Investigation on in vivo and in vitro Metabolites of Bromazolam

doi:10.19894/j.issn.1000-0518.240179

Chinese Journal of Applied Chemistry ›› 2024, Vol. 41 ›› Issue (12): 1770-1779.DOI: 10.19894/j.issn.1000-0518.240179

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Investigation on in vivo and in vitro Metabolites of Bromazolam

Yi-Ling TANG¹^,², Jun-Bo ZHAO¹, Miao ZHANG¹^,², Xiao-Yu QIAN¹^,², Ping XIANG¹, Hui YAN¹()

^1.Department of Forensic Toxicology，Academy of Forensic Science，Shanghai 200063，China
^2.School of Pharmacy，China Pharmaceutical University，Nanjing 211198，China

Received:2024-06-06 Accepted:2024-10-28 Published:2024-12-01 Online:2025-01-02
Contact: Hui YAN
About author:yanh501@163.com
Supported by:
the National Key Research and Development Program of China(2022YFC3302003);the Ministry of Finance, PR China(GY2022G-2);Shanghai Key Laboratory of Forensic Science(21DZ2270800);the Key Laboratory of Forensic Expertise of the Ministry of Justice and Shanghai Forensic Service Platform(19DZ2292700)

Abstract

Abstract:

The abuse of new psychoactive substances （NPS） shows a significant growth trend around the world， which has the characteristics of a wide variety， a wide epidemic range and the strong addiction. Bromazolam， which appears in the illegal market as a benzodiazepine NPS， is usually used to enhance the stimulating effect of opioids or to use its hypnotic and amnesia side effects to carry out drug sexual assault. In this study， the metabolic model of bromazolam in zebrafish and human liver microsomes was established. Phase Ⅰ and Ⅱ metabolites of bromazolam were identified by liquid chromatography-high resolution mass spectrometry （LC-HRMS）. Seven metabolites were identified， of which six were phase Ⅰ metabolites and one was a phase Ⅱ metabolite. The metabolic pathways of bromazolam include monohydroxylation （M1）， dihydroxylation （M2）， monohydroxylation+reduction （M3）， dealkylation （M4）， oxidative deamination （M5）， monohydroxylation+oxidative deamination （M6） and monohydroxylation+glucuronidation （M7）. Monohydroxylation metabolites （M1） are recommended as the possible biomarker of bromazolam. In this paper， the metabolism of bromazolam in vivo was studied for the first time， and the information of metabolites and metabolic pathways of bromazolam was improved， which is helpful to provide methods and data for its abuse.

Key words: New psychoactive substances, Bromazolam, Metabolism, Zebrafish, Human liver microsomes

CLC Number:

O657.6

Yi-Ling TANG, Jun-Bo ZHAO, Miao ZHANG, Xiao-Yu QIAN, Ping XIANG, Hui YAN. Investigation on in vivo and in vitro Metabolites of Bromazolam[J]. Chinese Journal of Applied Chemistry, 2024, 41(12): 1770-1779.

Figures/Tables 5

Fig.1 Chemical structure of bromazolam

Table 1 Metabolic information of bromazolam and its seven metabolites

Peak ID	Metabolic reaction	Time/min	Elemental composition	Exact mass （m/z）	Actual mass （m/z）	Mass error/（mg·L^-1）	Diagnostic product ions （m/z）	Rank^* in zebrafish	Rank in HLM
Bromazolam	/	7.79	C₁₇H₁₄ $B r N 4 +$	353.039 6	353.039 2	-1.1	95， 104， 129， 181， 205， 274， 285， 325	/	/
M1	Hydroxylation	7.07	C₁₇H₁₄BrN₄O⁺	369.034 6	369.034 0	-1.6	77， 95， 245， 282， 324， 351	3	2
M2	Dihydroxylation	6.64	C₁₇H₁₄BrN₄ $O 2 +$	385.029 5	385.028 7	-2.1	233， 340， 367	7	6
M3	Hydroxylation+reduction	6.09	C₁₇H₁₆BrN₄O⁺	371.050 2	371.050 3	0.3	91， 234， 342， 354	5	4
M4	Dealkylation	7.55	C₁₆H₁₂Br $N 4 +$	339.024 0	339.023 9	-0.3	205， 311	6	5
M5	Oxidative deamination	7.79	C₁₇H₁₅BrN₃O⁺	356.039 3	356.039 9	1.7	206， 220， 288， 328	1	1
M6	Hydroxylation+oxidative Deamination	7.08	C₁₇H₁₅BrN₃ $O 2 +$	372.034 2	372.033 8	-1.1	91， 217， 285， 327， 354	4	3
M7	Hydroxylation+glucuronidation	5.50	C₂₃H₂₂BrN₄ $O 7 +$	545.066 6	545.066 5	-0.2	324， 351， 369	2	7

Table 1 Metabolic information of bromazolam and its seven metabolites

Peak ID	Metabolic reaction	Time/min	Elemental composition	Exact mass （m/z）	Actual mass （m/z）	Mass error/（mg·L^-1）	Diagnostic product ions （m/z）	Rank^* in zebrafish	Rank in HLM
Bromazolam	/	7.79	C₁₇H₁₄ $B r N 4 +$	353.039 6	353.039 2	-1.1	95， 104， 129， 181， 205， 274， 285， 325	/	/
M1	Hydroxylation	7.07	C₁₇H₁₄BrN₄O⁺	369.034 6	369.034 0	-1.6	77， 95， 245， 282， 324， 351	3	2
M2	Dihydroxylation	6.64	C₁₇H₁₄BrN₄ $O 2 +$	385.029 5	385.028 7	-2.1	233， 340， 367	7	6
M3	Hydroxylation+reduction	6.09	C₁₇H₁₆BrN₄O⁺	371.050 2	371.050 3	0.3	91， 234， 342， 354	5	4
M4	Dealkylation	7.55	C₁₆H₁₂Br $N 4 +$	339.024 0	339.023 9	-0.3	205， 311	6	5
M5	Oxidative deamination	7.79	C₁₇H₁₅BrN₃O⁺	356.039 3	356.039 9	1.7	206， 220， 288， 328	1	1
M6	Hydroxylation+oxidative Deamination	7.08	C₁₇H₁₅BrN₃ $O 2 +$	372.034 2	372.033 8	-1.1	91， 217， 285， 327， 354	4	3
M7	Hydroxylation+glucuronidation	5.50	C₂₃H₂₂BrN₄ $O 7 +$	545.066 6	545.066 5	-0.2	324， 351， 369	2	7

Fig.2 Liquid chromatogram of bromazolam and its seven metabolites

Fig.3 MS2 spectra of bromazolam and its seven metabolites

Fig.4 Metabolic pathway of bromazolam in zebrafish and liver microsomes

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Investigation on in vivo and in vitro Metabolites of Bromazolam

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