MSACL 2018 US Abstract

Topic: Small Molecules / Tox

Quantitation of Seven Benzodiazepines and Metabolites in Urine by LC-MSMS

Yuzi (Emma) Zheng (Presenter)
Cleveland Clinic

Bio: I completed my PhD at the Centre for High-Throughput Biology at University of British Columbia (UBC), Canada, specializing in qualitative and quantitative proteomics. After my PhD, I was a postdoctoral research fellow in the Department of Pathology and Laboratory Medicine at UBC working in the clinical chemistry laboratory at St. Paul’s Hospital with research focused on translating biomarker research findings into quantitative mass spectrometry protein assays. Currently, I am a clinical chemistry fellow at Cleveland Clinic involved in development and validation of mass spectrometry assays for patient care.

Authorship: Yu Zi (Emma) Zheng, Dustin R Bunch, Katherine Lembright and Sihe Wang
Department of Laboratory Medicine, Cleveland Clinic, Cleveland, OH

Short Abstract

Benzodiazepines are central nervous system depressants that are prescribed to patients to prevent seizures, treat anxiety or simply help them to sleep. Benzodiazepines when overdosed can lead to addiction and sometimes cause death. Therapeutic drug monitoring of benzodiazepines in urine are used to help physicians identify the drugs used, especially in the pain management settings. Here we report a novel liquid chromatography-tandem mass spectrometry assay for measuring seven common benzodiazepines and active metabolites in urine, which has been validated for clinical testing.

Long Abstract


Benzodiazepines (BZD) are a class of psychotropic drugs, first introduced in the 1960s with the core chemical structure of a benzene ring fused with a diazepine ring [1]. Based on the 2011 Unites States national prescription rates, the most prescribed BZD are alprazolam, clonazepam, diazepam, lorazepam and temazepam which are also the most abused BZD in the United States [2]. Oxazepam and nordiazepam, while not commonly prescribed, are active metabolites of many different BZD [3]. Clinically, BZD measurement is important in emergency toxicological screenings, drug abuse testing, and forensic medical examinations [4, 5].

Traditional methods of BZD measurement like the immunoassays lack specificity meaning individual BZD and metabolites similar in structure are unable to be distinguished. Many of these assays do not have sufficient sensitivity to reliably measure the low clinical relevant concentrations [6-8]. Gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) has then been utilized to measure these analytes; however, requires extensive sample preparation and long analytical time [9].

Here, we report a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring seven BZD and metabolites in urine: 7-aminoclonazepam, α-hydroxyalprazolam, α-hydroxytriazolam, oxazepam, lorazepam, nordiazepam, and temazepam. Monitoring these BZD and metabolites can detect the use of most commonly prescribed BZD in the United States.


7-Aminoclonazepam, α-hydroxyalprazolam, α-hydroxytriazolam, oxazepam, lorazepam, nordiazepam and temazepam were purchased from Cerilliant and used for preparing the calibrators at 50, 100, 500, 1000 and 5000 ng/mL for all analytes. d4-7-aminoclonazepam, d5-α-hydroxyalprazolam, d5-oxazepam, d5-alprazolam and d5-temazepam also purchased from Cerilliant were used as internal standards at concentration of 300 ng/mL. Sample preparation included enzymatic hydrolysis using β-glucuronidase (Ango Science), centrifugation, and dilution before LC-MS/MS analysis. The LC-MS/MS system used was a Thermo Fisher Scientific TLX-2 Transcend LC system coupled to a Quantum Ultra mass spectrometer. The MS/MS was set in the multiple reaction monitoring (MRM) mode with two MRM transitions monitored for each analyte (quantifier and qualifier) and one MRM transition for each corresponding internal standard. The LC method had a total run time of 4.35 min between injections for one channel. The following experiments were included to validate the method: ion suppression, interferences, analytical measuring range (AMR), carryover, precision, stability and method comparison.


No ion suppression was observed for all seven analyte and their corresponding internal standards through post-column infusion. In the mixing experiment, we performed an extensive exogenous interference study where six commercial urine quality control materials containing common therapeutic drugs, drugs of abuse and their metabolites, chemicals and hormones were studied; no interferences were found. AMR was assessed by serial dilution of a spiked patient urine at twelve concentration levels. CV of the triplicate measurement was < 15 % for all analytes at each concentration level. The lower limit of quantitation (LLOQ) determined was between 30 ng/mL to 50 ng/mL and the upper limit of quantitation was at 10,000 ng/mL with analytical recovery ranged from 80.0 % to 119.5 % for all seven analytes. Carryover was assessed at 10,000 ng/mL over a low sample at 30 ng/mL, and there was no significant carryover observed. Clinically, it is rare to have a patient sample with a BZD level at 10,000 ng/mL. In the precision experiment, total CV ranged from 2.9 % to 9.4 % and intra-assay CV were found to be between 2.0 % and 8.3 %. For the un-extracted urine samples, we found six of the seven analytes (α-hydroxyalprazolam, α-hydroxytriazolam, oxazepam, lorazepam, nordiazepam and temazepam) were stable for 24 h at room temperature, 14 days at 4 °C, 3 month at -20 °C and -70 °C. 7-aminoclonazepam was less stable with 12 h at both room temperature and 4 °C, 7 days at -20 °C and 3 month at -70 °C. A reference laboratory utilizing GC-MS/MS was used for the method comparison. All negative urine samples (meaning below the LLOQ by our method) were also negative by the comparison method. For the positive urine samples, Deming regression showed correlation coefficient ranging from 0.9612 (α-hydroxyalprazolam) to 0.9974 (Lorazepam). To further evaluate the method accuracy, a set of commercial BZD urine toxicology controls at two different concentration levels were analyzed. The results from our LC-MS/MS method showed -15. 8 % to 17.3 % difference from the target values.

Conclusions & Discussion

We developed an LC-MS/MS assay for measuring seven benzodiazepines and active metabolites in urine. This assay was fully validated for clinical use with short analytical time and broad analytical measurement ranges.

References & Acknowledgements:

1. Lader, M., History of benzodiazepine dependence. J Subst Abuse Treat, 1991. 8(1-2): p. 53-9.

2. Division, D.C., BENZODIAZEPINES, Justice, Editor. 2013, Drug Enforcement Administration:

3. Luk, S., et al., Urinary diazepam metabolite distribution in a chronic pain population. J Anal Toxicol, 2014. 38(3): p. 135-42.

4. Centers for Disease, C. and Prevention, Emergency department visits involving nonmedical use of selected prescription drugs - United States, 2004-2008. MMWR Morb Mortal Wkly Rep, 2010. 59(23): p. 705-9.

5. Gudin, J.A., et al., Risks, management, and monitoring of combination opioid, benzodiazepines, and/or alcohol use. Postgrad Med, 2013. 125(4): p. 115-30.

6. Krasowski, M.D., et al., Using molecular similarity to highlight the challenges of routine immunoassay-based drug of abuse/toxicology screening in emergency medicine. BMC Emerg Med, 2009. 9: p. 5.

7. DeRienz, R.T., et al., Evaluation of four immunoassay screening kits for the detection of benzodiazepines in urine. J Anal Toxicol, 2008. 32(6): p. 433-7.

8. West, R., et al., Comparison of clonazepam compliance by measurement of urinary concentration by immunoassay and LC-MS/MS in pain management population. Pain Physician, 2010. 13(1): p. 71-8.

9. Perez, E.R., et al., Comparison of LC-MS-MS and GC-MS Analysis of Benzodiazepine Compounds Included in the Drug Demand Reduction Urinalysis Program. J Anal Toxicol, 2016. 40(3): p. 201-7.

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