Hopanoids are triterpenoids produced mainly by bacteria, are ubiquitous in the environment, and have many important applications as biological markers. A wide variety of related hopanoid structures exists, many of which are polyfunctionalized. These modifications render the hopanoids too involatile for conventional gas chromatography (GC) separation, so require either laborious oxidative cleavage of the functional groups or specialized high temperature (HT) columns. Here we describe the systematic evaluation and optimization of a HT-GC method for the analysis of polyfunctionalized hopanoids and their methylated homologs. Total lipid extracts are derivatized with acetic anhydride and no further treatment or workup is required. We show that acid or base hydrolysis to remove di- and triacylglycerides leads to degradation of several BHP structures. DB-XLB type columns can elute hopanoids up to bacteriohopane-tetrol at 350 °C, with baseline separation of all 2-methyl/desmethyl homologs. DB-5HT type columns can additionally elute bacteriohopaneaminotriol and bacteriohopaneaminotetrol, but do not fully separate 2-methyl/desmethyl homologs. The method gave 2- to 7-fold higher recovery of hopanoids than oxidative cleavage and can provide accurate quantification of all analytes including 2-methyl hopanoids. By comparing data from mass spectra with those from a flame ionization detector, we show that the mass spectromet (MS) response factors for different hopanoids using either total ion counts or m/z 191 vary substantially. Similarly, 2-methyl ratios estimated from selected-ion data are lower than those from FID by 10-30% for most hopanoids, but higher by ca. 10% for bacteriohopanetetrol. Mass spectra for a broad suite of hopanoids, including 2-methyl homologs, from Rhodopseudomonas palustris are presented, together with the tentative assignment of several new hopanoid degradation products.

中文翻译：

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高温气相色谱-质谱法鉴定和定量多官能藿类化合物


Hopanoids是主要由细菌产生的三萜类化合物，在环境中普遍存在，并且作为生物标记物具有许多重要的应用。存在多种相关的藿香类结构，其中许多是多官能化的。这些修饰使得藿类化合物对于传统气相色谱 (GC) 分离来说太不挥发，因此需要对官能团进行费力的氧化裂解或专门的高温 (HT) 柱。在这里，我们描述了用于分析多官能藿烷类化合物及其甲基化同系物的 HT-GC 方法的系统评估和优化。总脂质提取物用乙酸酐衍生化，无需进一步处理或后处理。我们表明，酸或碱水解去除二酰基甘油酯和三酰基甘油酯会导致几种 BHP 结构的降解。 DB-XLB 型色谱柱可在 350 °C 下洗脱高达细菌藿烷四醇的藿烷类化合物，并可对所有 2-甲基/去甲基同系物进行基线分离。 DB-5HT型色谱柱还可洗脱细菌藿氨基三醇和细菌藿氨基四醇，但不能完全分离2-甲基/去甲基同系物。该方法的藿香类化合物回收率比氧化裂解高 2 至 7 倍，并且可以对包括 2-甲基藿香类化合物在内的所有分析物进行准确定量。通过将质谱数据与火焰离子化检测器的数据进行比较，我们发现使用总离子计数或 m/z 191 的不同藿类化合物的质谱 (MS) 响应因子差异很大。类似地，对于大多数藿类化合物，根据选择离子数据估计的 2-甲基比率比 FID 估计的 2-甲基比率低 10-30%，但高出约 10%。 10% 用于细菌希望四醇。 展示了来自沼泽红假单胞菌的一系列藿香类化合物（包括 2-甲基同系物）的质谱，以及几种新的藿香类化合物降解产物的初步归属。