High Sensitivity EtG and EtS Method Reduces Costs and Analysis Time

• Increase retention for ethyl glucuronide and ethyl sulfate in half the overall analysis time.
• Improve LC/MS sensitivity with increased organic mobile phase.
• Reduce cost of materials with minimal sample prep and short analytical column. 

Interest in alcohol testing using methods based on alcohol metabolites, instead of ethanol itself, is growing as the demand for longer term monitoring programs, such as substance abuse, parole, and corporate testing, increases. Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are ethanol metabolites that can be detected in urine for up to 80 hours, much longer than ethanol itself can be detected in the body. Typical methods for EtG and EtS analysis employ long C18 or phenyl-linked ether columns, a highly aqueous mobile phase, and tandem mass spectrometry. While commonly used, this approach has several inherent problems. Adequate retention is difficult to achieve, due to the highly polar nature of EtG and EtS (Figure 1). In addition, MS sensitivity is poor, due the highly aqueous mobile phase. Acetonitrile can be added to the column effluent to improve desolvation and MS sensitivity; however, this further complicates methodology that still produces only marginal retention. Here we establish new methodology—based on ion-pairing and an Ultra II™ Biphenyl column—that provides higher retention, faster analysis times, and improved MS sensitivity.

Figure 1: Structures of ethyl glucuronide and ethyl sulfate, two alcohol metabolites.

Increasing Retention through Ion Pairing

In order to improve retention for these highly polar target analytes, dihexylammonium acetate, a volatile, LC/MS-friendly ion-pairing reagent, was added to the mobile phase. During analysis, the ion-pairing reagent associates with charged analytes, masking their polarity and increasing retention (Figure 2). Upon entering the LC/MS interface, the pair dissociates, and only the unbound target analyte is detected. Although the use of ion-pairing reagents in HPLC is more common with UV detectors, which allow for nonvolatile mobile phase additives, several volatile ion-pairing reagents suitable for LC/MS analyses are commercially available.

As shown in Figure 3, retention is significantly increased over typical methods by using ion pairing in combination with a 50mm Ultra II™ Biphenyl column. Even though retention is increased, overall analysis time is roughly half that of conventional methods, due to the shorter column length. Although retention times for the two compounds are less than 5 minutes, the k’ values for both EtG and EtS are greater than 2 using this new methodology. Analytes with k’ values greater than 2 are more likely to be separated from matrix components that may interfere with analysis through ion suppression in the LC/MS interface. Note that on the Ultra II™ Biphenyl column EtS is very well separated from an isobaric matrix component.

Figure 2: Ion pairing increases retention in reversed phase HPLC.

Higher Sensitivity and Faster Analysis Times

Since retention was increased significantly with ion pairing, sensitivity could also be increased by eluting compounds from the column using a mobile phase containing a higher percentage of organic solvent. The method presented here uses a mobile phase containing 40% methanol, which allows for higher desolvation efficiency, thus improving sensitivity. Both EtG and EtS showed excellent response as measured by intensity (Figure 3).

Another benefit of increased retention is that a shorter column can be used, speeding up analyses and increasing sample throughput. The total run time (including re-equilibration time) on the 50mm Ultra II™ Biphenyl column is approximately half the time typically required when using a 150mm column under conventional conditions. Using a longer column not only adds extra material costs to an analysis, but it also can reduce sample throughput since it takes longer for all matrix components to elute and for the column to re-equilibrate. Cutting analysis time can save money by increasing laboratory throughput and reducing solvent usage.

Figure 3: Analyze alcohol metabolites faster and with greater sensitivity using Ultra II™ Biphenyl columns and ion pairing.

Compound m/z k' Intensity 1. matrix 125 — — 2. ethyl glucuronide 221 2.75 220,408 3. ethyl sulfate 125 4.02 236,693 Sample: Inj.: 5µL Conc.: 5µg/mL each component Sample diluent: urine diluted 1:10 with mobile phase Column: Ultra II™ Biphenyl (cat.# RE9609552) Dimensions: 50mm x 2.1mm Particle size: 5µm Pore size: 100Å Conditions: Instrument: Shimadzu Prominence UFLCXR Mobile phase: A: 5mM dihexylammonium acetate in water B: 5mM dihexylammonium acetate in methanol Time (min.) %B 0.0 40 2.5 40 2.6 100 5.4 100 5.5 40 7.5 40 Flow: 0.3mL/min. Temp.: 40°C Det.: Shimadzu LCMS-2010EV Ion source: ESI- Mode: SIM Source temp.: 200°C Source voltage: -4,000V

The ion pairing LC/MS method described here increases retention, allowing faster, more sensitive analysis of both EtG and EtS than is typically achieved using conventional methods. Greater sensitivity is obtained with a less expensive column and minimal sample preparation. Using this method on the Ultra II™ Biphenyl column can reduce the cost of analysis and substantially increase sample throughput.