Analysis of In Vitro Mirtazapine Metabolites in Human Liver Microsomes by LC-HRMS

doi:10.12116/j.issn.1004-5619.2024.340606

Abstract

Abstract:

Objective To establish and optimize an in vitro incubation system with human liver microsomes and investigate the in vitro metabolites and possible metabolic pathways of mirtazapine. Methods Three major metabolites of mirtazapine were selected to optimize the incubation conditions of liver microsomes. The metabolites of mirtazapine were analyzed by liquid chromatography-high resolution mass spectrometry （LC-HRMS） to identify the in vitro metabolites and metabolic pathways of mirtazapine. Results Ten metabolites, including nine phase Ⅰ metabolites and one phase Ⅱ metabolite, were identified in the in vitro liver microsome incubation. Among them, five new metabolites and one new metabolic pathway were discovered. The pathways involved in phase Ⅰ metabolic included methylation, hydroxylation, oxidation, reduction, etc., while the phase Ⅱ biotransformation was mainly glucuronidation. Conclusion The metabolites discovered in this study are consistent with the main metabolites of mirtazapine reported in literature, which are N-desmethylmetazapine, 8-hydroxy mirtazapine and mirtazapine-N-oxide. The results can provide basis for the confirmation of mirtazapine cases and provide reference for the study of other substances.

Key words: forensic medicine, toxicological analysis, mirtazapine, human liver microsome, metabolite, metabolic pathway, liquid chromatography-high resolution mass spectrometry （LC-HRMS）

CLC Number:

Ying ZHANG, Wen-fang ZHANG, Duo-qi XU, Shi-yang QIN, Shi-yun YANG, Jing QIAO. Analysis of In Vitro Mirtazapine Metabolites in Human Liver Microsomes by LC-HRMS[J]. Journal of Forensic Medicine, 2024, 40(6): 569-574.

Figures/Tables 8

References 23

1	DE BOER T， RUIGT G S F. The selective α₂-adrenoceptor antagonist mirtazapine （Org 3770） enhances noradrenergic and 5-HT_1A-mediated serotonergic neurotransmission[J]. CNS Drugs，1995，4（S1）：29-38. doi：10.2165/00023210-199500041-00006 .
2	BERENDSEN H H G， BROEKKAMP C L E. Indirect in vivo 5-HT_1A-agonistic effects of the new antidepressant mirtazapine[J]. Psychopharmacology （Berl），1997，133（3）：275-282. doi：10.1007/s002130050402 .
3	HASSANEIN E H M， ALTHAGAFY H S， BARAKA M A， et al. Pharmacological update of mirtazapine： A narrative literature review[J]. Naunyn Schmiedebergs Arch Pharmacol，2024，397（5）：2603-2619. doi：10.1007/s00210-023-02818-6 .
4	WINGEN M， BOTHMER J， LANGER S， et al. Actual driving performance and psychomotor function in healthy subjects after acute and subchronic treatment with escitalopram， mirtazapine， and placebo[J]. J Clin Psychiatry，2005，66（4）：436-443. doi：10.4088/jcp.v66n0405 .
5	VIGNALI C， GROPPI A， BRANDOLINI F， et al. Mirtazapine fatal poisoning[J]. Forensic Sci Int，2017，276：e8-e12. doi：10.1016/j.forsciint.2017.04.025 .
6	NISHIO T， TOUKAIRIN Y， HOSHI T， et al. A case of lacosamide and mirtazapine self-poisoning[J]. Leg Med （Tokyo），2024，69：102457. doi：10.1016/j.legalmed.2024.102457 .
7	ANTTILA S A K， LEINONEN E V J. A review of the pharmacological and clinical profile of mirtazapine[J]. CNS Drug Rev，2001，7（3）：249-264. doi：10.1111/j.1527-3458.2001.tb00198.x .
8	TIMMER C J， SITSEN J M， DELBRESSINE L P. Clinical pharmacokinetics of mirtazapine[J]. Clin Pharmacokinet，2000，38（6）：461-474. doi：10.2165/00003088-200038060-00001 .
9	The role of mirtazapine in the pharmacotherapy of depression[J]. J Clin Psychiatry，2000，61（8）：609-616. doi：10.4088/jcp.v61n0812 .
10	COHEN M， PANAGIDES J， TIMMER C J， et al. Pharmacokinetics of mirtazapine from orally administered tablets： Influence of a high-fat meal[J]. Eur J Drug Metab Pharmacokinet，1997，22（2）：103-110. doi：10.1007/BF03189792 .
11	KELDER J， FUNKE C， DE BOER T， et al. A comparison of the physicochemical and biological properties of mirtazapine and mianserin[J]. J Pharm Pharmacol，1997，49（4）：403-411. doi：10.1111/j.2042-7158.1997.tb06814.x .
12	DELBRESSINE L P， VOS R M. The clinical rele-vance of preclinical data： Mirtazapine， a model compound[J]. J Clin Psychopharmacol，1997，17（S1）：29-33. doi：10.1097/00004714-199704001-00004 .
13	DAHL M L， VOORTMAN G， ALM C， et al. In vitro and in vivo studies on the disposition of mirtazapine in humans[J]. Clin Drug Investig，1997，13（1）：37-46. doi：10.2165/00044011-199713010-00005 .
14	STÖRMER E， VON MOLTKE L L， SHADER R I， et al. Metabolism of the antidepressant mirtazapine in vitro： Contribution of cytochromes P-450 1A2， 2D6， and 3A4[J]. Drug Metab Dispos，2000，28（10）：1168-1175.
15	DODD S， BOULTON D W， BURROWS G D， et al. In vitro metabolism of mirtazapine enantiomers by human cytochrome P450 enzymes[J]. Hum Psychopharmacol，2001，16（7）：541-544. doi：10.1002/hup.328 .
16	DELBRESSINE L P， MOONEN M E， KASPER-SEN F M， et al. Pharmacokinetics and biotransformation of mirtazapine in human volunteers[J]. Clin Drug Investig，1998，15（1）：45-55. doi：10.2165/00044011-199815010-00006 .
17	MARTÍNEZ-RAMÍREZ J A， VOIGT K， PETERS F T. Studies on the metabolism of five model drugs by fungi colonizing cadavers using LC-ESI-MS/MS and GC-MS analysis[J]. Anal Bioanal Chem，2012，404（5）：1339-1359. doi：10.1007/s00216-012-6212-3 .
18	ROBERTSON M D， DRUMMER O H. Postmortem drug metabolism by bacteria[J]. J Forensic Sci，1995，40（3）：382-386.
19	HAWKSWORTH D L， WILTSHIRE P E J. Forensic mycology： The use of fungi in criminal investigations[J]. Forensic Sci Int，2011，206（1/2/3）：1-11. doi：10.1016/j.forsciint.2010.06.012 .
20	DE SANTANA F J M， JABOR V A P， CESARINO E J， et al. Enantioselective analysis of mirtazapine， demethylmirtazapine and 8-hydroxy mirtazapine in human urine after solid-phase microextraction[J]. J Sep Sci，2010，33（2）：268-276. doi：10.1002/jssc.200900534 .
21	闵涛，刘永涛，徐子振，等. 常见有机磷农药稳定性研究进展[J].毒理学杂志，2018，32（2）：164-167.
	MIN T， LIU Y T， XU Z Z， et al. Research progress on the stability of common organophosphorus pesticides[J]. Dulixue Zazhi，2018，32（2）：164-167.
22	BECONI M G， YATES D， LYONS K， et al. Metabolism and pharmacokinetics of JM6 in mice： JM6 is not a prodrug for ro-61-8048[J]. Drug Metab Dispos，2012，40（12）：2297-2306. doi：10.1124/dmd.112.046532 .
23	NYKODEMOVÁ J， ŠULÁKOVÁ A， PALIVEC P， et al. 2C-B-Fly-NBOMe metabolites in rat urine， human liver microsomes and C. elegans： Confirmation with synthesized analytical standards[J]. Metabolites，2021，11（11）：775. doi：10.3390/metabo1111 0775 .

化合物	化学式	［M+H］⁺质量数			保留时间/min	二级特征碎片离子（m/z）
化合物	化学式	理论质量数（m/z）	实测质量数（m/z）	误差/×10^-6	保留时间/min	二级特征碎片离子（m/z）
米氮平	C₁₇H₂₀N₃⁺	266.165 17	266.164 79	1.43	3.71	209，195，72
M-252（N-去甲米氮平）	C₁₆H₁₈N₃⁺	252.149 52	252.149 12	1.59	3.64	235，209，195
M-282A（8-羟基米氮平）	C₁₇H₂₀N₃O⁺	282.160 61	282.159 42	-1.84	3.37	225，211，166，72
M-282B（米氮平氮氧化物）	C₁₇H₂₀N₃O⁺	282.160 09	282.159 94	0.53	3.82	264，208，195
M-268	C₁₆H₁₈N₃O⁺	268.144 44	268.143 86	4.10	3.74	250，225，211
M-280A	C₁₇H₁₈N₃O⁺	280.144 44	280.144 23	0.75	3.20	252，195，58
M-280B	C₁₇H₁₈N₃O⁺	280.144 44	280.144 20	0.86	3.86	252，209，195
M-280C	C₁₇H₁₈N₃O⁺	280.144 44	280.144 17	0.96	4.25	252，209，195，58
M-458	C₂₃H₂₈N₃O₇⁺	458.192 18	458.191 77	0.90	1.84	282，211，72
M-296	C₁₇H₁₈N₃O₂⁺	296.139 35	296.139 28	1.35	3.54	278，268，250，211，58
M-298	C₁₇H₂₀N₃O₂⁺	298.155 00	298.154 88	0.42	3.32	280，211，70

化合物	化学式	［M+H］⁺质量数			保留时间/min	二级特征碎片离子（m/z）
化合物	化学式	理论质量数（m/z）	实测质量数（m/z）	误差/×10^-6	保留时间/min	二级特征碎片离子（m/z）
米氮平	C₁₇H₂₀N₃⁺	266.165 17	266.164 79	1.43	3.71	209，195，72
M-252（N-去甲米氮平）	C₁₆H₁₈N₃⁺	252.149 52	252.149 12	1.59	3.64	235，209，195
M-282A（8-羟基米氮平）	C₁₇H₂₀N₃O⁺	282.160 61	282.159 42	-1.84	3.37	225，211，166，72
M-282B（米氮平氮氧化物）	C₁₇H₂₀N₃O⁺	282.160 09	282.159 94	0.53	3.82	264，208，195
M-268	C₁₆H₁₈N₃O⁺	268.144 44	268.143 86	4.10	3.74	250，225，211
M-280A	C₁₇H₁₈N₃O⁺	280.144 44	280.144 23	0.75	3.20	252，195，58
M-280B	C₁₇H₁₈N₃O⁺	280.144 44	280.144 20	0.86	3.86	252，209，195
M-280C	C₁₇H₁₈N₃O⁺	280.144 44	280.144 17	0.96	4.25	252，209，195，58
M-458	C₂₃H₂₈N₃O₇⁺	458.192 18	458.191 77	0.90	1.84	282，211，72
M-296	C₁₇H₁₈N₃O₂⁺	296.139 35	296.139 28	1.35	3.54	278，268，250，211，58
M-298	C₁₇H₂₀N₃O₂⁺	298.155 00	298.154 88	0.42	3.32	280，211，70

[1]	Jiao-jiao JI, Xin WANG, Jia-man LIN, Duo-qi XU, Hui YAN, Min SHEN. Research Progress on the Application of MALDI-MSI in Hair Analysis [J]. Journal of Forensic Medicine, 2024, 40(6): 542-549.
[2]	Zhen-shuo GUO, Wen-jia DUAN, Yu LIU, Yi-ling TANG, Hui YAN. Research Progress on the Analysis of Anabolic Androgenic Steroids in Biological Samples Based on High Resolution Mass Spectrometry [J]. Journal of Forensic Medicine, 2024, 40(6): 533-541.
[3]	Si-yang HE, Qian-ya DENG, Shui-qing ZHENG, Chun-fang NI, Wen-juan SUN, Fang-qi CAO, Chen LIANG, Fei-jun GONG. Identification of a Novel Synthetic Cathinone CMMP [J]. Journal of Forensic Medicine, 2024, 40(6): 550-556.
[4]	Xian-yu FAN, Xue-yan ZHU, Xin WANG, Ping XIANG, Jun-bo ZHAO. High Resolution Mass Spectrometry Fragmentation Rules of Etomidate and Its Structural Analogues [J]. Journal of Forensic Medicine, 2024, 40(6): 557-563.
[5]	Zhu-tao GUO, Qian-ya DENG, Si-yang HE, Shui-qing ZHENG, Rong WANG, Wen-juan SUN, Chen LIANG, Chun-fang NI. Rapid Screening of 60 Types of Natural Toxins in Whole Blood by UPLC-Q/Orbitrap HRMS [J]. Journal of Forensic Medicine, 2024, 40(6): 564-568.
[6]	Guo-qing GAO, Shuo YANG, Li-ying ZHOU, Shi-bei DU, Yan SHI. In Vivo Analysis of New Psychoactive Substances： Nitazenes [J]. Journal of Forensic Medicine, 2024, 40(6): 597-607.
[7]	Hong-xia HAO, Jie-min CHEN, Xiao-ying YU, Meng WANG, Zhi-lu ZHOU, Yan-liang SHENG, Wen-tao XIA. Estimation of Visual Acuity after Recovery from Different Degrees of Ocular Trauma Using Pattern Visual Evoked Potential [J]. Journal of Forensic Medicine, 2024, 40(5): 447-453.
[8]	Zhi YAN, Xun-ming JI, Xiao HE, Xiao-jing ZHANG, Lei WAN, Hong ZHANG, Mei TIAN, Bin CONG. Progress and Application Prospects of Forensic Molecular Imaging Technology in Living Individual Examination [J]. Journal of Forensic Medicine, 2024, 40(5): 476-483.
[9]	Tian-fu HE, Huan-hui ZHU, Yuan-yuan TIAN, Yin-shuang JIN, Xian-wen LIN, Song-cai WANG. Detection of Etomidate and Etomidate Acid in Urine Using HPLC-MS/MS Method [J]. Journal of Forensic Medicine, 2024, 40(5): 454-460.
[10]	Hao-jie QIN, Yuan-yuan LIU, En-hao FU, Ya-wen LIU, Zhi-ling TIAN, He-wen DONG, Tai-ang LIU, Dong-hua ZOU, Yi-bin CHENG, Ning-guo LIU. Intelligent Recognition and Segmentation of Blunt Craniocerebral Injury CT Images Based on DeepLabV3+ Model [J]. Journal of Forensic Medicine, 2024, 40(5): 419-429.
[11]	Jing-chen GE, Min SHANG, Ming-yang YAO, Ming-fei WEI, Jun-zhan SHI, Ze-wei YAO, Jia-yin SHI, Fan LI. Application of MSCT Image Post-Processing Techniques in the Identification of Rib Fracture Malunion [J]. Journal of Forensic Medicine, 2024, 40(4): 324-329.
[12]	Yu-jiao WU, Yue-qin ZHOU, Yi-wen SHEN, Jie-qing JIANG. Analysis of 39 Forensic Clinical Sex Identification Cases after Sex Reassignment Surgery [J]. Journal of Forensic Medicine, 2024, 40(4): 365-371.
[13]	Zong-yang NIE, Wei HU, Ling-yu LI, Qing-bo ZHANG, Xin HUANG, Bo LI. Structure Confirmation of Three New Psychoactive Substances and Qualitative Identification of Unknown Substances [J]. Journal of Forensic Medicine, 2024, 40(4): 340-351.
[14]	Chen-jun MA, Yong TAN, Qing-hua ZHANG. The Talent Training Model of Forensic Medicine in Japan and Its Reference [J]. Journal of Forensic Medicine, 2024, 40(3): 284-290.
[15]	Ze-ren YANG, Dong GAO, Qing XIA, Dan RAN, Yan-liang SHENG, Wen-tao XIA. Effects of Different Test Positions on Quantitative Muscle Strength of Wrist and Finger Flexor Muscle Groups and Its Standardization [J]. Journal of Forensic Medicine, 2024, 40(3): 237-244,253.