Aim: To investigate the efficiency of two new fast-acting enzymes, recombinant arylsulfatase (IMCS-PSF) and mutant β-glucuronidase from Escherichia coli (IMCSzyme), in hydrolyzing specific terbutaline metabolites. Materials & methods: Two purified novel enzymes are used to precisely determine the amount of each metabolite in urine at different time points after oral administration. After systematically evaluating the hydrolysis efficiency of the novel enzymes compared with commercially available enzymes, these recently developed enzymes were applied to establish the separate urine concentration profiles of terbutaline and each metabolite. Results & discussion: The results highlight the highly efficient arylsulfatase enzyme expressed from E. coli for urine analysis of terbutaline while suggesting sulfoconjugates as the main terbutaline metabolites. Conclusion: This study demonstrated the high efficiency of the IMCS-PSF enzyme in hydrolyzing terbutaline conjugates in comparison with other enzyme reagents typically used for the analysis of terbutaline and sulfoconjugates are the main terbutaline metabolites in urine.

Keywords:

Papers of special note have been highlighted as: • of interest

References

1 Sarah MD. Antenatal administration of corticosteroids for women at risk of preterm birth. BMJ 356, 1467 (2017).
Google Scholar
2 Haas DM, Benjamin T, Sawyer R, Quinney SK. Short-term tocolytics for preterm delivery – current perspectives. Int. J. Womens Health 6, 343–349 (2014).
MedlineGoogle Scholar
3 Jones GH, Scott SJ. Continuous infusions of terbutaline in asthma – a review. J. Asthma 48(8), 753–756 (2011).
Medline CASGoogle Scholar
4 Centner TJ, Alvey JC, Stelzleni AM. Beta agonists in livestock feed: status, health concerns, and international trade. J. Anim. Sci. 92(9), 4234–4240 (2014).
Medline CASGoogle Scholar
5 Kalsen A, Hostrup M, Soderlund K, Karlsson S, Backer V, Bangsbo J. Inhaled beta2-agonist increases power output and glycolysis during sprinting in men. Med. Sci. Sports Exerc. 48(1), 39–48 (2016).
Medline CASGoogle Scholar
6 Kalsen A, Hostrup M, Karlsson S, Hemmersbach P, Bangsbo J, Backer V. Effect of inhaled terbutaline on substrate utilization and 300-kcal time trial performance. J. Appl. Physiol. 117(10), 1180–1187 (2014).
Medline CASGoogle Scholar
7 Jessen S, Onslev J, Lemminger A, Backer V, Bangsbo J, Hostrup M. Hypertrophic effect of inhaled beta2 -agonist with and without concurrent exercise training: a randomized controlled trial. Scand. J. Med. Sci. Sports doi:10.1111/sms.13221 (2018) (Epub ahead of print).
MedlineGoogle Scholar
8 Hostrup M, Kalsen A, Onslev J et al. Mechanisms underlying enhancements in muscle force and power output during maximal cycle ergometer exercise induced by chronic beta2-adrenergic stimulation in men. J. Appl. Physiol. 119(5), 475–486 (2015).
Medline CASGoogle Scholar
9 World Anti-Doping Agency. The 2018 Prohibited List. https://www.wada-ama.org/sites/default/files/prohibited_list_2018_en.pdf.
Google Scholar
10 Smith DJ. The pharmacokinetics, metabolism, and tissue residues of beta-adrenergic agonists in livestock. J. Anim. Sci. 76(1), 173–194 (1998).
Medline CASGoogle Scholar
11 Orlovius AK, Guddat S, Parr MK et al. Terbutaline sulfoconjugate: characterization and urinary excretion monitored by LC/ESI-MS/MS. Drug Test. Anal. 1(11–12), 568–575 (2009). • Reveals terbutaline sulfoconjugates as major terbutaline metabolites by using in-house synthesized terbutaline sulfoconjugates as reference standards.
Medline CASGoogle Scholar
12 Hochhaus G, Mollmann H. Pharmacokinetic/pharmacodynamic characteristics of the beta-2-agonists terbutaline, salbutamol and fenoterol. Int. J. Clin. Pharmacol. Ther. Toxicol. 30(9), 342–362 (1992).
Medline CASGoogle Scholar
13 Morgan DJ. Clinical pharmacokinetics of beta-agonists. Clin. Pharmacokinet. 18(4), 270–294 (1990).
Medline CASGoogle Scholar
14 Davies DS, George CF, Blackwell E, Conolly ME, Dollery CT. Metabolism of terbutaline in man and dog. Br. J. Clin. Pharmacol. 1(2), 129–136 (1974).
MedlineGoogle Scholar
15 Dominguez-Romero JC, Garcia-Reyes JF, Martinez-Romero R, Martinez-Lara E, Del Moral-Leal ML, Molina-Diaz A. Detection of main urinary metabolites of beta2-agonists clenbuterol, salbutamol and terbutaline by liquid chromatography high resolution mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 923-924, 128–135 (2013).
Medline CASGoogle Scholar
16 Taylor LL, Flint NA, Ma V, Hill BM, Clark CJ, Strathmann FG. Internal hydrolysis indicator for sample specific monitoring of beta-glucuronidase activity. J. Anal. Toxicol. 41(5), 407–411 (2017).
Medline CASGoogle Scholar
17 Gonçalves VCDS, Santos KTJ, Zuniga NOC et al. Optimization of a multiresidue and multiclass analysis method for anabolic agents and β2-agonists in bovine urine by GC-MS/MS. Microchem. J. 133, 551–555 (2017).
CASGoogle Scholar
18 Li T, Cao J, Li Z, Wang X, He P. Broad screening and identification of beta-agonists in feed and animal body fluid and tissues using ultra-high performance liquid chromatography-quadrupole-orbitrap high resolution mass spectrometry combined with spectra library search. Food Chem. 192, 188–196 (2016).
Medline CASGoogle Scholar
19 Wang X, Guo T, Wang S, Yuan J, Zhao R. Simultaneous determination of 11 beta-agonists in human urine using high-performance liquid chromatography/tandem mass spectrometry with isotope dilution. J. Anal. Toxicol. 39(3), 213–218 (2015).
MedlineGoogle Scholar
20 Sitasuwan P(N), Marinova M, Lee AL. Utilizing purified β-glucuronidase and arylsulfatase to accurately quantitate metabolites in human urine. Presented at: 65th Anual ASMS Conference on Mass Spectrometry and Allied Topics. Indionapolis, IN, USA, 4–7 June 2017.
Google Scholar
21 Mastrianni KR, Lee LA, Brewer WE, Dongari N, Barna M, Morgan SL. Variations in enzymatic hydrolysis efficiencies for amitriptyline and cyclobenzaprine in urine. J. Anal. Toxicol. 40(9), 732–737 (2016).
Medline CASGoogle Scholar
22 Morris AA, Chester SA, Strickland EC, McIntire GL. Rapid enzymatic hydrolysis using a novel recombinant β-glucuronidase in benzodiazepine urinalysis. J. Anal. Toxicol. 38(8), 610–614 (2014).
Medline CASGoogle Scholar
23 Sitasuwan P, Melendez C, Marinova M et al. Degradation of opioids and opiates during acid hydrolysis leads to reduced recovery compared to enzymatic hydrolysis. J. Anal. Toxicol. 40(8), 601–607 (2016).
Medline CASGoogle Scholar
24 Mastrianni KR, Metavarayuth K, Brewer WE, Wang Q. Analysis of 10 beta-agonists in pork meat using automated dispersive pipette extraction and LC–MS/MS. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 1084, 64–68 (2018).
Medline CASGoogle Scholar
25 IMCSzyme™, package insert, catalog number 04-E1F-050K, Integrated Micro-Chromatography Systems Columbia, SC, USA (2014).
Google Scholar
26 Dyreborg A, Krogh N, Backer V, Rzeppa S, Hemmersbach P, Hostrup M. Pharmacokinetics of oral and inhaled terbutaline after exercise in trained men. Front. Pharmacol. 7, 150 (2016). • Offers pharmacokinetics detail of oral terbutaline in men after exercise.
MedlineGoogle Scholar
27 Elers J, Hostrup M, Pedersen L et al. Urine and serum concentrations of inhaled and oral terbutaline. Int. J. Sports Med. 33(12), 1026–1033 (2012). • Provides urine concentrations as well as pharmacokinetics of oral terbutaline in asthmatic and healthy men.
Medline CASGoogle Scholar
28 Hostrup M, Kalsen A, Hemmersbach P, Backer V. Intra-individual variability in the urine concentrations of inhaled salmeterol in male subjects with reference to doping analysis? Impact of urine specific gravity correction. J. Sports Med. Doping Stud. 2, 118 (2012).
Google Scholar
29 Sporer BC, Sheel AW, McKenzie DC. Dose response of inhaled salbutamol on exercise performance and urine concentrations. Med. Sci. Sports Exerc. 40(1), 149–157 (2008).
Medline CASGoogle Scholar
30 Nilsson HT, Persson K, Tegner K. The metabolism of terbutaline in man. Xenobiotica 2(4), 363–373 (1972).
Medline CASGoogle Scholar

Vol. 10, No. 22

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Received 29 May 2018

Accepted 2 October 2018

Published online 29 October 2018

Published in print November 2018

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Acknowledgements

The authors appreciate the helpful discussion with P Sitasuwan and LA Lee on designing the urine hydrolysis experiments. The enzyme samples are gifts from Integrated Micro-Chromatography Systems, LLC.

Financial & competing interests disclosure

Q Wang is a shareholder of Integrated Micro-Chromatography Systems (IMCS), LLC. The terms of this arrangement have been reviewed and approved by IMCS, LLC. in accordance with its policy on objectivity in research. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

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Advancing the analysis of terbutaline in urine samples using novel enzyme hydrolysis

Abstract

References