We have compared and assessed the suitability of several chromatographic methods for the analysis of long chain alkenones and long chain diols and the associated paleotemperature proxies (U^K’₃₇ and LDI). We evaluated the traditional methods for the analysis of the U^K’₃₇ and the LDI, gas chromatography (GC) − flame ionization detection (FID) and GC mass spectrometry (MS) using selected ion monitoring (SIM), respectively, and developed a new method using GC–MS/MS in multiple reaction monitoring mode (MRM) for the analysis of long chain diols as well as a method for automatic silylation of diols using a robot autosampler. Finally, we evaluated liquid chromatography (LC) methods to simultaneously measure the U^K’₃₇ and the LDI, using ultra high performance LC (UHPLC) with low (nominal mass) resolution MS in SIM mode, and UHPLC with high resolution MS (HRMS). Detection and quantification limits and reproducibility were assessed by means of serial dilutions of culture extracts.

Automated silylation by a robot autosampler showed similar reproducibility as off-line silylation while substantially decreasing sample preparation time. The novel MRM method had a slightly lower limit of quantification (LOQ; i.e. 0.3 pg C₂₈ 1,13-diol injected on-column) than the traditional method (0.5 pg) and improved reproducibility while allowing more unambiguous identification of LCDs in complex matrices. For diols, UHPLC–MS using SIM had the highest LOQ (i.e. 15 pg) and a comparable reproducibility as GC–MS. UHPLC–HRMS had a LOQ of ca. 1.5 pg, and an improved reproducibility for diol analysis. For alkenone analysis, both UHPLC–HRMS and UHPLC–MS using SIM were 2–3 orders of magnitude more sensitive (LOQ ca. 20 and 2 pg C_37:2 alkenone injected on-column, respectively) than GC-FID (LOD ca. 3 ng), with a similar reproducibility of the U^K’₃₇ index. Hence, UHPLC–HRMS allows simultaneous analysis of the U^K’₃₇ and LDI at an increased sensitivity. In addition, it allows simultaneous measurement of TEX₈₆, a temperature proxy based on the isoprenoid glycerol dialkyl glycerol tetraethers. This reduces the preparation time by excluding the need of derivatization and separation of the ketone (containing the long chain alkenones) and polar fractions (containing the long chain diols and GDGTs). However, synthetic standards are required to fully assess the accuracy of the new methods for determination of the LDI and U^K’₃₇.

我们比较和评估的长链烯酮和长链二醇和相关联的古温度代理（U的分析几个色谱方法的适用性^K” ₃₇和LDI）。我们评价了在U的分析的传统方法^K” ₃₇和LDI，气相色谱（GC） -使用所选择的离子监测（SIM），分别火焰离子化检测器（FID）和GC质谱（MS），并开发出一种在多反应监测模式（MRM）中使用GC-MS / MS进行长链二醇分析的新方法，以及使用自动进样器自动对二醇进行甲硅烷基化的方法。最后，我们评估了液相色谱（LC）方法来同时测量在U ^K” ₃₇和LDI，使用SIM模式下具有低（标称质量）分辨率MS的超高性能LC（UHPLC）和高分辨率MS（HRMS）的UHPLC。通过连续稀释培养物提取物来评估检测和定量限以及可重复性。

机器人自动进样器的自动甲硅烷基化显示了与离线甲硅烷基化相似的重现性，同时大大减少了样品制备时间。与传统方法（0.5 pg）相比，新的MRM方法的定量限（LOQ；即在柱上进样0.3 pg C ₂₈ 1,13-二醇）略低，并且具有更高的重现性，同时可以更明确地鉴定复杂基质中的LCD 。对于二醇，使用SIM的UHPLC-MS具有最高的LOQ（即15 pg），并且具有与GC-MS相当的重现性。UHPLC–HRMS的LOQ约为。1.5 pg，提高了二醇分析的重现性。对于烯酮分析，使用SIM的UHPLC-HRMS和UHPLC-MS灵敏度都提高了2-3个数量级（LOQ约为20和2 pg C _37：2烯酮分别注入柱上，）比GC-FID（LOD约3纳克），与U的一个类似的可重复性^K” ₃₇索引。因此，UHPLC-HRMS允许U形的同时分析^K” ₃₇以增加的灵敏度和LDI。此外，它还允许同时测量TEX ₈₆，TEX ₈₆是基于类异戊二烯甘油二烷基甘油四醚的温度替代物。通过排除对酮（含有长链烯酮）和极性级分（含有长链二醇和GDGT）的衍生和分离的需要，可以减少制备时间。然而，需要合成的标准，以充分评估的新方法的精度测定的LDI和U的^K” ₃₇。