DMPK Laboratory (Biology Division), GVK BIO, Nacharam, Hyderabad, Andhra Pradesh 500076, India.
J Chromatogr B Analyt Technol Biomed Life Sci. 2013 May 1;926:68-76. doi: 10.1016/j.jchromb.2013.02.021. Epub 2013 Mar 4.
A rapid sensitive and selective MRM based method for the determination of polyethylene glycol 400 (PEG 400) in rat plasma was developed using liquid chromatography/tandem mass spectrometry (LC-MS/MS). PEG 400 and telmisartan (Internal standard) were extracted from rat plasma with acetonitrile and analysed on C18 column (Waters Xbridge, 50×4.6 mm, 3.5 μm) with the mobile phase (A - 0.1% formic acid in water; B - methanol). A generic gradient method with a short run time of 3.5 min was developed for the analysis of PEG 400. A total of nine oligomers were identified for PEG 400. The most abundant ions corresponding to PEG 400 oligomers at m/z 327, 371, 432, 476, 520, 564, 608, 652 and 696 with daughter ion at m/z 89 were selected for multiple reaction monitoring (MRM) in electrospray mode of ionisation. Analyte peak area of the oligomers was summed up to calculate the plasma concentrations of total PEG 400. The standard curve was linear (0.9954) over the concentration range of 1.01-1013.40 μg/mL. The lower limit of quantitation for PEG 400 was 1.01 μg/mL using 50 μL plasma. The coefficient of variation and relative error for inter and intraassay at three QC levels were 2.31-13.34 and -7.99 to 0.37, respectively. The method was validated for various parameters such as extraction recovery, matrix effect, autosampler stability, benchtop stability, freeze thaw stability, long term stability and was proved to be consistent across three QC levels with overall %CV less than 15. The developed method was successfully applied to the absolute bioavailability study of PEG 400 in male Sprague Dawley rats. Plasma concentrations of PEG 400 was measured after administration through oral and intravenous routes in male Sprague Dawley rats at a dose of 3.38 g/kg. Pharmacokinetic (PK) parameters were characterised by performing the analysis using Phoenix Winnonlin software (v 6.3). PEG 400 has good oral bioavailability with mean absolute bioavailability of 47.23%. Plasma concentration profile/PK parameters of PEG 400 was established in both intravenous and oral routes, which helps to qualify the analytical batch of NCEs having spiky plasma concentration profiles/erratic results. Purity of the PEG 400 oligomers was estimated using ELSD detection. Differences in pharmacokinetics of oligomers was studied. It was found that with increase in molecular weight of the oligomer, a decrease in absolute bioavailability was observed.
建立了一种基于液相色谱-串联质谱（LC-MS/MS）的快速、灵敏、选择性的测定大鼠血浆中聚乙二醇 400（PEG 400）的方法。PEG 400 和替米沙坦（内标）用乙腈从大鼠血浆中提取，在 C18 柱（Waters Xbridge，50×4.6mm，3.5μm）上用流动相（A-0.1%甲酸水；B-甲醇）进行分析。开发了一种总运行时间为 3.5 分钟的通用梯度方法，用于 PEG 400 的分析。鉴定出 9 种聚乙二醇 400 低聚物。在电喷雾电离模式下，选择质荷比为 m/z 327、371、432、476、520、564、608、652 和 696 的最丰富离子及其子离子 m/z 89 进行多重反应监测（MRM）。将低聚物的分析物峰面积相加，计算总 PEG 400 的血浆浓度。PEG 400 的标准曲线在 1.01-1013.40μg/mL 浓度范围内呈线性（0.9954）。使用 50μL 血浆，PEG 400 的定量下限为 1.01μg/mL。三个 QC 水平的内、日间变异系数和相对误差分别为 2.31-13.34 和-7.99-0.37。该方法经过各种参数的验证，如提取回收率、基质效应、自动进样器稳定性、工作台稳定性、冻融稳定性、长期稳定性，并在三个 QC 水平上均一致，总体 CV 小于 15%。所建立的方法成功应用于雄性 Sprague Dawley 大鼠中 PEG 400 的绝对生物利用度研究。雄性 Sprague Dawley 大鼠以 3.38g/kg 剂量经口服和静脉途径给予后，测定 PEG 400 的血浆浓度。使用 Phoenix Winnonlin 软件（v6.3）进行分析，以确定药代动力学（PK）参数。PEG 400 具有良好的口服生物利用度，绝对生物利用度为 47.23%。在静脉和口服途径均建立了 PEG 400 的血浆浓度特征/ PK 参数，有助于确定具有尖峰血浆浓度特征/不稳定结果的新型化学实体的分析批次。使用蒸发光散射检测法（ELSD）估算 PEG 400 低聚物的纯度。研究了低聚物的药代动力学差异。结果发现，随着低聚物分子量的增加，绝对生物利用度降低。
