Elsevier

Journal of Chromatography A

Volume 1355, 15 August 2014, Pages 61-72
Journal of Chromatography A

Multiresidue analysis of 22 sulfonamides and their metabolites in animal tissues using quick, easy, cheap, effective, rugged, and safe extraction and high resolution mass spectrometry (hybrid linear ion trap-Orbitrap)

https://doi.org/10.1016/j.chroma.2014.05.078Get rights and content

Highlights

  • Development of new rapid analytical method for 22 compounds of sulfonamides and their metabolites.

  • Combination QuEChERS–LC–LTQ-Orbitrap was applied.

  • A comprehensive qualitative and quantitative method was proposed.

  • Full validation of proposed method according to 2002/657/EC decision criteria.

  • Method was applied to a certified reference material and real animal tissues samples.

Abstract

A new high performance liquid chromatography–high resolution mass spectrometry (HPLC–HRMS) method was developed for a simultaneous multi-residue analysis of 22 sulfonamides (SAs) and their metabolites in edible animal (pig, beef, sheep and chicken) tissues. Sample preparation was optimized on the basis of the “QuEChERS” protocol. The analytes were identified using their LC retention times and accurate mass; the identification was further confirmed by multi-stage high mass accuracy (<5 ppm) mass spectrometry. The performance of the method was evaluated according to the EU guidelines for the validation of screening methods for the analysis of veterinary drugs residues. Acceptable values were obtained for: linearity (R2 < 0.99), limit of detection (LOD, 3–26 μg/kg), limit of quantification (LOQ, 11–88 μg/kg), accuracy (recovery 88–112%), intra- and inter-day precision 1–14 and 1–17%, respectively, decision limit (CCα) and detection capability (CCβ) around the maximum residue limits (MRL) of SAs (100 μg/kg). The method was validated by analysis of a reference material FAPAS-02188 “Pig kidney” with ǀ Z-scoreǀ < 0.63. The method was applied to various matrices (kidney, liver, muscle) originated from pig, beef, sheep, and chicken) allowing the simultaneous quantification of target sulfonamides at concentration levels above the MRL/2 and the identification of untargeted compounds such as N4-acetyl metabolites using multi-stage high mass accuracy mass spectrometry.

Introduction

Veterinary drugs are recognized as newly emerging environmental contaminants. Residues of antibiotics and, in particular, sulfonamides are an essential part of this emerging group [1]. After tetracyclines, SAs are the most commonly used veterinary antibiotics in the EU owing to their low cost and relative efficiency in combating many common bacterial infections [2]. Sulfonamides are N-substituted derivatives of the p-aminobenzenesulfonic acid with amphoteric properties. The latter are essential for their antibacterial activity owing to their free amino (NH2) and sulfonamide (SO2NH) group. In the body (mainly in the liver), the chemical structure of SAs can be modified leading to the formation of metabolites. Biotransformation occurs mainly by phase I oxidation and phase II acetylation giving the N1 and N4 derivatives. Glucuronide conjugation and aromatic hydroxylation can also take place leading other metabolites such as sulfinamide, AZO-SAs or nitro-SAs (Fig. 1) [3], [4]. As a consequence of the extensive usage of SAs for farm animals, their unwanted residues (parent compounds or metabolites) can persist in many edible tissues [4], [5], [6]. The uncontrolled exposure of consumers to SAs can be harmful to human health; it may lead to allergic reactions, Stevens-Johnson syndrome and several hematological, gastrointestinal and neurological diseases [7], [8]. Because to the possible side effects, the use of SAs in animals is regulated in many countries in order to protect human health. According to the European Union (EU) regulation no. 37/2010, SAs are an authorized substances and only dapsone derivative is a prohibited one. The maximum residue limits (MRL) for the total amount of SAs in edible biological tissues, such as muscle, liver, kidneys and milk is 100 μg/kg [9] and requires the development of the relevant analytical monitoring methods.

Liquid chromatography coupled to mass spectrometry (MS) using a quadrupole mass analyser is a technique of choice for quantitative or confirmatory analysis of a wide range of chemical residues. The vast majority of these methods employed in routine analysis, use low-resolution mass spectrometry instruments in “multiple reaction monitoring” (MRM) or “selected reaction monitoring” (SRM) modes, allowing the detection of target analytes preselected prior to their mass signal acquisition (pre-target screening). In this mode, the analytes are usually detected by monitoring the ionic signal of at least two mass transitions, in combination with their chromatographic retention time. Despite the instruments’ sensitivity and their acquisition speed allowing the monitoring of a large number of analytes, an inherent limitation of targeted LC–MS/MS approaches concerning the inability to detect residues unknown contaminants, such as metabolites or other new substances that may be present in a biological sample [10], [11], [12], [13].

Recently, an “untargeted approach”, based on high resolution MS using a time-of-flight (TOF) [14], [15] or Orbitrap analyser [6], has been used in the determination of emerging organic contaminants in biological matrices. In this approach all ions obtained are monitored without any pre-selection and the identification is achieved according to the analytes’ exact mass. The method was successfully applied for the analysis of a wide range of veterinary antibiotics including 12 SAs [6]. To our knowledge, it has so far been the only method allowing the identification of an acetylated SA (Ac-SDM) in animal tissue using post-targeted approach. To better assess the occurrence of SAs in food, their metabolites should also be considered. This was the reason to conclude the evidence need for an analytical method capable to simultaneously determine parent compound as well as their metabolites.

Despite the advances in the detection techniques, the major problem in the determination of antibiotics in animal tissues is due to the presence of high amount of matrix fat and proteins which can interfere in HPLC–MS. Different extraction methods were proposed for multiresidue analysis of veterinary drugs in biological matrices including meat, liver and kidney. Most of the procedures were long and tedious, involving ultrasonic [16], microwave [17], solid–liquid [13] or solid phase extraction [12], dispersive solid-phase matrix [18], and pressurized solvent extraction [10]. More recently, “QuEChERS” (acronym of quick, easy, cheap, effective, rugged, safe), a simple and fast extraction method, firstly developed for the extraction of pesticides [19], has been introduced for the extraction of veterinary drugs from various types of matrix increasing sample throughput and reducing the cost of analysis. This method involves the extraction with 1% acetic acid in acetonitrile (MeCN), phase-separation assisted by an addition of salts, and followed by a cleanup step by dispersive solid-phase extraction. The subsequent modifications included the use of buffered salts (acetate or citrate) [20], [21], or an additional extraction solvent [22]. Although “QuEChERS” is a powerful technique, few studies have been reported for the extraction of veterinary drugs in foods and these methods concerned typically a limited number of compounds. Multiclass HPLC-triple quad MS/MS methods were developed for the analysis of SAs in poultry (6 [23] and 16 compounds [24]) and fish [22]. A specific method was developed for the quantification of 9 SAs in bovine liver [25] and 13 SAs in both poultry and pig feed [11]. To our knowledge, only one work reported the use of “QuEChERS” method combined to HR MS (TOFMS) for the analysis of SAs in animal (fish) tissues and it was limited to 7 SAs compounds [15]. Therefore, we were interested to apply this rapid and effective extraction method coupled with HRMS for the simultaneous analysis of SAs residues and their metabolites in animal tissues covering wide heaviest matrices like muscle, kidney, and liver.

The objective of this work was to develop a rapid multiresidue method for the simultaneous trace level analysis of 22 SAs and their metabolites in meat, based on QuEChERS-based extraction and Orbitrap mass spectrometry, using the “targeted” and “non-targeted” approaches. The principal difficulty on the sample preparation level was the higher level of fat in comparison with the literature studies of fish or poultry. The “targeted approach” was used to develop and validate the method and to quantify known SAs in real samples. A parallel “non-targeted” approach allowed the identification of several SAs and metabolites.

Section snippets

Reagents and samples

The structure and the physico-chemical properties of the studied SAs are summarized in Fig. 2. The standard SAs were obtained from Ehrenstorfer (Augsburg, Germany) (SGN, SD, STZ, SM, SMP, SMM, SDO, SNZ, SDM and SQX) and Sigma Aldrich (Ibra Haddad et Fils, Lebanon) (SAA, SIM, SP, SME, SMT, SMZ, SCP, SMX, SIX, SB, SNT, SCL, Da). The internal standard SMX-D4 was obtained from C/D/N Isotopes Inc. (Pointe-Claire, QC, Canada). All the standards were of high purity grade (>95.0%).

LC–MS grade methanol

Results and discussion

The aim of this work was to develop of a new multiresidue method for the simultaneous analysis of 22 SAs antibiotics in animal tissues. The selection of the analytes was based on their worldwide use. The optimization of the method was performed on muscle tissue and then applied to other matrices (kidney, liver).

Conclusion

A new multiresidue method for rapid simultaneous determination of 22 SAs and their metabolites in edible animal tissues by a modified QuEChERS procedure and HPLC Orbitrap MS analysis was developed. A satisfactory detection-quantification strategy with suitable sensitivity and selectivity required a resolving power of 60,000 FWHM and an extraction window of 5 ppm. The whole analytical procedure was validated according to the European Commission decision 2002/657/EC. The method provided

Acknowledgments

We thank the Region of Aquitaine, the European Union, the Lebanese National Council for Scientific Research (CNRSL) and the Lebanese Atomic Energy Commission (LAEC) for financial support.

References (35)

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