Italiya Kishan S, Sharma Saurabh, Kothari Ishit, Chitkara Deepak, Mittal Anupama
Industrial Research Laboratory (IRL), Department of Pharmacy, Birla Institute of Technology and Science (BITS-PILANI), Pilani, Rajasthan, 333031, India.
Industrial Research Laboratory (IRL), Department of Pharmacy, Birla Institute of Technology and Science (BITS-PILANI), Pilani, Rajasthan, 333031, India.
J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Sep 1;1061-1062:49-56. doi: 10.1016/j.jchromb.2017.06.043. Epub 2017 Jun 29.
Lisofylline (LSF) is an anti-inflammatory and immunomodulatory agent with proven activity in serious infections associated with cancer chemotherapy, hyperoxia-induced acute lung injury, autoimmune disorders including type-1 diabetes (T1DM) and islet rejection after islet transplantation. It is also an active metabolite of another anti-inflammatory agent, Pentoxifylline (PTX). LSF bears immense therapeutic potential in multiple pharmacological activities and hence appropriate and accurate quantification of LSF is very important. Although a number of analytical methods for quantification of LSF and PTX have been reported for pharmacokinetics and metabolic studies, each of these have certain limitations in terms of large sample volume required, complex extraction procedure and/or use of highly sophisticated instruments like LC-MS/MS. The aim of current study is to develop a simple reversed-phase HPLC method in rat plasma for simultaneous determination of LSF and PTX with the major objective of ensuring minimum sample volume, ease of extraction, economy of analysis, selectivity and avoiding use of instruments like LC-MS/MS to ensure a widespread application of the method. A simple liquid-liquid extraction method using methylene chloride as extracting solvent was used for extracting LSF and PTX from rat plasma (200μL). Samples were then evaporated, reconstituted with mobile phase and injected into HPLC coupled with photo-diode detector (PDA). LSF, PTX and 3-isobutyl 1-methyl xanthine (IBMX, internal standard) were separated on Inertsil® ODS (C18) column (250×4.6mm, 5μm) with mobile phase consisting of A-methanol B-water (50:50v/v) run in isocratic mode at flow rate of 1mL/min for 15min and detection at 273nm. The method showed linearity in the concentration range of 50-5000ng/mL with LOD of 10ng/mL and LLOQ of 50ng/mL for both LSF and PTX. Weighted linear regression analysis was also performed on the calibration data. The mean absolute recoveries were found to be 80.47±3.44 and 80.89±3.73% for LSF and PTX respectively. The method was successfully applied for studying the pharmacokinetics of LSF and PTX after IV bolus administration at dose of 25mg/kg in Wistar rat. In conclusion, a simple, sensitive, accurate and precise reversed-phase HPLC-UV method was established for simultaneous determination of LSF and PTX in rat plasma.
利索茶碱（LSF）是一种抗炎和免疫调节药物，已证实其在与癌症化疗相关的严重感染、高氧诱导的急性肺损伤、自身免疫性疾病（包括1型糖尿病（T1DM））以及胰岛移植后的胰岛排斥反应中具有活性。它也是另一种抗炎药物己酮可可碱（PTX）的活性代谢产物。LSF在多种药理活性方面具有巨大的治疗潜力，因此对LSF进行恰当且准确的定量分析非常重要。尽管已经报道了许多用于LSF和PTX定量分析的方法用于药代动力学和代谢研究，但这些方法在所需样本量大、提取过程复杂和/或使用如LC-MS/MS等高精尖仪器方面都存在一定局限性。本研究的目的是开发一种用于大鼠血浆中同时测定LSF和PTX的简单反相高效液相色谱法，主要目标是确保最小样本量、易于提取、分析经济、选择性好，并避免使用如LC-MS/MS等仪器，以确保该方法能广泛应用。采用以二氯甲烷为萃取溶剂的简单液-液萃取法从大鼠血浆（200μL）中萃取LSF和PTX。然后将样品蒸发，用流动相复溶并注入配有光电二极管检测器（PDA）的高效液相色谱仪中。LSF、PTX和3-异丁基-1-甲基黄嘌呤（IBMX，内标）在Inertsil® ODS（C18）柱（250×4.6mm，5μm）上分离，流动相由A-甲醇B-水（50:50v/v）组成，等度洗脱，流速为1mL/min，洗脱15min，检测波长为273nm。该方法在50 - 5000ng/mL的浓度范围内呈线性，LSF和PTX的检测限均为10ng/mL，定量下限均为50ng/mL。还对校准数据进行了加权线性回归分析。LSF和PTX的平均绝对回收率分别为80.47±3.44%和80.89±3.73%。该方法成功应用于研究Wistar大鼠静脉注射25mg/kg剂量后LSF和PTX的药代动力学。总之，建立了一种简单、灵敏、准确且精密的反相高效液相色谱-紫外法用于同时测定大鼠血浆中的LSF和PTX。
