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Extended Lifetime Ethanol Metabolites and Method Development for Ethyl Sulfate and Ethyl Glucuronide Separation

In this study methods are presented for ethanol metabolites ethyl glucuronide (EtG) and ethyl sulfate (EtS)

Alcohol abuse remains an epidemic causing widespread health, legal, and economic impacts. Extreme abuse has escalated in volume and cases of alcohol abuse are consistently increasing in younger and younger populations. It is estimated that in 2013 over 17 million citizens in the United States exhibited clinical Alcohol Use Disorders (AUDs).1 Only a fraction of these individuals seek treatment (near 8%) resulting in a rapid growth in long term health effects.1 In addition to the detrimental impact on human health due to long term abuse of alcohol, the acute impact on human lives resulting from alcohol misuse is dramatic. In 2014, over 1.1 million arrests were made for driving under the influence of alcohol or narcotics.2 It is estimated that approximately 25% of these arrests were repeat offenders.2  Approximately 88,000 deaths can be attributed to alcohol misuse annually in the United States.3 Increases in the number of addiction treatment centers and judicial pressure have started to shift the numbers in a more positive direction in the past several years. In addition, improved testing methodologies and discovery of longer lifetime ethanol biomarkers have contributed to this trend by improving the accuracy and testing timelines for court appointed abstinence and therapeutic abstinence monitoring..

The growing volume of chronic abuse, increased number of treatment centers/participants, and increased number of repeat offenders of DUI have significantly increased the volume of ethanol testing for both forensic and clinical implications. The short elimination timeline of ethanol in the body makes direct monitoring of ethanol in the blood or urine a non-ideal target when routine monitoring. Monitoring in a therapeutic environment or ensuring the lack of consumption for individuals prohibited from ethanol consumption by the courts can be accomplished through less frequent testing provided longer lifetime biomarkers.  Increasingly, long lifetime ethanol metabolites have become key biomarkers for alcohol treatment program monitoring and zero tolerance abstinence enforced by the courts. Several biomarkers have been demonstrated to have utility in these settings and each have unique benefits.

Ethanol Biomarkers

Ethanol consumption/effect monitoring can be accomplished by either examining ethanol and its metabolites directly or by monitoring the toxic impact that the consumption of ethanol has on the body and the body’s systems. These two classifications of ethanol biomarkers are commonly referred to as direct and indirect biomarkers respectively.4

Indirect Biomarkers

Indirect biomarkers are valuable when monitoring  ethanol abusers for the severity of a chronic disorder, relapse,  and the prognosis/diagnosis of organ damage that the excessive alcohol consumption has caused. The toxic effects of ethanol consumption can be seen in many organ systems but the obvious source of greatest impact is the liver. Increasing presence of liver enzymes in the blood, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma glutamyltransferase (GGT), are indicative of organ damage.5,6 These enzymes are in high concentration in hepatocytes and the release of these enzymes into the bloodstream can be due to liver injury and cell death.6,7 Ambiguity can arise from such elevated levels due to the heterogeneity in individuals as well as the determination of the source of the liver damage. Liver enzyme panels will exhibit elevated levels due to non-alcohol related disease states such as non-alcoholic fatty liver disease, liver cancer metastasis, Hepatitis etc. In addition, the liver damage is the result of longer term abuse and thus short term or binge drinking and abusive drinking in younger individuals may not be adequately monitored via these biomarkers.

Carbohydrate deficient transferrin (CDT) is a prevalent indirect biomarker as it exhibits enhanced specificity and sensitivity versus that of common liver enzyme levels in the blood. Transferrin is a glycoprotein responsible for transporting iron through the bloodstream. This glycoprotein can contain zero, one, or two branched carbohydrate chains terminated with varying levels of sialic acid residues.5,6,7 Alcohol abuse is speculated to negatively impact carbohydrate synthesis due to its short term metabolite, acetaldehyde, and in addition chronic alcohol abusers exhibit lower levels of sialic acid, affecting the transferrin structure. This biomarker demonstrates significant advantages in specificity as the levels are not typically elevated in other disease states. CDT also exhibits a long lifetime in the system of nearly a month and will remain after weeks of abstinence. However, CDT demonstrates a longer term onset and is a more clinically valuable biomarker for chronic abusers rather than short term binge drinking abusers as elevated levels are not distinguishable for subjects abusing alcohol in short spurts.7

Direct Biomarkers

Issues with the cause and effect determination for indirect biomarkers as biomarkers of ethanol consumption are overcome by the direct monitoring of ethanol and its metabolites. Ethanol and its metabolites are also rapidly detectable breath, urine, and/or blood allowing for positive indication immediately following consumption.

Most commonly, ethanol itself is monitored as the indicator of intoxication or very recent consumption. Elimination rates and fast metabolism of ethanol results in a very small detection window for direct monitoring of ethanol in any matrix. Breath analysis, urinalysis and blood analysis are only viable for assess a current level of intoxication and all matrices exhibit less than a 10 hour window after ingestion to provide an appreciable result.8,9 This detection window is not effective for monitoring subjects for court appointed abstinence or monitoring in a therapeutic environment for recurrence as testing would occur daily to be effective.

To accomplish a less frequent test course for continuous monitoring while ensuring continual coverage, longer lifetime biomarkers are required. Although the vast majority of ethanol is metabolized rapidly into compounds that have no clinical value for direct monitoring, a small number of secondary metabolism routes produce several uniquely beneficial biomarkers.

Ethanol Metabolites

Enzyme Pathways for Ethanol Metabolism

Figure 1: Enzyme Pathways for Ethanol Metabolism

 

The primary route of ethanol metabolism (nearly 95%) is the rapid oxidation of ethanol to acetaldehyde. This occurs by three mechanisms 1) Alcohol dehydrogenase in hepatocytes, 2) Catalase, 3) Cytochrome P450 Enzyme CYP2E1  followed by the further oxidation of acetaldehyde to acetic acid by aldehyde dehydrogenase. Acetic acid is then either eliminated or further metabolized through the TCA cycle. Nearly all of the remaining 5% of ethanol is excreted either in urine, sweat, or breath. However, a very small fraction (less than 1%) is metabolized by non-oxidative pathways.10 These minor metabolism mechanisms have been demonstrated to result biomarkers such as  fatty acid ethyl esters (FAEEs), ethyl glucuronide (EtG), ethyl sulfate (EtS), and phosphatidylethanol (PEth) which exhibit longer lifetimes in the system and can be detected in several  biological sample types. (Metabolism Pathways demonstrated in Figure 1)

Ethyl glucuronide is formed through the conjugation of glucuronic acid and ethanol through the activity of the enzyme UDP-glucuronosyltransferase which is then eliminated through the kidney.11 Similarly EtS is formed through enzyme catalyzed conjugation catalyzed by the enzyme sulfotransferase.12

 EtG and EtS has been demonstrated to be able to be measured in subjects within 24 hrs even in cases of light drinking (1-2 drinks) and for up to for up to 5 days after last drink due to slowed elimination rates of the metabolites and high sensitivity.11,12,13 EtG and EtS can also be measured in post-mortem subjects for a prolonged period of time and can be measured in hair, urine and blood effectively.14,15 These biomarkers are often detected using LC-MS methodologies or commercially available immunoassays.

Unfortunately, due to the high sensitivity of these two biomarkers, concern has arisen and case decisions overturned due to false positive results due to exposure to over-the-counter alcohol containing substances such as hand sanitizers and mouthwashes.15,16,17 Although the these biomarkers account for only less than 1% of the ethanol metabolized, EtG and EtS levels have been detected in samples from subjects that consumed 1 alcoholic drink or used hand sanitizer containing ethanol that day.15,16 Due to this potential for false positive results, cutoff points are still being evaluated however levels as high as 500 ng/mL can be caused by exposure to ethanol products and not necessarily due to consumption.4,5

Alternatively, Phosphatidylethanol (PEth)  metabolites are excellent biomarkers for monitoring relapse in patients in treatment programs as they are only formed through significant alcohol consumption and exhibit long windows of detection (up to as much as 2-3 weeks).5,18 PEth biomarkers are a collection of potentially 48 phospholipid homologs that are formed through the activity of phospholipase D on phospholipids in the presence of ethanol.5  In the absence of ethanol, phospholipase D activity results in the formation of phosphatidic acids but in the presence of ethanol, the enzyme has a strong binding affinity for ethanol over water that results in the formation of PEths instead of the hydrolysis product.5 Although 48 PEth biomarkers have been identified, most test for the two most prominent 16:0/18:1 and 16:0/18:1 homologs.4,5 The biomarkers rise in concentration in as short as 1-3 hrs after consumption but peak between 3 and 6 days after consumption.19 Levels of PEth biomarkers are significantly raised for chronic alcohol abusers versus short term binge drinks or moderate drinkers and thus PEth has been most frequently used as an indicator of relapse for chronic alcoholics.20 21,22

Analytical Method Development

Each of the described direct biomarkers demonstrate traits that make each uniquely suited for particular ethanol consumption monitoring situations. From abstinence zero-tolerance monitoring, to chronic abuse relapse monitoring, to transplant centers, and post-mortem forensic testing, the most appropriate biomarker must be chosen based on its sensitivity, onset time, window of detection, and sample source based on the situation.

Many have adopted immunoassay methods for the detection of direct biomarkers due the availability of the instrumentation in laboratories, commercial availability of biomarker kits,  and the low cost. However, mass spectrometry methods are gaining prominence to overcome shortcomings of immunoassay methods with regard to sensitivity and specificity. Gas chromatographic (GC) methods have demonstrated utility however GC requires derivatization and exhibits relatively long run times.  High performance liquid chromatography coupled with mass spectrometry (HPLC-MS) has become the emerging method of choice to improve throughput, sensitivity and accuracy.

The following applications that have been developed by our researchers in Bellefonte, PA demonstrate the benefit in the use of Hydrophilic Interaction Liquid Chromatography (HILIC) mode of separation for EtS, and EtG analysis from urine.

HILIC versus Reversed-Phase Chromatography for the Analysis of EtS and EtG

Reversed-phase liquid chromatography (RPLC) relies on the partitioning of analytes from the aqueous-rich mobile phase into the organic-rich environment of the stationary phase.  Under a given set of conditions, the more hydrophobic an analyte is, the more it will associate with the stationary phase.  Hydrophobic compounds, therefore will retain well using RPLC.  EtG and EtS, however, are highly polar molecules and therefore do not retain well under RPLC conditions.  HILIC chromatography is a complex system involving partition, polar and ion-exchange interactions. In HILIC, retention based on partition results when polar molecules partition from the organic-rich mobile phase into the aqueous-rich environment of the stationary phase.  Therefore in HILIC the more polar the molecule, generally, the more retention is possible.  Most methods currently in use are based on RPLC where EtS and EtG elute along with many other poorly retained urine components.  Although adequate results are often obtained, matrix effects from coeluting species is common.  In an attempt to improve robustness, repeatability and reliability, HILIC chromatography was explored.  EtS and EtG were found to retain and separate well using an Ascentis Express OH5 column.

The methods developed also took advantage of the benefits of the  Ascentis® Express line of analytical columns which is a superficially porous particle (SPP) platform that greatly improves the mass transfer efficiency of analytes in comparison to traditional porous particles while also improving the packing efficiency due to narrower particle size distribution. Columns using a similar particle platform were compared for the separation of EtG and EtS in a urine matrix to compare the selectivity of OH5 phase versus C18.

Comparison of Ascentis Express Fused-Core® Particle Technology to Traditional Porous Silica Particles.

Fig. 2  Comparison of Ascentis® Express Fused-Core® Particle Technology to Traditional Porous Silica Particles.

Experimental

The comparison was demonstrated for the analysis of EtG and EtS between a competitor’s C18 fused core particle analytical column versus the Ascentis Express OH5 column. A 10 cm X 2.1 mm column OH5 column (Product No. 53757-U) with  2.7 𝛍m particle size was compared to a competitor’s 15 cm X 2.1 mm column with a 1.8 𝛍m particle with C18 stationary phase. The separation conditions were optimized on standard preparations. The optimized conditions for the reversed-phase C18 and the HILIC OH5 separation are shown below:

 

HILIC Method Conditions
Instrument: Waters Acquity QDa
Column: Ascentis Express OH5, 10 cm x 2.1 mm, 2.7 µm particle size
Mobile phase: [A] 5 mM ammonium formate and 0.1% formic acid in 95:5, acetonitrile:water; [B] 5 mM ammonium formate and 0.1% formic acid in 80:20, acetonitrile:water
Gradient: 0 to 100% B in 1 min, held at 100% B for 4 min, re-equilibrate 0% B for 5 min
Flow Rate: 0.4 mL/min
Pressure: 1450 psi (100 bar)
Temperature: 30°C
Detection: MS, ESI(-), SIR,125 m/z, 221 m/z
Injection: 10 µL

 

Reversed-Phase Conditions
Instrument: Waters Acquity QDa
Column: Competitor C18, 15 cm x 2.1 mm, 1.8 µm particle size
Mobile phase: [A] water with 0.05% formic acid; [B] acetonitrile
Gradient: 1% B held for 1 min, to 10% B in 2.6 min, to 100% B in 0.01 min, held for 0.5 min, re-equilibrate 1% B for 2.9 min
Flow Rate: 0.4 ml/min
Pressure: 7010 psi (483 bar)
Temperature: 50°C
Detection: MS, ESI(-), SIR,125 m/z, 221 m/z
Injection: 10 µL

The example chromatograms and experimental parameters are depicted below in Figures 3 and 4.

Example Chromatogram of Ethyl Sulfate Standards on A) C18 and B) HILIC Columns

Figure 3. Example Chromatogram of Ethyl Sulfate Standards on A) C18 and B) HILIC Columns

 

Example Chromatograms of Ethyleglucuronide Standards on A) C18 and B) HILIC columns

Figure 4. Example Chromatograms of Ethyleglucuronide Standards on A) C18 and B) HILIC columns

 

The optimized methods were then utilized to assess the resolution, recovery, and reproducibility of the quantitation of EtS and EtG in synthetic urine. Deuterated standards were used to improve accuracy. For the reversed-phase C18 method the standards were diluted 1:9 in 0.05% formic acid. For the HILIC OH5 sample the calibration solutions were diluted 1:19 in acetonitrile. Calibration standards spanned from 50 to 2,000 μg/mL prior to dilution.

Results

The retention factors (k’) for both EtS and EtG are provided in Table 1.  Retention values were significantly increased using the HILIC conditions as compared to RPLC. Most significantly EtS peak exhibited an increase in retention factor from a non-ideal 0.89  to a more favorable value of 1.47.

Table 1. Comparison of Retention Factors (K') for EtS and EtG on RP and HILIC Columns

  Reversed-Phase (C18) HILIC (OH5)
EtS 0.89 1.47
EtG 1.24 4.39

 

The improved retention in HILIC mode allows one to more accurately quantitate the ethanol metabolites in a urine matrix as the increased retention allows for greater separation amongst the large amount of hydrophilic interferents found in a urine matrix and a higher probability of separation of endogenous species from the analytes of interest. This increased affinity for early eluting compounds resulted in baseline resolution for the two metabolite targets in the synthetic urine matrix. This improved retention and resolution in a urine matrix results in a more robust and reliable method when compared to C18 reverse phase separations and results in high recovery rates of both analytes. The chromatograms below (Figure 5) demonstrate this separation in a urine matrix monitoring two ions by selected ion monitoring.

Example Chromatogram Demonstrating the Separation and Detection of EtS and EtG in Urine

Figure 5. Example Chromatogram Demonstrating the Separation and Detection of EtS and EtG in Urine Matrix using HILIC LC/MS

 

The optimized methods were also utilized to evaluate the reproducibility and the consistency of calibration using the OH5 column for the quantitation of various concentrations of EtS and EtG. A calibration curve using the OH5 column is depicted below in Figure 6. While incorporating the matrix the analytical correlation was quite strong and robust demonstrated by the R2 of 0.9998 and 0.9986 for EtS and EtG respectively.

Calibration Curves for A) EtS and B) EtG on Using HILIC Method

Figure 6. Calibration Curves for A) EtS and B) EtG on Using HILIC Method

 

When the same calibration curve was taken using the C18 column the reproducibility of peak area consistency at low concentrations suffered significantly. The relative standards deviations of the peak areas of the deuterated standards in the synthetic urine for three separate samples for each analyte concentration are captured in Table 2. A high level of variability peak area of low concentrations of EtS for the C18 column is evident in comparison to consistent results across both analytes for the OH5 column. This is directly attributed to coelution of low retention matrix interferents and EtS on the hydrophobic C18 phase.

EtS EtG
Concentration before dilution (µg/mL) C18 RSD OH5 RSD Concentration before dilution (µg/mL) C18 RSD OH5 RSD
50 25.6% 3.0% 250 1.9% 0.2%
100 26.6% 1.8% 500 1.6% 2.2%
200 26.9% 3.4% 1000 2.2% 3.2%
500 25.9% 0.9% 2000 0.9% 2.7%
1000 5.9% 1.8% 5000 1.4% 1.0%
2000 3.9% 1.1% 10000 0.8% 0.1%

Conclusions

Ethanol biomarkers with extended lifetimes in the body have had a tremendous impact on the testing landscape for alcohol abuse treatment centers and forensic toxicologists in recent years. Several of the biomarkers demonstrate unique utility in the continuous and extended monitoring of patient ethanol consumption patterns. EtG and EtS are examples of two of the most prevalent longer lifetime biomarkers gaining attention in these testing communities. Method development for the analytical separation of these two key biomarkers has been demonstrated on HILIC mode and common reverse phase chromatography columns in a human urine matrix. Benefits of the stronger retention of polar analytes and interferents of the urine matrix are clearly demonstrated and baseline resolution is obtained for the two analytes while demonstrating a increased consistency in quantification in this complicated background. The methods developed here could improve the throughput, accuracy, and  consistency of many labs utilizing non-ideal separation conditions for these now high volume assays.

Legal Information

Ascentis is a registered trademark of Sigma-Aldrich Co. LLC
Fused-Core is a registered trademark of Advanced Materials Technology, Inc.

Materials

     

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