Journal of Chromatography B, 944 (2014) 25–29

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Determination of carbadox and olaquindox metabolites in swine muscle by liquid chromatography/mass spectrometry Tomasz Sniegocki ∗ , Malgorzata Gbylik-Sikorska, Andrzej Posyniak, Jan Zmudzki Department of Pharmacology and Toxicology, National Veterinary Research Institute, 24-100 Pulawy, Poland

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Article history: Received 3 July 2013 Accepted 28 September 2013 Available online 9 November 2013 Keywords: Carbadox Olaquindox Metabolites SelexIONTM

a b s t r a c t This paper presents LC–MS/MS method that was developed for the simultaneous determination and confirmation metabolites of carbadox (desoxycarbadox, quinoxaline-2-carboxylic) and olaquindox (3methylquinoxaline-2-carboxylic acid) residues in pig muscle tissues at concentrations ≤3.0 ␮g kg−1 . Pig muscle tissues were deproteinated with meta-phosphoric acid in methanol and then were extracted with ethyl acetate:dichloromethane (50:50, v/v). The whole extracts were evaporated to dryness in rotary evaporator at 45 ◦ C, and dry residues were re-dissolved in 0.5% isopropanol in 1% acetic acid. The LC separation was performed on a C8 column with a gradient system consisting of isopropanol/water/acetic acid and methanol as the mobile phase. Additionally SelexIONTM technology to reduce matrix effect was used. The decision limit (CC␣) ranged from 1.04 ␮g kg−1 to 2.11 ␮g kg−1 and the detection capability (CC␤) ranged from 1.46 ␮g kg−1 to 2.89 ␮g kg−1 . The total recoveries were from 99.8% to 101.2%. The results of validation fulfil the requirement of the confirmatory criteria according to the European Commission Decision 2002/657/EC. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Carbadox and olaquindox can be illegally used in swine feed for growth promotion, to improve feed efficiency, to increase the rate of weight gain, to control swine dysentery and/or bacterial enteritis in young swine. After administration to swine, the carbadox and olaquindox are quickly metabolized to more stable products, as follows: carbadox to desoxycarbadox (DCBX), quinoxaline-2-carboxylic (QCA) and olaquindox to 3-methylquinoxaline-2-carboxylic acid (mQCA). In 1991, the use of such agents in feeding stuffs was prohibited in the European Union because of their toxicity. However, the use of carbadox is still permitted in the USA and some other countries in swine feed for growth promotion. Regulatory laboratories are required to have suitably validated analytical methods to ensure compliance with the ban (Commission Regulation 1998) [1]. Traditionally, the use of such compounds has been controlled by the analysis of their respective metabolites, in pig muscle and liver. In 2007, European Reference Laboratory (Fougeres-France) proposed for DCBX, QCA and mQCA in meat a recommended concentration of 10 ␮g kg−1 as minimum requirement for analytical method [2,3]. Therefore, it appears necessary to have sensitive, simple and reliable analytical methods applicable for meat samples. A number of methods

have been reported for the markers and metabolites of CBX and OLQ. Usually high-performance liquid chromatography with ultraviolet detection [4–9], gas with mass spectrometry and electron capture detection [10–14] and liquid chromatography with mass spectrometry [14–18] were used. Most of reported methods are based on solid phase extraction alone or coupled with liquid–liquid extraction [4–6,8,10,15–19]. Additionally, the described procedures did not analyze DCBX, QCA and mQCA [4–13,16–19] or they use two separate chromatographic conditions for determination all these metabolites [14,15]. None of these methods describes the simultaneous determination of these compounds in a single run. Additionally, these methods take time, are laborious, and could not be used to cope with a large number of samples. The aim of the study was to develop and validate a fast and simple method for the determination of DCBX, QCA and mQCA metabolites in swine muscles after one single analytical protocol. The muscles were chosen to be analyzed, because these are a major commodity in the food trade, and therefore it is important to know about the meat on the market, even though the critical organ for the determination of DCBX, QCA and mQCA metabolites is liver. 2. Materials and methods 2.1. Reagents

∗ Corresponding author. Tel.: +48 81 889 31 92; fax: +48 81 886 25 95. E-mail address: [email protected] (T. Sniegocki). 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.09.039

DCBX, mQCA and QCA-d4 were obtained from RIVM (Bilthoven, Netherlands). QCA was obtained from Sigma–Aldrich (Steinheim,

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T. Sniegocki et al. / J. Chromatogr. B 944 (2014) 25–29

Table 1 Precursor ions and product ion transitions of QCA, mQCA and DCBX residues – mass spectrometry and SelexIONTM parameters. Analytes time (min)

Precursor ion (m/z)

Ion transition

Collision energy (eV)

DCBX (5.9)

228.9

141.0 200.9

−26 −16

−50 −50

3390 3390

2.6 2.6

QCA (7.2)

172.9

129.1 102.0

−16 −26

−40 −40

3100 3100

−1.7 −1.7

mQCA (8.9)

187.0

143.0 102.0

−17 −27

−145 −145

3400 3400

−0.4 −0.4

QCA-d4 (7.0)

176.9

133.1 106.0

−18 −28

−55 −55

3050 3050

−1.4 −1.4

Germany). Purified water was achieved with a Milli-Q apparatus (Millipore, Bedford, MA, USA); acetic acid, ethyl acetate, isopropanol, methanol, meta-phosphoric acid and dichloromethane were from J.T. Baker (Deventer, Netherlands). Acetonitrile was from Merck (Darmstadt, Germany). All reagents should be minimum of analytical grade or higher. 2.2. Standard solutions The QCA, mQCA and QCA-d4 stock solutions of 1 mg mL−1 were prepared in methanol, while DCBX stock solutions of 1 mg mL−1 were prepared in methanol and water (80:20, v/v) and stored in the dark at

mass spectrometry.

This paper presents LC-MS/MS method that was developed for the simultaneous determination and confirmation metabolites of carbadox (desoxycarbadox, qu...
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