Scientia Agricultura Sinica ›› 2026, Vol. 59 ›› Issue (9): 2016-2028.doi: 10.3864/j.issn.0578-1752.2026.09.013

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

In vitro Metabolites of Tenvermectin in Rat and Dog Liver Microsomes Analyzed by UPLC-Q-Exactive Orbitrap MS

LIU XiaoHua1,2(), LIANG JianPing1,2(), LIU Yan3, WU ChengYu2, ZHANG YiCheng2, HUANG XianHui1,2(), LI XiangMei1,2()   

  1. 1 Guangdong Provincial Key Laboratory of Food Quality and Safety/College of Food Science, South China Agricultural University, Guangzhou 510642
    2 Guangdong Key Laboratory for Veterinary Drug Development and Safety Evaluation/College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642
    3 Guangdong Centre for Agricultural Products Quality and Safety (Guangdong Green Food Development Center), Guangzhou 510523
  • Received:2025-11-17 Accepted:2026-03-18 Online:2026-05-01 Published:2026-05-06
  • Contact: HUANG XianHui, LI XiangMei

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Abstract:

【Objective】Tenvermectin (TVM) is a novel 16-membered macrolide antibiotic synthesized from genetically engineered bacteria that produce avermectin and milbemycin. The in vitro metabolites of the new veterinary drug Tenvermectin (TVM) in the liver microsomal model were analyzed and identified by high-resolution mass spectrometry. It directly reflects the in vitro metabolic characteristics of the drug, and at the same time provides a theoretical basis and important clues for in vivo metabolic studies.【Method】In this study, rat and dog liver microsomes were first prepared by differential centrifugation. The in vitro metabolic activities of both were determined by incubating them with specific substrates for rat liver microsomes (7-ethoxy coumarin) and dog liver microsomes (coumarin), respectively. An in vitro liver microsome metabolic model of TVM was established. Under in vitro conditions, the metabolites in the incubation system were extracted and purified by incubating rat and dog liver microsomes with TVM. The metabolites of TVM were detected by UPLC-Q-Exactive Orbitrap MS. The existence of the metabolites was determined through software prediction and instrument verification of the metabolites. At the same time, combined with the elemental composition and structure of the original tetraviridin, each metabolite and its fragment ions were rapidly and accurately identified.【Result】The findings indicated that nine TVM metabolites were identified in rat liver microsomes, comprising demethylated products (M2, M5, M8), oxidized products (M1, M3, M4, M9), ketone products (M6), and demonosaccharified products (M7). In contrast, ten TVM metabolites were detected in dog liver microsomes, including demethylated products (M2, M5, M8), oxidized products (M9, M10, M11, M13), ketone products (M6), demonosaccharified products (M7), and demethylated ketone products (M12). The metabolite types and contents in the liver microsomes of the two species were different.【Conclusion】UPLC-Q-Exactive Orbitrap MS was used to study the metabolites and metabolic pathways of TVM. The metabolic pathways of TVM in rat and dog liver microsome in vitro included demethylation, oxidation, ketonization, demonoglycation and ketonization after demethylation, among which demethylation and oxidation were the main ones. This research clarified the in vitro metabolic mode of tenvermectin and refined its metabolic pathway. The research results provide an important theoretical basis and reference for further studying the metabolic products, pharmacokinetics and pharmacodynamics of TVM in animals.

Key words: tenvermectin, metabolites identification, UPLC-Q-exactive orbitrap MS, liver microsomes, metabolic pathway

Fig. 1

Chromatographic behavior of TVM and possible structural fragmentation modes A: Extracted ion chromatogram of TVM; B: The proposed fragmentation pathways of TVM"

Fig. 2

Extracted ion chromatogram of TVM metabolites from rat liver microsomes"

Fig. 3

Extracted ion chromatogram of TVM metabolites from dog liver microsomes"

Fig. 4

MS/MS spectra of TVM and metabolites from rat liver microsomes A：TVM；B：M1；C：M2；D：M3；E：M4；F：M5；G：M6；H：M7；I：M8；J：M9"

Fig. 5

MS/MS spectra of TVM and metabolites from dog liver microsomes A：TVM；B：M2；C：M5；D：M6；E：M7；F：M8；G：M9；H：M10；I：M11；J：M12；K：M13"

Table 1

Identification of TVM metabolites from rat liver microsomes"

代谢产物编号
Metabolite ID		 代谢方式
Pathway		 分子式
Formula		 预测分子量
Neutral mass
(Da)		 m/z
[M+Na]+		 质量误差
Mass accuracy
(ppm)		 保留时间
R.T.
(min)		 峰面积
Peak area		 相对含量
Relative content (%)
TVM 原型Prototype C46H70O14 846.4760081 869.46417 -1.85 15.71 6.60E+09 -
M1 氧化Oxidation C46H70O15 862.4709227 885.45898 -1.93 15.46 1.08E+08 7.40
M2 去甲基化Demethylation C45H68O14 832.4603580 855.44836 -2.07 15.39 3.44E+08 23.57
M3 氧化Oxidation C46H70O15 862.4709227 885.45825 -2.76 15.35 3.35E+07 2.30
M4 氧化Oxidation C46H70O15 862.4709227 885.45821 -2.80 15.29 9.41E+06 0.64
M5 去甲基化Demethylation C45H68O14 832.4603580 855.44855 -1.84 15.27 3.12E+07 2.14
M6 酮化Ketone formation C46H68O15 860.4552726 883.44324 -2.04 15.23 3.08E+08 21.11
M7 去单糖化Loss of C7H12O3 C39H58O11 702.3973639 725.38541 -2.38 14.92 1.43E+07 0.98
M8 去甲基化Demethylation C45H68O14 832.4603580 855.44861 -1.77 14.84 8.82E+06 0.60
M9 氧化Oxidation C46H70O15 862.4709227 885.45898 -1.93 12.86 6.02E+08 41.25

Table 2

Identification of TVM metabolites from dog liver microsomes"

代谢产物编号
Metabolite ID		 代谢方式
Pathway		 分子式
Formula		 预测分子量
Neutral mass（Da）		 m/z
[M+Na]+		 质量误差
Mass accuracy
(ppm)		 保留时间
R.T.
(min)		 峰面积
Peak area		 相对含量
Relative content (%)
TVM 原型Prototype C46H70O14 846.4760081 869.46472 -1.22 15.69 7.15E+09 -
M2 去甲基化Demethylation C45H68O14 832.4603580 855.44855 -1.84 15.40 1.27E+09 60.00
M5 去甲基化Demethylation C45H68O14 832.4603580 855.44928 -0.99 15.26 3.68E+07 1.74
M6 酮化Ketone formation C46H68O15 860.4552726 883.44364 -1.59 15.23 8.58E+07 4.05
M7 去单糖化Loss of C7H12O3 C39H58O11 702.3976390 725.38568 -2.00 14.91 2.19E+07 1.03
M8 去甲基化Demethylation C45H68O14 832.4603580 855.44806 -2.42 14.84 2.99E+07 1.41
M9 氧化Oxidation C46H70O15 862.4709227 885.45892 -2.00 12.89 5.41E+07 2.56
M10 氧化Oxidation C46H70O15 862.4709227 885.45813 -2.89 15.42 2.23E+07 1.05
M11 氧化Oxidation C46H70O15 862.4709227 885.45911 -1.79 15.12 4.70E+08 22.20
M12 去甲基化后酮化Demethylation and ketone formation C45H66O15 846.4396226 869.42798 -1.62 14.94 2.30E+07 1.09
M13 氧化Oxidation C46H70O15 862.4709227 885.45874 -2.20 14.59 1.03E+08 4.87

Fig. 6

Proposed metabolic pathway of TVM metabolites from rat liver microsomes and dog liver microsomes"

[1]
ZHANG J, YAN Y J, AN J, HUANG S X, WANG X J, XIANG W S. Designed biosynthesis of 25-methyl and 25-ethyl ivermectin with enhanced insecticidal activity by domain swap of avermectin polyketide synthase. Microbial Cell Factories, 2015, 14(1): 152.

doi: 10.1186/s12934-015-0337-y
[2]
张魁, 陈安良. 天维菌素在杀虫杀螨方面的研究进展. 现代园艺, 2022, 45(11): 72-73, 76.
ZHANG K, CHEN A L. Research progress of Tianweijun in insecticidal and acaricidal activities. Contemporary Horticulture, 2022, 45(11): 72-73, 76. (in Chinese)
[3]
KIM M S, CHO W J, SONG M C, PARK S W, KIM K, KIM E, LEE N, NAM S J, OH K H, YOON Y J. Engineered biosynthesis of milbemycins in the avermectin high-producing strain Streptomyces avermitilis. Microbial Cell Factories, 2017, 16(1): 9.

doi: 10.1186/s12934-017-0626-8
[4]
YAN Y S, XIA H Y. Recent advances in the research of milbemycin biosynthesis and regulation as well as strategies for strain improvement. Archives of Microbiology, 2021, 203(10): 5849-5857.

doi: 10.1007/s00203-021-02575-1
[5]
THUAN N H, PANDEY R P, SOHNG J K. Recent advances in biochemistry and biotechnological synthesis of avermectins and their derivatives. Applied Microbiology and Biotechnology, 2014, 98(18): 7747-7759.

doi: 10.1007/s00253-014-5926-x pmid: 25104027
[6]
HUANG J, CHEN A L, ZHANG H, YU Z, LI M H, LI N, LIN J T, BAI H, WANG J D, ZHENG Y G. Gene replacement for the generation of designed novel avermectin derivatives with enhanced acaricidal and nematicidal activities. Applied and Environmental Microbiology, 2015, 81(16): 5326-5334.

doi: 10.1128/AEM.01025-15 pmid: 26025902
[7]
杨波, 张绍勇, 陈振, 黄隽, 王继栋, 陈安良. 新化合物天维菌素的杀虫杀螨活性. 农药学学报, 2016, 18(1): 124-129.
YANG B, ZHANG S Y, CHEN Z, HUANG J, WANG J D, CHEN A L. Insecticidal and acaricidal activity of novel compound tenvermectin. Chinese Journal of Pesticide Science, 2016, 18(1): 124-129. (in Chinese)
[8]
MCCALL J W, DICOSTY U, MANSOUR A, FRICKS C, MCCALL S, DZIMIANSKI M T, CARSON B. Inability of Dirofilaria immitis infective larvae from mosquitoes fed on blood from microfilaremic dogs during low-dose and short-treatment regimens of doxycycline and ivermectin to complete normal development in heartworm naïve dogs. Parasites & Vectors, 2023, 16(1): 199.
[9]
CIUCA L, VISMARRA A, CONSTANZA D, DI LORIA A, MEOMARTINO L, CIARAMELLA P, CRINGOLI G, GENCHI M, RINALDI L, KRAMER L. Efficacy of oral, topical and extended- release injectable formulations of moxidectin combined with doxycycline in Dirofilaria immitis naturally infected dogs. Parasites & Vectors, 2023, 16(1): 54.
[10]
LAING R, GILLAN V, DEVANEY E. Ivermectin-old drug, new tricks? Trends in Parasitology, 2017, 33(6): 463-472.

doi: 10.1016/j.pt.2017.02.004
[11]
KAPLAN R M. Drug resistance in nematodes of veterinary importance: A status report. Trends in Parasitology, 2004, 20(10): 477-481.

pmid: 15363441
[12]
SINGH N K, SINGH H, JYOTI, PRERNA M, RATH S S. First report of ivermectin resistance in field populations of Rhipicephalus (Boophilus) Microplus (Acari: Ixodidae) in Punjab districts of India. Veterinary Parasitology, 2015, 214(1/2): 192-194.

doi: 10.1016/j.vetpar.2015.09.014
[13]
EL-ASHRAM S, ABOELHADID S M, KAMEL A A, MAHROUS L N, FAHMY M M. First report of cattle tick Rhipicephalus (boophilus) annulatus in Egypt resistant to ivermectin. Insects, 2019, 10(11): 404.

doi: 10.3390/insects10110404
[14]
FERNÁNDEZ-SALAS A, RODRÍGUEZ-VIVAS R I, ALONSO- DÍAZ M A. First report of a Rhipicephalus microplus tick population multi-resistant to acaricides and ivermectin in the Mexican tropics. Veterinary Parasitology, 2012, 183(3/4): 338-342.

doi: 10.1016/j.vetpar.2011.07.028
[15]
BLANCKENHORN W U, PUNIAMOORTHY N, SCHEFFCZYK A, RÖMBKE J. Evaluation of eco-toxicological effects of the parasiticide moxidectin in comparison to ivermectin in 11 species of dung flies. Ecotoxicology and Environmental Safety, 2013, 89: 15-20.

doi: 10.1016/j.ecoenv.2012.10.030 pmid: 23273869
[16]
STRONG L. Avermectins: A review of their impact on insects of cattle dung. Bulletin of Entomological Research, 1992, 82(2): 265-274.

doi: 10.1017/S0007485300051816
[17]
LEWIS M J, DIDHAM R K, EVANS T A, BERSON J D. Experimental evidence that dung beetles benefit from reduced ivermectin in targeted treatment of livestock parasites. Science of the Total Environment, 2024, 945: 174050.

doi: 10.1016/j.scitotenv.2024.174050
[18]
ZHANG H, ZHANG S Y, ZHANG J, QI H, WANG H, ZHANG L Q, HUANG J, WANG J D. Acyltransferase domain swapping for the production of tenvermectin B metabolites in genetically engineered strain Streptomyces avermitilis HU02. Journal of Agricultural and Food Chemistry, 2022, 70(38): 11994-12003.

doi: 10.1021/acs.jafc.2c04482
[19]
李贵玉. 抗寄生虫候选药物天维菌素的成药性初步研究[D]. 兰州: 甘肃农业大学, 2023.
LI G Y. Preliminary evaluation on the druggability of the antiparasitic drug candidate, tenvermectin[D]. Lanzhou: Gansu Agricultural University, 2023. (in Chinese)
[20]
SHANU-WILSON J, EVANS L, WRIGLEY S, STEELE J, ATHERTON J, BOER J. Biotransformation: impact and application of metabolism in drug discovery. ACS Medicinal Chemistry Letters, 2020, 11(11): 2087-2107.

doi: 10.1021/acsmedchemlett.0c00202
[21]
CERNY M A, KALGUTKAR A S, OBACH R S, SPRACKLIN D K, WALKER G S. Effective application of metabolite profiling in drug design and discovery. Journal of Medicinal Chemistry, 2020, 63(12): 6387-6406.

doi: 10.1021/acs.jmedchem.9b01840 pmid: 32097005
[22]
U F D Administration. Safety testing of drug metabolites, guidance for industry. US Food & Drug Administration, 2020.
[23]
LEE CHIU S H, TAUB R, SESTOKAS E, LU A Y H, JACOB T A. Comparative in vivo and in vitro metabolism of ivermectin in steers, sheep, swine, and rat. Drug Metabolism Reviews, 1987, 18(2/3): 289-302.

doi: 10.3109/03602538708998309
[24]
CHIU S H, SESTOKAS E, TAUB R, SMITH J L, ARISON B, LU A Y. The metabolism of avermectin-H2B1a and-H2B1b by pig liver microsomes. Drug Metabolism and Disposition, 1984, 12(4): 464-469.

doi: 10.1016/S0090-9556(25)07749-9
[25]
TIPTHARA P, KOBYLINSKI K C, GODEJOHANN M, HANBOONKUNUPAKARN B, ROTH A, ADAMS J H, WHITE N J, JITTAMALA P, DAY N P J, TARNING J. Identification of the metabolites of ivermectin in humans. Pharmacology Research & Perspectives, 2021, 9(1): e00712.
[26]
SHEN Y M, LIU X W, YANG Y J, LI J Y, MA N, LI B. In vivo and in vitro metabolism of aspirin eugenol ester in dog by liquid chromatography tandem mass spectrometry. Biomedical Chromatography, 2015, 29(1): 129-137.

doi: 10.1002/bmc.v29.1
[27]
MATSUBARA T, OTSUBO S, YOSHIHARA E, TOUCHI A. Biotransformation of coumarin derivatives (2) oxidative metabolism of 7-alkoxycoumarin by microsomal enzymes and a simple assay procedure for 7-alkoxycoumarin o-dealkylase. Japanese Journal of Pharmacology, 1983, 33(1): 41-56.

pmid: 6603543
[28]
AITIO A. A simple and sensitive assay of 7-ethoxycoumarin deethylation. Analytical Biochemistry, 1978, 85(2): 488-491.

pmid: 565602
[29]
PEARCE R E, MCINTYRE C J, MADAN A, SANZGIRI U, DRAPER A J, BULLOCK P L, COOK D C, BURTON L A, LATHAM J, NEVINS C, PARKINSON A. Effects of freezing, thawing, and storing human liver microsomes on cytochrome P 450 activity. Archives of Biochemistry and Biophysics, 1996, 331(2): 145-169.

doi: 10.1006/abbi.1996.0294
[30]
NELSON A C, HUANG W, MOODY D E. Variables in human liver microsome preparation: Impact on the kinetics of L-α-acetylmethadol (LAAM)N-demethylation and dextromethorphan O-demethylation. Drug Metabolism and Disposition, 2001, 29(3): 319-325.
[31]
LIU Z Y, HUANG L L, DAI M H, CHEN D M, WANG Y L, TAO Y F, YUAN Z H. Metabolism of olaquindox in rat liver microsomes: structural elucidation of metabolites by high-performance liquid chromatography combined with ion trap/time-of-flight mass spectrometry. Rapid Communications in Mass Spectrometry, 2008, 22(7): 1009-1016.

doi: 10.1002/rcm.3422 pmid: 18320546
[32]
KAMATAKI T, ANDO M, YAMAZOE Y, ISHII K, KATO R. Sex difference in the O-dealkylation activity of 7-hydroxycoumarin O-alkyl derivatives in liver microsomes of rats. Biochemical Pharmacology, 1980, 29(7): 1015-1022.

pmid: 6770868
[33]
ZHOU D S, LINNENBACH A J, LIU R F, LUZIETTI R A, HARRIS J J, BOOTH-GENTHE C L, GRIMM S W. Expression and characterization of dog cytochrome P450 2A13 and 2A25 in baculovirus-infected insect cells. Drug Metabolism and Disposition, 2010, 38(7): 1015-1018.

doi: 10.1124/dmd.110.033068 pmid: 20382755
[34]
ZHANG H Y, ZHANG D L, RAY K, ZHU M S. Mass defect filter technique and its applications to drug metabolite identification by high-resolution mass spectrometry. Journal of Mass Spectrometry, 2009, 44(7): 999-1016.

doi: 10.1002/jms.1610 pmid: 19598168
[35]
QIU Y F, GUO J, CHEN J D, ZHANG W J, WANG W Y. Metabolic profiling of lumateperone in vitro and in vivo by UPLC-Q Exactive Orbitrap HRMS, and its pharmacokinetic study in rat plasma by LC-MS/MS. Journal of Pharmaceutical and Biomedical Analysis, 2024, 246: 116221.

doi: 10.1016/j.jpba.2024.116221
[36]
LI K L, LIU M J, ZHANG M, LI Q, YU K Q, LI J X, SHANG Z C, CAI W. Rapid characterization of the potential active metabolites of diacerein in rat plasma based on UHPLC-Q-exactive orbitrap mass spectrometry and molecular docking. Journal of Pharmaceutical and Biomedical Analysis, 2023, 233: 115447.

doi: 10.1016/j.jpba.2023.115447
[37]
HU W, ZHONG Z Y, REN X F, LIU H Y, TANG X J. The in vitro metabolism of GMDTC in liver microsomes of human, monkey, dog, rat and mouse: Metabolic stability assessment, metabolite identification and interspecies comparison. Journal of Pharmaceutical and Biomedical Analysis, 2023, 236: 115718.

doi: 10.1016/j.jpba.2023.115718
[38]
ZENG Z, ANDREW N W, ARISON B H, LUFFER-ATLAS D, WANG R W. Identification of cytochrome P4503A4 as the major enzyme responsible for the metabolism of ivermectin by human liver microsomes. Xenobiotica, 1998, 28(3): 313-321.

doi: 10.1080/004982598239597 pmid: 9574819
[39]
ZENG Z P, ANDREW N W, WODA J M, HALLEY B A, CROUCH L S, WANG R W. Role of cytochrome P450 isoforms in the metabolism of abamectin and ivermectin in rats. Journal of Agricultural and Food Chemistry, 1996, 44(10): 3374-3378.

doi: 10.1021/jf960222+
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