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"

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