Fast and sensitive LC–MS/MS method for the simultaneous determination of lisinopril and hydrochlorothiazide in human plasma

A sensitive and rapid liquid chromatography-tandem mass spectrometry(LC–MS/MS) method has been developed for the simultaneous determination of lisinopril(LIS) and hydrochlorothiazide(HCTZ) in human plasma using their labeled internal standards(ISs). Sample pre-treatment involved solid phase extraction on Waters Oasis HLB cartridges using 100 μL of plasma, followed by liquid chromatography on Hypersil Gold C_(18)(50 mm×3.0 mm, 5 μm) column. The analytes were eluted within 2.0 min using acetonitrile-5.0 m M ammonium formate, p H 4.5(85:15, v/v) as the mobile phase. The analytes and ISs were analyzed in the negative ionization mode and quantified using multiple reaction monitoring. The method showed excellent linearity over the concentration range of 0.50–250.0 ng/m L for both the analytes. The intra-batch and inter-batch precision(% CV) was ≤5.26% and their extraction recoveries were in the range of 96.6%–103.1%. Matrix effect evaluated in terms of IS-normalized matrix factors ranged from 0.97 to 1.03 for both the analytes. The validated method was successfully applied to determine the plasma concentration of the drugs using 10 mg lisinopril and 12.5 mg hydrochlorothiazide fixed dose formulation in 18 healthy Indian volunteers.

LC–MS/MS法检测黑色粮谷类食品中的苏丹黑B和苏丹红B

采用液相色谱串联质谱（LC-MS／MS）法测定黑色粮谷类食品中苏丹黑B和苏丹红B含量。用正己烷、饱和乙腈提取目标物，以乙腈饱和的正己烷去除油脂，以固相萃取柱富集净化处理，外标法定量。苏丹黑B和苏丹红B的质量浓度在5-100ng／mL范围内与色谱峰面积呈良好的线性，线性相关系数r^2〉0．999，检出限为5μg／kg。加标回收率为90．0％-96．0％，测定结果的相对标准偏差小于1．0％（n=6）。该方法分析时间短，准确性好，适用于黑色粮谷类食品中苏丹黑B和苏丹红B含量的检测分析。

Application of an LC–MS/MS method for the analysis of amlodipine,valsartan and hydrochlorothiazide in polypill for a bioequivalence study

A sensitive and selective method has been proposed for the simultaneous determination of amlodipine(AML),valsartan(VAL) and hydrochlorothiazide(HCTZ) in human plasma by liquid chromatography–tandem mass spectrometry(LC–MS/MS). The analytes and their deuterated analogs were quantitatively extracted from100 μL human plasma by solid phase extraction on Oasis HLB cartridges. The chromatographic separation of the analytes was achieved on a Chromolith RP18 e(100 mm × 4.6 mm) analytical column within 2.5 min. The resolution factor between AML and VAL, AML and HCTZ, and VAL and HCTZ was 2.9, 1.5 and 1.4, respectively,under isocratic conditions. The method was validated over a dynamic concentration range of 0.02–20.0 ng/m L for AML, 5.00–10,000 ng/m L for VAL and 0.20–200 ng/m L for HCTZ. Ion-suppression/enhancement effects were investigated by post-column infusion technique. The mean IS-normalized matrix factors for AML, VAL and HCTZ were 0.992, 0.994 and 0.998, respectively. The intra-batch and inter-batch precision(% CV) across quality control levels was ≤ 5.56% and the recovery was in the range of 93.4%–99.6% for all the analytes. The method was successfully applied to a bioequivalence study of 5 mg AML + 160 mg VAL + 12.5 mg HCTZ tablet formulation(test and reference) in 18 healthy Indian males under fasting. The mean log-transformed ratios of C max, AUC0–120 h and AUC0-inf and their 90% CIs were within 90.2%–102.1%. The assay reproducibility was demonstrated by reanalysis of 90 incurred samples.

LC and LC–MS/MS studies for the identification and characterization of degradation products of acebutolol

The aim of the present investigation was to demonstrate an approach involving use of liquid chromatography(LC) and liquid chromatography-mass spectrometry(LC–MS) to separate, identify and characterize very small quantities of degradation products(DPs) of acebutolol without their isolation from the reaction mixtures. The drug was subjected to oxidative, hydrolytic, thermal and photolytic stress conditions as per International Conference on Harmonization(ICH) guideline Q1 A(R2). Among all the stress conditions the drug was found to be labile in hydrolytic(acidic & basic) and photolytic stress conditions, while it was stable in water-induced hydrolysis, oxidative and thermal stress conditions. A total of four degradation products were formed. A C18 column was employed for the separation of all the DPs on a gradient mode by using high-performance liquid chromatography(HPLC). All the DPs were characterized with the help of their fragmentation pattern and the masses obtained upon LC–MS/MS and MSnanalysis. All the hitherto unknown degradation products were identified as 1-(2-(2-hydroxy-3-(isopropylamino)propoxy)-5-(amino)phenyl)ethanone(DP-I), N-(4-(2-hydroxy-3-(isopropylamino)propoxy)-3-acetylphenyl)acrylamide(DP-II), 1-(2-(2-hydroxy-3-(isopropylamino)propoxy)-5-(hydroxymethylamino)phenyl)ethanone(DP-III) and 1-(6-(2-hydroxy-3-(isopropylamino)propoxy)-2,3-dihydro-2-propylbenzo[d]oxazol-5-yl)ethanone(DP-IV). Finally the in-silico carcinogenicity and hepatotoxicity predictions of the drug and all the DPs were performed by using toxicity prediction softwares viz., TOPKAT, LAZAR and Discovery Studio ADMET. The results of in-silico toxicity studies revealed that acebutolol(0.967) and DP-I(0.986) were found to be carcinogenic, while acebutolol(0.490) and DP-IV(0.437) were found to be hepatotoxic.

Comparison of ESI–and APCI–LC–MS/MS methods:A case study of levonorgestrel in human plasma

Electrospray ionization(ESI) and atmospheric pressure chemical ionization(APCI) techniques for liquid chromatography–tandem mass spectrometry(LC–MS/MS) determination of levonorgestrel were evaluated.In consideration of difference in ionization mechanism,the two ionization sources were compared in terms of LC conditions,MS parameters and performance of method.The sensitivity for detection of levonorgestrel with ESI was 0.25 ng/m L which was lower than 1 ng/m L with APCI.Matrix effects were evaluated for levonorgestrel and canrenone(internal standard,IS) in human plasma,and the results showed that APCI source appeared to be slightly less liable to matrix effect than ESI source.With an overall consideration,ESI was chosen as a better ionization technique for rapid and sensitive quantification of levonorgestrel.The optimized LC–ESI–MS/MS method was validated for a linear range of 0.25–50 ng/m L with a correlation coefficient ≥0.99.The intra-and inter-batch precision and accuracy were within 11.72% and 6.58%,respectively.The application of this method was demonstrated by a bioequivalence study following a single oral administration of 1.5 mg levonorgestrel tablets in 21 Chinese healthy female volunteers.

Determination of fenticonazole in human plasma by HPLC–MS/MS and its application to pharmacokinetic studies

Two simple and sensitive high performance liquid chromatography–tandem mass spectrometry(HPLC–MS/MS) methods were developed and validated for the determination of fenticonazole in human plasma after percutaneous and intravaginal administration. Mifepristone was used as an internal standard(IS), and simple protein precipitation by acetonitrile containing 2% acetic acid was utilized for extracting the analytes from the plasma samples. Chromatographic separation was performed on a Kinetex XB-C_(18) column. The quantitation was performed by a mass spectrometer equipped with an electrospray ionization source in multiple reactions monitoring(MRM) positive ion mode using precursor-to-product ion transitions of m/z 455.2–199.1 for fenticonazole and m/z 430.2–372.3 for mifepristone. The validated linear ranges of fenticonazole were 5–1000 pg/m L and 0.1–20 ng/m L in plasma for the methods A and B, respectively. For the two methods, the accuracy data ranged from 85% to 115%, the intra- and inter-batch precision data were less than 15%, the recovery data were more than 90%, and no matrix interference was observed. The methods A and B were successfully validated and applied to the pharmacokinetic studies of fenticonazole gel in Chinese healthy volunteers after percutaneous and intravaginal administration, respectively.

Application of LC–MS/MS for quantitative analysis of glucocorticoids and stimulants in biological fuids

Liquid chromatography tandem mass chromatography （LC-MS/MS） is an important hyphenated technique for quantitative analysis of drugs in biological fluids. Because of high sensitivity and selectivity, LC-MS/MS has been used for pharmacokinetic studies, metabolites identification in the plasma and urine. This manuscript gives comprehensive analytical review, focusing on chromatographic separation approaches （column packing materials, column length and mobile phase） as well as different acquisition modes （SIM, MRM） for quantitative analysis of glucocorticoids and stimulants. This review is not meant to be exhaustive but rather to provide a general overview for detection and confirmation of target drugs using LC-MS/MS and thus useful in the doping analysis, toxicological studies as well as in pharmaceutical analysis.

Highly sensitive LC–MS/MS method to estimate doxepin and its metabolite nordoxepin in human plasma for a bioequivalence study

A selective, sensitive and rugged liquid chromatography–tandem mass spectrometry(LC–MS/MS) assay has been developed for the simultaneous determination of doxepin(Dox) and its pharmacologically active metabolite, nordoxepin(NDox) in human plasma. The analytes and their internal standards(IS)were extracted from 500 m L of human plasma by liquid-liquid extraction using methyl tert-butyl ether.Chromatographic separation was achieved on Hypurity C8 column(100 mm ? 4.6 mm, 5 mm) using a mixture of acetonitrile-methanol(95:5, v/v) and 2.0 mM ammonium formate in 93:7(v/v) ratio. Detection was accomplished by tandem mass spectrometry in the positive ionization and multiple reaction monitoring acquisition mode. The protonated precursor to product ion transitions studied for Dox, NDox,and their corresponding ISs, propranolol and desipramine, were m/z 280.1-107.0, 266.0-107.0,260.1-116.1 and 267.1-72.1, respectively. A linear dynamic range of 15.0–3900 pg/mL for Dox and 5.00–1300 pg/mL for NDox was established with mean correlation coefficient(r2) of 0.9991 and 0.9993, respectively. The extraction recovery ranged from 86.6%–90.4% and 88.0%–99.1% for Dox and NDox, respectively. The intra-batch and inter-batch precision(% CV) across quality control levels was r 8.3% for both the analytes. Stability evaluated under different storage conditions showed no evidence of degradation and the % change in stability samples compared to nominal concentration ranged from 4.7% to12.3%. The method was successfully applied to a bioequivalence study of 6 mg doxepin hydrochloride orally disintegrating tablet in 41 healthy Indian subjects under fasting and fed conditions.

QuEChERS前处理联合UPLC–MS/MS法检测花生中22种农药残留

建立了Qu ECh ERS(Quick,Easy,Cheap,Effective,Rugged,Safe)前处理联合UPLC–MS/MS法检测花生中22种农药残留的方法。样品用10 m L乙腈提取,以多壁碳纳米管、N-丙基乙二胺为吸附剂,对2 m L提取液进行净化,净化液稀释至2倍体积,以MRM扫描方式、正负离子模式同时分析。22种农药在10,20,50μg/kg 3个添加水平下,平均回收率为70.6%~121.2%,相对标准偏差小于10%(n=6);多菌灵、抗蚜威、扑草净在0.05~10μg/L,啶虫脒、氟虫腈砜、苯醚甲环唑、哒螨灵、嘧霉胺、嘧菌酯在0.5~20μg/L,烯酰吗啉、噻虫嗪、氟啶脲、灭幼脲、吡虫啉、甲维盐、除虫脲、氟虫腈、氟甲腈、氟虫腈亚砜、咪鲜胺、二甲戊灵在0.5~50μg/L之间,阿维菌素在0.5~100μg/L范围内线性良好,相关系数r2均大于0.995 0。22种农药的定量限在2μg/kg以下,远低于各待测农药最高残留限量标准(MRL)。该法适于花生中农药残留的同时快速检测。

Quantification of 17-desacetyl norgestimate in human plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and its application to bioequivalence study

A rapid and sensitive ultra-performance liquid chromatography–tandem mass spectrometry（UPLC–MS/MS） method was developed and validated for the estimation of 17-desacetyl norgestimate in human plasma using solid-phase extraction technique. 17-desacetyl norgestimate D6 was used as the internal standard. Simple gradient chromatographic conditions and mass spectrometric detection enabled accurate and precise measurement of 17-desacetyl norgestimate at sub-picogram levels. The proposed method was validated for a linear range of 20–5000 pg/m L with a correlation coefficient Z 0.9988. The intra-run and inter-run precision and accuracy were within 10%. The overall recoveries for 17-desacetyl norgestimate and 17-desacetyl norgestimate D6 were 96.30% and 93.90%, respectively. The total run time was 4.5 min. The developed method was applied for the determination of the pharmacokinetic parameters of 17-desacetyl norgestimate following a single oral administration of a norgestimate and ethinyl estradiol0.250 mg/0.035 mg tablets in 35 healthy female volunteers.

A validated UPLC–MS/MS method for simultaneous determination of imatinib, dasatinib and nilotinib in human plasma

A sensitive, rapid, simple and economical ultra-performance liquid chromatography-tandem mass spectrometric method （UPLC-MS/MS） was developed and validated for simultaneous determination of imatinib, dasatinib and nilotinib in human plasma using gliquidone as internal standard （IS）. Liquid-liquid extraction method with ethyl acetate was used for sample pre-treatment. The separation was performed on an Xtimate Phenyl column using isocratic mobile phase consisting of A （aqueous phase： 0.15% formic acid and 0.05% ammonium acetate） and B （organic phase： aeetonitrile） （A：B=40：60, v/v）. The flow rate was 0.25 mL/min and the total run time was 6 min. The multiple reaction monitoring （MRM） transitions, m/z 494.5-394.5 for imatinib, 488.7-401.5 for dasatinib, 530.7-289.5 for nilotinib and 528.5-403.4 for IS, were chosen to achieve high selectivity in the simultaneous analyses. The method exhibited great improvement in sensitivity and good linearity over the concentration range of 2.6-5250.0 ng/mL for imatinib, 2.0-490.0 ng/mL for dasatinib, and 2.4-4700.0 ng/mL for nilotinib. The method showed acceptable results on sensitivity, specificity, recovery, precision, accuracy and stability tests. This UPLC-MS/MS assay was successfully used for human plasma samples analysis and no significant differences were found in imatinib steady-state trough concentrations among the SLC22A5 -1889T 〉 C or SLCOIB3 699G 〉 A genotypes （P 〉 0.05）. This validated method can provide support for clinical therapeutic drug monitoring and pharmacokinetic investigations of these three tyrosine kinase inhibitors （TKIs）.

Sensitive and rapid determination of amantadine without derivatization in human plasma by LC–MS/MS for a bioequivalence study

A highly sensitive, rapid and rugged liquid chromatography-tandem mass spectrometw （LC-ESI-MS/MS） method was developed for reliable estimation of amantadine （AMD）, an antiviral drug in human plasma. The analyte and internal standard （IS）, amantadine-d6 （AMD-d6）, were extracted from 200μL plasma by solid phase extraction on Phenomenex Strata-X-C 33 ~t cartridges. Chromatography was performed on Synergi^TM Hydro-RP C18 （150 mm×4.6 mm, 4 μm） analytical column using a mixture of acetonitrile and 10 mM ammonium formate, pH 3.0 （80：20, v/v） as the mobile phase. Detection and quantitation was done by multiple reaction monitoring in the positive ionization mode for AMD （m/z 152.1→ 135.1 ） and IS （m/z 158.0 → 141.1） on a triple quadrupole mass spectrometer. The assay was linear in the concentration range of 0.50-500 ng/mL with correlation coefficient （r^2） 〉 0.9969. The limit of detection of the method was 0.18 ng/mL The intra-batch and inter-batch precisions were 〈 5.42% and the accuracy varied from 98.47% to 105.72%. The extraction recovery of amantadine was precise and quantitative in the range of 97.89%-100.28%. IS-normalized matrix factors for amantadine varied from 0.981 to 1.012. The stability of AMD in whole blood and plasma was evaluated under different conditions. The developed method was successfully applied for a bioequivalence study with 100 mg of AMD in 32 healthy volunteers. The reproducibility of the assay was determined by reanalysis of 134 subject samples.

Quantitative and qualitative analysis of common peaks in chemical fingerprint of Yuanhu Zhitong tablet by HPLC-DAD–MS/MS

A quality control （QC） strategy for quantitative and qualitative analysis of “common peaks” in chemical fingerprint was proposed to analyze Yuanhu Zhitong tablet （YZT）, using high performance liquid chromatography with diode array detector and tandem mass spectrometry （HPLC-DAD-MS/MS）. The chromatographic separation was achieved on an Agilent Eclipse plus C18 column with a gradient elution using a mixture of 0.4‰ ammonium acetate aqueous （pH 6.0 adjusted with glacial acetic acid） and acetonitrile. In chemical fingerprint, 40 peaks were assigned as the “common peaks”. For quantification of “common peaks”, the detection wavelength was set at 254 nm, 270 nm, 280 nm and 345 nm, respectively. The method was validated and good results were obtained to simultaneously determine 10 analytes （protopine, jatrorrhizine, coptisine, palmatine, berberine, xanthotoxin, bergapten, tetrahydropalmatine, imperatorin and isoimperatorin）. For qualification of “common peaks”, 33 compounds including 10 quantitative analytes were identified or tentatively characterized using LC-MS/MS. These results demonstrated that the present approach may be a powerful and useful tool to tackle the complex quality issue of YZT.

GPC–HPLC–MS/MS法测定食用油中天然辣椒素、二氢辣椒素和合成辣椒素含量

以动植物油脂为实验材料，建立了测定食用油中天然辣椒素、二氢辣椒素和合成辣椒素含量的凝胶渗透色谱–高效液相色谱–串联质谱(GPC–HPLC–MS／MS)法。样品经凝胶渗透色谱净化后，采用液相色谱串联质谱法(HPLC–ESI–MS／MS)分析，多反应监测模式（MRM）下外标法定量。在0.1～5.0μg／L范围内线性良好，天然辣椒素、二氢辣椒素和合成辣椒素的相关系数分别为0.9996，0.9998，0.9998，检出限为0.5μg／kg。在5μg／kg添加水平下，空白加标回收率为71.5%～82.5%，测定结果的相对标准偏差为3.0%～8.3%(n=6)。该方法样品处理过程简便快捷，测定结果准确，可满足实验室大量、快速分析的需求。

Improved simultaneous quantitation of candesartan and hydrochlorthiazide in human plasma by UPLC–MS/MS and its application in bioequivalence studies

A validated ultra-performance liquid chromatography mass spectrometric method （UPLC-MS/MS） was used for the simultaneous quantitation of candesartan （CN） and hydrochlorothiazide （HCT） in human plasma. The analysis was performed on UPLC-MS/MS system using turbo ion spray interface. Negative ions were measured in multiple reaction monitoring （MRM） mode. The analytes were extracted using a liquid-liquid extraction （LLE） method by using 0.1 mL of plasma volume. The lower limit of quantitation for CN and HCT was 1.00 ng/mL whereas the upper limit of quantitation was 499.15 ng/mL and 601.61 ng/mL for CN and HCT respectively. CN d4 and HCT-13Cd2 were used as the internal standards for CN and HCT respectively. The chromatography was achieved within 2.0 min run time using a C18 Pheno-menex, Gemini NX （100 mm ~ 4.6 mm, 5 mm） column with organic mixture：buffer solution （80：20, v/v） at a flow rate of 0.800 mL/min. The method has been successfully applied to establish the bioequivalence of candesartan cilexetil （CNC） and HCT immediate release tablets with reference product in human subjects.

Development of a sensitive and rapid method for quantitation of (S)-(-)- and (R)-(+)-metoprolol in human plasma by chiral LC–ESI–MS/MS

A selective, sensitive and high throughput liquid chromatography-tandem mass spectro-metry （LC-ESI-MS/MS） method has been developed for separation and quantification of metoprolol enantiomers on a chiral Lux Amylose-2 （250 mm＆#215;4.6 mm, 5 mm） column. Solid phase extraction of （S）-（-）- and （R）-（t）-metoprolol and rac-metoprolol-d6 as an internal standard （IS） was achieved on Lichrosep DVB HL cartridges employing 200 mL human plasma. Both the analytes were chromatographically separated with a resolution factor of 2.24 using 15 mM ammonium acetate in water, pH 5.0 and 0.1% （v/v） diethyl amine in acetonitrile （50：50, v/v） as the mobile phase within 7.0 min. The precursor-product ion transitions for the enantiomers and IS were monitored in the multiple reaction monitoring and positive ionization mode. The method was validated over the concentration range of 0.500-500 ng/mL for both the enantiomers. Matrix effect was assessed by post-column analyte infusion experiment and the mean extraction recovery was greater than 94.0% for both the enantiomers at all quality control levels. The stability of analytes was evaluated in plasma and whole blood under different storage conditions. The method was successfully applied to a clinical study in 14 healthy volunteers after oral administration of 200 mg metoprolol tablet under fasting conditions. The assay reproducibility is shown by reanalysis of 68 incurred samples. The suitability of the developed method was assessed in comparison with＆amp;nbsp;different chromatographic methods developed for stereoselective analysis of metoprolol in biological matrices.

Simultaneous determination of human plasma protein binding of bioactive favonoids in Polygonum orientale by equilibrium dialysis combined with UPLC–MS/MS

A simple and selective ultra performance liquid chromatography--electrospray ionization tandem mass spectrometry （UPLC-ESI-MS/MS） assay was developed for the determination of the human plasma protein binding of four bioactive ftavonoids （such as orientin and vitexin） in Polygonum orientale. Protein precipitation was used for sample preparation. Equilibrium dialysis technique was applied to determine the plasma protein binding under physiological conditions. The separation was achieved through a Waters C i s column with a mobile phase composed of 0.1% formic acid in acetonitrile and 0.1% aqueous formic acid using step gradient elution at a flow rate of 0.35 mL/min. A Waters ACQUITYTM TQD system was operated under the multiple reaction monitoring （MRM） mode of positive electrospray ionization. All of the recovery, precision, accuracy and stability of the method met the requirements. Good correlations （r 〉 0.99） of the four compounds were found, which suggested that these compounds can be simultaneously determined with acceptable accuracy. Results showed that the plasma protein bindings of the four bioactive flavonoids were in the range of 74-89% over the six concentrations studied. The binding parameters containing protein binding affinity, protein binding dissociation constant, and protein binding site were studied. The maximum ability to bind with protein was also determined in the assay in order to understand the drug-protein binding of each compound better.

Development and validation of a high throughput UPLC–MS/MS method for simultaneous quantification of esomeprazole,rabeprazole and levosulpiride in human plasma

A high throughput ultra pressure liquid chromatography-mass spectrometry （UPLC-MS/MS） method with good sensitivity and selectivity has been developed and validated for simultaneous quantification of esomeprazole, rabeprazole and levosulpiride in human plasma using lansoprazole as internal standard （IS）. The extraction method based on liquid-liquid extraction technique was used to extract the analytes and IS from of 50 μL of human plasma using methyl tert-butyl ether：ethyl acetate （80：20, v/v）, which offers a high recovery. Chromatographic separation of analytes and IS was achieved on a Hypersil gold C18 column using gradient mobile phase consisting of 2 mM ammonium formate/acetonitrile. The flow rate was set at 0.5 mL/min to elute all the analytes and IS within 1.00 min runtime. Detection of target compounds was performed on a triple quadruple mass spectrometer by multiple reaction monitoring （MRM） mode via positive electrospray ionization （ESI）. Method validation results demonstrated that the developed method has good precision and accuracy over the concentration ranges of 0.1-2000 ng/mL for each analyte. Stability of compounds was established in a battery of stability studies, i.e., bench top, autosampler, dry extract and long-term storage stability as well as freeze-thaw cycles. The validated method has been successfully applied to analyze human plasma samples for application in pharmaco- kinetic studies.

Determination of lercanidipine in human plasma by an improved UPLC–MS/MS method for a bioequivalence study

An improved and reliable ultra-performance liquid chromatography/tandem mass spectrometry （UPLC- MS/MS） method has been developed and validated for the determination of lercanidipine in human plasma. Plasma samples with lercanidipine-d3 as an internal standard （IS） were prepared by solid phase extraction on Phenomenex Strata-X cartridges using 100 μL of human plasma. Chromatographic analysis was performed on UPLC BEH C18 （50 mm × 2.1 mm, 1.7 μm） column under isocratic conditions. Linear calibration curves were obtained over a wide dynamic concentration range of 0.010-20.0 ng/mL. Matrix effect was assessed by post-column infusion, post-extraction spiking and standard-line slope methods. The mean extraction recovery was 〉 94% for the analyte and IS. Inter-batch and intra-hatch precision （% CV） across five quality controls was 〈 5.8%. Bioequivalence study was performed with 36 healthy sub- jects after oral administration of 10 mg of lercanidipine and the assay reproducibility was evaluated by reanalysis of 133 incurred samples.

Development and validation of a UPLC–MS/MS assay for the determination of gemcitabine and its L-carnitine ester derivative in rat plasma and its application in oral pharmacokinetics

A simple and rapid UPLC–MS/MS method to simultaneously determine gemcitabine and its L-carnitine ester derivative(2’-deoxy-2’, 2’-difluoro-N-((4-amino-4-oxobutanoyl) oxy)-4-(trimethyl amm-onio) butanoate-cytidine, JDR) in rat plasma was developed and validated.The conventional plasma sample preparation method of nucleoside analogues is solidphase extraction(SPE) which is time-consuming and cost-expensive. In this study, gradient elution with small particles size solid phase was applied to effectively separate gemcitabine and JDR, and protein precipitation pretreatment was adopted to remove plasma protein and extract the analytes with high recovery(>81%). Method validation was performed as per the FDA guidelines, and the standard curves were found to be linear in the range of 5–4000 ng/ml for JDR and 4–4000 ng/ml for gemcitabine, respectively. The lower limit of quantitation(LLOQ)of gemcitabine and JDR was 4 and 5 ng/ml, respectively. The intra-day and inter-day precision and accuracy results were within the acceptable limits. Finally, the developed method was successfully applied to investigate the pharmacokinetic studies of JDR and gemcitabine after oral administration to rats.