Simultaneous determination of ethanol's four types of non-oxidative metabolites in human whole blood by liquid chromatography tandem mass spectrometry

Highlights

• Ten ethanol non-oxidative metabolites in human whole blood with a wide range of polarity were analyzed simultaneously.

• Small sample consumption (50 μL) was used, making it suitable in cases with limited specimens.

• Different extraction procedures (liquid-liquid extraction, solid-phase extraction and protein precipitation) were compared.

• The proposed method was applied to two authentic samples, in which the metabolites provided critical information.

Abstract

The importance of ethanol non-oxidative metabolites as the specific biomarkers of alcohol consumption in clinical and forensic settings is increasingly acknowledged. Simultaneous determination of these metabolites can provide a wealth of information like drinking habit and history, but it was difficult to achieve because of their wide range of polarity. This work describes development and validation of a simple liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for 4 types of ethanol non-oxidative metabolites (ethyl glucuronide, ethyl sulfate, fatty acid ethyl esters and phosphatidylethanols) in 50 μL of human whole blood. Pretreatment method, column and MS conditions were optimized. For the first time, the four types of ethanol non-oxidative metabolites with enormous discrepancies of property were simultaneously extracted and analyzed in one run within 40 min. The limits of detections (LODs) were among 0.1–10 ng/mL, and good linearity was obtained. Deviations in precision and accuracy were all lower than 15% at three QC levels. This method was then applied to two forensic samples, resulting in information on drinking habits and drinking time which were very useful for the interpretation of the blood alcohol results.

Introduction

Alcohol is a substance that is abused worldwide. It is closely related to many social and public problems. In particular, alcohol plays a major role in civil and criminal litigation, especially in traffic accidents and crimes of violence [1]. The direct and standard way for evaluating alcohol consumption is the measurement of blood alcohol concentration (BAC) by headspace gas chromatography (HS-GC) [2]. Although this measurement can provide direct evidence to the cases related to alcohol consumption, a major limitation is the short ethanol detection window of <8 h in blood due to the rapid elimination [3], [4]. In traffic cases, some perpetrators choose to hit-and-run to avoid legal prosecution. Meanwhile, in alcohol facilitated sexual assault cases, victims often report the incident after a delayed period of time. Under such circumstances, BAC may decline to zero at the time of confession or police interception, resulting in an unreliable, negative alcohol test. Instead, determination of ethanol non-oxidative metabolites may solve this problem because of their high specificity and longer detection windows [5].

After intake, approximately 0.1% alcohol undergoes non-oxidative metabolism to form ethyl glucuronide (EtG), ethyl sulfate (EtS), fatty acid ethyl esters (FAEEs) and phosphatidylethanols (PEths) [6]. These metabolites allow detection of alcohol intake ranging from several hours to several days, or even up to weeks. EtG and EtS have longer detection windows (>18 h in blood, longer at higher dose) than ethanol [5], [7], which both provide high sensitivity and specificity for recent ethanol consumption [8]. FAEEs have detection windows of approximately 24 h in blood [5], and they have received much attention partly because of their cytotoxicity [9]. PEths are abnormal phospholipids formed only after alcohol consumption [10] and they possess detection windows longer than 7 days, and can be even longer for chronic alcohol abusers [5]. PEths are reliable markers of chronic and excessive alcohol consumption [11]. Because of their different detection windows compared to ethanol and other characteristics such as high specificity, these ethanol non-oxidative metabolites have received increased attention in recent years [12].

In current analytical methods of ethanol non-oxidative metabolites, only one or two types of metabolites can be determined [5]. The enormous discrepancies in physicochemical property between the four types of metabolite make their simultaneous detection very challenging. However, analysis of only one or two metabolites may not cover the whole relevant detection window or provide important information on evidence of drinking time and/or drinking habits. For example, positive EtG and EtS results can only provide evidence for recent drinking, but cannot provide information on drinking history or chronicity of alcohol consumption. To get more information, several different methods need to be conducted for determination of all relevant target compounds. This is time-consuming and labor demanding, and requires large sample consumption. It is not applicable for urgent cases or cases involving drug screening besides alcohol detection, which are often restricted by the amount of specimen and limit time for final statement/reporting all drug findings.

The objective of this study is therefore to establish a new method for the simultaneous analysis of ethanol non-oxidative metabolites, specifically EtG, EtS, six FAEEs (ethyl myristate (E14:0); ethyl palmitate (E16:0); ethyl stearate (E18:0); ethyl oleate (E18:1); ethyl linoleate (E18:2), ethyl arachidonate (E20:4)) and two PEths (PEth 16:0/18:1; PEth 18:1/18:1). Human whole blood was selected as the specimen because it is used for the BAC testing and it is the only suitable specimen for analyzing PEths, which are groups of abnormal phospholipids in cell membranes mainly located in erythrocytes [13]. The developed method was then validated and applied to samples from two special forensic cases. In these cases BAC could not provide more useful information whereas determination of ethanol's metabolites provided the necessary evidence. Concentrations of these metabolites were interpreted and estimations of drinking time and habit were also determined.