Fast multi-residue method for determination of nineteen benzimidimidazoles in meat tissues by liquid chromatography tandem mass spectrometry

A fast, sensitive and selective method has been developed for quantitative determination of residues of nineteen benzimidazoles in meat by speeding the productivity of the conventional liquid chromatographs. The analytes are extracted with phosphate buffer and acetonitrile. Extract is purifi ed by Strata X cartridges. Benzimidazoles are separated within 9 min with a conventional liquid chromatograph. The residues are detected by mass spectrometry at Selected Reaction Monitoring SRM. The separation of the compounds is done on a reversed phase RP-PFP column (50×2,1mm) with 2.6μm core-shell particle size at 50oC. The maximal pressure during analysis is 250 bars. The calculated decision limits (CCα) are between Maximum Residue Limits (MRL)+8% MRL and MRL+33% MRLs. Detection Capability (CCβ) values are in the range MRL+13% and MRL+66%. The detection capabilitys (CCβ) are between MRL and 8% MRL and MRL+66% MRL for the range of investigated benzimidazoles. The results of three separate assays (n= 3×6) show the mean recovery between 80% and 110%. Linearity of the method, assessed by coeffi cient of correlation r2, is between 0.990 and 0.995. The accuracy at MRL level of benzimidazoles is <10% with precision below 15%, expressed as Relative Standard Deviation (RSD). Research Article Fast multi-residue method for determination of nineteen benzimidimidazoles in meat tissues by liquid chromatography tandem mass spectrometry Milena Funeva-Peycheva* and Georgi Stoev Central Laboratory of Veterinary Control and Ecology (CLVCE), Iskarsko Shousse 5, Sofi a 1528, Bulgaria Received: 29 March, 2019 Accepted: 04 November, 2019 Published: 05 November, 2019 *Corresponding author: Milena Funeva-Peycheva, Central Laboratory of Veterinary Control and Ecology (CLVCE), Iskarsko Shousse 5, Sofi a 1528, Bulgaria, E-mail:


Introduction
Benzimidazoles are anti-parasitic agents used against endo parasites and anthelmintics, widely used when raising animals from which food is produced. Benzimidazoles are used as fungicidal agents too [1]. Some representatives of this drug's class have teratogenic and embryotoxic effects [2]. Large number of metabolites in edible animal tissues can be identifi ed, for example up to and more than 19 possible residues [3]. To ensure consumers the European Union has set Maximum Residue Limits (MRLs) for benzimidazoles and their metabolites in foods with animal origin. These limits range from 50 to 225μg/kg depending on compound and the type of matrixes (muscle, liver, kidney and fat) [4], (Table 1).
For most of the benzimidazoles, the marker residue is defi ned as the sum of the parent drug and/or its major (or most persistent) metabolite. A comprehensive review of properties and methodologies for determination of benzimidazole residues in biological matrices is presented by Danaher et al., [5]. Despite  There are many scientifi c works related to benzimidazoles analysis in animal tissues, milk and plant tissues. Numbers of reported methods relate to analysis of individual benzimidazoles and their metabolites in food products [6][7][8][9]. However, because of the large number of benzimidazoles licensed for use, multiresidue methods are more useful for the practice. They are more attractive and challenging for the analytical experts, too. These methods could provide more complete surveillance for the drugs. Methods for analysis of enlarged number of benzimidazole residues in milk [10][11][12][13][14] and tissues [3,[15][16][17][18] have been developed also. Methods for determination of 10 up to 14 benzimidazoles, using Liquid Chromatography-tandem mass spectrometry (LC-MS/MS) were published in the last decade [3,[19][20][21][22]. The separation of the enlarged number of benzimidazoles is realized for 30-40 min. Danaher et al., [23], separate 14 benzimidazoles simultaneously in a run-time of 60 tandem mass spectrometry. Open J Anal Bioanal Chem 3(1): 065-071. DOI: https://dx.doi.org/10.17352/ojabc.000013 min and gradient elution program using Xterra C18 column. Kinsella et al., [24], develops a LC -MS/MS multi-residue method for simultaneous identifi cation and quantifi cation of 38 residues of the most widely used anthelmintic veterinary drugs, including some benzimidazoles. Lugomer et al., analyzed 18 benzimidazoles in milk for 28 min [21]. Recently several papers present multiresidue screening methods, based on the increased effi ciency of Ultra-High Performance Liquid Chromatography (UHPLC) and High Resolution Mass Spectrometry (HRMS) [25][26][27][28]. The reliability of the methods using other types of mass spectrometry as TOF HRMS is higher than that of the Low Resolution Mass Spectrometry (LRMS) and Selected Reaction Monitoring (SRM) mode. These techniques are not applicable and suitable for confi rmatory purposes. The benefi cial effects of UHPLC with SRM-LRMS have been applied for determination and confi rmation of residues of 24 benzimidazoles in meat [29], where the samples are analyzed within 60 min by two runs at positive and negative ionization, respectively. UHPLC with sub-2μm particle size columns become as an attractive technique for improving the separation and/or reducing the time of analysis. The increased back pressure -above 500 bars -requires new generation of Liquid Chromatography (LC) equipment. In order to realize high effi ciency the equipment must possesses reduced delay and death volumes. This equipment, however, is more expensive. Horne et al., and later Kirkland, proposed core-shell particles with porous adsorption layer as a packing material for LC column for increasing the effi ciency [30,31]. Many companies have already produced columns with such type of particles, an internal diameter of 1.7 or 2.6μm and 0.23-0.35μm porous layers like C 18 , HFP or HILIC as packing materials. [32][33][34].

Method validation: The validation of the method is carried out according rules of Commission Decision 2002/657/EC [35]:
Specifi city, linearity, recovery, repeatability, accuracy, decision limit (CC  ) and detection capability (CC  ).

Validation study
The method is validated using porcine muscle as a model matrix at the specifi ed MRLs of each one of analytes. While most of benzimidazole compounds are expressed as the sum of the marker metabolites (Table 1), validation is provided applying MRL value for each individual marker residue. This Table 3: Gradient and pressure profi les used for separation of benzimidazoles.

LOD and LOQ: To verify Limits of Detection (LOD) and
Limits of Quantifi cation (LOQ) samples are fortifyed with all analytes on 0.100, 0.010 and 0.001 MRL-levels. LOD and LOQ are calculated on the basis of signal to noise ratio S/N=10 for LOD and S/N=20 for LOQ. The higher effi ciency of chromatographic column with core-shell particles aids the LOQ for all analytes to be below CC  and CC  (Table 3)