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A method is illustrated for the simultaneous analysis of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in human urine samples. The method described uses a simple dilution of the urine sample followed by fast (<7-min) liquid chromatography–tandem mass spectrometry (LC–MS-MS) analysis with detection below 25 ng/mL. When tested with a large number of patient samples, the method was found to be rugged, reproducible, and free of interference by matrix contaminants.

Monitoring illicit drug use through laboratory testing has become a cornerstone in reducing substance abuse and focusing efforts on those stricken with addiction issues. Although many methods have been developed for detecting commonly abused drugs, monitoring individuals for alcohol consumption is hampered by the rapid metabolism of alcohol which makes abuse undetectable. Until recently this rapid metabolism has limited alcohol testing to a few hours after drinking stops. New developments have made it possible to detect alcohol abuse in the 3–5 days preceding the test by identifying the presence of alcohol metabolites. This detetection is especially important in monitoring alcohol use in settings in which alcohol use is prohibited: alcoholism and drug abuse treatment, professional assistance programs, abstinence as a condition of probation, child custody issues, or for people under the legal drinking age (1).

Beverage alcohol, also known as ethanol, is metabolized by the body through several pathways. One pathway metabolizes ethyl alcohol to ethyl glucuronide (EtG), and another to ethyl sulfate (EtS). Normally when alcohol is metabolized in the body, most is eliminated by enzymes that are part of the glycolysis pathway that converts sugar to cellular energy. An enzyme associated with the pathway, alcohol dehydrogenase, "takes away" electrons (oxidation) from the alcohol molecules, which results in final products of water and carbon dioxide and gives no information regarding alcohol consumption. EtG and EtS, however, are formed by "nonoxidative" pathways that, instead of breaking down the alcohol molecules, make them more complex by combining them with larger molecules, which makes it nontoxic. These nonoxidative metabolites, in the case of alcohol, EtG, and EtS, are then slowly eliminated in the urine, making them excellent markers for recent alcohol consumption (1). 

The presence of EtG or EtS in urine is indicative that alcohol was consumed within the previous 3–4 days. Thus, EtG and EtS levels are a more accurate indicator of alcohol consumption over the course of several days, which makes the approach more amenable to long-term monitoring versus direct blood alcohol measurements. The detection of the combination of EtG and EtS in parallel offers greater sensitivity and accuracy for determination of recent ethanol ingestion than by detection of either biomarker alone. Finally, the identification of both EtG and EtS in a patient sample ensures that ethanol was ingested and was not produced as a result of 

Methods to detect EtG and EtS include immunoassay, gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS). Immunoassays have limited specificity and sensitivity with a limit of detection of approximately 1000 ng/mL. GC–MS offers more specificity than immunoassays with detection limits of approximately 500 ng/mL. LC–MS methods have improved specificity and detection limits to less than 

For alcohol, EtG and EtS cutoffs have become controversial because of the large number of widely used antimicrobial products (that is, gel hand cleaners) that contain alcohol. As a result, most regulatory entities have set cutoffs for EtG between 100 ng/mL and 1000 ng/mL and for EtS between 25 ng/mL and 100 ng/mL for reporting of an adverse finding. Our goal was to develop a specific and sensitive LC–MS-MS method for detecting EtG and EtS that fulfills these detection cutoffs.

All reagents and solvents were high performance liquid chromatography (HPLC) grade or analytical grade. HPLC-grade methanol and acetonitrile were purchased from Honeywell, Burdick & Jackson (Muskegon, Michigan). Purified water (Milli-Q, EMD Millipore, Billerica, Massachusetts) was used for reagent, sample, and mobile phase preparation. Ethyl  -D-glucuronide (EtG) and ethyl  -D-glucuronide-d5 (EtG-d5) were purchased from Cerilliant (Round Rock, Texas). Ethyl sulfate and ethyl sulfate-d5 (EtS-d5), were purchased from Athena Environmental Sciences, Inc. (Baltimore, Maryland). Ammonium formate (reagent grade, 97%) was purchased from Sigma-Aldrich (St. Louis, Missouri). Certified drug negative normal human urine was purchased from UTAK Laboratories, Inc. (Valencia, California). 

The individual deuterated internal standard (IS) working solutions (100 µg/mL for EtG-d5 and 50 µg/mL for EtS-d5) were prepared in methanol and stored at 4 °C until use. A 5-µL aliquot of each internal standard was added to a 0.5-mL final diluted human urine specimen, yielding final urine IS concentrations of 1000 ng/mL for EtG-d5 and 500 ng/mL for EtS-d5. For calibrators, a stock solution of 1 mg/mL of each analyte was prepared in acetonitrile and stored at 4 °C until use. A working solution of 100 µg/mL was prepared by dilution with methanol. A calibration curve (25, 50, 100, 250, 500, 1000, and 2000 ng/mL) was generated by spiking different aliquots of the working stock solution into a blank drug negative normal human urine specimen.  

Three quality control (QC) samples were prepared in triplicate with a different lot of working standard solution to yield QC concentrations of 80, 400, and 800 ng/mL in blank drug negative normal human urine specimens, respectively. The samples were prepared for analysis using "dilute and shoot" methodology.  

 Figure 1: EtG and EtS analysis (standard spiked in 1:10 diluted human urine; standard concentrations = 2000 ng/mL). Peaks: 1 = EtG, 2 = EtS. 			

LC–MS analysis was performed on an Agilent 1200 Series HPLC system (Agilent Technologies, Santa Clara, California) interfaced with an API 4000 MS system with electrospray ionization (ESI) (AB Sciex, Foster City, California) operated in multiple reaction monitoring (MRM) negative ionization mode (ESI–). The HPLC column used for all analyses was a 100 mm × 2.0 mm Synergi 2.5-µm Hydro-RP (Phenomenex, Torrance, California). Mobile phase A was 10 mM ammonium acetate, pH 7.6, in water and mobile phase B was a 50:50 mixture of methanol and acetonitrile. The flow rate was 0.5 mL/min. A gradient method was used from 0 to 80% B in 4 min followed by a 3-min equilibration. Urine samples were diluted 10-fold with mobile phase A before injection, and the sample injection volume was 10 µL. The HPLC column was maintained at 25 °C. For MS detection various parent–daughter transitions were evaluated (for EtG 221.2: 75.0 [EtG-1] or 221.2:85.1 [EtG-2] and for EtS 124.9:80.1 [EtS-1] or 124.9:97.0 [EtS-2]) to obtain the best sensitivity free of matrix interference. 			

The use of a small-particle polar functional HPLC phase allowed for a fast elution of EtG and EtS at less than 2 min (Figure 1) using a near-neutral mobile phase. In ESI negative mode, EtG and EtS were detected by monitoring the mass transitions and their deuterium-labeled internal standards. Quantitative and qualitative data were generated for both transitions of EtG (EtG-1 and EtG-2) and EtS (EtS-1 and EtS-2). Based on the instrumentation used and samples analyzed, one can determine the transition that works best for specific application requirements.  

Figure 2: Extracted ion chromatograms for EtG, EtS, and their internal standards (EtG-d5 and EtS-d5) at a 100-ng/mL concentration in 1:10 diluted human urine.			

The optimized HPLC method utilized a 7-min separation that included a gradient from a 100% aqueous solution to 80% organic in 4 min (Figure 2) followed by a 100% aqueous equilibration. The retention times of EtG and EtS are 1.10 and 1.30 min, respectively. Although the total run time is 7 min, the method's 2-min data window allows for multiplexing of HPLC runs to increase sample throughput and reduce MS idle time. This can be advantageous in a clinical setting where large numbers of samples need to be analyzed.  

Figure 3: Patient urine profiles: (a) Patient 2 (EtG and EtS concentrations are at relatively low levels); (b) Patient 3 (EtG and EtS concentrations are at relatively medium levels); (c) Patient 6 (EtG and EtS concentrations are at very high levels). 			

The highly polar nature of EtG and EtS made typical LC methods difficult to achieve adequate retention using traditional C18 or phenyl phase columns. In addition, these polar analytes require loading conditions utilizing close to 100% aqueous mobile phase to achieve adequate retention and separation from matrix contaminants. High aqueous mobile phase reduces MS sensitivity and raises concerns of "phase collapse" phenomena that could compromise method reproducibility. The polar endcapped C18 column used for this method provides improved polar selectivity for adequate retention of EtG and EtS and yields clear separations away from matrix contaminants. EtG is clearly separated from a potential isobaric matrix component. The method is very rugged and demonstrates adequate selectivity even for concentrated standards (Figure 1) and high-level patient samples (Figure 3c). Standard calibration curves were generated over the concentration range of 25–2000 ng/mL by plotting the relative response (peak area of EtG and EtS divided by the peak area of internal standards) versus concentration. A linear fit with weighting factor of 1/

 was used to counter the undue influence of high concentrations of EtG and EtS in the high calibrators. The standard calibration curves were linear over the calibration ranges with 

  values of 0.9989 for EtG-1 (Figure 4a), 0.9978 for EtG-2 (Figure 4b), 0.9969 for EtS-1 (Figure 4c), and 0.9959 for EtS-2 (Figure 4d).  

Figure 4: Standard curves from 25 ng/mL to 1000 ng/mL for EtG and EtS in human urine: (a) EtG-1, (b) EtG-2, (c) EtS-1, and (d) EtS-2.			

Sensitivity of the method was evaluated by determining the lowest level concentration with a signal-to-noise ratio of at least 10:1 for limit of quantification (LOD). At the lowest level standard concentration (25 ng/mL) the signal-to-noise ratios were 47.5 for EtG-1, 48.5 for EtG-2, 88.2 for EtS-1, and 205.3 for EtS-2 (Figure 5 and Table I). Thus, the LOQ values were estimated to be <25.0 ng/mL for EtG and EtS (Table I) which is well below the requirements for this assay. QC samples were prepared at levels of 80, 400, and 800 ng/mL. These concentrations were selected to represent low, medium, and high concentration across the calibration range for each analyte. The QC samples were diluted in the same way as the actual sample described above and analyzed in triplicate to assess reproducibility. The mean expected recovery of the lowest-level QC samples (80 ng/mL) were 118% for EtG-1, 103% for EtG-2, 113% for EtS-1, and 112% for EtS-2 (Table I). The percentage coefficients of variation (%CV) for the intra-assay precision ranged from 1.83% to 9.97% for levels of 80 ng/mL, 4.58 to 7.20% for levels of 400 ng/mL, and 0.98% to 2.33% for levels of 800 ng/mL (Table I). No endogenous signal was found in three normal urine specimens, which demonstrated the selectivity of the method. There was no carryover observed when blank urine samples were injected after the highest calibrator (2000 ng/mL). This optimized LC–MS procedure allows for the best recovery of the urinary EtG and EtS, and demonstrates good potential for high-throughput sample preparation and automation. 

Figure 5: Extracted ion chromatograms: (a) EtG-1, (b) EtG-2, (c) EtS-1, and (d) EtS-2.			

A rapid analysis of urinary EtG and EtS was developed that is rugged and accurate and can be easily automated and multiplexed for a high-throughput environment. The use of a polar endcapped C18 phase for this separation provided a shorter chromatographic analysis time and still separated both the EtS and EtG analytes away from matrix contaminants, which is of great importance for such "dilute-and-shoot" methods. In addition, by employing multiple parent–daughter ion transitions, the method provides additional flexibility based on the samples being analyzed and instruments being employed in the method. 

Table I: Statistical data for EtG and EtS in urine obtained by LCâMS

 are with Phenomenex Inc., Torrance, California.

(1) R.L. DuPont, G.E. Skipper, and W.L. White. 

Articles

A method is illustrated for the simultaneous analysis of ethyl glucuronide and ethyl sulfate in human urine samples.

A Sensitive, Specific, Accurate, and Fast LC–MS-MS Method for Measurement of 42 Ethyl Glucuronide and Ethyl Sulfate in Human Urine

Chronic pain affects approximately 86 million people in the United States (1). Pain management centers have been established all over the country to help treat these chronic conditions. During treatment, routine testing is critical to monitor compliance and prevent abuse. Because the average age of healthcare patients continues to increase, the demand for pain management is growing rapidly. To keep up with demand, less expensive, faster, more accurate, and reproducible methods for analyzing the presence and concentration of pain management medications in vivo are needed. 

Urine is commonly used for pain management testing as it is a copiously available bodily fluid that can be collected easily. The constituents in urine and their concentration vary significantly between patients and are dependent upon diet, hydration, and health condition. Variations in the endogenous components in urine can impact quantitation in methods using liquid chromatography with mass spectrometric detection (LC–MS-MS). Coelution of endogenous urine components with the analytes of interest can cause matrix effects that result in enhancement or suppression of the MS signal, which impacts the quantitative accuracy of the assay. Removal of endogenous components using solid-phase extraction (SPE) is an effective method for reducing potential matrix interferences before LC–MS-MS analysis.  

Typically SPE and gas chromatography (GC)–MS have been used to perform the analysis of opioids in urine. Most GC–MS methods require 7 min or longer from injection to injection. The method described here couples sample cleanup and concentration using strong-cation-exchange SPE with rapid LC–MS-MS analysis (<2 min) for 13 commonly prescribed pain management compounds in human urine.  

A bioanalytical validation for a new SPE and LC–MS-MS procedure for the analysis of pain management drugs in human urine was performed. Five different lots of urine samples were spiked at therapeutically relevant concentrations with 13 commonly prescribed drugs, including hydrocodone, naltrexone, tramadol, and fentanyl. All drug standards were obtained from Cerilliant Corporation (Round Rock, Texas). The lower limit of quantitation (LLOQ) was targeted at 10× below the therapeutic level with less than 20% RSD at LLOQ. The upper limit of quantitation (ULOQ) was set at 10× above the therapeutic cutoff. 

Urine samples were first hydrolyzed to convert the glucuronide metabolites to native form using β-glucuronidase. This was done by first weighing 1 g of β-glucuronidase and diluting with 20 mL of deionized water. A 100-µL aliquot of this solution was added to 1 mL of urine and vortexed to mix (6200 units in 1 mL of urine). Samples were incubated for 2 h at 60 °C and then were centrifuged for 10–15 min at 9500–13,000 rpm to form a pellet of protein and solids. The clarified supernatant (~1 mL) was then removed and diluted with 1 mL of 100 mM ammonium acetate buffer (pH 5.95).  

Cleanup was performed using polymer-based strong-cation-exchange media (Strata-X-C 30 mg/3 mL, Phenomenex, Torrance, California) to remove endogenous matrix components. Analysis was performed using a high performance liquid chromatography  (HPLC) system (1200SL, Agilent, Santa Clara, California) coupled to a 4000 QTRAP MS system with a Turbo V ion source (Applied Biosystems, Foster City, California). The chromatographic column used was a 50 mm × 2.1 mm, 2.6-µm C18 (Kinetex, Phenomenex, Torrance, California), which is based upon new core-shell particle technology.  

Equilibrate: 1 mL 100 mM ammonium acetate buffer (pH 5.95) 

Load: Pretreated sample diluted with 1 mL 100 mM ammonium acetate buffer (pH 5.95) 

Wash: 2 × 1 mL 100 mM ammonium acetate buffer (pH 5.95) 

Dry: Full vacuum for 2–5 min to remove residual water and methanol 

Elute: 2 × 0.5 mL 60:24:16 ethyl acetate–isopropanol–ammonium hydroxide 

Evaporate to dryness using nitrogen gas and a heating block at 45 °C, then reconstitute in 1 mL of initial mobile phase for LC–MS-MS analysis. 

The analysis of all 13 pain management drugs was completed in less than 1.5 min with a total gradient cycle time from injection to injection of less than 2.2 min (Figure 1). The use of the core-shell HPLC column provided extremely narrow chromatographic bands with average peak widths at base of ~1 s. These extremely narrow peak widths challenged the scan rate of the MS system operated in triple-quadrupole mode and required the use of scheduled MRM transitions to ensure enough scans per peak to maintain chromatographic resolution. The narrow peak widths also translated into significant increases in MS sensitivity, requiring the dilution of some samples by 20-fold to bring concentrations within the dynamic range of the system. 

The columns used provided efficiencies similar to sub-2 µm particles at back pressures that allow them to be used on conventional HPLC systems. Figure 2 shows the core-shell particle geometry. These particles are manufactured by growing a 0.35-µm porous layer on a 1.9-µm nonporous core. The result is 2.6-µm particles.. 

Table I summarizes the results obtained using the procedure described. The chromatographic flow rate of 1.2 mL/min enabled the fast separation of the 13 compounds and the internal standards, with retention times ranging from 0.22 min (2 #3215; void, 0.075 min) for morphine to 1.2 min for methadone. Some internal standards were used as reference for analytes where the deuterated analog was not readily available. The analytical method gave an LLOQ of 2.5 ng/mL with accuracy of 83–98% for all compounds with the exception of fentanyl, which had an LLOQ of 0.05 ng/mL due to its low therapeutic concentration. At LLOQ and 3 × LLOQ, most RSDs were well below 15%, indicating a wide linear working range for all compounds. Method performance at cutoff was better than at the 3 × LLOQ, indicating a possible dilution error during sample preparation. Future work will be done to confirm this. The cutoff concentrations are defined as 300 ppb for all compounds except for fentanyl. Due to the relatively high pharmacological efficacy of fentanyl, ~50 times greater than morphine, the linear analytical range of fentanyl was 50× lower than the other opioids. At cutoff concentrations, all compounds were analyzed with 84–102% accuracy and 5% or less RSD values. Matrix effects were evaluated by determining lot-to-lot precision and accuracy using five different urine donors. At the cutoff concentration the RSD values were less than 7% with high accuracy across all donors. Figure 3 shows an example calibration graph spanning the linear range of 2.5–2500 ppb for morphine, with 1/

Table I: LCâMS-MS results from bioanalytical validation protocol 			

The high selectivity of LC–MS-MS greatly reduces interferences that affect analyte quantitation. This high selectivity can reduce sample cleanup requirements; however, reduced sample cleanup allows more endogenous material to accumulate in the MS source, which ultimately can increase the frequency of maintenance. The use of SPE for sample clean-up greatly reduces matrix interferences while also significantly reducing the contaminant load on the MS source. The effect of SPE was most dramatic for fentanyl due to its 5 ng/mL cutoff concentration. Using a 30-mg bed mass significantly reduces the solvent volume required for the conditioning, wash, and elution steps. This reduced solvent volume translated into significant cost and time savings for the subsequent blowdown steps.  

The SPE protocol utilized has been optimized for the 13 analytes and internal standards. The basic p

 values of the cationic analytes are all close to 8. The buffer chosen, ammonium acetate, is adjusted to pH 5.95 in order to protonate and charge the cationic analytes at 2 pH units below their p

 values. This pH ensures the target analytes are fully protonated maximizing retention and recovery with the sulfonic acid ligand on the SPE medium. Use of ion-exchange retention allows 100% methanol wash to more effectively remove endogenous contaminants including those with lower basic p

 values, such as urobilin, which, when present, may appear yellow. The selectivity of the elution solvent composition, 60:24:16 ethyl acetate–isopropanol–ammonium hydroxide, was optimized to allow elution of the analytes of interest while leaving any remaining endogenous components on the sorbent. Optimized wash and elution solutions yielded clearer and cleaner extracts (Figure 4) and more reproducible and accurate chromatography. The sorbent mass was also optimized to 30 mg of media in a 3 mL tube. The high ion-exchange capacity of the media allows processing small sample volumes even at low concentrations. Due to the high sensitivity of the method, only 1 mL of urine was required for SPE. Using less sorbent reduces cost and solvent consumption and reduces elution volumes, thereby saving time during evaporation, an important consideration for high sample throughput and increased productivity. 

Ultrahigh efficiency and fast chromatographic separations result in very narrow peak widths, enhancing resolution and sensitivity. However, these very narrow peaks also present detection challenges when using MS. This is especially true for this method, in which two MRM transitions must be monitored concurrently for each of the thirteen compounds and one for each of the eight internal standards. To optimize the acquisition rate on the MS system, a scheduled MRM window of 6 s was used to get ~20 data points per second (15–18 Hz experimentally). Sufficient sampling of the chromatographic peak is important to maintain resolution between closely eluted bands including other analytes as well as possible interferences. The MRM settings used here are highly recommended because the separation provided by the column produces very narrow, low-volume peaks. 

Additionally, when using the columns described here, LC–MS-MS instrumentation should be optimized so the narrow analyte bands are not compromised by extracolumn band broadening. The instrumentation used for this experiment was optimized by bypassing the MS grounding union, eliminating the diverter valve, and utilizing a near-zero dead volume in-line column filter (KrudKatcher Ultra, Phenomenex) to protect the column. It is recommended that the narrowest internal diameter and shortest lengths of connecting tubing possible be used. The effects of dispersion also can be minimized by using high flow rates, injecting the sample in weaker solvent than the initial mobile phase, and running steep gradients when possible.  

A bioanalytical method validation was performed for 13 commonly prescribed pain-management drugs in human urine. Accuracy, precision, and lot-to-lot matrix effects were all within expectations for bioanalytical mthods. The use of the polymeric SPE discussed here greatly reduces matrix interferences while also providing clean sample extracts. The 30 mg/3 mL format reduced the amount of solvent used for processing and the time required for blowdown steps. The columns used provided fast run times and improved method sensitivity. This prevalidated solution greatly reduces the time required for implementation in any laboratory interested in analyzing pain management samples. 

13 pain management opioids were resolved in 1.5 min with 1-s wide peaks at base on a 50 mm × 2.1 mm, 2.6-µm 

 C18 column. Sensitivity, reproducibility, and productivity are dramatically improved after efficient sample cleanup and concentration using the SPE described here. The combined SPE LC–MS-MS analysis provides significant reductions in analysis time, labor requirements, and cost per sample while increasing sensitivity, accuracy, and precision.  

 are with Phenomenex, Inc., Torrance, California.  

(1) American Chronic Pain Association website. 

Fast turnaround time is critical in the clinical testing environment. Here, fast liquid chromatography (LC) technologies were utilized for the comprehensive assay of commonly prescribed pain management drugs in under 2 min. The use of fast LC also provided significantly improved sensitivity. A mini-validation for these analytes in human urine was performed and acceptable values for accuracy, precision, linearity, lot-to-lot variability, and matrix effects were demonstrated for each analyte.

Sky Countryman

Articles

A Sensitive, Specific, Accurate, and Fast LC–MS-MS Method for Measurement of 42 Ethyl Glucuronide and Ethyl Sulfate in Human Urine

Development of a High-Throughput LC–MS-MS Assay for 13 Commonly Prescribed Pain Management Drugs from Urine with Cleanup Using Solid-Phase Extraction