05),流速为1.0mL.min-1,检测波长为254nm。结果:人血浆中表柔比星检测浓度在0.12～4.80mg.L-1范围内线性关系良好(r=0.9984);最低血浆检测浓度为0.01mg.L-1;日内RSD<4.40%、日间RSD<5.60%,平均提取回收率为99.40%(RSD=4.02%);样品溶液在室温下12h内稳定。结论:本方法简便、灵敏、准确和重现性好。     

反相高效液相色谱测定人体甲氧苄胺嘧啶血药浓度分析

目的探讨反相高效液相色谱测定人体甲氧苄胺嘧啶血药浓度的方法与效果。方法选择Agilent1100反相高效液相色谱仪测定人体甲氧苄胺嘧啶的血药浓度。结果甲氧苄胺嘧啶的线性方程为Y=6.25×10-4X＋1.08×10-2,线性范围为0.05～10.00μg/ml。甲氧苄胺嘧啶日内和日间精密度的相对标准偏差分别为4.4%和4.5%,提取回收率为92.1%,平均血药浓度为0.260μg/ml。结论采用反相高效液相色谱测定人体甲氧苄胺嘧啶血药浓度,方法简单、可行、灵敏度高,具有良好的回收率、准确度和精密度。     

Validation of an automated liquid chromatographic method for omeprazole in human plasma using on-line solid-phase extraction

An automated system using on-line solid-phase extraction and HPLC with UV detection has been validated in order to determine omeprazole in human plasma. The extraction was carried out using C18 cartridges. After washing, omeprazole was eluted from the cartridge with mobile phase onto an Inertsil ODS-2 column. The developed method was selective and linear for drug concentrations ranging between 5 and 500 ng ml(-1). The recovery of omeprazole ranged from 88.1 to 101.5%, and the limit of quantitation (LOQ) was 5 ng ml(-1). The intraday accuracy ranged from 93.1 to 106.2% and the interday accuracy varied from 95.4 to 105.1%. For the LOQ, good values of precision (8.7 and 17.5% for intraday and interday, respectively) were also obtained. This automated system has been applied to determine omeprazole in human plasma samples from bioequivalence studies.     

RP-HPLC法测定人血浆中氟比洛芬的浓度

目的:建立以反相高效液相色谱法测定人血浆中氟比洛芬浓度的方法。方法:采用外标法,以乙腈沉淀法处理样品后进样测定,色谱柱为Symmetry shield C_(18),流动相为磷酸盐缓冲液(pH7.0):乙腈=75:25,流速为1.0 mL·min~(-1),紫外检测波长为247 nm。结果:氟比洛芬血药浓度在0.05～20μg·mL~(-1)范围内线性关系良好(r=0.999 1),检测限为0.05μg·mL~(-1);低、中、高浓度的回收率在96.1%～107.1%之间,日内及日间RSD均<10%,符合方法学要求。结论:本方法简便、准确、快速,适用于人血浆中氟比洛芬浓度监测及药动学研究。     

高效液相色谱法测定三羟乙基芦丁的血药浓度

目的：建立三羟乙基芦丁的血药浓度测定方法。方法：将三羟乙基芦丁转变成四羟乙基芦丁,经高效液相色谱分离后,用荧光检测器测定。结果：血药浓度标准曲线在１２５～４０ｎｇ·ｍＬ－１间,ｒ＝０９９９４;最低检测限为１ｎｇ·ｍＬ－１;高、中、低３种浓度平均回收率为９１５２％～９５８３％。日内和日间平均ＲＳＤ小于４３％和５２％。结论：本法可为羟乙基芦丁的药动学研究提供一检测方法。     

Potential marker for smoked methamphetamine hydrochloride based on a gas chromatography-mass spectrometry quantification method for trans-phenylpropene

Residue from smoked methamphetamine hydrochloride contains pyrolytic products that are detectable by gas chromatography-mass spectrometry (GC-MS). A validated GC-MS method was developed for the determination of trans-phenylpropene, a pyrolytic product of methamphetamine HCl, in residue of smoked drug as well as in human urine. trans-Phenylpropene and an isomeric internal standard, 2-phenylpropene, were extracted from urine using n-hexane. The method was validated for linearity over a range of 0.1-10 microg/mL with a limit of detection of 0.05 microg/mL, limit of quantification of 0.1 microg/mL, interday accuracy within 10.5%, intraday accuracy better than 3.7%, interday precision of 15.4%, intraday precision of 14.4%, and recovery of 89.1%. The method was applied to the detection of trans-phenylpropene found in the residue of methamphetamine HCl heated beyond its melting temperature on aluminum foil under simulated smoking conditions. The method is applicable to the detection of trans-phenylpropene in urine as a potential marker for smoked methamphetamine HCl abuse.     

超高效液相色谱-串联质谱法测定大鼠血浆中替诺福韦的浓度

目的:建立大鼠血浆中替诺福韦浓度的测定方法。方法:采用超高效液相色谱-串联质谱法。取6只大鼠单次灌胃给予富马酸替诺福韦酯100mg.kg-1,检测给药45min后血浆中药物浓度,以恩替卡韦为内标,选用阳离子固相萃取柱除去血浆中的蛋白质。色谱柱为AcquitybenUPLC-C18,流动相为0.2%甲酸水溶液(含40mmo.lL-1醋酸铵)-乙腈=97:3,流速为0.25mL.min-1,柱温为40℃,电喷雾正离子(ESI+)源,监测离子对分别为质荷比(m/z)287.9→175.7(替诺福韦)和m/z277.9→151.7(恩替卡韦)。结果:替诺福韦检测浓度的线性范围为10～800ng.mL-1(r=0.9992),最低检测限为10ng.mL-1,回收率为(100.22±9.47)%～(102.76±4.99)%,日间、日内RSD均小于9.4%;给药45min后血浆样品中替诺福韦的平均浓度为819.2ng.mL-1。结论:该方法操作简便、专属性强、灵敏度高,可用于血浆中替诺福韦的浓度测定。     

Simultaneous determination of dextrorphan and guaifenesin in human plasma by liquid chromatography with fluorescence detection

A sensitive liquid chromatographic (LC) method was developed and validated for the simultaneous determination of dextrorphan and guaifenesin in human plasma using fluorescence detection. Dextrorphan and guaifenesin were extracted from plasma by a liquid-liquid extraction procedure using chloroform containing laudanosine as the internal standard. A cyano column (15 cm × 46 mm i.d., Spherisorb 5-CN) and a mobile phase containing acetonitrile-triethylamine-distilled water (10:1:89, v/v/v) (pH 6) were used. The concentration-response relationship for dextrorphan was found to be linear over a concentration range of 23–515 ng ml⁻¹ with a lower limit of detection of 20 ng ml⁻¹; the accuracy of the method would fall (95% confidence limit) within 9.53% and 11.07% of the true value for the inter-and intra-day, respectively; the inter- and intra-day precision, as measured by RSD, ranged from 1.88% to 30.07% (mean 2.28%) and from 4.69% to 7.51% (mean 5.67%) over the dynamic concentration range of the method (33–326 ng ml⁻¹). The concentration-response relationship for guaifenesin was found to be linear over a concentration range of 181–8136 ng ml⁻¹ with a lower detection limit of 30 ng ml⁻¹; the accuracy of the method would fall (95% confidence limit) within 9.78% and 8.04% of the true value for the inter- and intra-day, respectively; the inter- and intra-day precision, as measured by the RSD, ranged from 2.55 to 6.07% (mean 3.90%) and from 3.12 to 3.90% (mean 3.52%) over the dynamic concentration range of the method (435–6430 ng ml⁻¹). The relative percentage recovery of dextrorphan, guaifenesin, and laudanosine was found to be 96%, 94%, and 88%, respectively. Benchtop and storage stability of the plasma samples were found to be adequate. In addition, the frozen plasma samples were submitted to three filter freeze/thaw cycles without significant change in the stability of guaifenesin and dextrorphan.     

Development of HPLC method for the determination of levosulpiride in human plasma

A rapid and simple high performance liquid chromatography (HPLC) method was developed and validated for determination of levosulpiride in human plasma. After extraction with ethylacetate/methylene chloride (5:1, v/v), analysis of levosulpiride in plasma samples was carried out using a reverse phase C18 column with fluorescence detector (maximum excitation at 300 nm and maximum emission at 365 nm) for separation and quantification. A mixture of methanol-20 mM phosphate buffer (pH 3.5, 16:84, v/v) was used as a mobile phase. The method was specific and sensitive with a limit of quantification of 5 ng/ml. This HPLC method was validated by examining the precision and accuracy for inter- and intra-day analysis in the concentration range of 5-150 ng/ml. The relative standard deviation (R.S.D.) in inter- and intra-day validation were 8.16-19.75 and 3.90-11.69%, respectively. In stability tests, levosulpiride in human plasma was stable during the storage and assay procedure. The method was applied to the bioequivalence study of two levosulpiride tablet formulations (25 mg) after a single oral administration.     

Selective determination of tenofovir in human plasma by LC-MS-MS method

In the present study, we developed and validated a selective, specific and sensitive liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS/MS) method for the determination of tenofovir in human plasma. Entecavir was used as an internal standard, and plasma samples were prepared by solid-phase extraction performed on Phenomenex Strata cartridges (30 mg). The mobile phase consisted of 10 mM ammonium acetate in water and methanol (60:40, v/v). The chromatographic separation was performed isocratically on a Phenomenex C₁₈ (4.6 mm×150 mm, 5 μm), and analytes were analyzed in multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI) interface using the respective [M+H]⁺ ions, m/z 288.2→m/z 176.1 for tenofovir and m/z 278.1→m/z 152 for entecavir. The calibration curve (r² = 0.9962) of tenofovir was established within the range of 4.096–1000 μg/L. The intra- and inter-day precisions were less than 10%. This validated method was successfully applied to a pharmacokinetic study in 12 healthy Chinese volunteers after the oral administration of tenofovir disoproxil fumarate.     

Comparison of solid-phase microextraction and liquid-liquid extraction in 96-well format for the determination of a drug compound in human plasma by liquid chromatography with tandem mass spectrometric detection

Two sensitive and selective methods based on solid phase microextraction (SPME) and liquid-liquid extraction (LLE) in 96-well format, in combination with high performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection have been developed to determine a model drug compound in human plasma. Both assays were performed on an Applied Biosystems-Sciex API 4000 tandem mass spectrometer interfaced with a turbo ion-spray probe and operated in the negative ionization mode. A lower limit of quantitation (LLOQ) of 1 ng/mL achieved when 0.25 mL of human plasma was processed. In both methods, a stable isotope labeled internal standard was utilized. The methods were validated in the concentration range of 1-500 ng/mL. The intraday precision (%C.V.) of the method using LLE was 0.8% at LLOQ, and was equal to or lower than 3.3% at all other concentrations, while the intraday precision (%C.V.) of the method using SPME was 6.9% at LLOQ, and was equal to or lower than 5.7% at all other concentrations. Based on the direct comparison of the two methods and their successful applications in clinical sample analysis, it may be concluded that SPME may be considered and used as an alternative approach for quantitative determination of drugs in pharmacokinetic studies.     

A simple high-performance liquid chromatographic method for detection of hydroxyzine in human plasma after overdose

INTRODUCTION: Hydroxyzine, a piperazine H1-receptor antagonist, is effective in generalised anxiety disorder. For toxicological purposes, a simple reversed-phase high-performance liquid chromatographic assay was developed for the detection of hydroxyzine in human plasma. METHODS: A liquid-liquid procedure was used to extract the drug from plasma in the presence of an internal standard (clothiapine). The analysis was performed on a Spherisorb S5 C(8) analytical column with UV detection. RESULTS: A linear response was observed over the concentration range 20-1500 ng/ml. A good accuracy (bias<7.3%) was achieved for all quality controls, with intraday and interday variation coefficients inferior to 8.5%. The limit of detection was 10 ng/ml, without interference of endogenous components. DISCUSSION: This rapid method (run time<13 min) is currently used for poison management involving hydroxyzine.     

Simultaneous determination of zidovudine and nevirapine in human plasma by RP-LC

A simple method for the simultaneous determination of zidovudine and nevirapine in human plasma by reversed-phase liquid chromatography with UV detection at 265 nm was developed. A solid–liquid extraction procedure with internal standard was applied to the samples prior to analysis. The system requires a Zorbax SB-C18 column, 250×4.6 mm I.D. and a mobile phase composed of potassium dihydrogen phosphate (10 mM; pH 6.5)-acetonitrile (83:17, v/v). Peak-areas are linear; correlation coefficients are better than 0.999; both inter- and intra-day accuracy and precision are lower than 15%. Extraction recoveries are higher than 90% for both zidovudine and nevirapine. The method proposed was employed to determine the levels of the two retroviral drugs in plasma from HIV infected human subjects.     

液相色谱—质谱联用法测定人血浆中多潘立酮及其在健康中国志愿者中的药物动力学

目的:测定人血浆中多潘立酮的浓度,并研究在中国志愿者中单剂量口服20mg后的药物动力学过程.方法:用液-液萃取法提取血浆中药物.采用液相色谱-质谱联用分析手段,建立测定人血浆中多潘立酮浓度的分析方法.结果:多潘正酮在0.52—154.5μg/L浓度范围内线性良好(r=0.9999),其在人血浆中的回收率大于75％,低浓度(0.52μg/L)的日内、日间差异分别为9.4％和7.6％.多潘立酮的血药浓度-经时曲线符合二房室模型,其主要药物动力学参数为:T_(max)=(0.8±0.7)h,C_(max)=(50±32)μg/L,T_(1 2)=(7.8±1.6)h.结论:本方法专属性强,灵敏度高,可准确测定多潘不酮在人血浆中的浓度.     

High-performance liquid chromatographic determination of methotrexate in plasma

A procedure for the determination of methotrexate in human plasma is reported. The analysis involved extraction of methotrexate as an ion pair in ethyl acetate. Reconstituted residue was analyzed using reverse phase C-18 column and a mobile phase consisting of acetate buffer (87%), methanol (6.5%), and acetonitrile (6.5%). The methotrexate recovery range was 95-97%. Theophylline was used as internal standard with a recovery of 96%. The intraday coefficient of variation for the assay ranged from 1.8-3.0%, while interday variation coefficient range was 3.5-3.7%. The method is selective, reproducible, and covers a wide range of methotrexate concentrations in patient's plasma.