LC-MS methods for analyzing anti-inflammatory drugs in animal-food products

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LC-MS methods for analyzing anti-inflammatory drugs in animal-food products Alessandra Gentili To improve the well-being of food-producing animals, the use of antiinflammatory drugs (AIDs) for pain management has become common practise in cattle breeding. Because of the frequency of treatment and inadequate withdrawal times, it is probable that residues of these pharmaceuticals will be found in human food, such as milk, meat and eggs. The two classes of anti-inflammatories most used in veterinary medicine are corticosteroids and non-steroidal AIDs (NSAIDs); the potentially adverse effects of these compounds has induced the governing authorities to regulate their residue levels in different animal-food products. Because of the chemical properties of AIDs, liquid chromatography (LC) coupled to mass spectrometry (MS) is the technique of choice for performing multi-residue analysis in complex matrices and confirming their identity. This article reviews the current status of applying LC-MS in analyzing AIDs. We address instrumental aspects (e.g., ionization sources and analyzers) as well as extraction procedures, the complexity of which is linked to the sensitivity and the selectivity of LC-MS and to matrix effects. ª 2007 Elsevier Ltd. All rights reserved. Keywords: AID; Anti-inflammatory drug; Chromatographic technique; Corticosteroid; LC-MS; Liquid chromatography; Mass spectrometry; Meat; Milk; Non-steroidal antiinflammatory drug; NSAID

1. Introduction Alessandra Gentili* Dipartimento di Chimica, Universita` ‘‘La Sapienza’’ di Roma, Piazzale Aldo Moro no. 5, P.O. Box 34, Posta 62, 00185 Roma, Italy

*

Fax: +39 06 490631; E-mail: [email protected]

The idea that food-producing animals housed in well-designed and comfortable environments have longer, healthier, and more productive lives has been well supported by formal research studies. One of the measures aimed at improving animals welfare is pain management by using antiinflammatory drugs (AIDs) [1]. These substances can be effective in suppressing or preventing inflammation, treating allergy, lowering fever and reducing pain; they can be used in conjunction with other drugs, such as antibiotics [1,2]. The classes of AIDs [1–3] differ on the basis of their actions towards the biochemical mediators (peptides, histamine, serotonin, cytokines, eicosanoids,

0165-9936/$ - see front matter ª 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2007.01.013

and platelet-activating factor) that are released during inflammation and that propagate the inflammatory response. Some drugs antagonize the action of the biochemical mediators, others inhibit their elaboration. The main classes of AIDs are the antagonists of bradykinin and kallidin, the antihistamines, the non-steroidal AIDs (NSAIDs) and the corticosteroids, but the two latter are the most used in the veterinary field. There has been plenty of attention to the occurrence of hormone and antibiotic residues in animal-food products. However, from analysis of a slaughter survey, conducted in the USA some years ago [4,5], it emerged that a significant number of animals were contaminated with unacceptable levels of NSAIDs. The US Food and Drug Administration (FDA) and Food Safety Inspection Service (FSIS) are very concerned about this issue and are considering several measures to curb the presence of NSAIDs. However, the use of NSAIDs in this field [1] has recently increased significantly because their shortterm side-effects are negligible; only their prolonged use induces gastric intestinal ulceration, sometimes accompanied by anemia and disturbances in platelet function. Owing to their effectiveness in suppressing or preventing inflammation, corticosteroids continue to be the most commonly used (and misused) AIDs in veterinary medicine, administered at high doses because they are well tolerated by cattle [1]. These two classes of AIDs have, to different degrees, the potential for adverse [1,3] effects towards animals and humans and the introduction of harmful residues

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Figure 1. Liquid chromatography in multi-reaction monitoring (MRM) chromatogram (2 transitions for each analyte), acquired by electrospray source in positive-ion mode, of an extract from bovine milk, spiked at 1 ppb (200 pg injected) for triamcinolone (TRI), dexamethasone (DEXA), prednisolone-hemisuccinate (PREDLO-HS), prednisolone-acetate (PREDLO-AC), hydrocortisone-acetate (HCORT-AC), at 3 ppb for prednisolone (PREDLO), flumethasone (FLU) and at 5 ppb for hydrocortisone (HCORT), prednisone (PRED) and cortisone (CORT).

into the human food chain should be avoided. Many authors have proposed methods based on gas chromatography-mass spectrometry (GC-MS) to detect corticosteroids and NSAIDs [6,7], including chemical oxidation or derivatization. Although providing good sensitivity, these methods are not easy to use and they modify the chemical structure of the molecule, reducing information and specificity. 596

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Liquid chromatography coupled to multidimensional mass spectrometry (LC-MSn) provides a powerful alternative, combining rapidity, specificity and sensitivity. The principles and the instrumentation of LC-MSn have been explained by different authors in books and reviews [8–11]. We devote this article, which complements other recent reviews on pharmaceutical-residue analysis, particularly on some classes of drugs as emerging

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Figure 2. The product-ion scan mass spectra, acquired by electrospray source in negative-ion mode, for phenylbutazone (panel A), carprofen (panel B), meloxicam (panel C) and etodolac (panel D); in Q1 scan, carprofen shows, besides the [MH] pseudomolecolare ion, an intense adduct with formiate.

environmental contaminants [12–15], to the use of LCMS for identifying residues of AIDs, which are much used in veterinary medicine but which have scarcely been investigated in edible products (see Figs. 1 and 2).

2. LC-MS instrumentation The numerous advantages of LC coupled to MS are well known and widely recognized for the analysis of

environmental and food contaminants. The sensitivity, selectivity and identification make LC-MS suitable for confirmation of multi-residue analysis at sub-ppb concentration levels and for simplifying extraction from complex matrices. The introduction of a new atmospheric pressure ionization (API) interface – atmospheric pressure photoionization (APPI) [16] – and of other approaches to extend the applicability of LC-MS [17] (e.g., coordination-ionspray (CIS) or electron-capture negative-ion chemical ionization (ECNICI)) allow http://www.elsevier.com/locate/trac

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direct detection of an analyte, so that the method is quicker. With the advent of the Ultra Performance LC system (UPLC) [18,19], a new technology able to operate at high pressure (1000 bar, compared with 400 bar of high-pressure LC (HPLC)), it is possible to use 1mm i.d. columns with packing in the 1–2 lm range at high flow rates. In this way, the performance of the LCMS technique is improved: the times for multi-residue analysis are shortened (around 10 min or less), the resolution and the sensitivity are higher. Nevertheless, the very narrow chromatographic peaks require the UPLC system to be coupled to a fast mass analyzer (i.e. with a high duty cycle) (e.g., time-of-flight (TOF)). On the basis of the data reported in literature, good results are achieved also by coupling UPLC with a triple quadrupole (QqQ) operating in multi-reaction monitoring (MRM) mode. Besides the general performance requirements, Commission Decision 2002/657/EC [20] has defined additional criteria for confirmatory methods by introducing the concept of identification points (IPs). For confirmation of banned substances belonging to group A (that includes most corticosteroids), four IPs are required, while, for substances with established maximum residue limits (MRLs), belonging to group B, three IPs are enough. The number of ‘‘earned IPs’’ depends on the mass analyzer and the acquisition mode (the one with the best duty cycle has to be chosen) (e.g., with a QqQ, a confirmation analysis has be carried out by selecting two MRM transitions (1 point for the precursor ion + 1.5 points · 2 product ions = 4 IPs). The resolution and the mass accuracy of instruments (e.g., quadrupole-quadrupoleTOF (QqTOF) [21]) achieve high-power recognition and confirm the presence of molecules that, subjected to collision-induced decomposition (CID), generate only one product ion (2 points for the precursor ion + 2.5 points for the product ion = 4.5 IPs). The very fast duty cycle of the QqQ-linear ion trap (QqQ-LIT) [22] provides sensitivity superior to that of traditional QqQ and IT and records product-ion-scan spectra for confirmation without compromising the signal-to-noise (S/N) ratio; also the resolution and the accuracy are higher and these characteristics improve the selectivity. This instrument, combining the robustness of a QqQ with the full scan MS2 sensitivity of an IT, could be a valid tool to carry out confirmation analysis quantitatively; it is used in metabolomic studies and could be applied to find new metabolites of veterinary drugs in different animal products.

3. Matrices and sample preparation There are two main reasons to develop and to validate analytical methods for confirming xenobiotic residues in animal-food products: 598

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(1) at the retail level (butcher shop, supermarket) or on import or export, when sampling is restricted to muscular tissue, milk, eggs and honey (not to biological fluids); (2) the need to verify that drug concentrations in the consumable parts of an animal are below the MRLs, so these matrices are of most interest together with the target organs (e.g., liver and kidney) in which xenobiotic substances concentrate. Analysis of the scientific literature within this ambit shows that liquid extraction (LE) and liquid-solid extraction (LSE) are used most as sample-treatment techniques. LSE is almost always performed in the form of solid-phase extraction (SPE) on various types of sorbent materials; C18 continues to be the most used material for corticosteroid clean-up [23–26], while there have been good performances on Oasis-HLB cartridges (a hydrophilic-lipophilic balanced copolymer of N-vinylpyrrolidone and divinylbenzenes) for NSAIDs [15,27,28] without acidification. In several works for extracting flunixin from milk, a strong cation-exchange column was used [27,29,30], taking advantage of protonation of the aliphatic aminic group due to acidification of the sample used to induce protein precipitation. Often LE and LSE have been used in combination: after analyte isolation by LE, the drugs were subsequently enriched using one or more SPE procedures [23–25,27– 31]. Application of matrix solid-phase dispersion (MSPD), which combines sample blending, clean-up and extraction, has not been reported for this class of veterinary drugs. Another sample-preparation technique, pressurized liquid extraction (PLE), has been applied to corticosteroid extraction from liver [32]; it is also well known as accelerated solvent extraction (ASE) [33,34]. In the case of antimicrobials, PLE ha been a very effective technique for isolating analytes from fat-containing matrices [35,36], as it can use water at high pressure and high temperature to extract polar drugs. This innovative extraction technique is based on using high pressure and high temperature to keep the extracting solvent in the liquid state at temperatures above its boiling point; advantages are rapidity, simplicity, small amounts of solvent waste, automated procedures and the possibility of using water as extractant (non-toxic, non-pollutant and cheap).

4. LC-MS for determining AIDs 4.1. Corticosteroids Corticosteroids [1–3] include two classes of steroid hormones naturally synthesized in the adrenal cortex from cholesterol: mineralocorticoids and glucocorticoids. Chemical modifications to the cortisol molecule (the main natural glucocorticoid) have generated derivatives with increased therapeutic potency but

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reduced mineralocorticoid effects. Table 1 gives the names, the chemical structures and the monoisotopic masses of the corticosteroid drugs used most in the veterinary field. The European Union (EU) has strictly regulated the use of corticosteroids in livestock breeding for two reasons: (1) potential misuse due to their broad pharmacologic and physiologic effects; and, (2) collateral effects linked with suppression of the immune response and interference with other hormone systems. The EU banned their administration for fattening purposes, according to the 96/22/EC Directive [37], and established MRLs (see Table 2) for some of them [38,39]. The fragmentation patterns and the explanation of the fragments of corticosteroids have been discussed by several authors [6,40–42]. Their polarity and functionalities allow use of ESI, APCI and APPI sources, in positive-ion or negative-ion modes, and different modes of instrumental acquisition (MRM, product ion scan (ProIS), precursor ion scan (PreIS) and neutral loss scan (Nloss)) for MS detection. Antignac et al. [6] summarized the studies with CID of these compounds, ionized by ESI and APCI sources in positive-ion and negative-ion modes. In positive-ion ESI, the fragmentation at low collision energies of the pseudomolecular ion [M+H]+ induced losses of H2O for each hydroxyl group fixed on the molecule, as well as the loss of HF or HCl for halogenated compounds; at

higher collision energies, fragmentations of the B, C and D rings led to product ions at m/z 121, 171, 237 and 279. In negative-ion ESI, the fragmentation of the precursor ions type [M+base] produced two ions, [MH] and [MHCH2O]; the latter resulted from the loss of formaldehyde involving cleavage of C20–C21. High collision energies induced losses identical to those produced in positive-ion mode (H2O, HF, HCl). Using an APCI source, some authors [41,43] observed, besides pseudomolecular ions MH+, solvent adducts; Fiori et al. [43] recorded loss of water, formaldehyde and glycoaldehyde from the protonated molecules. Antignac et al. [40] verified the sensitivity of the MRM acquisition mode on Nloss (Q1 and Q3 were synchronised with a 90-Da difference corresponding to the loss of acetic acid plus formaldehyde) and ProIS. Sangiorgi et al. [41] developed a screening method in the Nloss scan: in presence of acetonitrile, the APCI source generated adducts MHCH3CN+, which, subjected to declustering, give pseudomolecular ions MH+, with the same neutral loss (41 Da) for all glucocorticoids analyzed. Greig et al. [42] used Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), coupled with different API sources, to analyze some corticosteroids. The analytes showed excellent response using APPI, when compared with both ESI and APCI. APPI has the advantage of requiring less heat for desolvation, resulting in less thermal degradation of these compounds and higher S/N than APCI; APPI also resulted in greater

Table 1. Names, structures and monoisotopic mass of main corticosteroids used in veterinary medicine

CH2OH 18

R11 19

CH3

2 3

11 9

12

C

10 5

O

4

R9 B

20

17

13

D 14

1

A

C CH3

O R17

16

R16

15

8 7

6

R6 Compound Beclomethasone Bethamethasone Cortisone Dexamethasone Fludrocortisone Fluorometholone Flumethasone Hydrocortisone Methylprednisolone Prednisolone Prednisone Triamcinolone

Monoisotopic mass 408.17 392.20 360.19 392.20 380.20 376.20 410.19 362.21 374.21 360.19 358.18 394.18

Double-bond positions 1,4

D D1,4 D4 D1,4 D4 D1,4 D1,4 D4 D1,4 D1,4 D1,4 D1,4

R6

R9

R11

R16

R17

– – – – – –CH3 –F – –CH3 – – –

–Cl –F – –F –F –F –F – – – – –F

–OH –OH ‚O –OH –OH –OH –OH –OH –OH –OH ‚O –OH

–CH3 –CH3 (b) – –CH3 (a) – – – – – – – –OH

–OH –OH –OH –OH –OH –OH –OH –OH –OH –OH –OH –OH

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Table 2. Maximum Residuum Limits (MRLs) established by the European Union for the most used veterinary anti-inflammatory agents Pharmacologically active substance

Marker residue

Animal species

Corticosteroids Bethamethasone

Bethamethasone

Bovine, Porcine

Bovine Dexamethasone

Dexamethasone

Bovine, Porcine

Bovine Methylprednisolone

Methylprednisolone

All food-producing species

All milk-producing species for human consumption Prednisolone

Prednisolone

Bovine, Porcine

All milk-producing species for human consumption

MRLs (lg/kg)

Target tissue

2 0.75 0.75 0.3

Liver Kidney Muscle Milk

2 0.75 0.75 0.3

Liver Kidney Muscle Milk

10

Not used

10 10 4 4 6

Liver Kidney Muscle Fat Milk

Liver Kidney Muscle Fat Milk

NSAIDs Carprofen

Carprofen

Bovine All milk-producing species for human consumption

500 Not used

Muscle Milk

Flunixin

Flunixin

Bovine

5-Hydroxyflunixin Flunixin

Bovine Porcine

300 100 30 20 40 200 50 30 10

Liver Kidney Fat Muscle Milk Liver Muscle Kidney Skin+Fat

Meloxicam

Meloxicam

Bovine

65 65 20 15

Liver Kidney Muscle Milk

Tolfenamic acid

Tolfenamic acid

Bovine, porcine

400 100 50 50

Liver Kidney Muscle Milk

Bovine

sensitivity than ESI and produced, besides pseudomolecular ion [M+H]+, radical-cation MH+. For other biological matrices, a limited number of papers deals with corticosteroid analysis in food products of animal origin: a review on the MS techniques applied to the determination of corticosteroid residues in biological matrices [6]; and, another on the analytical strategies for detecting growth-promoter residues in food-producing animals [44]. 600

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Draisci et al. [32] combined an LC-APCI(-)-MRM method with ASE for confirming residues of three fluorinated synthetic corticosteroids from liver samples. The application of ASE allowed extraction of each sample in 35 min and processing of up to 24 samples sequentially with quantitative recoveries and relative standard deviations <7.4%. A chromatographic flow rate of 100 lL/min would not seem very appropriate for an ionization source that uses the mobile phase as reagent gas

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and is designed for higher flow rates (1–2 mL/min), despite the concentration effect of the narrow-bore column (see Table 3). Antignac et al. [23] determined 11 corticosteroids in meat (hair and urine) by means of LC-ESI(-)-MRM. They gave particular attention to the extraction procedure for reducing the number of interfering compounds in the final extract and to improving the limits of detection (LODs); nevertheless, the extraction efficiency cannot be estimated because the authors did not report recovery percentages. The selection of the only MRM transition [M+acetate]/[MHCH2O], common to all analytes, guarantees high sensitivity but does not satisfy the confirmation criteria required by the Commission Decision 2002/657/CE. In 2003 [24], the same authors submitted this analytical method to a validation protocol that respected the requirements of Commission Decision 2002/657/CE to confirm the occurrence of triamcinolone acetonide in meat samples; the quantitative performances of the confirmation method were evaluated on the basis of the less intense transition. Van Den Hauwe et al. [25] performed a field study to assess the drug-residue level in edible tissues after a therapeutic application of the synthetic glucocorticoids. Three different tissues (muscle, kidney and liver) were analyzed by enzyme immunoassay and the higher contents, found in the liver, were confirmed by LC-MS2. The results indicated that liver tissue provides a suitable matrix to monitor the presence of illegal residues of synthetic glucocorticoids in slaughtered animals and that the conventional therapeutic use of dexamethasone causes drug concentrations in edible tissues that may exceed the MRLs by more than an order of magnitude. Even after the introduction of LC-MS, differentiating between betamethasone and dexamethasone, two isomeric corticosteroids, has remained something of a problem. One solution, proposed by Baiocchi et al. [45], used a porous graphite column whose resolution allowed the two forms to be distinguished. The authors achieved the same results on a conventional C18 column by using acetonitrile as organic modifier; the disadvantage of this option was the increase in the LODs. The other advantages of the method, proposed for the routine analysis of eight corticosteroids in bovine liver by LC-IT-MS2, were the extraction procedure, based on only one step, and the short chromatographic run, carried out in isocratic conditions without the need for re-equilibration. Cherlet et al. [26] developed an LC-APCI(+)-MRM method in line with Decision 2002/657/CE for determining dexamethasone residues in bovine milk samples. The clean-up procedure was based on SPE on C18 cartridge, after protein precipitation with trichloroacetic acid; not having diluted the milk sample before precipitation, the analyte was recovered with a low yield, probably because of partial entrapment in the abundant precipitate that forms on acidifying milk.

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Cui et al. [31] coupled UPLC to ESI-MS2 for the simultaneous determination of 17 glucocorticoids in milk and eggs. Analytes separated within 13 min and each chromatographic peak had a duration of about 12 s. The low selectivity of methanol as organic modifier produced several coelutions and the two peaks observed for the triamcinolone were probably due to an isomerization induced by heat in the same chromatographic column thermostatted at 40C. The authors adopted a very tedious, time-consuming extraction procedure that annulled the advantage of this method (i.e. the rapidity of separation); cortisol was chosen as internal standard, so the analytical answer could be altered by natural levels of this hormone in food.

4.2. NSAIDs NSAIDs [1–3] is the term used to describe compounds that are not ‘‘steroidal’’ and that suppress inflammation. Unlike corticosteroids, which inhibit numerous pathways, NSAIDs act primarily to reduce the biosynthesis of prostaglandins (responsible for swelling and pain) by inhibiting the cyclooxygenases (COX-1 and COX-2). Because of their activities, they are sometimes referred as non-narcotic analgesics or aspirin-like drugs (aspirin is the progenitor of this drug class). Structurally, they show great variation, but they can generally be classified according to the selectivity for inhibition of the cyclooxygenases: (1) non-selective COX inhibitors; and, (2) selective COX-2 inhibitors [2]. All NSAIDs are acid compounds with pKa in the range 3–5; the acidic group is essential for COX inhibitory activity and for their inactivation mainly as glucuronides. Table 4 shows the classes, the names, the chemical structures and the monoisotopic masses of the NSAIDs most used in veterinary medicine. In the group of non-selective COX inhibitors are the anilides that, with the exception of nimesulide, do not properly belong to NSAIDs (they do not have an acidic group). Because of the polar nature of NSAIDs, the ESI source is particularly suitable for their MS detection, even if there are some applications with APCI sources in literature [46,47]. All NSAIDs respond in negativeion mode following the deprotonation of the carboxylic functional group; the same site can be protonated, also allowing the detection of these compounds in positiveion mode [48]. Investigation of the CID of NSAIDs highlighted interesting behaviour: the same fragment ion was formed in either ionization mode and was called a ‘‘twin ion’’ because of having the same nominal mass despite having opposite charge. Accurate mass measurements showed that the loss was due to CO2 in negative-ion mode and to HCOOH in positive-ion mode and that the twin ions differ in mass by two electrons [48]. http://www.elsevier.com/locate/trac

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Table 3. LC-MS2 methods for determining corticosteroid and NSAID residues in different food matrices Anti-inflammatory drug

Corticosteroids Dexamethasone and b-epimer betamethasone

Matrix

Analytical technique

Separative conditions

Extraction procedure

Method performance Recovery (%)

Limit

Authors, Year [Reference]

HPLC-APCI(-)-QqQ. 3 MRM transitions: m/z 361/307, m/z 361/292, m/z 361/325; flumethasone as internal standard (IS)

Kingsorb C18 column (250 · 2 mm, 5 lm); isocratic elution with a mobile phase of acetonitrile–5 mM ammonium acetate– methanol (35:60:5, v/v)

Accelerated solvent extraction Two steps: (1) defatting with hexane at 60C and 1.0 104 kPa; (2) extraction with hexane: ethyl acetate (1:1, v/v) at 50C and 1.0 104 kPa

75–77

Limit of quantitation (LOQ) = 1 ppb

Draisci et al, 2001 [32]

Beclomethasone, betamethasone, cortisol, cortisone, desoxycortisone, dexamethasone, flumethasone, methylprednisolone, prednisolone, prednisone, triamcinolone

Bovine meat

HPLC-ESI(-)-QqQ. 1 MRM transition:[M+acetate]/ [MHCH2O]; fludrocortisone (IS)

Nucleosil C18AB column (50 · 2 mm, 5 lm); gradient elution with methanol (A) and 0.5% (v/v) acetic acid

15 g of bovine fresh meat were dry-frozen and pulverised; Extraction with methanol and acetate buffer; enzymatic hydrolysis (15 h at 52C); solid phase extraction (SPE) on C18 cartridge; liquidliquid clean-up; SPE on SiOH cartridge

Not reported

Limit of detection (LOD) = 40–70 ppt, except for beclomethasone (380 ppt) and triamcinolone (500 ppt)

Antignac et al., 2001 [23]

Triamcinolone acetonide

Bovine meat

HPLC-ESI(-)-QqQ. 2 MRM transitions: m/z 493/413, m/z 493/375; triamcinolone acetonide-d6 (IS)

97

Detection capability (CCb) = 110 ppt

Antignac et al., 2003 [24]

Dexamethasone and flumethasone

Bovine liver

HPLC-ESI(±)-QqQ. 2 MRM transitions; fluorometholone (IS)

Hypercarb column (100 · 2.1 mm, 5 lm); isocratic elution

Hydrolysis, extraction with acetronitrile, SPE on C18 cartridge

82

Van den Hauwe, 2003 [25]

Dexamethasone, betamethasone, flumethasone, flunisolide, prednisolone, prednisone, triamcinolone and triamcinolone acetonide

Bovine liver

HPLC-APCI(+)-ion-trap MS2

Porous graphitic carbon column (Hypercarb 5l, 125 · 4.6 mm, 5lm); isocratic elution: 85% methanol, 15% dichloromethane

2.5 g liver added to ammonium acetate and sonicated in a ultrasonic bath at 30% of maximum power (50 W)

60–75

Dexamethasone CCb = 2.13 ppb; flumethasone CCb = 0.19 ppb LOQs = 0.33–40 ppb

See [28]

Baiocchi et al., 2003 [45]

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Bovine liver

Bovine milk

HPLC-APCI(+)-ion trap MS2; desoximetasone (IS)

PLRP-S polymeric reversed phase column (250 Æ 4.6mm; 8 lm) thermostatted at 30C; Gradient elution with 0.1% acetic acid in water (A) and acetonitrile (B)

Protein precipitation with tricloroacetic acid; clean up on C18 cartridge

56

CCb = 0.76 ng/mL.

Cherlet et al., 2004 [26]

Prednisone, prednisolone, cortisone, aldosterone, hydrocortisone, methylprednisolone, fluorometholone, dexamethasone, triamcinolone, becomethasone, flumethasone, fludrocortisone acetate, budesonide, triamcinolone acetonide, fluocinolone acetonide, clobetasol propionate, clobetasone butyrate

Milk and eggs

UPLC-ESI(-)-QqQ; 2 MRM transitions; cortisol (I.S.)

ACQUITY UPLC BEH C18 column (100 · 2.1 mm;1.7 lm) thermostatted at 40C; Gradient elution with water containing 0.1% formic acid (A), and methanol (B).

Enzymatic hydrolysis (overnight); extraction with methanol; wash with hexane; SPE on HLB OASIS cartridge; SPE on Sep-Pak silica cartridge; SPE on aminopropyl cartridge.

66–112

LOQs = 0.04–1.27 ppb

Cui et al., 2006 [31]

Bovine milk

HPLC-ESI(+)-QqQ; 1 MRM transition:

Zorbax Eclipse XDB-C18 column (150 · 2.1 mm; 5 lm) thermostatted at 40C; isocratic elution with 0.4% acetic acid in water (A) and acetonitrile:methanol (95:5) (B).

Protein precipitation with HCl 0.1 N and liquid-liquid extraction; acidification of the extract and clean-up on strong cation-exchange (SCX) SPE cartridge.

Not reported

LOQ = 1ppb

Feely et al., 2002 [27]

Bovine liver, kidney, fat and muscle

HPLC-ESI(+)-QqQ; 1 MRM transition:

Zorbax Eclipse XDB-C18 column (150 · 2.1 mm; 5 lm) thermostatted at 40C; isocratic elution with 0.4% acetic acid in water (A), methanol (B), 0.4% acetic acid in acetonitrile (C) and A/acetonitrile/B (D)

Acid hydrolysis; liquid extraction with ethyl acetate; SPE on SCX cartridge.

86–95

Liver and kidney LOD = 0.1 ppb; Muscle and fat LOD = 0.2 ppb.

Boner et al., 2003[29]

NSAIDs Flunixin,5-hydroxyflunixin

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Flunixin

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Dexamethasone

92–98%

Van Hoof et al., 2004 [28] CCb = 2–68 ppb

Protein precipitation with acetonitrile; concentration of supernatant 60C; filtration

2 g of minced tissue extracted with acetonitrile; SPE on Oasis HLB cartridge

Simmetry C18 column (150 · 2.1 mm; 5 lm); gradient elution with water (A) acetonitrile (B) each containing 0.1% formic acid.

Alltima HP C18 column (150 · 2.1 mm, 5lm); gradient elution with 0.1% acetic acid in water (A) and methanol (B).

HPLC-ESI(+)-QqQ; 2 MRM transition

HPLC-ESI(±)-ion trap MS2 and MS3; desoximethasone (IS)

Bovine milk

Bovine muscle

Flunixin, 5-hydroxyflunixin and ketoprofen

Acetylsalicylic acid, ketoprofen, flunixin, tolfenamic acid, phenylbutazone, meloxicam

See [42] Zorbax Eclipse XDB-C18 column (150 · 2.1 mm; 5 lm) thermostatted at 40C; gradient elution with 0.4% acetic acid in water (A) and 0.2% acetic acid in acetonitrile:methanol (B) HPLC-ESI(+)-QqQ; 1 MRM transition Bovine milk 5-hydroxy-flunixin

Not reported

Daeseleire et al., 2003 [50] Decision limit (CCa) = 0.5–1 ppb

Limit Recovery (%)

Method performance Extraction procedure Separative conditions Analytical technique Matrix Anti-inflammatory drug

Table 3 (continued)

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Ngoh et al., 2003 [30]

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Because of the acidic nature of these drugs, their chromatographic separation can be realized by ion suppression or ion-pair reversed-phase chromatography [15]; Quintana and Reemtsma [49] used tri-n-butylamine (10 mM) as a volatile ion-pairing agent to retain the more polar compounds (e.g., salicylic acid); in order to counterbalance the strong retention of the analytes, the chromatographic column was thermostatted at 55C. Several research groups performed studies on the metabolism of flunixin in lactating dairy cows to identify metabolites and to define an adequate withdrawal period [27,30,50]. Four residues – flunixin, 5-OH flunixin, 4 0 OH flunixin and 2 0 -OH methylflunixin – were positively identified by LC-ESI(+)-QqQ in the milk samples examined and the most abundant, 5-OH flunixin, was regarded as the marker residue [27]. The same research group developed an LC-MS2 method [29] for the determination of flunixin in all edible bovine tissues, but all these methods identified the analytes by selecting only one MRM transition. Daeseleire et al. [50] developed and partially validated, in line with Commission Decision 2002/657/EC, an LCESI(+)-MS2 method for confirming traces of flunixin, its 5-hydroxy metabolite and ketoprofen in raw milk. The sensitivity and the selectivity of the LC-MS2 technique allowed a simple, rapid method of extraction to be applied to achieve low LODs. The first multi-residue method for the simultaneous identification of six NSAIDs in bovine muscle by LC-ESIIT was proposed by Van Hoof et al. [28]. Ketoprofen was detected in positive-ion mode, while the other drugs were detected in negative-ion mode; in addition, second-generation product ions were acquired for the confirmation of salicylic acid, tolfenamic acid and ketoprofen that, in their MS2 spectra, showed only one product ion. The authors did not report the recovery values and underlined the necessity of further investigations on extraction procedure because of a presupposed strong bond of NSAIDs to proteins.

5. Conclusions The number of publications on the analysis of AIDs in animal-food products by LC-MS has been limited (see Table 4); however, many works have addressed research on corticosteroid residues in biological matrices or NSAIDs as emerging contaminants in environmental samples. The low cost of NSAIDs, their antipyretic and analgesic action make them therapeutic agents specifically designed for pain management in cattle together with anaesthetic agents and sedatives; their use has become common in cattle breeding. The interest shown in the environmental field should also be applied to food contamination and a greater number of works should be

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Table 4. Chemical classification, names, structures, monoisotopic mass and metabolites of NSAIDs

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dedicated to developing LC-MS methods for determining the residue levels in the different food matrices. Because of the polar nature of NSAIDs, ESI is the source of choice for their MS detection; a problem linked to ESI is ion suppression due to the adverse influence of matrix constituents, also known as the matrix effect [51]. Often, the main interfering substances occurring in food are fat material; as reported in the literature [52], solvophobic compounds, dispersed in water with a volatile cosolvent, spontaneously form on the surface of aerosol droplets a film that inhibits ion evaporation. To reduce the amount of matrix components that are injected into the LC-MS system, some authors have adopted a more selective analyte-extraction procedure or a more extensive sample clean-up [23,24,27,29,30,50]. However, to perform a reliable quantitative analysis, the calibration curve should be always constructed in the

matrix and an adequate internal standard should be used to correct the ESI response during analysis [51,53]. For a long time, methods based on GC-MS were preferred for the determination of corticosteroids; today, there is increasing interest in LC-MS-based procedures. These compounds are detectable by ESI, APCI and APPI sources in both ionization modes, but there are differing opinions on the best performances. In 2004, Antignac et al. [6] reviewed 10 years of experience with a variety of GC-MS and LC-MS techniques and concluded that LC-QqQ and LC-IT-MSn are currently the most suitable tools for monitoring corticosteroids. In general, for routine analysis of food contaminants, the mass analyzers most commonly used are the QqQs and the IT. This latter is characterized by high-power identification but the best performances in quantitative analysis are by QqQ, in terms of sensitivity and width of

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linear dynamic range. More recent instruments (e.g., QqTOF and QqQ-LIT) are gaining widespread acceptance in other fields, but their use in food analysis is still restricted. Because the confirmation of identity during residue analysis of contaminants in animal-food products is the most important application of LC-MS, these hybrid mass spectrometers could be useful for accurate mass measurements and increased resolution.

References [1] The Merck Veterinary manual. Anti-inflammatory Agents, (http://www.merckvetmanual.com/mvm/index.jsp?cfile=htm/bc/ toc_191600.htm). [2] H.R. Adams (Editor), Veterinary Pharmacology and Therapeutics, 8th ed., Blackwell Publishing, Oxford, UK, 2001. [3] in J.G. Hardman, L.E. Limbird (Editors), Goodman & Gilmans The Pharmacological Basis of Therapeutics, 10th International Edition, McGraw-Hill Medical Publishing Division, New York, USA, 2001. [4] The New York State Cattle Health Assurance Program, Beef Quality Assurance Module, Non-steroidal anti-inflammatory drug use in food producing animals (http://nyschap.vet.cornell.edu/ module/beefquality/section1/NSAIDS.pdf). [5] L.P. Vogel, E. Curry-Galvin, J. Am. Vet. Med. Assoc., News, October 1, 2000 (http://www.avma.org/onlnews/javma/oct00/ s100100a.asp). [6] J.-P. Antignac, F. Monteau, J. Ne´griolli, F. Andre´, B. Le Bizec, Chromatographia 59 (2004) S13. [7] A. Gentili, Anal. Bioanal. Chem. (2006) (in press) (DOI: doi:10.1007/s00216-006-0821-7) (http://www.springerlink.com/ content/1618-2650/?k=Alessandra+gentili). [8] A.L. Yergey, C.G. Edmonds, I.A.S. Lewis, M.L. Vestal, Liquid Chromatography–Mass Spectrometry, Techniques and Applications, Plenum Press, New York, USA, 1990. [9] R.B. Cole (Editor), Electrospray Ionization Mass Spectrometry – Fundamentals, Instrumentation and Applications, Wiley, New York, USA, 1997. [10] W.M.A. Niessen, J. Chromatogr. A 856 (1999) 179. [11] W.M.A. Niessen, J. Chromatogr. A 1000 (2003) 413. [12] S.D. Richardson, Anal. Chem. 76 (2004) 3337. [13] I. Ferrer, E.M. Thurman, Analysis of emerging contaminants, in: I. Ferrer, E.M. Thurman (Editors), Liquid Chromatography/Mass Spectrometry, MS/MS and Time of Flight MS – Analysis of Emerging Contaminants, ACS Symposium Series 850, Orlando Meeting, American Chemical Society, Washington, DC, USA, 2003, p. 2. [14] M. Gros, M. Petrovic, D. Barcelo´, Anal. Bioanal. Chem. 386 (2006) 941. [15] M. Petrovic´, M.D. Hernando, M.S. Dı´az-Cruz, D. Barcelo´, J. Chromatogr., A 1067 (2005) 1. [16] A. Raffaelli, A. Saba, Mass Spectrom. Rev. 22 (2003) 318. [17] H. Hayen, U. Karst, J. Chromatogr., A 1000 (2003) 549. [18] M.E. Swartz, Separation Science Redefined, Waters Corporation Supplement, LC-GC, May 2005. [19] E. Barcelo´-Barrchina, E. Moyano, M.T. Galceran, J.L. Lliberia, B. Bago´, M.A. Cortes, J. Chromatogr., A 1125 (2006) 195. [20] European Commission, Commission Decision 2002/657/CE implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results, Off. J. Eur. Commun. L 221 (2002) 8. [21] I.V. Chernushevich, A.V. Loboda, B.A. Thomson, J. Mass Spectrom. 36 (2001) 849. [22] J.W. Hager, Rapid Commun. Mass Spectrom. 16 (2002) 512.

608

http://www.elsevier.com/locate/trac

[23] J.-P. Antignac, B. Le Bizec, F. Monteau, F. Poulain, F. Andre, J. Chromatogr., B 757 (2001) 11. [24] J.-P. Antignac, B. Le Bizec, F. Monteau, F. Andre, Anal. Chim. Acta 483 (2003) 325. [25] O. Van Den Hauwe, M. Schneider, A. Sahin, C.H. Van Peteghem, H. Naegeli, J. Agric. Food Chem. 51 (2003) 326. [26] M. Cherlet, S. De Baere, P. De Backer, J. Chromatogr., B 805 (2004) 57. [27] W.F. Feely, C. Chester-Yansen, K. Thompson, J.W. Campbell, P.L. Boner, D.D.W. Liu, L.S. Crouch, J. Agric. Food Chem. 50 (2002) 7308. [28] N. Van Hoof, K. De Wasch, S. Poelmans, H. Noppe, H. De Brabander, Rapid Commun. Mass Spectrom. 18 (2004) 2823. [29] P.L. Boner, D.D.W. Liu, W.F. Feely, R.A. Robinson, J. Wu, J. Agric. Food Chem. 51 (2003) 7555. [30] M.A. Ngoh, P.G. Wislocki, K. Thompson, T. Katz, A. Weingarten, T. Terhune, B. Hurshman, J. Agric. Food Chem. 51 (2003) 4701. [31] X. Cui, B. Shao, R. Zhao, Y. Yang, J. Hu, X. Tu, Rapid Commun. Mass Spectrom. 20 (2006) 2335. [32] R. Draisci, C. Marchiafava, L. Palleschi, P. Cammarata, S. Cavalli, J. Chromatogr., B 753 (2001) 217. [33] B.E. Richter, B.A. Jones, J.L. Ezzell, N.L. Porter, Anal. Chem. 68 (1996) 1033. [34] E. Bjo¨rklund, T. Nilsson, Trends Anal. Chem. 19 (2000) 434. [35] S. Bogialli, R. Curini, A. Di Corcia, M. Nazzari, R. Saperi, Rapid Commun. Mass Spectrom. 17 (2003) 1146. [36] A. Gentili, D. Perret, S. Marchese, M. Sergi, C. Olmi, R. Curini, J. Agric. Food Chem. 52 (2004) 4614. [37] European Commission, Council Directive 96/22/EC of 29 April 1996 concerning the prohibition on the use in stock farming of certain substances having a hormonal or thyrostatic action and of ß-agonists, and repealing Directives 81/602/EEC, 88/146/EEC and 88/299/EEC. Off. J. Eur. Commun. L 125 (1996) 3. [38] European Commission, Council Regulation 2377/90/EEC laying down a Community procedure for the establishment of maximum residue limits veterinary medicinal products in foodstuffs of animal origin, Off. J. Eur. Commun. L 224 (1990) 1. [39] European Commission, Commission Regulation 508/1999/EC amending Annexes I to IV to Council Regulation (EEC) No 2377/ 90 laying down Community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin, Off. J. Eur. Commun. L 224 (1999) 1. [40] J.-P. Antignac, B. Le Bizec, F. Monteau, F. Poulain, F. Andre´, Rapid Commun. Mass Spectrom. 14 (2000) 33. [41] E. Sangiorgi, M. Curatolo, W. Assini, E. Bozzoni, Anal. Chim. Acta 483 (2003) 259. [42] M.J. Greig, B. Bolanos, T. Quenzer, J.M.R. Bylund, Rapid Commun. Mass Spectrom. 17 (2003) 2763. [43] M. Fiori, E. Pierdominici, F. Longo, G. Brambilla, J. Chromatogr., A 807 (1998) 219. [44] A.A.M. Stolker, U.A.Th. Brinkman, J. Chromatogr., A 1067 (2005) 15. [45] C. Baiocchi, M. Mussino, M. Pazzi, C. Medana, C. Marini, E. Genta, Chromatographia 58 (2003) 11. [46] M.E. Abdel-Hamid, L. Novotny, H. Hamza, J. Pharm. Biomed. Anal. 24 (2001) 587. [47] D. Lo¨ffer, T.A. Ternes, J. Chromatogr., A 1021 (2003) 133. [48] I. Ferrer, E.M. Thurman, Anal. Chem. 77 (2005) 3394. [49] J.B. Quintana, T. Reemtsma, Rapid Commun. Mass Spectrom. 18 (2004) 765. [50] E. Daeseleire, L. Mortier, H. De Ruyck, N. Geerts, Anal. Chim. Acta 488 (2003) 25. [51] W.M.A. Niessen, P. Manini, R. Andreoli, Mass Spectrom. Rev. 25 (2006) 881. [52] J.B. Fenn, J. Am. Soc. Mass Spectrom. 4 (1993) 524. [53] D. Zimmer, Chromatographia 57 (2003) S–325.