Department of Pharmaceutics, Virginia Commonwealth University, Richmond, VA 23298, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Aug 15;903:102-11. doi: 10.1016/j.jchromb.2012.06.047. Epub 2012 Jul 20.
Polypeptide therapeutics present a challenge for quantitative analysis when using immunoassays or recently, liquid chromatography-tandem mass spectrometry because of their structural similarities to endogenous proteins and peptides in plasma. In this assay, a Waters Oasis® mixed-mode anion exchange (MAX) microelution modified solid phase extraction (SPE) method coupled with two-dimensional reversed phase ion pair chromatography-tandem mass spectrometry was used for the validation and analysis of oxyntomodulin in rat plasma. Oxyntomodulin (OXM) and its isotope labeled internal standard were extracted from rat plasma and analyzed with a chromatographic run time of 8 min. Modified SPE, two-dimensional liquid chromatography coupled with 3-nitrobenzyl alcohol as a mobile phase additive, and monitoring of multiply charged SRM transitions (+7 charge state) of OXM were necessary to achieve a lower limit of quantification of 1 ng/mL. The method was validated with a linear range of 1-1000 ng/mL, with average R² of 0.992, and reversed calculated residuals between -8.6% and 6.0%. Precision and accuracy for inter- and intra-day were determined to be ±17%. Following a complete validation, the method was applied to show utility using rat plasma samples that were intravenously dosed with oxyntomodulin.
当使用免疫测定法或最近的液相色谱-串联质谱法时，多肽治疗药物因其与血浆中内源性蛋白质和肽的结构相似性而对定量分析提出了挑战。在该测定法中，使用 Waters Oasis®混合模式阴离子交换（MAX）微洗脱改良固相萃取（SPE）方法与二维反相离子对色谱-串联质谱法结合，用于验证和分析大鼠血浆中的胰高血糖素样肽-1（OXM）。OXM 和其同位素标记的内标从大鼠血浆中提取，并在 8 分钟的色谱运行时间内进行分析。改良 SPE、二维液相色谱与 3-硝基苄醇作为流动相添加剂，以及监测 OXM 的多重电荷 SRM 跃迁（+7 电荷态），是实现 1ng/mL 下限定量的必要条件。该方法的线性范围为 1-1000ng/mL，平均 R²为 0.992，反向计算残差在-8.6%和 6.0%之间。日内和日间的精密度和准确度均确定为±17%。在完成全面验证后，该方法应用于显示使用静脉注射给予 OXM 的大鼠血浆样品的实用性。
