Alkali Soil Natural Environmental Science Center, Northeast Forestry University, Key Laboratory of Saline-Alkali Vegetation Ecology Restoration in Oil Field, Ministry of Education, Harbin 150040, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2013 Nov 15;939:38-44. doi: 10.1016/j.jchromb.2013.09.005. Epub 2013 Sep 10.
Neutral pentacyclic triterpenes with only one or two hydroxyl groups, such as betulin, are not easily ionized by electrospray ionization (ESI). However, because betulin is reactive and neutral, derivatization may improve ionization efficiency. In the present study, the potency of different derivatization reagents was evaluated and p-toluenesulfonyl isocyanate (PTSI) was proven to be the optimal. The derivative generated by the reaction of betulin with PTSI was ionizable and fragmentable in the negative mode by liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI/MS/MS). Based on this chemical derivatization, an LC-ESI/MS/MS method was developed and validated for the quantification of betulin in rat plasma. The sample was extracted with ethyl acetate, derivatized with PTSI, separated on an ACQ UPLC BEH phenyl column, and analyzed in negative multiple reaction monitoring (MRM) mode. The calibration curve was linear over the betulin concentration range 2.5-200ng/mL. The lower limit of quantification was 2.5ng/mL. The inter- and intra-day accuracy and precision were within ±15%. Betulin recoveries were 86.7% or higher at three quality control levels (5, 50, and 160ng/mL). This validated method was subsequently applied to a pharmacokinetic study of betulin in rat plasma after oral administration.
仅含有一个或两个羟基的中性五环三萜类化合物，如白桦脂醇，不易通过电喷雾电离（ESI）进行离子化。然而，由于白桦脂醇具有反应性和中性，因此衍生化可能会提高离子化效率。在本研究中，评估了不同衍生化试剂的效力，证明对甲苯磺酰异氰酸酯（PTSI）是最佳的。白桦脂醇与 PTSI 反应生成的衍生物在负模式下可通过液相色谱-电喷雾电离/串联质谱（LC-ESI/MS/MS）进行电离和碎片化。基于这种化学衍生化，建立并验证了一种用于大鼠血浆中白桦脂醇定量的 LC-ESI/MS/MS 方法。样品用乙酸乙酯提取，用 PTSI 衍生化，在 ACQ UPLC BEH 苯基柱上分离，并在负多重反应监测（MRM）模式下进行分析。白桦脂醇的浓度范围为 2.5-200ng/mL 时，校准曲线呈线性。定量下限为 2.5ng/mL。日内和日间准确度和精密度均在±15%范围内。在三个质控水平（5、50 和 160ng/mL）下，白桦脂醇的回收率均在 86.7%或更高。该验证方法随后应用于大鼠口服给药后白桦脂醇的药代动力学研究。
