Confirmatory identification of sixteen non‐steroidal anti‐inflammatory drug residues in raw milk by liquid chromatography coupled with ion trap mass spectrometry

Abstract

The European Council Decision 2002/657/EC established that group B substances detected in foods must be identified and confirmed on the basis of their molecular structure. To this aim, we have developed a panel of methods for unambiguous determination of sixteen non-steroidal anti-inflammatory drugs (NSAIDs) in cattle and buffalo raw milk. A multi-residue reversed-phase high-performance liquid chromatography method with photodiode array detection is described for quantitative screening analysis. For confirmatory purposes, two multi-residue reversed-phase ion trap liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) methods were developed: the former to identify salicylic acid, naproxen, carprofen, flurbiprofen, ibuprofen, meclofenamic acid, niflumic acid, flunixin and its metabolite 5-hydroxyflunixin in the negative ion mode; the latter to identify ketoprofen, suxibutazone, diclofenac, mefenamic acid, tolfenamic acid, phenylbutazone and its metabolite oxyphenbutazone in the positive ion mode. These drugs are representative of different subclasses of NSAIDs not chemically related. The methods were in-house validated, evaluating specificity and calculating the mean recoveries, repeatability, within-laboratory reproducibility, and limits of quantification. For all the NSAIDs, apart from salicylic acid and 5-hydroxyflunixin, mean recoveries ranging between 69.0% and 96.7% were measured. The qualitative identification of all drugs was attained by their MS/MS spectra in the concentration range studied. Similarly, at 5 microg/kg all NSAIDs, apart from flurbiprofen, were unambiguously confirmed.

Analysis of non-steroidal anti-inflammatory drugs in milk using QuEChERS and liquid chromatography coupled to mass spectrometry: triple quadrupole versus Q-Orbitrap mass analyzers

“…A Thermo Accela UHPLC system coupled to a Maylab Switch column manager and to the Q-Exactive mass spectrometer was used. The chromatographic system was coupled to the MS with a heated electrospray ionization source II (HESI II (12,70). Total runtime was 12 min.…”

“…Acetonitrile, the effect of which on protein precipitation facilitates efficient extraction, is the most widely used solvent [4][5][6]11]. However, methanol [7] as well as acetonitrile/methanol [12] or acetonitrile/ethyl acetate mixtures [9] are also employed, and good overall recovery values have been reported for all systems. A controversial issue concerning the extraction step is the addition of ascorbic acid to prevent oxidation of pyrazolones (e.g., phenylbutazone and oxyphenbutazone [11,12].…”

“…However, methanol [7] as well as acetonitrile/methanol [12] or acetonitrile/ethyl acetate mixtures [9] are also employed, and good overall recovery values have been reported for all systems. A controversial issue concerning the extraction step is the addition of ascorbic acid to prevent oxidation of pyrazolones (e.g., phenylbutazone and oxyphenbutazone [11,12]. Some authors report that it does not improve recovery [5] and may even have a detrimental effect on MS detection of other analytes [7].…”

“…Since LC-MS is susceptible to matrix effects, and milk is quite a complex matrix, most methods include some clean-up of the extracts. Several strategies have been described, such as a liquid-liquid extraction with hexane to eliminate fat content [4,9], but most of the methods apply solid phase extraction (SPE) with different sorbents, depending on the set of compounds included in the method, such as amino [6], octadecyl [12], or polymeric phases [5]. In general, the clean-up step is the bottleneck of the procedure.…”

Analyses of Non-steroidal Anti-inflammatory Drugs in Environmental Water Samples with Microemulsion Electrokinetic Chromatography

“…2,3,[6][7][8][9][10][11][12][13][14] Microemulsion electrokinetic chromatography (MEEKC), which has a similar separation mechanism as micellar electrokinetic chromatography (MEKC), has been demonstrated to be a reliable separation technique for a wide range of analytes, including NSAIDs compounds. [15][16][17][18][19][20][21] For all CE derived techniques, including MEKC and MEEKC, on-capillary UV detection is still employed as the major detection mode.…”