Phosphatidylethanol (PEth) in human blood samples is a marker for alcohol usage. Typically, PEth is detected by reversed-phase liquid chromatography coupled with negative ion tandem mass spectrometry, investigating the fatty acyl anions released from the precursor ion upon collision-induced dissociation (CID). It has been established that in other classes of asymmetric glycerophospholipids the unimolecular fragmentation upon CID is biased depending on the relative position (known as sn-position) of each fatty acyl chain on the glycerol backbone. As such, the use of product ions in selected-reaction-monitoring (SRM) transitions could be prone to variability if more than one regioisomer is present in either the reference materials or the sample. Here, we have investigated the regioisomeric purity of three reference materials supplied by different vendors, labelled as PEth 16:0/18:1. Using CID coupled with ozone-induced dissociation, the regioisomeric purity (% 16:0 at sn-1) was determined to be 76%, 80% and 99%. The parallel investigation of the negative ion CID mass spectra of standards revealed differences in product ion ratios for both fatty acyl chain product ions and ketene neutral loss product ions. Furthermore, investigation of the product ion abundances in CID spectra of PEth within authentic blood samples appears to indicate a limited natural variation in isomer populations between samples, with the cannonical, PEth 16:0/18:1 (16:0 at sn-1) predominant in all cases. Different reference material isomer distributions led to variation in fully automated quantification of PEth in 56 authentic dried blood spot (DBS) samples when a single quantifier ion was used. Our results suggest caution in ensuring the regioisomeric composition of reference materials are well-matched with the authentic blood samples.

中文翻译：

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合成和生物衍生的磷脂酰乙醇的相对异构体丰度变化及其对可靠定量的影响

人体血液样本中的磷脂酰乙醇（PEth）是酒精使用的标志。通常，PEth通过反相液相色谱与负离子串联质谱联用进行检测，研究碰撞诱导解离（CID）后从前体离子释放的脂肪酰基阴离子。已经确定，在其他类型的不对称甘油磷脂中，CID上的单分子片段会根据相对位置（称为sn-位）在甘油主链上的每个脂肪酰基链。因此，如果在参考物质或样品中存在一种以上的区域异构体，则在选择反应监测（SRM）过渡中使用产物离子可能会产生变异性。在这里，我们研究了由不同供应商提供的三种参考物质（标记为PEth 16：0/18：1）的区域异构纯度。使用CID结合臭氧诱导的解离，区域异构体纯度（在sn-1处为％16：0确定为76％，80％和99％。对标样的负离子CID质谱的平行研究显示，脂肪酰基链产物离子和乙烯酮中性损失产物离子的产物离子比率存在差异。此外，对真实血液样品中PEth的CID谱中产物离子丰度的研究似乎表明样品之间异构体群体的自然变化有限，其中规范的PEth为16：0/18：1（sn-1为16：0）在所有情况下都占主导。当使用单个定量离子时，不同的参考物质异构体分布会导致56个真实的干血斑（DBS）样品中PEth的全自动定量变化。我们的结果建议谨慎，以确保参考物质的区域异构体组成与真实的血液样本完全匹配。