Identification and assay of wild-type and modified nucleoside mixtures by turbo ionspray ionization tandem mass spectrometry

A method is presented which allows the identification and assay of a nucleoside in the presence of other analogues and homologues. The method is based on the conventional multiple reaction monitoring approach performed on the [M + H]+ ions of wild-type and modified nucleosides produced by the turbo ionspray ionization method on a triple-quadrupole mass spectrometer. The accuracy of the quantitative determination relies on the evaluation of a response factor rho, which takes into account the kinetics of dissociation of the parent ions into the protonated [B + 2H]+ nucleobase ions. The evaluation of the absolute concentration of each analyte in the examined mixture does not require any previous chromatographic separation.     

Rapid and simple liquid chromatography tandem mass spectrometry method for the quantification of zidovudine in rat plasma

A high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the quantification of zidovudine in rat plasma. Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reverse phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 268/127 for zidovudine and m/z 230/112 for the internal standard. The method exhibited a linear dynamic range of 5-500 ng/mL for zidovudine in rat plasma. The lower limit of quantification was 5 ng/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 1.5 min for each sample made it possible to analyze more than 400 plasma samples per day. The validated method was applied for pharmacokinetic studies of the novel drug delivery systems of zidovudine in rats.     

Validation of a sensitive LC/MS/MS method for the determination of zidovudine and lamivudine in human plasma

A sensitive LC/MS/MS assay for determining zidovudine (ZDV) and lamivudine (3TC) in human plasma was validated to support antiretroviral pharmacology research programs. After addition of stable labeled isotopic zidovudine (ZDV‐IS) and lamivudine (3TC‐IS) as internal standard, a solid‐phase extraction was performed with an Oasis HLB 1 cm³ cartridge, with recoveries of 92.3% for ZDV and 93.9% for 3TC. A Phenomonex Synergi Hydro‐RP (2.0 × 150 mm) reversed‐phase analytical column was utilized for chromatographic separation. The mobile phase consisted of an aqueous solution of 15% acetonitrile and 0.1% acetic acid. Detection was accomplished by ESI/MS/MS in the positive ion mode, monitoring 268/127, 271/130, 230/112 and 233/115 transitions, for ZDV, ZDV‐IS, 3TC and 3TC‐IS, respectively. The method was linear from 1 to 3000 ng/mL with a minimum quantifiable limit of 1 ng/mL when 100 μL of plasma was analyzed. Validation results demonstrated high accuracy (≤8.3% deviation) and high precision (≤10% CV) for the quality control samples. The method was also shown to be specific and reproducible. The value of the high sensitivity was demonstrated by quantitation of approximately 100 existing samples that had ZDV below the limit of quantitation using a previously validated, less sensitive HPLC‐UV method utilized in the laboratory. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous quantitation of zidovudine and zidovudine monophosphate from plasma, amniotic fluid and tissues by micellar capillary electrophoresis

Zidovudine (AZT) therapy given during pregnancy has been shown to reduce the vertical transmission of the human immunodeficiency virus (HIV) from mother to fetus. In order to investigate the efficacy of AZT, it is important to know the concentration of its active phosphorylated metabolites. We have developed the first CE method for the simultaneous quantitation of AZT and zidovudine monophosphate (AZT-MP) from rat plasma, amniotic fluid and fetal tissues. Sample extractions were performed by protein precipitation using acetonitrile for the plasma and amniotic fluids, while in fetal tissues solid phase extraction using Waters Oasis HLB extraction cartridges was used. Recoveries ranged from 78 to 92% for AZT, AZT-MP and 3'-azidouridine (internal standard, AZDU), in the three matrices. The optimum separation conditions were achieved using a 40 mm sodium dodecylsulfate (SDS) in 50 mm phosphate buffer (pH 7) with a run voltage of 15 kV. The CE system consists of a 75 microm i.d., 50 cm effective length uncoated fused silica capillary. The method was validated over the range 0.5-100 microg/ml ( micro g/g for tissues). Intra-day precision (RSD) and accuracy (%error) for AZT ranged from 0.13 to 11 and 0.68 to 11.1%, respectively, while for AZT-MP it ranged from 2.05 to 11.1 and 4.22 to 11.7%. Inter-day precision and accuracy for AZT ranged from 3.82 to 11.2 and 3.14 to 9.01%, while for AZT-MP it ranged from 3.9 to 9.32 and 3.44 to 9.37%, respectively. We also report the enzymatic dephosphorylation of AZT-MP in the placental tissue of rats. This new enzymatic pathway provides increased understanding of the mechanism of anti-viral transport in the rat during pregnancy.     

Determination of Abacavir, Lamivudine and Zidovudine in Pharmaceutical Tablets, Human Serum and in Drug Dissolution Studies by HPLC

A simple, accurate, precise and fully automated method for the simultaneous determination of abacavir, lamivudine and zidovudine in pharmaceutical tablets, human serum samples and drug dissolution studies has been developed. Separation was performed on a 5 μm Zorbax^® C₁₈ column (150 × 4.6 mm ID) with methanol:water:phosphate buffer at pH 5.65 (80:10:10; v/v/v) isocratic elution in less than 7 min with a flow rate of 0.6 mL min^?1.Good sensitivity for all analytes was observed with UV detection at 275 nm. The method allowed quantitation over the 500–3,000 ng mL^?1 range for abacavir and 500–5,000 ng mL^?1 range for lamivudine and zidovudine. The method has been applied, without any interference from excipients or endogenous substances, for the simultaneous determination of these three compounds in tablets. Human serum and drug dissolution studies.     

核磁共振法测定依帕列净含量

分别以盐酸吉西他滨和齐多夫定为内标,采用定量核磁共振法对依帕列净含量进行测定。以氘代二甲亚砜和重水混合液为溶剂,确定氢定量核磁共振方法(1H-q NMR)的测试条件为:激发脉冲角度30°;时间域数据点32 K;测定温度303 K;脉冲延迟时间20 s;采样次数32;窗函数0.3 Hz。在此实验条件下,结果专属性良好,稳定性可达24 h,耐用性符合要求。以样品与内标的峰面积比对其摩尔比绘制标准曲线,结果显示,依帕列净与内标盐酸吉西他滨的摩尔比在0.512 5~1.953 8范围内,依帕列净与内标齐多夫定的摩尔比在0.494 7~1.966 0范围内线性关系良好,相关系数(r2)均为0.999 9。以盐酸吉西他滨和齐多夫定为内标时,依帕列净的含量测定结果分别为99.83%和99.77%,相对标准偏差(RSD)分别为0.06%和0.19%。2种内标方法的测定结果一致,所建立的方法专属、准确、简便、快捷,适用于新药的含量测定。     

Salt‐assisted liquid–liquid extraction in microchannel

In this study, for the first time, salt‐assisted liquid–liquid extraction was performed in a microchannel system. The proposed design is based on the increase of contact surface area between target analytes and extracting phase during the sample and extracting phase transfer in microchannel. In this method, first sample solution, extracting solvent, and salt were mixed by stirrer and simultaneously delivered into a microchannel using a syringe pump. In order to optimize the influential parameters on the extraction efficiency of the proposed method, zidovudine and tenofovir disoproxil fumarate were selected as model analytes. The main parameters such as extracting solvent and its volume, salt amount, pH of sample solution, and microchannel shape, length, and its inner diameter were investigated and optimized. Under the optimized conditions, the proposed method was linear in the range of 0.1–30 µg/mL and R² coefficients were equal to 0.9922 and 0.9947 for zidovudine and tenofovir disoproxil fumarate, respectively. Extraction efficiency of the proposed method was compared with conventional salt‐assisted liquid–liquid extraction. The results show that the proposed design has higher extraction efficiency than conventional salt‐assisted liquid–liquid extraction. Finally, the proposed method was successfully applied for the determination of zidovudine and tenofovir disoproxil fumarate in plasma samples.     

Ultrasensitive method to quantify intracellular zidovudine mono‐, di‐ and triphosphate concentrations in peripheral blood mononuclear cells by liquid chromatography–tandem mass spectrometry

Although zidovudine (AZT) is not the preferred antiretroviral drug for adult HIV‐infected patients, it is still widely used in infants for both prevention of mother‐to‐infant HIV‐1 transmission and treatment of HIV‐infected children. However, it is difficult to measure intracellular concentrations of AZT metabolites in small blood samples due to their extremely low concentrations in peripheral blood mononuclear cells and interference by endogenous nucleotide triphosphates, residual plasma phosphates and electrolytes. We developed an ultrasensitive assay using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for measurement of intracellular concentrations of zidovudine (AZT)‐monophosphate (AZT‐MP), ‐diphosphate (AZT‐DP) and ‐triphosphate (AZT‐TP). The high sensitivity was due to the improvement of peripheral blood mononuclear cells extraction for complete removal of plasma and electrolytes, alkalization of LC buffer and use of alkaline‐stable high performance liquid chromatography column and tetrabutylammonium hydroxide as the ion pair. Using this method, the lower limits of quantification of AZT, AZT‐MP, ‐DP and ‐TP were 6, 6, 10 and 10 fmol per sample, respectively. Accuracy ranged 89–115% and precision was lower than 15% in the quantification range of 6–6000 fmol/sample for plasma AZT and intracellular AZT‐MP and 10–10 000 fmol/sample for AZT‐DP and ‐TP. The validation parameters met the international requirements. Among nine AZT‐treated HIV‐infected adult patients, five had low AZT‐TP levels (<10 fmol/10⁶ cells). Our assay has high sensitivity and is advantageous for evaluation of AZT phosphates in children and infants based on minimum blood sampling requirement. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous determination of zidovudine and lamivudine from rat plasma, amniotic fluid and tissues by HPLC

A sensitive HPLC method has been developed and validated for the simultaneous quantification of zidovudine (AZT) and lamivudine (3TC) in rat plasma, amniotic fluid and placental and fetal tissues. Samples were processed by solid-phase extraction using C2 cartridges. Chromatography was performed using a phenyl column (5 microm, 150 x 2 mm i.d.) under a flow rate of 0.2 mL/min. The mobile phase consisted of 8% acetonitrile in 5 mM 1-heptane sulfonic acid dissolved in 30 mM ammonium formate buffer (pH 3.3). The method was validated in the range 0.25-50 microg/mL for both 3TC and AZT in the four biological matrices. Finally, the method was applied to a study involving fetal transport of co-administration of these compounds in a pregnant rat.