Comprehensive Analysis of Tiamulin Metabolites in Various Species

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Comprehensive Analysis of Tiamulin Metabolites in Various Species of Farm Animals Using Ultra-High-Performance Liquid Chromatography Coupled to Quadrupole/Time-of-Flight Feifei Sun, Shupeng Yang, Huiyan Zhang, Jinhui Zhou, Yi Li, Jinzhen Zhang, Yue Jin, Zhanhui Wang, Yanshen Li, Jianzhong Shen, Suxia Zhang, and Xingyuan Cao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04377 • Publication Date (Web): 07 Dec 2016 Downloaded from http://pubs.acs.org on December 13, 2016

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Journal of Agricultural and Food Chemistry

1

Comprehensive Analysis of Tiamulin Metabolites in

2

Various

3

Ultra-High-Performance

4

Coupled to Quadrupole/Time-of-Flight

5

Feifei Sun†, ‖, Shupeng Yang‡,‖, Huiyan Zhang†, Jinhui Zhou‡, Yi Li‡, Jinzhen Zhang‡,

6

Yue Jin‡, Zhanhui Wang†, Yanshen Li§, Jianzhong Shen†, Suxia Zhang*,†, Xingyuan

7

Cao*,†

8 9

Species

of

Farm

Animals

Liquid

Chromatography

†

College of Veterinary Medicine, China Agricultural University, Beijing Laboratory

for Food Quality and Safety, Beijing Key Laboratory of Detection Technology for

10

Animal-Derived

11

National Reference Laboratory of Veterinary Drug Residues,

12

People’s Republic of China

13

Using

Food

Safety, Beijing

100193,

‡

Bee Research Institute, Chinese Academy of Agricultural Sciences, Bee Product

14

Quality Supervision and Testing Center, Laboratory of Risk Assessment for Quality

15

and Safety of Bee Products, Ministry of Agriculture, Beijing 100093, People’s

16

Republic of China

17

§

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The short title：Metabolic Pathways of Tiamulin

19

College of Life Science, Yantai University, Yantai, Shandong, 264005, P. R. China

‖

F.S. and S.Y. contributed equally to this work

20

*

21

*

22

Tel: +86-10-6273-2332; Fax: +86-10-6273-1032;

23

E-mail: [email protected]

Both authors are corresponding authors

Author to whom correspondence should be addressed:

ACS Paragon Plus Environment


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ABSTRACT: Tiamulin is an antimicrobial widely used in veterinary practice to treat

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dysentery and pneumonia in pigs and poultry. However, knowledge about the

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metabolism of tiamulin is very limited in farm animals. To better understand the

27

biotransformation of tiamulin, in present study, in vitro and in vivo metabolites of

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tiamulin in rats, chickens, swine, goats, and cows were identified and elucidated using

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Ultra-High

30

Quadrupole/Time-of-Flight (UHPLC-Q/TOF). As a result, a total of 26 metabolites of

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tiamulin identified in vitro and in vivo, and majority of metabolites were revealed for

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the first time. In all farm animals, tiamulin undergo phase I metabolic routes of

33

hydroxylation in the mutilin part (the ring system), S-oxidation and N-deethylation on

34

side chain, and no phase II metabolite was detected. Among these, 2β- and

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8α-hydroxylation and N-deethylation were the main metabolic pathways of tiamulin

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in farm animals. In addition, we have put forward that 8a-hydroxy-tiamulin and

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8a-hydroxy-N-deethyl-tiamulin could be hydroxylated into 8a-hydroxy-mutilin, the

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marker residue of tiamulin in swine. Furthermore, a significant interspecies difference

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was observed on the metabolism of tiamulin among various farm animals. The

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possible marker residue for tiamulin in swine was 8α-hydroxy-tiamulin,

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N-deethyl-tiamulin and 8α-hydroxy-N-deethyl-tiamulin, which was consistent with

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the hypothesis proposed by the European Agency for the Evaluation of Medicinal

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Products (EMEA). However, results in present study indicated that three metabolites

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(2β-hydroxy-tiamulin, N-deethyl-tiamulin and 2β-hydroxy-N-deethyl-tiamulin) of

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tiamulin in chickens had larger yields, implying that 2β-hydroxy-mutilin or

Performance

Liquid

Chromatography


coupled

to

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N-deethyl-tiamulin was more likely to be regarded as the potential marker residue of

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tiamulin in chickens.

48

KEYWORDS:

49

microsomes; farm animals; in vivo

Tiamulin;

comparative

metabolism;

50


UHPLC-Q/TOF;

liver


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 INTRODUCTION

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Tiamulin (TIA), produced by fungus Pleurotusmutilis or Clitopilus, is a

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semisynthetic derivative of antibiotic pleuromutilin.1 Tiamulin, valnemulin and

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retapamulin are the only three pleuromutilin derivates available on the market, while

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the former two drugs have been solely used in veterinary practice.2,3 The

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pleuromutilin exhibit their antimicrobial activities by binding to the 50S ribosomal

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subunit inhibiting protein synthesis.1,3,4 Owing to their unique mechanism of action,

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pleuromutilin derivatives have rarely cross-resistance with other antimicrobial

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agents.3 In addition, tiamulin has good activity against Gram-positive and

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Gram-negative bacteria.5 Therefore, tiamulin has been widely used for the treatment

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of different disease in various species, mainly including Mycoplasma infections in

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poultry, swine dysentery caused by Brachyspirahyodysenteriae, enzootic pneumonia

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infected by hyopneumoniae.6-10 Moreover, tiamulin could also be used as growth

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promoter to enhance growth.11,12 However, due to its extensive use in practice, its

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potential public health hazard by intake of it has received widespread attention and

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interests.13,14 In the present, the Maximum Residue Limits (MRLs) of tiamulin in

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edible tissues (100 µg kg-1 for muscle and 500 µg kg-1 for liver, respectively) has been

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established by the European Commission (EC), United States and China.15

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Furthermore, 8α-hydroxy-mutilin is likely to be the marker residue of tiamulin in

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swine and rabbits proposed by the EC.15


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So far as the authors’ know, some studies have been reported previously upon the

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pharmacokinetic and pharmacodynamic characteristics of tiamulin following different

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administration in various species including swine, chickens, sheep, calves and

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dogs.16-19 These results showed that tiamulin achieved high tissue concentrations in

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the enteric and respiratory tracts as well as in the tonsil. However, limited information

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could be available on the metabolism of tiamulin. Generally speaking, the metabolic

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process of veterinary drugs would markedly influence their efficacy, toxicity and

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residual level in tissues, and metabolism plays a vital role in the identification of the

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metabolites residue marker of veterinary drugs.20 The European Agency for the

80

Evaluation of Medicinal Products (EMEA) has given a fair report on metabolism and

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metabolites residue marker of tiamulin in 1998: after oral administration, total of 15

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metabolites were detected in pigs, and the drug prototype accounted for 7%, which

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indicated that tiamulin embraced an extensive metabolism in vivo.15 The sum of

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metabolites that could be hydrolyzed into 8α-hydroxy-mutilin were regarded as its

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residue marker. However, the chemical structures of the metabolites have not been

86

fully elucidated, and the origin of 8α-hydroxy-mutilin has not been put forward.

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Berneret.al put forward the possible hydroxylation position in mutilin part for the

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pleuromutiline: 1β-,2β- and 8α-.21 After that, Lykkeberg et.al did an investigation on

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the metabolism of tiamulin incubated with swine liver microsomes.22 As a result,

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three critical metabolites were detected and identified which were 2β-, 8α-, and

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N-deethyl-TIA, respectively, and their structures were characterized by Liquid

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Chromatography Coupled to Mass Spectrometry (LC-MS) and Nuclear Magnetic



93

Resonance (1H and

13

C-NMR).22 Furthermore, another two tiny metabolites

94

(8α-OH-N-deethyl-TIA and 2β-OH-N-deethyl-TIA) were identified by the complete

95

analysis of fragment ions. Moreover, the results also give the possibilities of several

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metabolic positions: N-deethyl-TIA, 2β-TIA and 8α-TIA accounted for 20%, 10% and

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7%, respectively. As for the likely marker residue (8α-hydroxy-mutilin) of tiamulin

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proposed by EC, it was not observed in the study of Lykkeberg et.al, implying that

99

8α-hydroxy-mutilin is only present in trace amount. The information mentioned above

100

showed that tiamulin has extensive metabolism in vivo. However, metabolism of

101

tiamulin in swine and the metabolic differences in various species have also not yet

102

been reported.

103

Metabolism of valnemulin in different species has been conducted well in our

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team.23 The results indicated that there were significant metabolic differences for

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valnemulin in various species, which was far more complicated than common

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conscience.23 Tiamulin, a homologous compound of valnemulin, drew our interest that

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whether tiamulin would also undergo extensive metabolism and exhibit interspecies

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difference? Thus, the following investigation was conducted: metabolism of tiamulin

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in rats, chickens, swine, cows and goats liver microsomes. The metabolites were

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detected and identified by UHPLC-Q/TOF method combined with metabolites

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analyzing software, MetabolynxXS. Their chemical structures were preliminarily

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identified by analyzing MS/MS spectra of tiamulin and its metabolites. Furthermore,

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the additional in vivo metabolism studies of tiamulin in rats, chickens and swine were

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investigated in order to verify the reliability of in vitro studies.


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 MATERIALS AND METHODS Reagents and Instruments. The analytical standard of tiamulin was provided by β-Nicotinamide

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Qilu-pharma

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phosphates (NADPH) was purchased from Sigma (German). Acetonitrile and formic

119

acid were of LC-MS grade which were purchased from Fisher Chemical Co.

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(USA).Water in present study was obtained by Milli-Q system (Millipore, USA).The

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other reagents were of the analytical grade. Tris-HCl (0.05 M, pH 7.4): 6.06 g of Tris

122

was weighed and dissolved with purified water then adjusted to 7.4 by HCl to final

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volume of 1.0 L. Homogenization buffer:0.15 g of EDTA and 42.75 g of sucrose were

124

added into volumetric flask and then 0.05 M Tris-HCl was added to a constant

125

volume of 500 mL. 0.01 mol L-1 PBS (pH 7.4): 8.0 g of NaCl, 3.58 g of

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Na2HPO4·12H2O, 0.24 g of KH2PO4, 0.2 g of KCl were added into1.0 L volumetric

127

flask and purified water was added till the constant volume of 1.0 L, and then stored

128

at 4 oC.

Co.

(Shandong,

China).

adenine

dinucleotide

129

Animals. Six Wistar rats (6-week, 3 males and 3 females) with the weight of

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approximately 200-220 g were purchased from Vital River Laboratory Animal

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Technology Co. Ltd. (Beijing, China). Six Avian chickens (4-week, male and female,

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each half) weighing 1,000-1,200 g were purchased from Beijing Huadu Co. Ltd.

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(Beijing, China). Four Changbai swine with the weight of 20-25 kg (2-month, half

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male and half female), four goats (2-month, weighing 20-25 kg, half male and half

135

female) and four cows (18-month, weighing 350-380 kg，half male and half female)



136

were purchased from Zhoukou Village Nursery (Beijing, China). Animals were

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acclimatized for 1 week under standardized condition.

138

Preparation of liver microsomes. Fresh livers of rats, swine, chickens, cows and

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goats were collected and their liver microsomes were obtained by differential

140

centrifugation. Fully description has been given in our study reported previously.23

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Here, a brief statement would be given as follows: fresh livers were cleaned several

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times with ice-cold 0.1 M PBS (pH 7.4) and 0.05 M HCl buffer to remove the

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remaining blood in livers. Then the liver would be minced thoroughly with scissors

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into fragments approximately 1 cm×1 cm and immediately one part of liver be

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homogenized with 3 parts of Tris-HCl buffer containing 1 mmol L-1 EDTA and 0.25

146

M sucrose. The homogenate was centrifuged at 10,000 g for 20 min at 4 °C, the

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supernatant (S9 fraction) was collected and centrifuged at 100,000 g for 60 min at 4

148

o

149

of the prepared microsomes was evaluated by crystalline Bovine Serum Albumin

150

(BSA) method proposed by Lowry et.al.24 To estimate the activities of liver

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microsomes, the prepared microsomes were incubated with coumarin and its

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metabolites were detected by UHPLC-Q/TOF. The results showed that coumarin was

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metabolized to 7-hydroxycoumarin, which implied that the liver microsomes had the

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biological activities.

C. The microsomal pellets were suspended with 0.05 M HCl buffer. Protein content

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Optimization of incubation condition of tiamulin. Liver microsomes of rats,

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chickens swine, goats and cows were incubated with tiamulin, individually. The

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incubation system was conducted in 0.05 M HCl buffer containing 2 mg L-1 liver


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microsomes, 50 mM tiamulin and 1 mM NADPH with a total volume of 500 µL.

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Then the mixture was incubated for 2 h at 37 oC in a shaker and terminated by 500 µL

160

ice-cold acetonitrile. Subsequently, it was centrifuged at 12,000 g for 15 min at 4 oC.

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Finally, the supernatant was filtrated through a 0.22 µm microbore cellulose

162

membrane into auto-sampler vial and analyzed by UHPLC-Q/TOF to identify

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metabolites. Experiments in present study were all in triplicate and control groups

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were set as follows: in an absence of NADPH or TIA, respectively.

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Preparation of in vivo samples. Animals (3 male rats and 3 female rats, 3 male

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chickens and 3 female chickens, 2 male swine and 2 female swine) would go through

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a 12 h fasting before experiment. Then they were separately orally administrated with

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tiamulin with a single dosage of 20 mg kg-1 b.w. The urine and feces samples were

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collected prior to experiment and during 0-12 h, 12-24 h and 24-48 h following

170

administration. The samples were frozen at -20 oC until analysis. 15 milliliters of

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ethyl acetate was added into 2.0 g of feces or 2.0 mL of urine sample, then the

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mixture was vortexed for 5 min and centrifuged at 4,500 g for 15 min at 4 oC. The

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supernatant was collected and evaporated to dryness with a gentle nitrogen flow at 50

174

o

175

re-dissolved solution was filtrated through 0.22 µm microbore cellulose membrane

176

and analyzed by UHPLC-Q/TOF.

C. The residue was reconstituted with 1.0 mL acetonitrile/water (15:85, v/v), then the

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Instrumental conditions. The established method in present study was based on an

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ACQUITY UPLC system (Waters Co., USA) coupled with a hybrid Q/TOF-MS

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SYNAPT HDMS (Waters, UK). The separation of tiamulin metabolites was achieved



180

through an Acquity BEH RP18 column (50mm×2.1mm i.d., 1.7 µm particle size)

181

(Waters, USA) at the operating flow rate of 0.3 mL min-1. Mobile phase A: aqueous

182

solution containing 0.1% formic acid and mobile phase B, acetonitrile containing 0.1%

183

formic acid. Gradient elution program was performed as follows: 0-3.0 min (5 %-15%

184

B), 3.0-16.0 min (15%-40% B), 16.0-17.0 min (40%-100% B), 17.0-19.0 min

185

(100%-5% B), 19.0-20.0 min (5% B). The injection volume was 10 µL.

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The system was operated in positive electrospray ionization mode (ESI+) with the

187

following typical parameters for mass spectrometry: capillary voltage, 3.0 kV; source

188

temperature, 100 oC; de-solvation temperature 350 oC; de-solvation gas flow rate and

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cone gas flow rate were 600 L h-1 and 30 L h-1, respectively; while the collision

190

energy and cone voltage were 30 eV and 30 V, individually. Additionally, internal

191

reference consisting of 1 ng µL-1 solution of leucineencephalin was infused at 50 µL

192

min-1 during acquisition, generating a reference ion at m/z 556.2771 in ESI+ mode.

193

Low energy data and high energy data were obtained with collision energy of 5 eV

194

and 15-35 eV, respectively.

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 RESULTSANDDISCUSSION

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Parsing strategy. High Resolution Mass Spectrum (HRMS) has been widely used

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for the identification of metabolites.25,26 As for the parent drug and its metabolites, the

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full scan and all ion framentation acquisition were performed. After that, the acquired

199

MSE data of samples and controls were further analyzed by MetabolynxXS software to

200

identify and characterize metabolites, which was mainly based on the data-mining


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201

techniques of mass defect filtering (MDF), background subtraction (BS) and extracted

202

ion chromatogram (EIC) analysis.27,28 In addition, fragments were obtained by

203

collision induced dissociation (CID). In present study, the metabolites of tiamulin

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were identified using UHPLC-Q/TOF along with the above-mentioned data-mining

205

techniques.

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Fragment Ions parsing of tiamulin. It is more likely to be protonated for tiamulin

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(m/z 494.3317) in ESI+ mode when formic acid is added into mobile phase. When the

208

drug is metabolized in liver, some modifications are intended to occur upon the

209

chemical structure of the drug, and its main metabolic pathways include oxidation,

210

reduction, hydrolysis and glucuronic acid conjugation etc. Compared with parent

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nuclear structure, its metabolites similarly have the same parent nuclear structure.

212

Thus, the structures of metabolites could be deduced by fully clarifying their fragment

213

ions, which provides responsible support for the following structurally identification

214

of metabolites. The MS/MS spectrum of protonated tiamulin was shown in Figure 1.

215

High resolution and mass accuracy were provided by Q/TOF while the error was

216

within 5 ppm, indicating the reliability. Protonated tiamulin (m/z 494) was likely to

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generate a pair of complementary fragment ions (m/z 303 and m/z 192) by the

218

cleavage of ester bond under collision induced dissociation (CID) condition

219

meanwhile m/z 192 was the superior ion with high response. The fragment ion of m/z

220

119 can be generated with the loss of ethylamine ether (C4H11N) from m/z 192,

221

meanwhile m/z 285 and m/z 267 could be generated subsequently by the loss of H2O

222

from m/z 303. According to the results obtained, a fragmentation pathway for TIA is



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proposed in figure 1, which will be regarded as the primary guidance for subsequent

224

structure elucidation of the metabolites of TIA.

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Metabolism profiling of tiamulin in swine liver microsomes. The metabolism of

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tiamulin in swine liver microsomes has been fully reported. In order rapidly

227

characterize the metabolism of TIA, the metabolism of TIA in swine liver

228

microsomes was conducted in present study. The positive and control samples were

229

analyzed by UHPLC-Q/TOF. Consequently, a total of 18 possible metabolites were

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observed in positive samples while they were not detected in control samples. Figure

231

2 showed their extracted ion chromatograms (EICs) by processing the full-scan MS

232

data within 5 ppm mass tolerance. In addition, to verify the structure of potential

233

metabolites, the MS/MS spectra and proposed fragmentation scheme of the detected

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metabolites were shown in Figure 1. The retention time, m/z value, main fragment

235

ions and some other information of each metabolite were shown detailed in table 1.

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The structure of metabolites were characterized or identified according to the

237

chromatographic behaviors, accurate mass, distributions of fragment ions, basic rules

238

of drug metabolism and structure of tiamulin.

239

Hydroxylated metabolites M1-M4, M6-M8, M10-M12. All these metabolites have

240

an m/z value of 510.3248 with the error of 1.1-3.8 ppm between measured and

241

calculated values, suggesting that all these metabolites mentioned above were the

242

products based upon hydroxylation of tiamulin or the oxidation at the nitrogen or the

243

sulphur part. Due to the different reactive positions of the metabolites, their retention

244

time varied in chromatogram. In comparison to the MS/MS spectrum of metabolite


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with other metabolites, M6 differed markedly, whose characteristic fragment ion was

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m/z 208, gaining 16 Da compared with the fragment of tiamulin, m/z 192, which

247

inferred that M6 was the product of oxidation on side chain. According to the

248

chemical structure of side chain, S-part is the position which is most likely to be

249

oxidized. Thus, M6 was regarded as the metabolite of S-oxide on side chain.

250

Fragment ion m/z 192, m/z 119, appeared in the chromatogram of the other

251

metabolites, implying their side chain maintained unchanged. 16 Da were individually

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gained comparing m/z 319, m/z 301 and m/z 283 with m/z 303, m/z 285 and m/z 267 of

253

TIA, which suggested that the hydroxylation were occurred on mutilin motif.

254

Considering the complexity of mutilin, the exact hydroxylated position could not be

255

fully demonstrated solely according to MS/MS spectra. Earlier researches have

256

indicated that hydroxylation was more intended to be position 1β-, 2β- and 8α-, which

257

complied with the results of metabolism research of TIA in swine liver microsomes

258

conducted by Lykkeberg et.al..21,22 Moreover, Lykkeberg also put forward that

259

2β-OH-TIA and 8α-OH-TIA were the main hydroxylated metabolites, which

260

accounted approximately for 10% and 7%, respectively.22 In present study,

261

considering the greater responses in LC-MS spectra along with the reported results,

262

the two main metabolites were identified as 2β-OH-TIA and 8α-OH-TIA, respectively.

263

As for other hydroxylated metabolites (M1, M7, M8, M10, M11 and M12), their exact

264

hydroxylated position were temporarily unable to be determined. Figure 1 showed

265

MS/MS fragment ion analysis of each metabolite.



266

N-deethylation of metabolite M13. The elemental component of M13 (m/z

267

466.2986) is C26H44NO4S+. Compared with tiamulin, M13 dropped an ethyl (C2H4),

268

suggesting that M13 was likely to be de-ethyl-TIA. Characteristic fragment ion of

269

M13, m/z 164, was 28 Da lower than m/z 192, indicating a de-ethylation at the amino

270

group, which was further confirmed as N-deethyl-TIA.

271

N-deethylation based on hydroxylated metabolites M14, M16-M19 and M22.

272

These metabolites were all composed of C26H44NO5S+ with the same m/z value of

273

482.2935 (monoisotopic mass), which was 16 Da higher than M13, indicating that

274

these metabolites were the hydroxylated or oxidized products based on M13. One of

275

the MS/MS fragment ion m/z 180 is 16 Da higher than m/z 164, fragment ion of M13,

276

indicating the oxidation occurred on side chain. Therefore, M17 was identified as

277

N-deethyl-S-oxidized-TIA.

278

The fragment ion m/z 319, m/z 301 and m/z 283 were 16 Da higher than those of

279

parent drug, respectively, implying that hydroxylation appeared on the mutilin part.

280

The fragment ion m/z 164 indicated N-deethylation occurred on side chain. During

281

these metabolites, M14 and M16 have higher responses, in the meantime, taking their

282

retention times and hydroxylation information into consideration, they were identified

283

to be 2β-OH-N-deethyl-TIA and 8α-OH-N-deethyl-TIA. As for other metabolites,

284

their hydroxylated position on mutilin part could not be soundly identified.

285

N-di-deethyl-TIA, M23. M23 consists of C24H40NO4S+ with protonated ion m/z

286

value of 438.2673, which was 56 Da lower than tiamulin, suggesting M23 might lose

287

two ethyl groups. Both M23 and tiamulin had fragment ion of m/z 303, m/z 285 and


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288

m/z 267, indicating that the mutilin part remained unchanged. Characteristic fragment

289

ion m/z 136 was 56 Da lower than m/z 192 of tiamulin, which firmly demonstrated the

290

occurrence of N-di-deethylation on side chain. Thus, M23 was identified as

291

N-di-deethyl-TIA.

292

Comparative metabolic study of tiamulin in liver microsomes of rats, chickens,

293

swine, cows and goats. The variation of metabolic enzymes in various species may

294

account for the metabolic differences. To investigate the metabolism of tiamulin in

295

different species, metabolic study of tiamulin was conducted in liver microsomes of

296

rats, chickens, swine, cows and goats. Figure 2 showed the accurate extracted ion

297

chromatogram (EICs) of tiamulin metabolites in liver microsomes of different species.

298

Table 1 showed metabolites of tiamulin in different species. As shown in Figure 2 and

299

Table 1, 23, 17, 18, 21 and 21 kinds of metabolites were, respectively, detected in

300

liver microsomes of rats, chickens, swine, cows and goats. With the exception of 18

301

kinds of metabolites reported already, another 5 kinds of metabolites were identified

302

and characterized soundly in microsomes of swine, which were M3, M5, M9, M15

303

and M21. Seen from the accurate m/z values and MS/MS spectra, M3, M5 and M9

304

were identified as metabolites of tiamulin hydroxylated on mutilin part while M15

305

and M21 were metabolites of tiamulin with deethylation and hydroxylation. As for the

306

four hydroxylated metabolites, their hydroxylated position on mutilin part could not

307

be exactly determined solely according to the acquired data. In order to compare the

308

metabolic differences in various species, the accurate extracted ion chromatogram

309

was integrated for each metabolite, then the peak area was utilized to estimate



310

metabolic differences in various species, the results could be seen in Figure 4. Figure

311

4 indicated that certain metabolic differences did exist in liver microsomes of

312

different animals and their major metabolites vary as well: M2 (2β-OH-TIA), M4

313

(8α-OH-TIA), M8 and M13 (N-deethly-TIA) in rats; M2 (2β-OH-TIA) and M13

314

(N-deethly-TIA) in chickens; M2 (2β-OH-TIA), M4 (8α-OH-TIA) and M13

315

(N-deethly-TIA) in swine and goats; M2 (2β-OH-TIA), M4 (8α-OH-TIA), M6

316

(Sulfoxide TIA), M10, M11, M12 and M13 (N-deethly-TIA) in cows. In spite of the

317

differences of the metabolites in various species, there were still three major

318

metabolites in common, which were M2 (2β-OH-TIA), M4 (8α-OH-TIA) and M13

319

(N-deethly-TIA). According to the remained non-metabolic tiamulin, the metabolic

320

capability of different species for tiamulin could be concluded that rats and cows own

321

the best metabolic ability for tiamulin, followed by goats, chickens and swine, the

322

worst.

323

In vivo metabolism of TIA in rats, chickens and swine. Generally speaking, results

324

gained from metabolism in vivo could be more authentic to reflect the

325

biotransformation in vivo for drugs than metabolism in vitro. In addition to liver

326

enzymes, there are some other factors which will affect the biotransformation of drugs

327

such as intestinal microbial, intestinal epithelial cells, kidney and so forth. To further

328

verify the results obtained from in vitro metabolism, in vivo metabolism of tiamulin

329

was carried out in rats, chickens and swine. Tiamulin was administrated to animals by

330

gavage at the dose of 20 mg kg-1 b.w.. Then urine and feces samples were collected

331

after oral dose. Subsequently, samples were extracted and cleaned up (HLB cartridge)


Page 16 of 33

Page 17 of 33


332

for analysis by UHPLC-Q/TOF. Results showed that tiamulin had extensive

333

metabolism, and 19, 8 and 9 kinds of metabolites had been detected in rats, chickens

334

and swine, individually. Furthermore, total of three new metabolites were detected in

335

the urine of rats, which were M24, M25 and M26. By analyzing their accurate m/z

336

values and MS/MS spectra, Metabolites of M24, M25 and M26 were identified as

337

di-hydroxylated metabolites of TIA on mutilin part. Figure 1 showed their analytical

338

process of MS/MS spectra. Similar to analysis of in vitro metabolism, peak area of

339

accurate extracted ion was used to better compare the yield of each metabolite. Figure

340

5 described results of tiamulin in vivo, from which we could draw a conclusion that

341

tiamulin had gone through extensive metabolism in vivo, only few non-metabolized

342

tiamulin being detected. Consist with results of metabolism in vitro, metabolism of

343

tiamulin in vivo also differed in different species to some extend: M2 (2β-OH-TIA),

344

M4 (8α-OH-TIA) and M14 (2β-OH-N-deethly-TIA) were the major metabolites in

345

rats; M2 (2β-OH-TIA), M4 (8α-OH-TIA), M13 (N-deethly-TIA) and M14

346

(2β-OH-N-deethly-TIA) in chickens; M2 (2β-OH-TIA), M4 (8α-OH-TIA), M13

347

(N-deethly-TIA), M14 (2β-OH-N-deethly-TIA) and M16 (8α-OH-N-deethly-TIA) in

348

swine. The results showed that hydroxylation and N-deethylation were the main

349

metabolic pathways for tiamulin.

350

Different metabolic pathways of tiamulin in various species. Results showed that

351

tiamulin had more thorough metabolism in vivo than in vitro, especially for M14

352

(2β-OH-N-deethly-TIA) and M16 (8α-OH-N-deethly-TIA), hydroxylation based on



Page 18 of 33

353

N-deethylation. Different metabolic pathways in detail for tiamulin in various species

354

were shown in Figure 3.

355

Potential marker residue of tiamulin in swine. EMEA has reported that over 15

356

metabolites were detected in swine after oral administration of 3H-tiamulin at a dose

357

of 10 mg kg-1 body weight, while these metabolites mainly remained in liver and no

358

individual metabolite accounted for 5% of the total residue.15 Another research

359

showed that in pigs, following oral administration, 35% of TIA along with its

360

metabolites was eliminated in urine while 65% of those were excreted in feces.15

361

Besides, EMEA recommended 8α-hydroxy-mutilin as residue markers for tiamulin in

362

edible animal tissues.15 In present study, the origin of 8α-hydroxy-mutilin, for the first

363

time,

It

came

from

364

8α-hydroxy-N-deethylation-tiamulin,

which

represented

365

phenomenon was consistent with the proposed opinion that 8α-hydroxy-mutilin was

366

likely to be the marker residue.15 EMEA described that sum of the residues that can be

367

hydrolyzed to be 8α-hydroxy-mutilin were identified as the marker residue in tissues

368

of pigs, chickens and turkeys.15 However, Figure 5 represented another phenomenon

369

that given the larger yields, 8α-hydroxy-N-deethyl-TIA (M16), 8α-hydroxy-TIA (M4)

370

or N-deethyl-TIA (M13) would be better to be the marker residue.

was

put

forward.

8α-hydroxy-tiamulin larger

yields.

and This

371

Possible marker residue of tiamulim in chickens. In laying hens, broilers and

372

turkeys, following oral administration of 3H-tiamulin at a dosage of 10 mg kg-1 body

373

weight for five consecutive days, more than 15 metabolites were detected where 4

374

metabolites represented over 30% of the total residue.15 EMEA has depicted that


Page 19 of 33


375

although tiamulin represented a larger amount of the total residues than the metabolite,

376

8α-hydroxy-mutilin, this metabolite was still regarded as an appropriate marker

377

residue for chicken tissues because there was a validated analytical method.15

378

However, in chickens, 8α-hydroxy-mutilin merely represented 3% of the total residue,

379

whereas

380

N-deethylation-TIA (M13) had large yields. Results showed that the yield of

381

2β-hydroxy-mutilin

382

8α-hydroxy-mutilin. 2β-hydroxy-mutilin or N-deethylation-TIA (M13) thus is more

383

likely to the potential marker residue. To elucidate our hypothesis, some other

384

experiments surely need to be conducted.

2β-hydroxy-TIA

was

(M2),

2β-hydroxy-N-deethylation-TIA

approximately

4

times

higher

(M14)

than

that

and

of

385

In summary, liver microsomal incubation technology was used in present study for

386

investigating the in vitro metabolism of tiamulin in rats, chickens, swine, goats and

387

cows. Finally, 23,17,18,21 and 21 kinds of metabolites individually identified in liver

388

microsomes of rats, chickens, swine, goats and cows by UHPLC-Q/TOF and

389

MetabolynxTM software. The chemical structures of metabolites were initially

390

identified by analyzing their MS/MS spectra. To elucidate the results obtained from in

391

vitro metabolism, in vivo metabolism studies of tiamulin in rats, chickens and swine

392

were carried out, where 19, 8 and 9 kinds of metabolites were detected, respectively.

393

In this study, the origin of 8α-hydroxy-mutilin, was firstly proposed. The results

394

indicated that tiamulin would undergo extensively metabolism in various animals,

395

while the main metabolic pathways were the hydroxylation on the nucleus part (2β,

396

8α, etc.), S-oxidation on the side chain and N-deethylation on side chain. Additionally,



397

the metabolic pathways of tiamulinin different species were illustrated and total of 26

398

metabolites were identified, most of which were reported for the first time.

399

Furthermore, the correlation of tiamulin between in vivo and in vitro metabolism was

400

compared in present study and the interspecies metabolic differences of tiamulin were

401

further investigated, which would provide a leading function for the residue detection

402

and risk assessment of tiamulin in animal-derived products. What’s more, the results

403

indicated that 8α-hydroxy-mutilin was likely to be the marker residue of tiamulin in

404

swine, but not in chickens. 2β-hydroxy-mutilin or N-deethylation-TIA is more

405

suitable to be the marker residue, which needs further confirmation.

406

 AUTHOR INFORMATION

407

Corresponding Author

408

*Xingyuan Cao: Tel: +86-10-6273-2332; E-mail: [email protected]

409

*Suxia Zhang: Tel: +86-10-6273-4565; E-mail: [email protected]

410

Funding

411

This work was supported by Natural Science Foundation of China (No. 31672599)

412

and Evaluation of Scientific Establishment of Maximum Residual Limits in Animal

413

Products Foundation of China (No. GJFP201600706). Yanshen Li was supported by

414

the National Natural Science Foundation of China (Grant No. 31402246).

415

Notes


Page 20 of 33

Page 21 of 33


416

The authors declare no competing ﬁnancial interest.

417

 ACKNOWLEDGMENTS

418

We are grateful to Ruiwei Fan, Lingling Xiong, Jingyuan Kong and Yalong Lin for

419

their timely help during the in vivo metabolism of tiamulin in swine and chickens.

420

 ABBREVIATIONS USED

421

TIA, tiamulin; MRL, maximum residue limit; EC, European Commission; EMEA,

422

European Agency for the Evaluation of Medicinal Products; NMR, nuclear magnetic

423

resonance;

424

chromatography-quadrupole/time-of-flight coupled to mass spectrometry; NADPH,

425

β-Nicotinamide adenine dinucleotide phosphates; LC-MS, liquid chromatography

426

coupled to mass spectrometry; PBS, phosphate buffer solution; EDTA, ethylene

427

diaminetetraacetic acid; BSA, bovine serum albumin; CID, collision induced

428

dissociation; MDF, mass defect filtering; BS, background subtraction; EIC, extracted

429

ion chromatograms; OH, hydroxy.

430

 SUPPORTING INFORMATION

431

Chemical structures of Tiamulin, 8α-hydroxy-mutilin, Pleuromutilin, Valnemulin and

432

Retapamulin has been attached in the supplementary material. This material is

433

available free of charge via the Internet at http://pubs.acs.org.

UHPLC-Q/TOF,

ultra-high


performance

liquid


434

 REFERENCES

435

1. Schlunzen, F.; Pyetan, E.; Fucini, P.; Yonath, A.; Harms, J. M., Inhibition of

436

peptide bond formation by pleuromutilins: the structure of the 50S ribosomal subunit

437

from Deinococcusradiodurans in complex with tiamulin. Mol. Microbiol. 2004, 54,

438

1287-1294.

439

2. Novak, R.; Shlaes, D. M., The pleuromutilin antibiotics: a new class for human

440

use. Curr. OpinInvestig. Drugs. 2010, 11, 182-191.

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3. Tang, Y. Z.; Liu, Y. H.; Chen, J. X., Pleuromutilin and its derivatives-the lead

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443

4. Poulsen, S. M.; Karlsson, M.; Johansson, L. B.; Vester, B., The pleuromutilin

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drugs tiamulin and valnemulin bind to the RNA at the peptidyltransferasecentre on the

445

ribosome. Mol. Microbiol. 2001, 41, 1091-1099.

446

5. Hunt, E., Pleuromutilin antibiotics. Drug Future. 2000, 25, 1163-1168.

447

6. Hannan, P. C.; Windsor, H. M.; Ripley, P. H., In vitro susceptibilities of recent

448

field isolates of Mycoplasma hyopneumoniae and Mycoplasma hyosynoviae to

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valnemulin (Econor), tiamulin and enrofloxacin and the in vitro development of

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resistance to certain antimicrobial agents in Mycoplasma hyopneumoniae. Res. Vet.

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Sci. 1997, 63, 157-160.

452

7. Pringle, M.; Landen, A.; Franklin, A., Tiamulin resistance in porcine

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Brachyspirapilosicoli isolates. Res. Vet. Sci. 2006, 80, 1-4.


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8. Islam, K. M.; Klein, U.; Burch, D. G., The activity and compatibility of the

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antibiotic tiamulin with other drugs in poultry medicine--A review. Poult. Sci. 2009,

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88, 2353-2359.

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9. Burch, D.; Young, S.; Watson, E., Treatment of histomonosis in turkeys with

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tiamulin. Vet. Rec. 2007, 161, 864.

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10. Wilberts, B. L.; Arruda, P. H.; Warneke, H. L.; Erlandson, K. R.; Hammer, J. M.;

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Burrough, E. R., Cessation of clinical disease and spirochete shedding after tiamulin

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treatment in pigs experimentally infected with "Brachyspirahampsonii". Res. Vet. Sci.

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11. Cromwell, G. L.; Stahly, T. S., Efficacy of Tiamulin as a Growth Promotant for

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Growing Swine. J. Anim. Sci.1985, 60, 14-19.

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12. Dibner, J. J.; Richards, J. D., Antibiotic growth promoters in agriculture: history

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and mode of action. Poult. Sci. 2005, 84, 634-643.

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13. Nozal, M. J.; Bernal, J. L.; Martin, M. T.; Jimenez, J. J.; Bernal, J.; Higes, M.,

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array-electrospray ionization mass spectrometry detection. J. Chromatogr. A. 2006,

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1116, 102-108.

471

14. De Baere, S.; Devreese, M.; Maes, A.; De Backer, P.; Croubels, S.,

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Quantification of 8-alpha-hydroxy-mutilin as marker residue for tiamulin in rabbit

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tissues by high-performance liquid chromatography-mass spectrometry. Anal. Bioanal.

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Chem. 2015, 407, 4437-4445.

analysis

of

tiamulin

in

honey

by

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chromatography-diode


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15. European Commission (EC), C. f. V. M. P., Tiamulin, summary report. 2000,

476

339-398.

477

16. Laber, G., Investigation of pharmacokinetic parameters of tiamulin after

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intramuscular

479

Pharmacol.Ther. 1988, 11, 45-49.

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17. Riond, J. L.; Schreiber, F.; Wanner, M., Influence of tiamulin concentration in

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feed on its bioavailability in piglets. Vet. Res.1993, 24, 494-502.

482

18. Gatne, M. M.; Badole, P. C.; Somkuwar, A. P.; Ranade, V. V., Pharmacokinetics

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of Tiamulin in Calves after Single Intramuscular Administration. Indian Vet. J. 1994,

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71, 1148-1149.

485

19. Gatne, M. M.; Badole, P. C.; Ranade, V. V., Pharmacokinetics of Tiamulin in

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Sheep after Single Intramuscular Administration. Indian Vet. J.1995, 72, 287-289.

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20. Xu, C.; Li, C. Y.; Kong, A. N., Induction of phase I, II and III drug

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metabolism/transport by xenobiotics. Arch. Pharm. Res. 2005, 28, 249-268.

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21. Berner, H., Vyplel, H., Schulz, G., Stuchlik, P., Inversion of configuration of the

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methylgroup at carbon 6 in the tricyclic skeleton of the diterpenepleuromutilin

491

(chemistry of pleuromutilins, IX). MonatsheftefürChemie Chemical Monthly. 1983,

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114, 1125-1136.

493

22. Lykkeberg, A. K.; Cornett, C.; Halling-Sorensen, B.; Hansen, S. H., Isolation and

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structural elucidation of tiamulin metabolites formed in liver microsomes of pigs. J.

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Pharm. Biomed. Anal. 2006, 42, 223-231.

and

subcutaneous

administration

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23. Yang, S.; Shi, W.; Hu, D.; Zhang, S.; Zhang, H.; Wang, Z.; Cheng, L.; Sun, F.;

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Shen, J.; Cao, X., In vitro and in vivo metabolite profiling of valnemulin using

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ultraperformance liquid chromatography-quadrupole/time-of-flight hybrid mass

499

spectrometry. J. Agric. Food Chem. 2014, 62, 9201-9210.

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24. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J., Protein measurement

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with the Folin phenol reagent. J. Biol. Chem. 1951, 193, 265-275.

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25. Yang, S. P.; Li, Y. S.; Cao, X. P.; Hu, D. F.; Wang, Z. H.; Wang, Y.; Shen, J. Z.;

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Zhang, S. X., Metabolic Pathways of T-2 Toxin in in Vivo and in Vitro Systems of

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Wistar Rats. J. Agric. Food Chem. 2013, 61, 9734-9743.

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26. Yang, S. P.; De Boevre, M.; Zhang, H. Y.; De Ruyck, K.; Sun, F. F.; Wang, Z. H.;

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Cao, X. Y.; Shen, J. Z.; De Saeger, S.; Zhang, S. X., Unraveling the in vitro and in

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vivo metabolism of diacetoxyscirpenol in various animal species and human using

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ultrahigh-performance liquid chromatography-quadrupole/time-of-flight hybrid mass

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spectrometry. Anal. Bioanal. Chem. 2015, 407, 8571-8583.

510

27. Nathanail, A. V.; Varga, E.; Meng-Reiterer, J.; Bueschl, C.; Michlmayr, H.;

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Malachova, A.; Fruhmann, P.; Jestoi, M.; Peltonen, K.; Adam, G.; Lemmens, M.;

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Schuhmacher, R.; Berthiller, F., Metabolism of the Fusarium Mycotoxins T-2 Toxin

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and HT-2 Toxin in Wheat. J. Agric. Food Chem. 2015, 63, 7862-7872.

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28. Stanic, A.; Uhlig, S.; Sandvik, M.; Rise, F.; Wilkins, A. L.; Miles, C. O.,

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Characterization of Deoxynivalenol-Glutathione Conjugates Using Nuclear Magnetic

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Resonance Spectroscopy and Liquid Chromatography-High-Resolution Mass

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Spectrometry. J. Agric. Food Chem. 2016, 64, 6903-6910.



Figure caption Figure 1 The MS/MS spectra and their proposed fragment ions pattern of tiamulin and its metabolites. Figure 2 Accurate extracted ion chromatograms (EICs, the extraction window is 50 mDa) of Tiamulin (TIA) metabolites from the incubated rat, chicken, pig, goat and cow liver microsomes. Figure 3 The proposed metabolic pathways of tiamulin in vitro and in vivo of different species animals. Figure 4 The peak areas of extraction ion chromatograms on tiamulin metabolites detected in rats, chickens, pigs, goats and cows liver microsomes Figure 5 The peak areas of extraction ion chromatograms of tiamulin metabolites detected in in vivo of rats, chickens and pigs


Page 26 of 33

Page 27 of 33


Figure 1



Figure 2


Page 28 of 33

Page 29 of 33


Figure 3



Figure 4


Page 30 of 33

Page 31 of 33


Figure 5



Page 32 of 33

Table 1 Summary of metabolites of TIA detected in in vivo and in vitro of different species of animals NO.

Description

Composition

In vitro

[M+H] + (m/z)

Error (ppm)

Retention Time(min)

rat

chicken swine √ ND

In vivo goat

cow

rat

√ √

√ √

√ √

√ ND

TIA M1

TIA TIA+O

C28H48NO4S+ C28H48NO5S+

494.3298 510.3248

1.3 1.7

9.01 4.62

√ √

M2

2β-OH-TIA

C28H48NO5S+

510.3248

1.2

4.77

√

√

√

√

√

√

M3

TIA+O

C28H48NO5S+

510.3248

2.4

5.34

√

ND

ND

√

√

√

M4

8α-OH-TIA

C28H48NO5S+

510.3248

1.1

5.86

√

√

√

√

√

√

+

Major fragments

chicken swine √ ND

√ ND

494, 303, 285, 267, 192*, 119 510, 319, 301, 283, 192*, 119

√

√

510, 319, 301, 283, 192*, 119

ND

ND

510, 319, 301, 283, 192*, 119

√

√

510, 319, 301, 283, 192*, 119 510, 319, 301, 283, 192*, 119

M5

TIA+O

C28H48NO5S

510.3248

3.8

5.97

√

ND

ND

ND

ND

√

ND

ND

M6

Sulfoxide TIA

C28H48NO5S+

510.3248

3.1

6.22

√

√

√

√

√

√

√

√

M7

TIA+O

C28H48NO5S+

510.3248

2.9

6.67

√

√

√

√

√

ND

ND

ND

510, 319, 301, 283, 192*, 119

M8

TIA+O

C28H48NO5S+

510.3248

1.5

6.98

√

√

√

√

√

√

ND

√

510, 319, 301, 283, 192*, 119

M9

TIA+O

C28H48NO5S+

510.3248

4.1

7.26

√

√

ND

√

√

ND

ND

ND

510, 319, 301, 283, 192*, 119

M10

TIA+O

+

C28H48NO5S

510.3248

2.4

7.77

√

√

√

√

√

ND

ND

ND

510, 319, 301, 283, 192*, 119

M11

TIA+O

C28H48NO5S+

510.3248

2.2

8.00

√

√

√

√

√

ND

ND

ND

510, 319, 301, 283, 192*, 119

M12

TIA+O

C28H48NO5S+

510.3248

1.8

8.53

√

√

√

√

√

ND

ND

ND

510, 319, 301, 283, 192*, 119

M13

N-deethly-TIA

C26H44NO4S+

466.2986

1.5

8.11

√

√

√

√

√

√

√

√

466, 303, 285, 267,164*, 119

M14

2β-OH-N-deethly-TIA

C26H44NO5S+

482.2935

2.3

4.08

√

√

√

√

√

√

√

√

482, 319, 301, 283, 164*, 119

M15

N-deethly-TIA+O

C26H44NO5S+

482.2935

3.9

4.53

√

ND

ND

ND

√

√

ND

ND

482, 319, 301, 283, 164*, 119

M16

8α-OH-N-deethly-TIA

C26H44NO5S+

482.2935

2.3

5.00

√

√

√

√

√

√

√

√

482, 319, 301, 283, 164*, 119

M17

Sulfoxide N-deethly-TIA

C26H44NO5S+

482.2935

1.1

5.56

√

√

√

√

√

√

√

√

482, 303, 285, 267, 180, 162*, 102

M18

N-deethly-TIA+O

C26H44NO5S+

482.2935

1.6

5.75

√

√

√

√

√

√

ND

ND

482, 319, 301, 283, 164*, 119

M19

N-deethly-TIA+O

C26H44NO5S+

482.2935

3.3

6.10

√

√

√

√

ND

√

ND

ND

482, 319, 301, 283, 164*, 119

M20

N-deethly-TIA+O

C26H44NO5S+

482.2935

2.8

6.92

√

√

√

√

√

√

ND

ND

482, 319, 301, 283, 164*, 119

M21

N-deethly-TIA+O

+

C26H44NO5S

482.2935

1.8

7.19

√

ND

ND

√

√

√

ND

ND

482, 319, 301, 283, 164*, 119

M22

N-deethly-TIA+O

C26H44NO5S+

482.2935

4.0

7.64

√

ND

√

√

√

ND

ND

ND

482, 319, 301, 283, 164*, 119

M23

N-di-deethly-TIA

C24H40NO4S+

438.2673

1.9

7.20

√

√

√

√

√

√

√

√

466, 303, 285, 267, 136, 119*

C28H48NO6S+

526.3197

1.3

2.6-4.4

ND

ND

ND

ND

ND

√

ND

ND

526, 335, 317, 299, 192*, 119

M24-26 TIA+2O +

510, 492, 303, 285, 267, 208, 190*, 119

The [M+H] (m/z) values were calculated from the proposed structural formulae. The Error (ppm) is the difference between the calculated and observed m/z values. * The base peak in the MS/MS spectra; √, detected; and ND, not detected.


Page 33 of 33



Comprehensive Analysis of Tiamulin Metabolites in Various Species

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