Multiresidue determination of anabolic agent residues: steroids

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New Analytical Methods

Multiresidue determination of anabolic agent residues: steroids, stilbenes and resorcylic acid lactones, in bovine urine by GC-MS/MS employing microwave assisted derivatization Amanda Lemes Silveira, Mauro Lucio Goncalves de Oliveira, Diego Gomes Rocha, Sérgio Dracz, Thiago Freitas Borgati, Mary Ane Gonçalves Lana, Rodinei Augusti, and Adriana Faria J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02439 • Publication Date (Web): 18 Jul 2018 Downloaded from http://pubs.acs.org on July 19, 2018

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Journal of Agricultural and Food Chemistry

Multiresidue determination of anabolic agent residues: steroids, stilbenes and resorcylic acid lactones, in bovine urine by GC-MS/MS employing microwave assisted derivatization

Amanda L. Silveira†, Mauro Lúcio G. de Oliveira‡, Diego G. Rocha†,§, Sérgio Dracz§, Thiago F. Borgati§, Mary Ane G. Lana§, Rodinei Augusti†, Adriana F. Faria†*

†

Department of Chemistry - Institute of Exact Sciences - Federal University of Minas

Gerais. ‡

Laboratory of Pesticides of the National Agricultural Laboratory of Minas Gerais.

§

Laboratory of Residues of Veterinary Drugs of the National Agricultural Laboratory of

Minas Gerais.

Corresponding Author *Tel.: +55 031 3409 5750. Fax: +55 031 3409 5720. E-mail: [email protected]

1 ACS Paragon Plus Environment


1

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Abstract

2 3

In this work, a GC-MS/MS method was developed for the determination of anabolic

4

agent residues in bovine urine. Optimized sample preparation was as follows: enzymatic

5

hydrolysis by β-glucuronidase/sulfatase enzyme from Helix pomatia for 16 h at 37.5 °C,

6

liquid-liquid extraction with diethyl ether, solid phase extraction with HLB and

7

aminopropylsilane cartridges and microwave assisted derivatization using 25 µL of

8

MSTFA/NH4I/ethanethiol and full microwave power for 2 min. The method was

9

validated according to the Decision 657/2002/EC, Codex Alimentarius and Manual da

10

Garantia da Qualidade Analítica guidelines. The acceptability criteria for quantitative

11

analysis were met for α-ethinylestradiol, α-nandrolone, β-estradiol, β-zearalanol, β-

12

zearalenol, drostanolone, ethisterone, dienestrol, diethylstilbestrol, hexestrol, megestrol,

13

methyltestosterone and zearalenone. The analytes, α-zearalenol, α-zearalanol and

14

norethandrolone, were validated for qualitative analysis.

15 16 17

18

Keywords: anabolic steroids, stilbenes, resorcylic acid lactones, microwave assisted

19

derivatization, GC-MS/MS, validation


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20

INTRODUCTION

21

Anabolic agents are substances that increase protein synthesis and, consequently,

22

improve the feed conversion efficiency and increase production of lean meat. The

23

consumption of meat and contaminated derivatives with anabolic residues has been the

24

cause of a public health alert, because of the suspected adverse effects on human

25

health1.

26

Since the end of the 1980s, the use of steroids as animal growth promoters has been

27

banned by the European Community2,3 and Group A substances (stilbenes, steroids,

28

antithyroid agents, resorcylic acid lactones and β-agonists) have been banned through

29

Council Directive 96/23/EC4. Many other countries, such as Brazil, the importation,

30

production, commercialization and use of hormonal substances with anabolic activities

31

for the purpose of growth and mass increase in slaughter cattle is prohibited5.

32

In general, anabolic agents are metabolized to less active and more hydrophilic

33

substances, and excreted in urine and feces. Urine is a matrix widely used for abuse

34

evaluation of these substances due to its homogeneity and easy collection3.

35

Urine is a matrix with many interferents and the anabolic residues, when present, are

36

at low levels. Therefore, the methods described in the literature commonly use liquid-

37

liquid extraction (LLE), followed by solid phase extraction (SPE) for extraction and

38

clean up. Gas or liquid chromatography tandem mass spectrometry (GC-MS or LC-MS)

39

is often employed for identification and quantification of these substances. A brief

40

description of the works available in the literature, describing analysis of anabolic

41

agents in bovine urine, is shown in Table 13,6-16.

42

{Insert Table 1}

43

The present study describes the GC-MS/MS multiresidue method optimization to

44

determine 17 anabolic agents of three classes: steroids, stilbenes and resorcylic acid 3 ACS Paragon Plus Environment


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45

lactones, in bovine urine. In this work some additional studies were performed: a

46

comparison was made between two incubation conditions used in the enzymatic

47

hydrolysis, the necessity of washing with hexane and the SPE steps in the extraction

48

procedure were evaluated. Moreover, microwave assisted derivatization was employed

49

for the first time for these analytes in bovine urine, which significantly reduced time,

50

and megestrol and drostanolone were quantitatively validated for the first time in bovine

51

urine. The optimized method was validated according to the Decision 657/2002/EC17,

52

Codex Alimentarius18 and Manual da Garantia da Qualidade Analítica 19 guidelines.

53


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54

MATERIAL AND METHODS

55

Reagents and Buffer Solutions: All reagents were of analytical grade. Methanol was

56

acquired from Tedia (Fairfield, USA), n-hexane from Vetec (Rio de Janeiro, Brazil),

57

acetone from J. T. Baker (Philipsburg, USA), diethyl ether, 2,2,4-trimethylpentane

58

(isooctane), N-methyl-N-(trimethylsilyl)-trifluoracetamide activated with ammonium

59

iodide and ethanethiol (MSTFA/NH4I/ethanethiol), enzyme β-glucuronidase from Helix

60

pomatia (type-2, ≥100,000 units/mL) and acetic acid were acquired from Sigma-Aldrich

61

(Saint Louis,

62

hydroxymethylpropane-1,3-diol (TRIS) were acquired from Êxodo Científica (São

63

Paulo, Brazil) and anhydrous sodium acetate from Neon Comercial (São Paulo, Brazil).

64

Ultrapure water was generated by Milli-Q Millipore system (Billerica, USA). The

65

sodium acetate buffer (2 mol L-1) was prepared by dissolving 164 g of sodium acetate in

66

1000 mL ultrapure water and the pH 5.2 was adjusted by acetic acid addition. TRIS

67

buffer (2 mol L-1) was prepared by dissolving 242 g of TRIS in 1000 mL ultrapure

68

water and the pH 9.5 was adjusted by hydrochloric acid addition.

USA),

sodium

hydroxide, hydrochloric

acid and

2-amino-2-

69 70

Standards and Standard Solutions: The anabolic standards: drostanolone (DRO),

71

megestrol (MEG), α-nandrolone (αNAN), β-zearalanol (TAL), α-zearalenol (αZE), β-

72

zearalenol (βZE) and zearalenone (ZEA) were acquired from Australian NMI (North

73

Ryde, Australia); α-ethinylestradiol (αEE), hexestrol (HEX), methyltestosterone (MTT)

74

and norethandrolone (NOT) were acquired from LGC Standards (Augsburg, Germany);

75

ethisterone (ETN), diethylstilbestrol (DES), dienestrol (DIE) and α-zearalanol (ZER)

76

were acquired from Dr Ehrenstorfer (Augsburg, Germany); β-estradiol (βES) was

77

acquired from Cambridge Isotope Lab (Massachusetts, USA). The internal standards:

78

diethylstilbestrol-D8 (DES-D8) and nandrolone-D3 (NOR-D3) were acquired from



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79

Cambridge Isotope Lab (Massachusetts, USA); megestrol-D3 (MEG-D3) and

80

methyltestosterone-D3 (MTT-D3) were acquired from RVIM National Institute for

81

Public Health and the Environment (Bilthoven, The Netherlands); hexestrol-D4 (HEX-

82

D4) was acquired from Toronto Research Chemicals (North York, Canada).

83

Diethylstilbestrol glucuronide (DESG) was acquired from Toronto Research Chemicals

84

(North York, Canada) and β-estradiol glucuronide (βESG) from Sigma (Saint Louis,

85

USA).

86

Individual stock standard solutions were prepared at a concentration of 200 µg mL-1

87

by dissolving the mass of each compound in methanol. The anabolic working standard

88

solution was prepared by mixing the individual stock solutions and diluting them with

89

methanol to a final concentration of 0.05 µg mL-1 for DES and 0.10 µg mL-1 for the

90

other analytes. The working internal standard solution was prepared by mixing the

91

individual stock solutions and diluting them with methanol to a final concentration of

92

0.05 µg mL-1 for DES-D8 and 0.10 µg mL-1 for the other internal standards. The

93

glucuronide working standard solution was prepared by mixing the individual stock

94

solutions of βESG and DESG and diluting them with methanol to a final concentration

95

of 0.10 µg mL-1. All solutions were stored at -20°C.

96 97

Instrumentation and Materials: Chromatographic analysis was performed on a gas

98

chromatography Agilent Technologies 7890B with triple quadrupole mass spectrometer

99

Agilent Technologies 7000C (Santa Clara, USA). The samples were incubated in an

100

incubator TE-420EI Tecnal (Piracicaba, Brazil), centrifuged in a centrifuge CR4i

101

Thermo Electron Corporation (Ohio, USA), next, the samples were shaken in a vortex

102

Ika Genius 3 (Wilmington, USA), dried in a shaking bath BT-25 Yamato (Tokyo,

103

Japan) and, finally, dried in a sample concentrator Dri-Block DB-3 Techne (Stone, UK).


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104

HLB Supel cartridge (200 mg/6 mL) was purchased from Sigma-Aldrich (Saint

105

Louis, USA) and aminopropylsilane cartridge Sep-Pak (200 mg/6 mL) was purchased

106

from Waters (São Paulo, Brazil).

107 108

Samples: Blank bovine urine samples were collected at the National Agricultural

109

Laboratory of Minas Gerais, Brazil. Prior to collection, hair was cut and asepsis of the

110

prepuce was performed. Urine samples were collected in clean and dry plastic bottles.

111

The samples were centrifuged at 3000 rpm for 5 min, filtered through a funnel with

112

glass wool and stored at -20 °C.

113 114

GC-MS/MS Analysis: The GC-MS/MS method conditions were optimized by injecting

115

diluted solutions, which were prepared by diluting the stock solutions with methanol to

116

a concentration of 50 µg mL−1 for each analyte. The solutions were injected, using the

117

full scan mode, to determine retention times and select the precursor ions. In order to

118

determine the product ions and collision energies, the solutions of each analyte were

119

injected in the product ion mode. The solvent vent injector temperature was 60 °C (for

120

1.37 min), heated up to 325 °C at 600 °C min-1 with vent time of 0.37 min and injection

121

volume of 10 µL. The column used was a HP-5MS (30 m, 0.25 mm I.D., film thickness

122

0.25 µm) from J & W Columns Agilent Technologies (Santa Clara, USA). Helium,

123

acquired from White Martins (Belo Horizonte, Brazil), was used as carrier gas at 2.0

124

mL min-1 (0-1.37 min) and 1.5 mL min-1 (1.37-14.17 min). Initial oven temperature was

125

50 °C (for 1.37 min) and was then heated up to 200 °C at 75 °C min-1, to 280 °C (for 3

126

min) at 40 °C min-1 and, finally, to 300 °C (for 4.5 min) at 5 °C min-1. The transfer line,

127

ion source and quadrupole temperatures were 300, 300 and 180 °C, respectively. The

128

electronic beam on the mass spectrometer was set at 70 eV in the electron ionization



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129

(EI) mode and was operated in the selected reaction monitoring (SRM) mode. The

130

precursor ions, product ions and collision energies, as well as the retention times of the

131

analytes and internal standards are shown in Table 2. {Insert Table 2}

132 133 134

Sample Preparation

135

Enzymatic Hydrolysis Optimization: A 5.0 mL aliquot of blank bovine urine was

136

fortified with working glucuronide standard solution at a level of 1.0 ng mL-1. Next, 2.0

137

mL sodium acetate buffer (2 mol L-1, pH 5.2) and 50 µL β-glucuronidase from Helix

138

pomatia were added and the extract was incubated under gentle stirring at 55 °C for 2 h

139

or at 37.5 °C for 16 h. The procedure was performed in triplicate.

140 141

Extraction and Clean up Optimization: A 5.0 mL aliquot of blank bovine urine was

142

fortified with anabolic working standard solution at a level of 1.0 ng mL-1 for DES and

143

2.0 ng mL-1 for the other analytes. Next, 2.0 mL sodium acetate buffer (2 mol L-1, pH

144

5.2) and 50 µL β-glucuronidase from Helix pomatia were added and the enzymatic

145

hydrolysis was performed in an incubator under gentle stirring at 37.5 °C for 16 h.

146 147

Five different procedures were evaluated for extraction and clean up optimization:

148

(1) LLE with diethyl ether;

149

(2) LLE with diethyl ether and SPE with HLB cartridge;

150

(3) LLE with diethyl ether, SPE with the HLB and aminopropylsilane cartridges;

151

(4) LLE with diethyl ether, hexane wash, SPE with HLB and aminopropylsilane

152

cartridges.


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153 154

(5) Evaluation of acetone volume used in the elution of the cartridges in the SPE by means of four consecutive elutions with 5 mL of solvent.

155

The optimum extraction and clean up procedure was as follows: after enzymatic

156

hydrolysis, the pH was adjusted by adding 4.0 mL TRIS buffer (2 mol L-1, pH 9.5). The

157

samples (pH 9.2) were extracted with 10.0 mL diethyl ether, the aqueous phase was

158

frozen with liquid nitrogen and the organic phase was transferred to 15 mL flask. The

159

extracts were evaporated to dryness under nitrogen stream in shaking bath at 50 °C. The

160

residues were re-dissolved with 1.5 mL methanol and 3.0 mL ultrapure water. HLB

161

cartridges were pre-conditioned with 5.0 mL methanol and 5.0 mL ultrapure water. The

162

samples were transferred to the cartridges and washed with 5.0 mL ultrapure water and

163

5.0 mL methanol-water (55:45, v/v) solution. Aminopropylsylane cartridges were pre-

164

conditioned with 5.0 mL methanol and 5.0 mL acetone. HLB cartridges were coupled to

165

aminopropylsilane cartridges and the analytes were eluted with 10.0 mL acetone. The

166

eluates were evaporated to dryness under nitrogen stream in shaking bath at 50 °C, next,

167

the eluates were re-dissolved with 300 µL methanol and 150 µL were transferred to

168

insert vials.

169 170

Derivatization Optimization: The derivatization procedure was optimized by a 33 Box-

171

Benhken factorial design. The factors evaluated were: microwave power, reaction time

172

and derivatization reagent volume, at levels shown in the Table 3. The central point was

173

assayed in authentic duplicates (experiments 13 and 14).

174

{Insert Table 3}

175

The optimum condition was: the extracts were evaporated to dryness under

176

nitrogen stream in shaking bath at 50 °C. Then, 25 µL MSTFA/NH4I/ethanethiol was

177

added and the vials were shaken for 30 s. Derivatization was carried out in a



Page 10 of 34

178

conventional microwave oven for 1.5 min at 900 W. The mixture was evaporated to

179

dryness under nitrogen stream in a sample concentrator at 60 °C and the derivatized

180

residue was re-dissolved in 100 µL of isooctane. Finally, 10 µL derivatized extract was

181

injected in the GC-MS/MS system.

182 183

Method Validation: Method validation was performed according to Decision

184

657/2002/EC17, Codex Alimentarius18 and Manual da Garantia da Qualidade Analítica19

185

guidelines. The validation parameters evaluated were: linearity range, selectivity,

186

trueness, precision, decision limit (CCα), detection capability (CCβ), limit of

187

quantification (LQ) and measurement uncertainty.

188

The calibration curves were prepared at six concentration levels (0.5; 0.75; 1.0;

189

1.25; 1.5 and 2.0 µg kg-1 for DES; 1.0; 1.5; 2.0; 2.5; 3.0 and 4.0 µg kg-1 for the other

190

analytes) by fortifying the blank bovine urine samples with volumes of anabolic

191

working standard solution ranging from 0 to 200 µL and also 100 µL of working

192

internal standard solution. To adjust the statistical models, the F-test was applied at 95%

193

confidence level to evaluate the homogeneity of the response variances. The quality of

194

linear fit was evaluated by applying the t-test (Equation 1). − 2 = || (1) 1 −

195

Where:

196

|| is the module of the correlation coefficient of the calibration curve;

197 198

is the number of concentration levels used to construct the calibration curve;

is the determination coefficient of the calibration curve.

199

Selectivity evaluation was assessed by spiking nine urine blank samples with the

200

addition of 100 µg kg-1 avermectins at each level evaluated (1.0, 1.5 and 2.0 x MRPL).


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201

A separate set of nine blank samples were spiked only with the anabolic agents solution

202

in levels 1.0, 1.5 and 2.0 x MRPL. Sample concentrations with and without addition of

203

the avermectins were calculated by interpolating the analyte peak areas in the respective

204

calibration curves. Then, the recoveries (R) were calculated and these were compared

205

by F-test for evaluation of the variance homogeneity and t-test for comparison of

206

averages.

207

To evaluate repeatability and trueness, aliquots of 5.0 mL blank bovine urine

208

were fortified at 1.0, 1.5 and 2.0 x MRPL, in six replicates for each level. The

209

experiment was repeated by the same analyst on a second day. A calibration curve was

210

prepared for each day of analysis. The experiment was repeated by a second analyst on

211

a third day to evaluate intermediate precision. R and relative standard deviation (RSD)

212

were estimated for these fortified samples.

213

The CCα (alpha error 1%) and CCβ (beta error 5%) were obtained by combining

214

the data from three calibration curves of fortified matrices with the analytes in

215

intermediate precision conditions. The values of CCα and CCβ are given by Equations 2

216

and 3. α = 2,33 u (2)

β = α + 1,64 u (3) 217

Where:

218

is the combined uncertainty at the lowest concentration level of the calibration

219

curve, taking into account the uncertainties of intermediate precision, recovery and

220

calibration at the zero concentration level (standard deviation of the intercept obtained

221

from the calibration curve of the three-day of validation).

222 223

LQ was considered the first concentration level on the calibration curves, and R and RSD at the LQ level were calculated for six replicates.



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224

The measurement uncertainty was estimated by Top-Down approach,

225

considering the uncertainty from the calibration curve and intermediate precision in the

226

combined uncertainty estimative (Equation 4) for the level of 1.0 MRPL19. = . #$%& ' + %( (4) !

227

Where:

228

is standard measurement uncertainty;

229 230 231 232

is the analyte concentration in the sample;

!

is the analyte concentration estimated by the calibration curve;

#$%& is the uncertainty of the calibration curve;

%( is the uncertainty of the intermediate precision of the analytical method.

233


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234

RESULTS AND DISCUSSION

235

Enzymatic Hydrolysis Optimization: Enzymatic hydrolysis optimization was

236

performed by comparing two incubation conditions: 55 °C for 2 h and 37.5 °C for 16 h.

237

Fig. 1 shows DES and βES peak areas obtained after hydrolysis in the studied

238

conditions. {Insert Fig. 1}

239

240

The peak area obtained for DES after hydrolysis for 16 h was about 300 times

241

greater than the area obtained after hydrolysis for 2 h. For βES, the peak area after

242

hydrolysis for 2 h was about 10 times smaller than the area obtained after hydrolysis for

243

16 h. Therefore, after hydrolysis at 55 °C for 2 h with β-glucuronidase/sulfatase from

244

Helix pomatia, a remaining amount of DES and βES in the glucuronide form was

245

observed. This result indicated that hydrolysis under these conditions was not as

246

effective as hydrolysis at 37.5 °C for 16 h for the analytes studied. Thus, the enzymatic

247

hydrolysis at pH 5.2, with 50 µL of β-glucuronidase/sulfatase from Helix pomatia, at

248

37.5 °C for 16 h, under gentle stirring, was selected.

249 250

Extraction and Clean up Optimization: When only LLE with diethyl ether was

251

performed (assay 1), the extract showed a dark coloration and presence of particulate

252

material. Therefore, it was not injected in the GC-MS/MS system and the condition was

253

disregarded.

254

When LLE with diethyl ether and SPE with HLB cartridge (assay 2) were

255

employed in the sample preparation, it was observed that after consecutive injections of

256

the replicates, sensitivity decreased and noise increased, preventing the quantification of

257

the analytes. Therefore, an additional clean up step was necessary.



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258

When LLE with diethyl ether followed by SPE with HLB and aminopropylsilane

259

cartridges were employed (assay 3), all analytes showed signals with good intensity and

260

repeatability, even after consecutive injections of the replicates. Figure 2 shows the

261

chromatograms for ETN and MTT under conditions of assays 2 and 3.

262

{Insert Fig. 2}

263

The results of assays 3 and 4 were statistically compared through the t-test (Fig. 3).

264

Although assay 3 provided a larger peak area for DIE and assay 4 for DES2, ETN and

265

MTT, these peak area variations were not statistically significant at 95% confidence

266

level (calculated t-value < critical t-value). Thus, the additional step of washing with

267

hexane was not used.{Insert Fig. 3}

268

The acetone volume used in the elution of the HLB and aminopropylsilane

269

cartridges was studied in assay 5. Four consecutive elutions were carried out using 5 mL

270

of acetone and the results showed that 10 mL was sufficient for the complete elution of

271

the analytes (Fig. 4). Therefore, this volume was employed.

272 273 274

The optimized extraction and clean up procedures are described in the subsection Extraction and clean up optimization. {Insert Fig. 4}

275 276

Derivatization Optimization: The identification and quantification of steroids,

277

stilbenes and resorcylic acid lactones, by GC-MS/MS, require preliminary

278

derivatization due to the presence of polar groups in their structures, which makes them

279

non-volatile. Trimethylsilylation, using MSTFA/NH4I/ethanethiol as derivatization

280

reagent, was optimized by 33 Box-Benhken design. For each experiment, the multiple

281

responses were calculated as the sum of the ratios between the individual area and the


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282

greater area of the analytes. The multiple responses were used to fit the statistical model

283

by the least squares method (Table 3).

284

The factors that may influence more significantly the derivatization procedure

285

efficiency were: microwave power and reaction time. The response surface showed that

286

the increase in microwave power and time reaction enhanced the response (Fig. 5). As

287

can be seen in Table 3, the highest response value was observed for experiment 4, with

288

reaction time of 2 min, 100% microwave power and 25 µL of derivatization reagent

289

volume. Thus, this condition was selected for the derivatization procedure, presenting as

290

advantages the use of a lower volume of the derivatization agent and a much lower

291

derivatization time than that reported in Table 1.

292

{Insert Fig. 5}

293 294

Method Validation: In order to evaluate linearity, the F-test was applied initially to

295

verify the homogeneity of the area variances. For MTT, ZER and ZEA, the variances

296

were homogeneous and the ordinary least squares method was used in the regression of

297

the calibration curves. For the other analytes, the variances were heterogeneous and the

298

weighted least squares method was applied, using the inverse of the variances as

299

weighting factor. Next, the t-test (Equation 1) was applied to verify the adequacy of the

300

linear adjustment. For all calibration curves, the t-values calculated were greater than

301

the critical value (t(0.05;4)=2.776), ranging from 2.9 to 13.6. Therefore, the fit of the

302

linear regression was adequate for all analytes. The parameters: slope, intercept, R2 and

303

t-values calculated are shown in Table 4.

304

{Insert Table 4}

305

The selectivity of the method was evaluated against the addition of avermectins,

306

antiparasites widely used in cattle. The analyte recoveries, with and without avermectin



Page 16 of 34

307

addition, were statistically compared. For samples with homogeneous variances

308

(calculated F-value < critical F-value), the pooled variance t-test was applied (Table 5).

309

When the variances were heterogeneous (calculated F-value >critical F-value), the non-

310

pooled variance t-test was used (Table 5). Since the recovery averages, with and without

311

avermectin addition, did not present significant difference at 95% confidence level

312

(calculated t-value < critical t-value), the method showed good selectivity against these

313

interferents.

314

{Insert Table 5}

315

Trueness was verified by recovery, which ranged from 88.2 to 119.2% and met

316

the Codex Alimentarius criteria18, except for ZER (127.0%), αZE (121.2%) and NOT

317

(137.7%) (Table 6). The RSD values in repeatability conditions ranged from 2.9 to

318

43.0% and RSD values in intermediate precision conditions ranged from 3.6 to 36.4%

319

(Table 6). For multiresidue methods, the Codex Alimentarius recommends the use of

320

the acceptability criterion18 for repeatability the same as intermediate precision.

321

Method repeatability and intermediate precision were lower than 45%, which is the

322

recommended RSD limit.

323

{Insert Table 6}

324

The estimated CCα and CCβ values for all analytes were lower than the MRPLs

325

(Table 4), therefore, met the criterion of acceptability of the Decision 657/2002/EC17.

326

LQ was the first concentration level on the calibration curves: 0.5 µg kg−1 for DES and

327

1.0 µg kg−1 for the other analytes. R at the LQ level ranged from 58.8 to 114.7% and

328

were adequate18, except for ZEA (133.6%), αZE (128.1%) and βZE (138.8%) (Table 4).

329

RSD at the LQ level ranged from 5.6 to 41.5% and were adequate for all analytes18

330

(Table 4). Therefore, LQ selected for ZEA and βZE was the second concentration level


Page 17 of 34


331

of the calibration curve, since this concentration presented adequate trueness and

332

precision.

333

The -value did not exceed four-thirds of the RSD value under conditions of

334

intermediate precision (Table 4). Therefore, measurement uncertainty met the

335

acceptability criterion of the Manual da Garantia da Qualidade Analítica19.

336

The method was validated for quantitative analysis of DES, HEX, DIE, αNAN,

337

αES, βES, DRO, αEE, MTT, ETN, TAL, ZEA, βZE and MEG, and validated for

338

qualitative analysis of ZER, αZE and NOT.

339

Finally, the optimized and validated method provided a wide scope for the

340

monitoring of anabolic agent residues in bovine urine, allowing the determination of

341

substances (ZER and βES) permitted in some countries, others (DES, HEX, DIE and

342

ETN) banned in several countries, and some (αNAN, MTT, NOT, DRO and MEG)

343

commonly used illegally.

344



345

Page 18 of 34

ACKNOWLEDGMENTS

346

The authors are grateful to the Laboratory of Residues of Veterinary Drugs of

347

the National Agricultural Laboratory of Minas Gerais for providing its infrastructure

348

and supplies for the development of this work.


Page 19 of 34


REFERENCES

1. C.M. Zhao, Z.F. Yue, H. Wu, F.R. Lai. Simultaneous determination of fourteen steroid hormone residues in beef samples by liquid chromatography-tandem mass spectrometry. Analytical Methods, 6 (2014) 8030-8038. 2. Z.M. Zhang, H.B. Duan, L. Zhang, X. Chen, W. Liu, G.N. Chen. Direct determination of anabolic steroids in pig urine by a new SPME-GC-MS method. Talanta, 78 (2009) 1083-1089. 3. S. Impens, J. Van Loco, J.M. Degroodt, H. De Brabander. A downscaled multiresidue strategy for detection of anabolic steroids in bovine urine using gas chromatography tandem mass spectrometry (GC-MS3). Analytica Chimica Acta, 586 (2007) 43-48. 4. Council of the European Union. Council Directive 96/22/EC of 29 April 1996 on measures to monitor certain substances and residues in live animals and animal products. J. Eur. Communities, L 125 (1996) 10-32. 5. Brasil, Ministério da Agricultura, Pecuária e Abastecimento. Secretaria de Defesa Agropecuária. Instrução Normativa N° 55, de 01 de dezembro de 2011. Proíbe do uso de substâncias anabolizantes em bovinos. 6. V. C. S. Gonçalves, K. T. J. Santos, N. O. C. Zuniga, V. V. de Lima, M. C. Padilha, C. Y. S. Siqueira, F. R. A. Neto. Optimization of a multiresidue and multiclass analysis method for anabolic agents and beta(2)-agonists in bovine urine by GC-MS/MS. Microchemical Journal, 133 (2017) 551-555. 7. Á. Tölgyesi, E. Barta, A. Simon, T. J. McDonald, V. K. Sharma. Screening and confirmation of steroids and nitroimidazoles in urine, blood, and food matrices: Sample preparation methods and liquid chromatography tandem mass spectrometric separations. Journal of Pharmaceutical and Biomedical Analysis, 145 (2017) 805-813. 8. B. Woźniak, I. Matraszek-Żuchowska, S. Semeniuk, A. Kłopot, J. Żmudzki. Screening and confirmatory GC-MS methods for the detection of trenbolone in bovine urine. Bulletin of the Veterinary Institute in Pulawy, 57 (2013) 559-566. 9. I. Matraszek-Zuchowska, B. Wozniak, J. Zmudzki. Determination of zeranol, taleranol, zearalanone, α-zearalenol, β-zearalenol and zearalenone in urine by LCMS/MS. Food Additives and Contaminants part A, 30 (2013) 987-994. 10. M. Gasparini, M. Curatolo, W. Assini, E. Bozzoni, N. Tognoli, G. Dusi. Confirmatory method for the determination of nandrolone and trenbolone in urine samples using immunoaffinity cleanup and liquid chromatography-tandem mass spectrometry. Journal of Chromatography A, 1216 (2009) 8059-8066. 11. G. Kaklamanos, G. Theodoridis, T. Dabalis. Determination of anabolic steroids in bovine urine by liquid chromatography–tandem mass spectrometry. Journal of Chromatography B, 877 (2009) 2330–2336. 19 ACS Paragon Plus Environment


Page 20 of 34

12. K. Schmidt, C. Stachel, P. Gowik. Development and in-house validation of an LCMS/MS method for the determination of stilbenes and resorcylic acid lactones in bovine urine. Analytical and Bioanalytical Chemistry, 391(2008) 1199-1210. 13. P.R. Kootstra, P.W. Zoontjes, E.F. van Tricht, S.S.Sterk. Multi-residue screening of a minimum package of anabolic steroids in urine with GC-MS. Analytica Chimica Acta, 586 (2007) 82-92. 14. C. S. Aman, A. Pastor, G. Cighetti, M. de la Guardia. Development of a multianalyte method for the determination of anabolic hormones in bovine urine by isotope-dilution GC-MS/MS. Analytical and Bioanalytical Chemistry, 386 (2006) 18691879. 15. C. Akre, R. Fedeniuk, J. D. MACNEIL. Validation of a simple, sensitive method for the determination of beta-estradiol in bovine urine using gras-chromatography negative ion chemical ionization mass spectrometry. Analyst, 129 (2004) 145-149. 16. R. Draisci, L. Palleschi, E. Ferretti, L. Lucentini, F. delli Quadri. Confirmatory analysis of 17b-boldenone, 17a-boldenone andandrosta-1,4-diene-3,17-dione in bovine urine by liquid chromatography–tandem mass spectrometry. Journal of Chromatography B, 789 (2003) 219–226. 17. Official J. European Communities, Diario Oficial de las Comunidades Europeas (DOCE) 2002/657/EC, August 12, 2002. 18. Codex Alimentarius. Guidelines for the design and implementation of national regulatory food safety assurance programme associated with the use of veterinary drugs in food producing animals. CAC/GL 71-2009. Adopted 2009. Revision 2014. 19. Manual da Garantia da Qualidade Analítica – Resíduos e Contaminantes em Alimentos. Ministério da Agricultura Pecuária e Abastecimento. Secretaria de Defesa Agropecuária, Brasília, 2011.

Notes The authors acknowledge the National Council for Scientific and Technological Development (process: 446278/2014-9, MCTI/CNPQ/Universal 14/2014), Minas Gerais Research Funding Foundation (process: CAG-APQ-01049-15) and Coordination for the Improvement of Higher Education Personnel (CAPES) for the financial support and MA scholarship.


Page 21 of 34


Figure Captions Fig. 1 - DES and βES areas obtained after enzymatic hydrolysis under two conditions.

Fig. 2 - Extracted ion chromatograms for MTT and ETN in assays 2 and 3.

Fig. 3 - Area comparison of DIE, DES2, DRO, ETN, MTT and ZER obtained in assays 3 and 4. Critical t-value = 3.18.

Fig. 4 - Area ratio of DIE, DES2, MTT, DES1, HEX, ETN and MEG by the areas of their internal standard versus the acetone aliquot used in the elution of the SPE in assay 5.

Fig. 5 - Response surface of the multiple response (MR) as function of the derivatization reagent volume (DRV) and reaction time (T) employed for derivatization procedure optimization in bovine urine. Percentage of variation explained of the model= 81.94% and Flack of fit = 10.28, lower than critical F(0,05;3;1)=215.7.



Page 22 of 34

Table 1. Brief Summary of the Methods Described in the Literature for the Analysis of Anabolic Agents in Bovine Urine Analytes

Sample Preparation

Derivatization

Quantification

Reference

Resorcylic acid lactones (2); stilbenes (2); steroids (4 and 4 metabolities); β-agonists (3) Steroids (10 and 1 metabolite)

Enzymatic hydrolysis: β-glucuronidase from Helix pomatia (1 h at 50 °C) LLE: TBME Enzymatic hydrolysis: β-glucuronidase from Helix pomatia (16 h at 37 °C) SLE: Novum or SPE: XL-A

100 µL MSTFA/NH4I/2mercaptoethanol (20 min at 60 °C)

GC-MS/MS

6

-

LC-MS/MS

7

Steroids (2)

Enzymatic hydrolysis: β-glucuronidase-arylsulfatase from Helix pomatia (overnight at 37 °C) LLE: diethyl ether Wash: carbonate buffer and distilled water SPE: C18 and NH2 Enzymatic hydrolysis: β-glucuronidase-arylsulfatase from Helix pomatia (overnight at 37 °C) LLE: diethyl ether SPE: C18 and NH2 Enzymatic hydrolysis: β-glucuronidase/arylsulfatase from Helix pomatia (overnight at 37 ºC) Clean up: immunoaffinity column

30 µL MSTFA/I2 (3 min at room temperature) and 30 µL MSTFA (40 min at 60 ± 2 °C)

GC-MS and GC-MS/MS

8

-

LC-MS/MS

9

-

LC-MS/MS

10

Resorcylic acid lactones (5); stilbenes (3); steroids (6 and 1 metabolite)

Enzymatic hydrolysis: Helix pomatia juice (2 h at 50 ºC) LLE: TBME Wash: hexane SPE: HLB and NH2

-

LC-MS/MS

11

Stilbenes (3); resorcylic acid lactones (6)

Enzymatic hydrolysis: Helix pomatia juice (16 h at 37 °C or 3 h at 50 °C) LLE: diethylether Wash: n-hexane SPE: HLB and NH2

-

LC-MS/MS

12

Resorcylic acid lactones (6)

Steroids (4)

22

ACS Paragon Plus Environment

Page 23 of 34


Stilbenes (3); steroids (13 and 2 metabolities)

Stilbenes (3); steroids (15); resorcylic acid lactones (2)

Stilbenes (3); steroids (6); resorcylic acid lactones (2)

Enzymatic hydrolysis: Helix pomatia juice (2 h at 55 °C) SPE: C18 LLE: η-pentane SPE: HLB SPE: C18 Hydrolysis: 12000 units of abalone acetone powder (2 h at 62 ± 2 °C) LLE: diethyl ether Wash: sodium carbonate and ultrapure water SPE: NH2 Enzymatic hydrolysis: β-glucuronidase/arylsulfatase from Helix pomatia (2.5 h at 40 ± 5 °C) SPE: C18 LLE: diethyl ether: petroleum ether

Steroid (1)

Enzymatic hydrolysis: β-glucuronidase from Helix pomatia (overnight at 37 °C) SPE: HLB LLE: 1-chlorobutane

Steroids (3)

Enzymatic hydrolysis: β-glucuronidase/arylsulfatase from Helix pomatia (12 h at 37 °C) SPE: C18

50 µL HFAA or 30 µL MSTFA++ (1 h at 60 °C).

GC-MS/MS

13

25 µL MSTFA/NH4I/ethanethiol (1 h at 60 ± 2 °C).

GC-MSn

3

100 µL BSTFA with 1% trimethylchlorosilane and 100 µL acetonitrile or 50 µL HFBA (1h at 60 ± 5 °C)

GC-MS/MS

14

85 µL dry ethyl acetate, 500 µL pyridine:ethyl acetate (10% v/v) and 15 µL pentafluorobenzoyl chloride (20 min at 60 °C). LLE: hexane -

GC-MS

15

LC-MS/MS

16

23



Page 24 of 34

Table 2. Retention Times, Precursor Ions, Product Ions and Collision Energies of the Quantification and Confirmation Transitions Optimized for Studied Analytes Retention Analyte

Time (min)

DESD81 DES1 HEXD4 HEX

Precursor Product Ion

Ion

420.0 6.681

6.695

6.998

7.005

c

DESD82 DES2

7.040

7.035

7.045

8.176

389.3 a

NORD3 βES

DRO

8.421

8.441

8.649

8.687

15

383.2

15

411.6b

217.1b

25

c

c

209.1

180.1

209.1

193.1

a

a

207.0

b

207.0

179.1

b

151.1

a

5 5 5 15

409.9

380.2

15

409.9b

394.9b

15

c

c

420.0

405.3

420.0

389.3

a

a

411.6

b

411.6 417.9

b

417.9 416.0

b

416.0

420.9

c

420.9 a

416.0

b

416.0

a

447.7

b

447.7

Analyte

383.2

b

217.1

a

194.0

b

182.0

a

284.9

b

231.9

c

194.1

182.1 a

284.9

b

231.9

a

141.0

b

405.4

Time (min)

15

411.6

a

αES

405.3

a

a

αNAN

Energy

Retention

(eV) c

420.0

a

DIE

Collision

15 15 15 25 25 25 15 25

15 5 25 15 15 5

MTT-D3

MTT

αEE

9.203

9.220

9.319

Precursor

Product

Ion

Ion

9.310

TAL

9.392

9.469

169.1

35

449.0

301.2

5

a

a

9.751

301.2

15

446.1b

356.3b

5

a

a

25

b

25

a

25

b

15

432.9

a

389.3

15

432.9b

295.1b

15

a

a

5

b

25

a

15

b

5

a

5

b

15

a

25

b

15

a

25

b

15

425.0

b

425.0 456.0

b

456.0

432.9

b

432.9 461.9

b

461.9

a

NOT

9.771

445.9

b

445.9

a

αZE

9.810

445.9

b

445.9

a

βZE

MEG-D3

MEG

9.939

10.760

10.785

c

446.1

a

ZEA

(eV)

449.0

a

ZER

Energy

c

a

ETN

Collision

445.9

b

445.9

205.0 231.1

301.2 316.3

389.3 295.1 151.1 333.3

287.1 356.2

317.1 333.2

317.1 333.2

c

c

244.2

5

373.9

284.3

5

373.9

a

370.9

b

370.9

a

5

b

5

241.2 281.3

a

Quantification transition; bConfirmation transition; cTransition used for internal pattern in area ratio; DES1: cisdiethylstilbestrol; DES-D81: cis-diethylstilbestrol-D8; DES2: trans-diethylstilbestrol; DES-D82: transdiethylstilbestrol-D8

.


Page 25 of 34


Table 3. 33 Box–Behnken Design Employed in the Derivatization Procedure Optimization Experiment

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Microwave power

-1

+1

-1

+1

-1

+1

-1

+1

0

0

0

0

0

0

Time

-1

-1

+1

+1

0

0

0

0

-1

+1

-1

+1

0

0

Derivatization reagent volume

0

0

0

0

-1

-1

+1

+1

-1

-1

+1

+1

0

0

2.93

3.68

3.31

12.40

2.67

5.18

4.96

10.11

5.80

4.19

5.03

5.62

7.61

6.51

Multiple response

Microwave power (%): (-1) 40, (0) 70 and (+1) 100; Time (s): (-1) 60, (0) 90 and (+1) 120; Derivatization reagent volume (µL): (-1) 13, (0) 25 and (+1) 38.

25



Page 26 of 34

Table 4. Parameters of the Calibration Curves, t-values, CCα, CCβ, LQ and Measurement Uncertainty by the Optimized Method in Bovine Urine Analyte

DES1

HEX

DIE

DES2

DESsum

αNAN

αES

βES

DRO

αEE

MTT

ETN

ZER

TAL

ZEA

αZE

βZE

MEG

NOT

Slope

0.140

0.133

0.463

0.914

1.06

0.51

15836471

189474

77052

28104

1.69

0.355

32834

16340

0.120

0.196

0.79

0.93

0.26

Intercept

0.000

0.034

-0.004

0.08

0.08

0.13

601838

-13729

-8574

-724

0.4

0.003

-2186

-147

-0.03

-0.039

-0.59

-0.04

0.05

R2

0.944

0.979

0.958

0.951

0.951

0.915

0.966

0.956

0.962

0.964

0.901

0.957

0.917

0.952

0.861

0.950

0.904

0.974

0.934

t-value

8.3

13.6

9.5

8.8

8.8

6.5

10.7

9.3

10.0

10.4

6.0

9.4

6.6

8.9

4.9

3.0

2.9

12.3

3.2

CCα (µg kg-1)

0.08

0.52

0.11

0.14

0.11

0.30

0.03

0.31

0.39

0.20

0.21

0.09

0.44

0.25

0.99

0.55

0.29

0.17

0.78

CCβ (µg kg-1)

0.13

0.88

0.19

0.24

0.19

0.50

0.05

0.53

0.67

0.33

0.36

0.15

0.75

0.43

1.68

0.93

0.49

0.29

1.26

RLQ (%)

94.1

99.0

107.8

104.6

95.7

70.7

104.3

104.8

107.1

101.9

102.8

93.7

108.2

114.7

133.6a

128.1a

138.8a

101.8

58.8

RSDLQ (%)

20.8

5.6

7.1

29.8

31.9

41.5

13.3

12.4

14.2

13.1

18.1

23.8

11.0

11.7

23.0

27.5

18.2

5.8

49.5

uc(µg kg-1)

0.141

0.130

0.236

0.128

0.124

0.769

0.351

0.342

0.318

0.401

0.514

0.160

0.456

0.447

0.484

0.488

0.598

0.160

0.805

a

RLQ

values that did not meet the minimum criteria of acceptability18

26


Page 27 of 34


Table 5. Critical and Calculated F-values and t-values for Bovine Urine Samples with and without Avermectin Addition Analyte a

a

DES1 HEX

DIE

DES2

DESsum

αNAN

αES

βES

DRO αEE MTT ETN ZER TAL ZEA αZE

βZE

MEG NOT

Calculated F-value

1.29

3.28

1.09

1.39

1.20

1.53

16.7

10.2

13.4

16.8

1.08

1.36

13.4

7.32

2.86

1.58 1.95

4.82

1.69

Critical t-value

2.12

2.12

2.12

2.12

2.12

2.12

2.26

2.23

2.31

2.26

2.12

2.12

2.26

2.23

2.12

2.12 2.12

2.20

2.12

Calculated t-value

1.26

0.61

1.59

0.33

0.42

1.34

1.02

0.41

0.78

1.03

0.51

1.09

1.11

0.49

0.37

0.17 0.39

0.54

2.11

Critical F-value = 3.44

.

27



Page 28 of 34

Table 6. Recovery and Intermediate Precision of the Optimized Method in Bovine Urine 1.0 MRPL

1.5 MRPL

2.0 MRPL

Analyte a

b

a

RSD

R

b

RSD

a

R

b

RSD

DES1

93.3

13.2

112.7

11.3

107.2

10.0

HEX

99.9

5.9

98.4

4.7

97.4

3.6

DIE

106.0

11.4

112.3

9.0

111.1

6.5

DES2

99.6

10.3

108.3

5.5

104.9

10.8

DESsum

101.2

10.3

114.8

7.8

112.0

9.1

αNAN

106.0

19.7

98.3

19.6

106.4

21.2

αES

92.0

16.8

113.1

25.2

107.5

29.2

βES

92.4

16.4

103.4

17.4

108.5

27.7

DRO

88.2

14.2

105.0

20.9

92.9

25.4

αEE

89.6

18.4

105.7

26.3

106.5

26.0

MTT

111.0

22.9

92.5

13.2

101.8

8.6

ETN

100.5

7.0

105.6

10.9

99.3

7.8

ZER

88.5

18.8

c

127.0

28.1

114.4

32.4

TAL

91.2

14.9

111.0

22.4

105.6

28.2

ZEA

101.9

18.7

115.2

30.5

110.2

24.7

αZE

101.2

18.2

c

121.2

28.9

106.1

21.7

βZE

107.2

26.2

119.2

34.2

103.6

28.2

MEG

94.4

7.4

97.6

6.5

98.5

4.3

137.7

27.4

113.7

24.9

116.9

36.4

NOT a

R

c

b

Recovery (%); Relative standard deviation (%); Values that did not meet the minimum criteria of acceptability18.

c


Page 29 of 34


Fig. 1 55 °C, 2 h 37.5 °C, 16 h

4

8.0x10

9.965

4

Peak area

6.0x10

4

4.0x10

7.593

4

2.0x10

2

2x10

2

1x10

0 DES

BES

Analyte



Page 30 of 34

Fig. 2


Page 31 of 34


Fig 3. Assay (3) Assay (4)

5

4.5x10

2.60 5

4.0x10

5

Peak area

3.5x10

5

3.0x10

5

2.5x10

5

2.0x10

1.39

2.27

5

1.5x10

5

1.0x10

4

5.0x10

1.30

2.32

DRO

ETN

0.09

0.0 DIE

DES2

MTT

ZER

Analyte



Page 32 of 34

2

4

3

Pe a k ar

6 2

ali Acetone

1

ea

8

quot

Fig. 4

MTT 2 DES MEG

ETN

HEX 1 DES

0 DIE

4


Page 33 of 34


Fig. 5

> < < <

Multiresidue determination of anabolic agent residues: steroids

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