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Open Access 05-03-2025 | Amitriptyline | Short Communication

Investigation of commercially available recombinant and conventional β-glucuronidases to evaluate the hydrolysis efficiencies against O-glucuronides and N-glucuronides in urinary drug screening

Authors: Akira Namera, Takeshi Saito, Masataka Nagao

Abstract

Purpose

To achieve the rapid analysis of drug metabolites in urine, we examined the differences in the hydrolysis efficiencies against O-glucuronide and N-glucuronide by two commercially available glucuronidases and three commercially available recombinant ones.

Methods

The metabolites analyzed included oxazepam-O-glucuronide, amitriptyline-N-glucuronide, and diphenhydramine-N-glucuronide. Hydrolysis was performed using commercially available five enzymes at two different temperatures, and the reaction progress was monitored for up to 360 min. The amount of hydrolyzed product was quantified using liquid chromatography–tandem mass spectrometry.

Results

Although no enzyme selectivity was observed for the hydrolysis of O-glucuronide, the hydrolysis efficiency against N-glucuronide varied significantly, depending on the enzyme and reaction temperature. Among the enzymes evaluated, IMCSzyme 3S and the enzyme derived from E. coli demonstrated superior hydrolysis of N-glucuronides under optimal conditions. For IMCS RT, good results were also obtained by adding twice the amount of enzyme specified.

Conclusions

Suitable enzymes and hydrolysis conditions were determined for the rapid and systematic screening of drug metabolites in human urine. These findings are expected to streamline the analytical workflow and reduce the need for tedious sample preprocessing.

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Tanibuchi Y, Omiya S, Usami T, Matsumoto T (2024) Clinical characteristics of over-the-counter (OTC) drug abusers in psychiatric practice in Japan: comparison of single and multiple OTC product abusers. Neuropsychopharmacol Rep 44:176–186. https://doi.org/10.1002/npr2.12415CrossRefPubMedPubMedCentral

Hashimoto T, Kaneda Y, Ozaki A, Hori A, Tsuchiya T (2022) Eleven-year trend of drug and chemical substance overdose at a local emergency hospital in Japan. Cureus 14:e32475. https://doi.org/10.7759/cureus.32475CrossRefPubMedPubMedCentral

Nemanich A, Liebelt E, Sabbatini AK (2021) Increased rates of diphenhydramine overdose, abuse, and misuse in the United States, 2005–2016. Clin Toxicol (Phila) 59:1002–1008. https://doi.org/10.1080/15563650.2021.1892716CrossRefPubMed

Bishop M, Schumann JL, Gerostamoulos D, Wong A (2021) The impact of codeine upscheduling on overdoses, Emergency Department presentations and mortality in Victoria. Australia Drug Alcohol Depend 226:108837. https://doi.org/10.1016/j.drugalcdep.2021.108837CrossRefPubMed

Schwartz RH (1988) Urine testing in the detection of drugs of abuse. Arch Intern Med 148:2407–2412. https://doi.org/10.1001/archinte.1988.00380110059012CrossRefPubMed

Drummer OH (2004) Pharmacokinetics and metabolism. In: Moffat AC, Osselton MD, Widdop B (eds) Clarke’s analysis of drugs and poisons. Pharmaceutical Press, London, pp 172–188

Spiehler V, Levine BS (2020) Pharmacokinetics. In: Levine BS, Kerrigan S (eds), Principles of forensic toxicology. Springer, Switzerland, p 91–100. https://doi.org/10.1007/978-3-030-42917-1_7

Kaushik R, Levine B, LaCourse WR (2006) A brief review: HPLC methods to directly detect drug glucuronides in biological matrices (Part I). Anal Chim Acta 556:255–266. https://doi.org/10.1016/j.aca.2005.09.012CrossRef

Scheidweiler KB, Desrosiers NA, Huestis MA (2012) Simultaneous quantification of free and glucuronidated cannabinoids in human urine by liquid chromatography tandem mass spectrometry. Clin Chim Acta 413:1839–1847. https://doi.org/10.1016/j.cca.2012.06.034CrossRefPubMedPubMedCentral

10.

Cao Z, Kaleta E, Wang P (2015) Simultaneous quantitation of 78 drugs and metabolites in urine with a dilute-and-shoot LC–MS-MS Assay. J Anal Toxicol 39:335–346. https://doi.org/10.1093/jat/bkv024CrossRefPubMed

11.

Suzuki O, Hattori H, Asano M, Takahashi T, Brandenberger H (1987) Positive and negative ion mass spectrometry of benzophenones, the acid-hydrolysis products of benzodiazepines. Z Rechtsmed 98:1–10. https://doi.org/10.1007/BF00200380CrossRefPubMed

12.

Johnson-Davis KL (2018) Opiate and benzodiazepine confirmations: To hydrolyze or not to hydrolyze is the question. J Appl Lab Med 2:564–572. https://doi.org/10.1373/jalm.2016.022947CrossRefPubMed

13.

Jufer-Phipps RA, Levine BS (2020) Benzodiazepines. In: Levine BS, Kerrigan S (eds), Principles of forensic toxicology. Springer, Switzerland, p 317–331. https://doi.org/10.1007/978-3-030-42917-1_20

14.

Flanagan RJ, Cuypers E, Maurer HH, Whelpton R (2020) Fundamentals of analytical toxicology: Clinical and forensic. John Wiley & Sons, New York. https://doi.org/10.1002/9781119122357CrossRef

15.

Hackett LP, Dusci LJ, Ilett KF, Chiswell GM (2002) Optimizing the hydrolysis of codeine and morphine glucuronides in urine. Ther Drug Monit 24:652–657. https://doi.org/10.1097/00007691-200210000-00012CrossRefPubMed

16.

Wang P, Stone JA, Chen KH, Gross SF, Haller CA, Wu AHB (2006) Incomplete recovery of prescription opioids in urine using enzymatic hydrolysis of glucuronide metabolites. J Anal Toxicol 30:570–575. https://doi.org/10.1093/jat/30.8.570CrossRefPubMed

17.

Yang HS, Wu AHB, Lynch KL (2016) Development and validation of a novel LC-MS/MS opioid confirmation assay: Evaluation of β-glucuronidase enzymes and sample cleanup methods. J Anal Toxicol 40:323–329. https://doi.org/10.1093/jat/bkw026CrossRefPubMed

18.

Lin Z, Lafolie P, Beck O (1994) Evaluation of analytical procedures for urinary codeine and morphine measurements. J Anal Toxicol 18:129–133. https://doi.org/10.1093/jat/18.3.129CrossRefPubMed

19.

Hufschmid E, Theurillat R, Martin U, Thormann W (1995) Exploration of the metabolism of dihydrocodeine via determination of its metabolites in human urine using micellar electrokinetic capillary chromatography. J Chromatogr B Biomed Appl 668:159–170. https://doi.org/10.1016/0378-4347(95)00046-lCrossRefPubMed

20.

Abraham TT, Lowe RH, Pirnay SO, Darwin WD, Huestis MA (2007) Simultaneous GC-EI-MS determination of delta9-tetrahydrocannabinol, 11-hydroxy-delta9-tetrahydrocannabinol, and 11-nor-9-carboxy-delta9-tetrahydrocannabinol in human urine following tandem enzyme-alkaline hydrolysis. J Anal Toxicol 31:477–485. https://doi.org/10.1093/jat/31.8.477CrossRefPubMed

21.

Gray TR, Barnes AJ, Huestis MA (2010) Effect of hydrolysis on identifying prenatal cannabis exposure. Anal Bioanal Chem 397:2335–2347. https://doi.org/10.1007/s00216-010-3772-yCrossRefPubMedPubMedCentral

22.

Aizpurua-Olaizola O, Zarandona I, Ortiz L, Navarro P, Etxebarria N, Usobiaga A (2017) Simultaneous quantification of major cannabinoids and metabolites in human urine and plasma by HPLC-MS/MS and enzyme-alkaline hydrolysis. Drug Test Anal 9:626–633. https://doi.org/10.1002/dta.1998CrossRefPubMed

23.

Fischer D, Breyer-Pfaff U (1995) Comparison of procedures for measuring the quaternary N-glucuronides of amitriptyline and diphenhydramine in human urine with and without hydrolysis. J Pharm Pharmacol 47:534–538. https://doi.org/10.1111/j.2042-7158.1995.tb05845.xCrossRefPubMed

24.

Dalgaard L, Larsen C (1999) Metabolism and excretion of citalopram in man: Identification of O-acyl- and N-glucuronides. Xenobiotica 29:1033–1041. https://doi.org/10.1080/004982599238092CrossRefPubMed

25.

Hawes EM (1998) N+-Glucuronidation, A common pathway in human metabolism of drugs with a tertiary amine group. Drug Metab Dispos 26:830–837PubMed

26.

Göschl L, Gmeiner G, Gärtner P, Stadler G, Enev V, Thevis M, Schänzer W, Guddat S, Forsdahl G (2021) Stanozolol-N-glucuronide metabolites in human urine samples as suitable targets in terms of routine anti-doping analysis. Drug Test Anal 13:1668–1677. https://doi.org/10.1002/dta.3109CrossRefPubMed

27.

Kemp PM, Abukhalaf IK, Manno JE, Manno BR, Alford DD, Abusada GA (1995) Cannabinoids in humans. I. Analysis of delta 9-tetrahydrocannabinol and six metabolites in plasma and urine using GC-MS. J Anal Toxicol 19:285–291. https://doi.org/10.1093/jat/19.5.285CrossRefPubMed

28.

Kul A, Sagirli O (2022) Determination of enzymatic hydrolysis efficiency in detection of cannabis use by UPLC-MS-MS. J Anal Toxicol 46:257–263. https://doi.org/10.1093/jat/bkab017CrossRefPubMed

29.

Meatherall R (1994) Optimal enzymatic hydrolysis of urinary benzodiazepine conjugates. J Anal Toxicol 18:382–384. https://doi.org/10.1093/jat/18.7.382CrossRefPubMed

30.

Maurer HH (1999) Systematic toxicological analysis procedures for acidic drugs and/or metabolites relevant to clinical and forensic toxicology and/or doping control. J Chromatogr B 733:3–25. https://doi.org/10.1016/S0378-4347(99)00266-2CrossRef

31.

Tsujikawa K, Kuwayama K, Kanamori T, Iwata Y, Ohmae Y, Inoue H, Kishi T (2004) Optimized conditions for the enzymatic hydrolysis of α-hydroxytriazolam-glucuronide in human urine. J Health Sci 50:286–289. https://doi.org/10.1248/jhs.50.286CrossRef

32.

Pirnay SO, Abraham TT, Lowe RH, Huestis MA (2006) Selection and optimization of hydrolysis conditions for the quantification of urinary metabolites of MDMA. J Anal Toxicol 30:563–569. https://doi.org/10.1093/jat/30.8.563CrossRefPubMed

33.

Morris AA, Chester SA, Strickland EC, McIntire GL (2014) Rapid enzymatic hydrolysis using a novel recombinant β-glucuronidase in benzodiazepine urinalysis. J Anal Toxicol 38:610–614. https://doi.org/10.1093/jat/bku083CrossRefPubMed

34.

Sempio C, Scheidweiler KB, Barnes AJ, Huestis MA (2018) Optimization of recombinant β-glucuronidase hydrolysis and quantification of eight urinary cannabinoids and metabolites by liquid chromatography tandem mass spectrometry. Drug Test Anal 10:518–529. https://doi.org/10.1002/dta.2230CrossRefPubMed

35.

Kang MG, Lin HR (2019) Systematic evaluation and validation of benzodiazepines confirmation assay using LC–MS-MS. J Anal Toxicol 43:96–103. https://doi.org/10.1093/jat/bky071CrossRefPubMed

36.

Matsuo T, Ogawa T, Iwai M, Kubo K, Kondo F, Seno H (2024) Development of an LC–MS/MS method for the determination of five psychoactive drugs in postmortem urine by optimization of enzymatic hydrolysis of glucuronide conjugates. Forensic Toxicol 42:181–190. https://doi.org/10.1007/s11419-024-00685-1CrossRefPubMedPubMedCentral

37.

Fu S, Lewis J, Wang H, Keegan J, Dawson M (2010) A novel reductive transformation of oxazepam to nordiazepam observed during enzymatic hydrolysis. J Anal Toxicol 34:243–251. https://doi.org/10.1093/jat/34.5.243CrossRefPubMed

38.

Sitasuwan P, Melendez C, Marinova M, Mastrianni KR, Darragh A, Ryan E, Lee LA (2016) Degradation of opioids and opiates during acid hydrolysis leads to reduced recovery compared to enzymatic hydrolysis. J Anal Toxicol 40:601–607. https://doi.org/10.1093/jat/bkw085CrossRefPubMed

39.

Skov K, Johansen SS, Linnet K, Rasmussen BS, Nielsen MKK (2023) Exploring enzymatic hydrolysis of urine samples for investigation of drugs associated with drug-facilitated sexual assault. Pharmaceuticals (Basel) 17:13. https://doi.org/10.3390/ph17010013CrossRefPubMed

40.

Fareed Y, Braun D, Flasch M, Globisch D, Warth B (2022) A broad, exposome-type evaluation of xenobiotic phase II biotransformation in human biofluids by LC-MS/MS. Exposome. https://doi.org/10.1093/exposome/osac008CrossRef

Title: Investigation of commercially available recombinant and conventional β-glucuronidases to evaluate the hydrolysis efficiencies against O-glucuronides and N-glucuronides in urinary drug screening
Authors: Akira Namera
Takeshi Saito
Masataka Nagao
Publication date: 05-03-2025
Publisher: Springer Nature Singapore
Keywords: Amitriptyline
Diphenhydramine
Published in: Forensic Toxicology
Print ISSN: 1860-8965
Electronic ISSN: 1860-8973
DOI: https://doi.org/10.1007/s11419-025-00715-6

Podcast | Sexual health in older age

Springer Medicine

Investigation of commercially available recombinant and conventional β-glucuronidases to evaluate the hydrolysis efficiencies against O-glucuronides and N-glucuronides in urinary drug screening

Abstract

Purpose

Methods

Results

Conclusions

Podcast | Sexual health in older age

Springer Medicine

Abstract

Purpose

Methods

Results

Conclusions

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