Helander Anders, Zheng Yufang
Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden.
Clin Chem. 2009 Jul;55(7):1395-405. doi: 10.1373/clinchem.2008.120923. Epub 2009 May 7.
The alcohol biomarker phosphatidylethanol (PEth) comprises a group of ethanol-derived phospholipids formed from phosphatidylcholine by phospholipase D. The PEth molecular species have a common phosphoethanol head group onto which 2 fatty acid moieties are attached. We developed an electrospray ionization (ESI) LC-MS method for qualitative and quantitative measurement of different PEth species in human blood.
We subjected a total lipid extract of whole blood to HPLC gradient separation on a C4 column and performed LC-ESI-MS analysis using selected ion monitoring of deprotonated molecules for the PEth species and phosphatidylpropanol (internal standard). Identification of individual PEth species was based on ESI-tandem mass spectrometry (MS/MS) analysis of product ions.
The fatty acid moieties were the major product ions of PEth, based on comparison with PEth-16:0/16:0, 18:1/18:1, and 16:0/18:1 reference material. For LC-MS analysis of different PEth species in blood, we used a calibration curve covering 0.2-7.0 micromol/L PEth-16:0/18:1. The lower limit of quantitation of the method was <0.1 micromol/L, and intra- and interassay CVs were <9% and <11%. In blood samples collected from 38 alcohol patients, the total PEth concentration ranged between 0.1 and 21.7 micromol/L (mean 8.9). PEth-16:0/18:1 and 16:0/18:2 were the predominant molecular species, accounting for approximately 37% and 25%, respectively, of total PEth. PEth-16:0/20:4 and mixtures of 18:1/18:1 plus 18:0/18:2 (not separated using selected ion monitoring because of identical molecular masses) and 16:0/20:3 plus 18:1/18.2 made up approximately 13%, 12%, and 8%.
This LC-MS method allows simultaneous qualitative and quantitative measurement of several PEth molecular species in whole blood samples.
酒精生物标志物磷脂酰乙醇（PEth）是一组由磷脂酶D作用于磷脂酰胆碱形成的乙醇衍生磷脂。PEth分子种类具有一个共同的磷酸乙醇头部基团，其上连接有两个脂肪酸部分。我们开发了一种电喷雾电离（ESI）液相色谱 - 质谱法，用于定性和定量测定人血液中不同的PEth种类。
我们将全血的总脂质提取物在C4柱上进行HPLC梯度分离，并使用对PEth种类和磷脂酰丙醇（内标）的去质子化分子进行选择离子监测来进行LC - ESI - MS分析。单个PEth种类的鉴定基于产物离子的ESI串联质谱（MS/MS）分析。
与PEth - 16:0/16:0、18:1/18:1和16:0/18:1参考物质相比，脂肪酸部分是PEth的主要产物离子。对于血液中不同PEth种类的LC - MS分析，我们使用了覆盖0.2 - 7.0微摩尔/升PEth - 16:0/18:1的校准曲线。该方法的定量下限<0.1微摩尔/升，批内和批间变异系数分别<9%和<11%。在从38名酒精患者采集的血液样本中，总PEth浓度范围为0.1至21.7微摩尔/升（平均8.9）。PEth - 16:0/18:1和16:0/18:2是主要的分子种类，分别占总PEth的约37%和25%。PEth - 16:0/20:4以及18:1/18:1加18:0/18:2的混合物（由于分子量相同，使用选择离子监测未分离）和16:0/20:3加18:1/18.2约占13%、12%和8%。
这种LC - MS方法能够同时对全血样本中的几种PEth分子种类进行定性和定量测定。
