Authors: Linda Côté

Agilent Technologies, Inc. Montréal, QC


Determination of Urinary Ethyl Glucuronide and Ethyl Sulfate by LC/MS/MS for Clinical Research


Liquid chromatography triple quadrupole mass spectrometry (LC/MS/MS) is ideally suited for the rapid analysis of multiple analytes. A highly sensitive and specific LC/MS/MS analytical method has been developed for the quantitation of ethyl glucuronide and ethyl sulfate. A dilution procedure and a solid phase extraction (SPE) procedure are evaluated and compared based on ease of use, analyte recovery and post-extraction cleanliness.

Calibrators were created by spiking synthetic urine (Surine-Cerilliant) with various concentrations of EtG and EtS standards (Cerilliant). The chromatographic system consists of a Polaris 3 C18-Ether column coupled with a guard column and a mobile phase comprised of acetonitrile and water containing 0.1% formic acid. Quantifier and qualifier transitions were monitored. EtG-D5 and EtS-D5 internal standards (Cerilliant) were included to ensure accurate and reproducible quantitation. Urine controls (UTAK Laboratories) were used and samples were kindly supplied by collaborators. The separation of EtG and EtS from isobaric interferences is especially critical; without proper separation by retention time, impurities present in both compounds can cause interferences with one another and lead to inaccurate quantitation.


Authors: Peter J. Simms & Steven V. Kozmary, MD

Lux Laboratories, Las Vegas, NV 89102


A Novel Separation for Ethyl Glucuronide and Ethyl Sulfate using a Multi-Mode Reversed-Phase Column

Ethyl glucuronide and ethyl sulfate were separated using a multi-mode C18 column. The analytes were eluted of the column using an formic acid/ammonium formate/acetonitrile gradient mobile phase. The separation was primarily affected by the mobile phase buffer pH, salt concentration and organic modifier composition. Increasing the pH and the buffer concentration caused a decrease in retention time for both analytes. In addition, changing the composition of the organic modifier affected the retention of ethyl glucuronide and ethyl sulfate. Changing the initial concentration of the organic modifier gave a U-shaped retention for ethyl glucuronide. Greater retention of ethyl glu-curonide and ethyl sulfate was achieved using this system compared to traditional the retention of the these analytes using reversed-phase methods.


Ethanol (alcohol) is one of the most commonly used drugs in the United States. When ethanol is used in conjunction with prescription narcotics, it can result in adverse affects to the person. Therefore, when testing pain management patients for prescription compliance, it is im-portant to make sure they are not using alcohol in conjunction with their prescription. When ethanol is consumed 90% of the dose that is con-sumed is metabolized. The primary urine metabolites are ethyl glucu-ronide and ethyl sulfate. Traditional confirmation LC/MS methods use a C18 stationary phase along with an organic modifier and an aqueous buffer as the mobile phase. Under these conditions, both analytes elute off the column very early and can often give broad peaks. The resulting poor chromatography can lead to poor results for both analytes. Using this mixed mode C18 column we were able to obtain greater retention of both ethyl sulfate and ethyl glucuronide. The separation was affected by mobile phase pH , buffer concentration and organic modifier composi-tion. Mobile phase pH and buffer concentration had the greatest effect on the retention of the analytes under reversed phase conditions. We now report on a chromatographic method for ethyl sulfate and ethyl glu-curonide using these conditions. This method gave better retention over traditional reversed phase methods.

Authors: Geza S. Bodor, MD

VA Eastern Colorado Health Care System & University of Colorado Denver, School of Medicine


Introducing Liquid Chromatography – Mass Spectrometry into the Clinical Laboratory: Using Vitamin D Testing as a Practical Example


Five years ago our vitamin D (VitD) testing volume and the associated reference laboratory cost increased exponentially. We were unable to identify an automated VitD immunoassay for our existing chemistry instrumnts and were not satisfied with the performance of other immunoassay platforms. Although this hospital laboratory had no previous experience with mass spectrometry, we decided to open an LCMSMS laboratory section for CitD testing and, depending on the success of our experiment, expand testing in the future.


We developed our in-house LCMSMS assay for measurement of serum 25(OH) vitamin-D2 and-D3 (OHD2, OHD3, respectively). Our method was calibrated to the NIST SRM 2972 standard by value assignment to the albumin based calibrators. Sample preparation involves addition of hexa-deuterated internal standard (IS) and protein crash, followed by reversed phase separation on a 2.1×50 mm C18 column. MS analysis is performed by APCI in the positive mode. Two MRM transitions are monifored for the analytes and one for the IS.


Assay development, validation and technologist training took approximately 8 months and we had to train a second MT shortly after going live to assure continuity of testing. Injection to injection time is 6.5 minutes, allowing testing of 120-130 patient samples a day and we perform testing 4-5 days per week. Sample preparation takes place during the day shift and the LCMSMS is run overnight unattended. Each batch contains a 6-point calibration curve and 3 levels of QC material. Each run is reviewed by a technologist according to set criteria before reporting results. Calibrator lots are prepared every three months and assay drift is monitored by NIST reference material and CAP proficiency subscription. No calibration drift has been detected during >4 years of continuous operation. Assay AMR are 6-200 and 4-200 ng/mL for OHD2 and OHD3, respectively. Assay CVs are 4% – 10% throughout the AMR. Total VitD is reported as the sum of OHD2 and OHD3. The assay has no interference from lipemia or hemolysis. When compared to CDC target, our in-house VitD assay remains within acceptable limits, regardless if OHD2 is present or absent in the proficiency samples while the commercially available VitD immunoassays all exhibit variable degree of bias depending on the VitD composition of the samples. Although the LCMSMS method has high start-up cost, return on investment (ROI) is extremely low (<<1 year) when compared to reference lab costs. ROI would be even faster in a commercial laboratory setting due to high dollar value of CMS reimbursement for VitD testing.

Authors: Deborah French

Department of Laboratory Medicine, University of California-San Francisco, San Francisco, CA 94107


Development and validation of a serum total testosterone liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay calibrated to NIST SRM 971

Background:At our institution, serum testosterone in adult males is measured by immunoassay while female and pediatric specimens are sent to a reference laboratory for liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis due to low concentrations. As this is of significant cost, a testosterone LC–MS/MS assay was developed in-house.

Methods:A 5500 QTRAP® using electrospray ionization and a Shimadzu Prominence with a C18 column were used. Gradient elution with formic acid, water and methanol:acetonitrile at 0.5 ml/min had a 7-min run-time. A liquid–liquid extraction with hexane:ethyl acetate was carried out on 200 μl of serum. Multiple reaction monitoring was employed.

Results:Sample preparation took ~80 min for 21 samples. Six calibrators were used (0–1263 ng/dl; concentration assigned by NIST SRM 971) with 3 quality controls (9, 168 and 532 ng/dl). The limits of detection and quantitation were 1 and 2 ng/dl respectively. Extraction recovery was ~90% and ion suppression ~5%. Within-run and total precision studies yielded b15% CV at the limit of quantitation and b7% CV through the rest of the linear range. Isobaric interferences were baseline separated from testosterone. Method comparisons between this assay, an immunoassay, and another LC–MS/MS assay were completed.

Conclusions: An accurate and sensitive LC–MS/MS assay for total testosterone was developed. Bringing this
assay in-house reduces turnaround time for clinicians and patients and saves our institution funds.

Authors: Trevor J. Pitcher, Haleh A. Jortani, William W. Tucker, HediyehA. Jortani, Thomas Kampfrath, and Saeed Jortani

Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, KY


Antiarrhythmic Drugs Reverse Bath Salts Induced Tachycardia In Vivo

Designer stimulant drugs are an emerging public health problem that is confounded by the lack of rapid diagnostics and specific treatment regiments. Among the many types of designer drugs on the market, synthetic cathinones (“bath salts”) have become increasingly popular. Typically bath salts are consumed by insufflation, ingestion or application to mucous membranes and are known to cause severe adverse effects including tachycardia, hypertension and respiratory distress. However, little is known regarding pharmacodynamic properties of bath salts in humans, specifically regarding their cardiogenic effects.

Authors: Thomas Kampfrath, Angela Sibio, Lee Blum, Saeed Jortani

Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, Kentucky; NMS Labs, WIllow Grove, PA


Cross-Reactivity Assessment of Bath Salts in Different Immunoassays


Bath salts, a class of synthetic molecules known as cathinones, are the latest drugs of abuse becoming increasingly popular in the United States. They are very popular among younger abusers trying to avert detection by the standard drug screening procedures. With little information known on their risks and effects by the medical community, frequent overdoses, hallucinations, and even death have been reported. Currently, the different cathinones are analyzed by gas chromatography – mass spectrometry by referral and highly specialized laboratories. Since he structures of cathinones are similar to amphetamines, cross-reactivities with common immunoassays are expected. Herein, we report on the cross-reactivity analysis of three different synthetic cathinones in varies commercial immunoassays used in routine drug screening practice.


The selected synthetic cathinones for cross-reactivity assessment were Mephedrone, MDPV (3,4-methylenedioxypyrovalerone), and Methylone. These substances are currently the most prevalent members of the bath salts in the United States and were kindly provided by Utak Laboratories Inc. (Valencia, CA). Those compounds were added to aliquots of normal human urine at a concentration of 10000 ng/mL a typical concentration utilized for cross-reactivity studies with unrelated drugs. In addition, one sample contained a mixture of all three cathinones at a concentration of 10000 ng/mL each as street drugs are rather a mixture and rarely pure. The tested immunoassays were the Roche Integra (Roche Diagnostics GmbH), Triage® 8 Drugs of Abuse Panel (Inverness Medical, San Diego, CA), Siemens Viva E (Malvern, PA) and Beckman Coulter Unicel DxC 800 (Brea, CA).


The Roche amphetamine screen was the only assay that we tested that showed cross-reactivity with bath salts. At first, we tested a mixture of Mephedrone, MDPV, and Methylone providing a positive result as indicated by a reaction rate of 1642 while 1000 is set for the cutoff (equivalent in assay reactivity to 500 ng/mL). Next, we tested each cathinone separately at a concentration of 10000 ng/mL. Here, only mephedrone was able to cross-react and provide a positive result (1060), while MDPV (417) and methylone (651) were well below the cutoff limit. None of those compounds gave positive results at the 10000 ng/mL cutoff in the Triage® 8 panel (amphetamine, barbiturate, cocaine, opiate, benzodiazepine, THC, methadone, PCP), Siemens Viva E and in the Beckman Coulter Unicel DxC 800 (amphetamine, barbiturate, cocaine, opiate, benzodiazepine, THC, methadone). Conclusion: Out of the three popular bath salts tested, only mephedrone cross-reacted in the Roche’s amphetamine screen. Neither of the bath salts tested either alone or as a mixture cross-reacted in the Triage or Beckman amphetamine assays. All other immunoassay screens resulted in negative results when bath salts were added to the urine. Considering the long turn-around time for sending samples for testing bath salts, the observed cross-reactivity in the Roche’s amphetamine assay may be an advantage since the clinical management of amphetamine and bath salts overdoses are similar.