Suryawanshi Satyendra, Asthana R K, Gupta R C
Pharmacokinetics and Metabolism Division, Central Drug Research Institute, Lucknow 226001, India.
J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Oct 15;858(1-2):211-9. doi: 10.1016/j.jchromb.2007.08.034. Epub 2007 Aug 31.
Extracts from Swertia chirata (family Gentianaceae) have antidiabetics and antioxidant activity, largely attributed to the flavonoids and secoiridoids, which are a major class of functional components in methanolic extracts from aerial part of plants. In order to facilitate analysis of systemic exposure to S. chirata derived products in animals, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) based method that is capable of routinely monitoring plasma levels of flavonoids and secoiridoids. An LC-MS/MS-based method has been developed for the simultaneous estimation of two bioactive markers, mangiferin and amarogentin along with three other components, amaroswerin, sweroside and swertiamarin in rat plasma. All the analytes including the internal standard (kutkoside) were chromatographed on RP-18 column (250 mm x 4 mm i.d., 5 microm.) coupled with guard column using acetonitrile: 0.5 mM ammonium acetate buffer, pH approximately 3.0 as mobile phase at a flow rate of 1 ml/min in gradient mode. The final flow to source was splitted in 1:1 ratio. The detection of the analytes was performed on API 4000 LC-MS/MS system in the multiple reaction-monitoring (MRM) mode. The quantitation for analytes other than the pure markers was based on relative concentration. The method was validated in terms of establishing linearity, specificity, sensitivity, recovery, accuracy and precision (Intra- and Inter-day), freeze-thaw stability, peltier stability, dry residue stability and long-term stability. The recoveries from spiked control samples were >90% for all analytes and internal standard except mangiferin where recovery was >60%. Intra- and inter-day accuracy and precision of the validated method were within the acceptable limits of <15% at low and <10% at other concentrations. The quantitation method was successfully applied to generate pharmacokinetic (PK) profile of markers as well as to detect other components in plasma after intravenous dose administration of herbal preparation in male Sprague-Dawley (SD) rats.
獐牙菜（龙胆科）提取物具有抗糖尿病和抗氧化活性，这主要归因于黄酮类化合物和裂环烯醚萜类化合物，它们是植物地上部分甲醇提取物中的一类主要功能成分。为便于分析动物体内獐牙菜衍生产品的全身暴露情况，我们开发了一种基于液相色谱串联质谱（LC-MS/MS）的方法，该方法能够常规监测黄酮类化合物和裂环烯醚萜类化合物的血浆水平。已开发出一种基于LC-MS/MS的方法，用于同时测定大鼠血浆中两种生物活性标志物芒果苷和苦味质，以及其他三种成分，即苦苷、獐牙菜苷和獐牙菜苦苷。所有分析物包括内标（库特可苷）都在RP-18柱（250 mm×4 mm内径，5μm）上进行色谱分离，连接保护柱，以乙腈：0.5 mM醋酸铵缓冲液（pH约3.0）作为流动相，以1 ml/min的流速进行梯度洗脱。最终进入源的流速以1:1的比例分流。分析物的检测在API 4000 LC-MS/MS系统上以多反应监测（MRM）模式进行。除纯标志物外，其他分析物的定量基于相对浓度。该方法在建立线性、特异性、灵敏度、回收率、准确性和精密度（日内和日间）、冻融稳定性、珀耳帖稳定性、干残渣稳定性和长期稳定性方面得到了验证。加标对照样品中所有分析物和内标的回收率均>90%，除芒果苷外，其回收率>60%。验证方法的日内和日间准确性和精密度在低浓度时<15%、其他浓度时<10%的可接受范围内。该定量方法已成功应用于生成标志物的药代动力学（PK）曲线，以及在雄性Sprague-Dawley（SD）大鼠静脉注射草药制剂后检测血浆中的其他成分。
