Talanta 144 (2015) 686–695
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
High-throughput method for macrolides and lincosamides antibiotics residues analysis in milk and muscle using a simple liquid–liquid extraction technique and liquid chromatography–electrospray–tandem mass spectrometry analysis (LC–MS/MS) Louise Jank a,b,n, Magda Targa Martins a,c, Juliana Bazzan Arsand a,b, Tanara Magalhães Campos Motta b, Rodrigo Barcellos Hoff a,b, Fabiano Barreto a,c, Tânia Mara Pizzolato a a
Programa de Pós-Graduação em Química – PPGQ, Universidade Federal do Rio Grande do Sul – UFRGS, Porto Alegre, RS, Brazil Laboratório Nacional Agropecuário – LANAGRO/RS, Ministério da Agricultura, Pecuária e Abastecimento, Porto Alegre, RS, Brazil c Programa de Pós-Graduação em Ciências Farmacêuticas – PPGCF, Universidade Federal do Rio Grande do Sul – UFRGS, Porto Alegre, RS, Brazil b
art ic l e i nf o
a b s t r a c t
Article history: Received 13 March 2015 Received in revised form 22 June 2015 Accepted 24 June 2015 Available online 4 July 2015
A fast and simple method for residue analysis of the antibiotics classes of macrolides (erythromycin, azithromycin, tylosin, tilmicosin and spiramycin) and lincosamides (lincomycin and clindamycin) was developed and validated for cattle, swine and chicken muscle and for bovine milk. Sample preparation consists in a liquid–liquid extraction (LLE) with acetonitrile, followed by liquid chromatography–electrospray–tandem mass spectrometry analysis (LC–ESI-MS/MS), without the need of any additional cleanup steps. Chromatographic separation was achieved using a C18 column and a mobile phase composed by acidified acetonitrile and water. The method was fully validated according the criteria of the Commission Decision 2002/657/EC. Validation parameters such as limit of detection, limit of quantification, linearity, accuracy, repeatability, specificity, reproducibility, decision limit (CCα) and detection capability (CCβ) were evaluated. All calculated values met the established criteria. Reproducibility values, expressed as coefficient of variation, were all lower than 19.1%. Recoveries range from 60% to 107%. Limits of detection were from 5 to 25 mg kg 1.The present method is able to be applied in routine analysis, with adequate time of analysis, low cost and a simple sample preparation protocol. & 2015 Published by Elsevier B.V.
Keywords: LC–MS/MS Antibiotics Milk Muscle Residues Macrolides Lincosamides
1. Introduction Antibiotics are widely used in livestock production with many purposes, such as disease treatment (therapeutic application), disease prevention (prophylactic application) and improve feeding efficiency (as growing promoters). Many antibiotic classes are used with this last objective, including macrolides and lincosamides. Macrolides are used to treat respiratory diseases and enteric infections in cattle, sheep, swine and poultry, once they are effective against gram-positive and some gram-negative bacteria [1]. They can be defined as lipophilic molecules, constituted by a macrocyclic lactone formed by 14 (erythromycin), 15 (azithromycin) or 16 carbons (spiramycin, tilmicosin and tylosin) linked to sugar n Corresponding author at: Programa de Pós-Graduação em Química – PPGQ, Universidade Federal do Rio Grande do Sul – UFRGS, Porto Alegre, RS, Brazil. E-mail address:
[email protected] (L. Jank).
http://dx.doi.org/10.1016/j.talanta.2015.06.078 0039-9140/& 2015 Published by Elsevier B.V.
molecules, through glycosidic bonds [2]. Erythromycin, spyramicin and tylosin are produced by different microorganisms (Streptomyces erythaeus, Streptomyces ambofaciens and Streptomyces fradiae, respectively), while tilmicosin is a semi-synthetic compound, obtained from tylosin [2]. Azithromycin is synthesized from erythromycin oxime, and is the first and presently the one member of 15-membered macrolide antibiotic present in market. Lincosamides are also a group of antibiotics commonly used, in both human and veterinary medicine. Their structure consists in a pyranose ring, an amide moiety and a pyrrolidine ring. The main members of lincosamides class are lincomycin and clindamycin. Lincomycin is naturally produced by Streptomyces lincolnensis, while clindamycin is a semisynthetic compound, produced from lincomycin, and it is around twenty times more effective than its precursor [3]. Due to the widespread use, residues of macrolides and lincosamides may remain in food. The presence of residues are
L. Jank et al. / Talanta 144 (2015) 686–695
687
Table 1 Analytes properties. Compound
Class
Physico-chemical properties
Erythromycin CAS number: 114-07-8
Macrolides
log kow: 3.6 S: 1440 (25 ºC) pKa:8.8
Azithromycin CAS number: 117772-70-0
Macrolides
log kow: 4.02 S: 2.37x10 3 mg L 1 pKa: 8.7
Tylosin CAS number: 1401-69-0
Macrolides
log kow: 3.27 S: 50 mg mL 1 pKa: 7.2
Tilmicosin CAS number: 108050-54-0
Macrolides
log kow: 3.80 S: 1.5x10 2 mg L 1 pKa: 8.18
688
L. Jank et al. / Talanta 144 (2015) 686–695
Table 1 (continued ) Compound
Class
Physico-chemical properties
Spiramicin CAS number: 8025-81-8
Macrolides
log kow: 3.80 S: 1.5x10 2 mg L 1 pKa: 7.9
Lincomycin CAS number: 154-21-2
Lincosamides
log kow: 0.20 S: 927 mg L 1 pKa: 7.6
Clindamycin CAS number:
Lincosamides
log kow: 2.16 S: 30.61 mg L 1 pKa: 7.79
S¼ solubility. Log k¼ octanol/water partition coefficient. pKa ¼Constant of dissociation.
implicated in several health issues, e.g. antimicrobial bacterial resistance [4]. For human health safety, maximum residue limits (MRLs) are set as the residue level that can remain in tissues or
animal origin food, after treatment with pharmaceutical compounds. It is considered that this residue level has no adverse health effects if ingested daily by humans throughout lifetime
Table 2 Mass spectrometry and chromatography parameters. Compound
Molecular weight (Da)
Precursor ion (m/z)
Fragments (m/z)
DP (V)
CE (V)
CXP (V)
Retention time (min)
LNC
406.2 424.9
ERT
733.4
AZT
748.5
SPR
842.5
TLM
868.5
TYL
915.5
126.0 359.0 126.1 377.3 158.1 576.0 158.2 116.0 174.0 101.0 174.0 143.3 125.8 174.0 101.0
11 11 136 151 126 126 271 271 31 31 80 80 80 56 56
37 23 35 23 37 23 51 61 47 51 30 26 44 47 59
16 14 12 40 16 22 24 14 18 22 15 15 15 18 22
6.49
CLN
407.0 407.0 425.3 425.3 734.0 734.0 749.5 749.5 843.0 843.0 435.5 435.5 435.5 916.0 916.0
7.10 7.42 6.94 6.90 7.13
7.46
Bold transitions corresponds to quantifier transition; other transitions are used as qualifier ions. DP ¼declustering potential. CE ¼ collision energy. CXP¼ collision cell exit potential.
L. Jank et al. / Talanta 144 (2015) 686–695
Table 3 MRLs and validation levels selected for macrolides and lincosamides in milk and bovine muscle. Milka
Bovine muscleb
MRL (mg kg 1)
MRL (mg kg 1)
ERT
40
200
SPR
200
200
TYL
50
100
LNC
150
100
CLN
–
–
AZT
–
–
TLM
50
50
Analyte
VLc (mg kg 1)
100 100 100 100 50 50 50 a
b
and : Given by Commission regulation (EU) No. 37/2010 of 22 December 2009. c Validation level.
[5,6]. Regulatory agencies and government authorities set MRLs for veterinary drugs in food worldwide [7]. In Brazil, the National Residue Control Plan (NRCP) defines which residues must be monitored and their MRLs, aiming mainly to monitor the incidence of residues and prevent potential risk to the population exposed to those products [8]. The NRCP is an attribution of Ministry of Agriculture, Livestock and Food Supply (MAPA) [9,10]. Many studies have reported methodologies based on liquid chromatography couple to mass spectrometry in single or tandem mode (LC–MS and LC–MS/MS, respectively) to determine macrolides and lincosamides residues in milk [11–14] and muscle [15– 21], and also in different matrices, such as honey [22,23], eggs [24], fish [25] and kidney [26,27]. These compounds are routinely included in multiresidue methodologies [14,16,19,22–24,28–31], and there is minor publications of macrolides class only [32,33] or macrolides and lincosamides only [21]. For instance, Freitas et al use extraction with acetonitrile to analyze milk and bovine muscle to screening of several antibiotics, including four macrolides [34,35]. Considering the complex nature of food matrices, it is expected that the majority of these reports include one or more clean-up steps to make the extract adequate for analysis in LC–MS methods. Even in single class or multiclass methods, several authors preconize the use of solid-phase extraction (SPE) [27,33,36–39], SPE associated with ion-pairing [40] or even on-line clean-up systems [41]. Generally, less complex steps are added, as fat removal using hexane, microfiltration and evaporation [34,35]. One or another of these steps are present in almost all reports dealing with macrolides and/or lincosamides residues analysis [24,27,36]. In Brazil, the veterinary drugs residues surveillance in raw milk and animal muscle is an issue of MAPA, accomplished through the NRCP. With the exception of the spiramycin in bovine muscle, residues of macrolides and lincosamides are monitored using kidney as target matrix [8]. The present report describes the development and validation of an analytical method for the analysis of major members of macrolides and lincosamides antibiotics groups in bovine milk and muscle of bovine, swine and poultry. The overall aim was to obtain a simple, fast and cheap sample preparation protocol allied with the high specificity and detection capability of LC–MS/MS.
689
2. Experimental 2.1. Chemicals and reagents Erythromycin (ERT), spiramycin (SPR), tylosin (TYL), azithromycin (AZT), tilmicosin (TLM), lincomycin (LNC) and clindamycin (CLN) standards were obtained from Sigma-Aldrich Logistik (Scnelldorf, Germany) all with 4 95% certified purity. Stock standard solutions were prepared dissolving 0.010 g of each compound in 10.0 mL of ultrapure water, in order to obtain a concentration of 1.0 mg mL 1. Aqueous working solutions of analytes (pool) for use in muscle samples were prepared using 125 mL of stock solutions of ERT, SPR, LNC and TYL, and 62.5 mL of TLM, AZT and CLN, with final volume of 10 mL. Addition of 16 mL of this work solution to 2.0 g of muscle sample is equivalent to MRL concentration for all analytes. For milk samples, aqueous working solution was prepared using the following volumes of stock solutions: 50 mL for ERT, 62.5 mL for TYL and TLM, 125 mL for AZT, 250 mL for SPR mL and 187.5 mL for LNC and CLN, with a final volume of 10 mL. Addition of 16 mL of the work solution to 2.0 mL of milk sample is equivalent is equivalent to MRL concentration for all analytes. Stability evaluation of these working solutions showed that they are stable for 3 months when kept between 10 and 30 °C. The stock solutions are stable for 6 months when kept in freezer ( 10 to 30 °C). Table 1 shows the molecular structure and major physicochemical parameters of the analytes included in the present method. Acetonitrile (ACN) HPLC grade was purchased from Merck (Darmstadt, Germany) and formic acid (FA) was obtained from J.T. Baker (Phillipsburg, NJ, USA). Deionized ultra pure water (o18.2 MΩ cm resistivity) was obtained from the Milli-Q SP Reagent Water System (Millipore, Bedford, MA, US). 2.2. Samples and sample extraction Bovine milk samples were obtained from local producers, from non-medicated dairy cows. Muscle samples from bovine, swine and poultry were collected in slaughterhouses by Federal Inspection Service (SIF), immediately frozen and send to the laboratory. Before analysis, samples were minced and homogenized and apparent fat tissue was removed. For milk analysis, an aliquot of 2.0 mL of homogenized fluid milk was taken and putted in a 50 mL polypropylene tube. Extraction procedure consisted in to add 4.0 mL of ACN, divided in 3 aliquots (2.0 mL þ1.0 mLþ 1.0 mL) to promote precipitation of milk proteins, shaking the tubes between each addition. After that, tubes were mixed in a shaker during 20 min and centrifuged during 5 min, at approximately 3000 g, at 5 °C. Aliquots of supernatant were transferred to HPLC vials and submitted to LC–MS/MS analysis. A volume of 2 mL of extract was injected in analytical system. For muscle samples, aliquots of 2.0 g were exactly weighted. Analytes were extracted from tissues using 10.0 mL of ACN. Sample and solvent were homogenized using a mechanical mixer (Ultra-Turrax), to promote complete tissue disruption. Then, samples were mixed in a shaker during 20 min and centrifuged during 5 min, at approximately 3000 g, at 5 °C. In the same way described to milk, aliquots of supernatant were transferred to HPLC vials and submitted to LC–MS/MS analysis. A volume of 2 mL of extract was injected in analytical system. 2.3. LC–MS/MS analysis LC–MS/MS
system
used
was
composed
by
a
liquid
690
L. Jank et al. / Talanta 144 (2015) 686–695
Fig. 1. Coefficients of variation (CV, in %) obtained for each analyte in 3 distinct sample injection volumes: 2.0, 5.0 and 10.0 mL (n¼ 7). To TLM, the CV value obtained for 10.0 mL was 25.4% and it is out of rane in the CV axis.
chromatograph Agilent 1100 Series and an API 5000 mass spectrometer (AB Sciex, Foster City, CA). As stationary phase, an HPLC column AgellaDurashell RP (100 mm 2.1 mm, 5 μm) was used preceded by a guard column filled with C18 (4.0 mm x 3.0mm, 5 μm, from Phenomenex). A binary mobile phase system was used, with a flow of 0.3 mL min 1. Mobile phase component A was an aqueous solution of 0.1% formic acid and component B was ACN with 0.1%
formic acid. The gradient optimized for the separation starts keeping 98% of A during 1 min, and then decreasing linearly to 5% of A during 4 min. This condition is hold for 3 min. Finally, A% increases linearly until 10 min, returning to 98%, and this condition is kept during 2 min, with a total run time of 12 min. Between each analysis, 3 min of equilibration time is applied, using the initial gradient conditions (98% A). Typical retention times for each analyte were described in Table 2. Electrospray ionization (ESI) in positive mode was used for MS analysis of targeted antibiotics. Data acquisition was performed in multiple reaction monitoring (MRM) mode to obtain sufficient quantification points to confirmation of each analyte. Mass spectrometry parameters were optimized by infusion of compounds, in a concentration of 250 ppb for each one, via syringe pump at a flow rate of 10 mL min 1, in mobile phase (component A:component B, 50:50). After identification of more abundant fragments ions for all compounds, as well as the ionization parameters for each particular transition, MRM chromatograms were obtained, indicating the retention order for the selected compounds. Flow injection analysis (FIA) was then performed for all compounds, to optimize the conditions of ion source in the mass spectrometer: source temperature at 550 °C, curtain gas (CUR) at 20 psi, ion spray voltage (IS) at 5500 V, ion source gas 1 (GS1) at 55 psi and gas 2 (GS2) at 45 psi, interface heater on, collision gas (CAD) 4 mTorr, entrance potential (EP) at 10 V and dwell time were 100 ms. All data were processed by software Analyst version 1.4.2 (AB Sciex). MRM conditions, retention time and optimal declustering
Fig. 2. Extracted chromatograms for each analyte at the MRL concentration level in bovine milk (1–7) and a total ion chromatogram of all analytes (8).
L. Jank et al. / Talanta 144 (2015) 686–695
691
Table 4 Summarized validation parameters for macrolides and lincosamides analysis in bovine milk. Analyte
LOD (mg L 1)
LOQ (mg L 1)
Repeatability (CV, %)
Reproducibility (CV, %)
Accuracy (%)
Recovery (%)
CCα (mg L 1)
CCβ (mg L 1)
AZT ERT SPR TLM TYL CLN LNC
12.5 5.0 25.0 6.2 6.2 18.7 18.7
25.0 10.0 50.0 12.5 12.5 37.5 37.5
7.9 12.8 8.7 1.7 14.4 6.8 0.5
12.2 16.3 12.2 10.2 18.9 10.2 9.8
104 92 101 101 91 104 101
60 66 67 73 71 68 60
119.6 50.2 236.0 61.2 64.7 178.9 184.3
139.1 60.5 272.1 72.3 79.4 207.7 218.3
potential (DP), collision cell exit potential (CXP) and collision energies (CE) in the MS/MS mode are demonstrated in Table 2, as well as typical product ions generated under these conditions.
Table 6 Decision limit (CCα) and detection capability (CCβ) for poultry and swine muscle. Analyte
2.4. Maximum limit residue and adopted validation level
Poultry CCα (mg kg
For macrolides and lincosamides, MRL values set by European Community, Brazil and Codex Alimentarius showed several differences [7,8]. Currently, Brazil has only a MRL value set for SPR in bovine muscle, since the monitoring of macrolides and lincosamides is performed using kidney as target tissue [8]. Considering that European Union recently approve the recognizon of the Brazilian NRCP, for the purposes of the present study, MRLs values set by EU were adopted as validation levels [42]. However, for those analytes without a MRL definition, as AZT and CLN, a validation level based on the lowest concentration level with satisfactory performance has been adopted. Moreover, for ERT and SPR, validation levels below the MRL were chosen, to harmonize the concentrations levels with the other analytes. Table 3 shows the MRLs and validation levels that have been selected to carry out the validation. 2.5. Validation procedure Method validation was carried out following Commission Decision 2002/657/EC [53] requirements for veterinary drugs residues methods and the Brazilian manual of analytical quality assurance for residues control [43,44]. Limit of detection (LOD), limit of quantification (LOQ), CCα, CCβ, specificity, linearity, accuracy, recovery, repeatability and reproducibility were evaluated. Complete validation sets were proceeded to bovine milk and muscle samples; for swine and chicken muscle, scope extension and performance evaluation were carried out, based on the procedures proposed by Hoff et al. [45]. Validation procedures for bovine milk and bovine muscle included the analysis of 21 blank samples spiked in 3 different concentration level (corresponding to 50%, 100% and 150% of MRL), along with a six-point calibration curve (which include 0, 25, 50, 100, 150 and 200% of MRL), 3 “tissue standards” (extracts of blank samples to which was added an amount of standard solution to obtain a concentration at MRL), blank samples and a calibration curve prepared in pure solvent. This procedure was repeated thrice
AZT ERT SPR TLM TYL CLN LNC
Swine 1
)
61.8 120.5 137.5 59.9 123.6 62.3 123.5
CCβ (mg kg
1
73.5 141.0 175.3 69.9 147.2 74.7 146.9
)
CCα (mg kg 1)
CCβ (mg kg 1)
56.9 11.9 135.5 62.5 116.8 58.2 122.3
63.7 123.9 171.0 74.9 133.6 66.3 144.6
times, in 3 different days. Moreover, 20 different blank samples were analyzed to evaluate specificity. Tissue standards are samples in which the spike procedure is performed after the whole extraction procedure, i.e., a blank tissue extract fortified with the standards, whereas regular blank samples are always spiked before the application of extraction techniques. In order to include swine and chicken muscle as additional matrices, the scope extension validation was carried out using 1 batch per matrix. Each batch was composed by 21 blank samples spiked in MRL concentration for each analyte, 2 calibrations curves (one of them prepared with matrix-matched method and the other prepared in pure solvent), 3 “tissue standards” and a blank sample, besides 20 blank samples used to evaluate the specificity/ selectivity. 2.6. Matrix effect Matrix effects (ME) were estimated by the comparison between a curve prepared using matrix-matched approach and one prepared in pure solvent, as previously describe elsewhere [46–48]. ME was evaluated using slope ratios comparison according to the approach proposed by Romero-Gonzáles et al. and Sulyok et al. in a modified application of the quantitative approach of Matuszewski et al [49–51]. Slopes are compared between each pair of curves obtained in the linear calibration curves prepared by spiking mobile phase (S), blank sample (R), and extract of blank sample (TS). Slope ratios below 0.9 or above 1.1 were associated with ion suppression and ion enhancement, respectively. For
Table 5 Summarized validation parameters for macrolides and lincosamides analysis in bovine muscle. Analyte
LOD (mg kg 1)
LOQ (mg kg 1)
Repeatability (CV,%)
Reproducibility (CV, %)
Accuracy (%)
Recovery (%)
CCα (mg kg 1)
CCβ (mg kg 1)
AZT ERT SPR TLM TYL CLN LNC
6.2 12.5 12.5 6.2 12.5 6.2 12.5
12.5 25.0 25.0 12.5 25.0 12.5 25.0
15.0 3.9 5.8 2.3 8.8 4.8 7.3
19.1 7.5 12.5 8.4 11.5 9.4 10.6
109 106 110 103 110 104 65
69 80 79 73 83 74 107
66.4 114.0 120.9 57.9 124.7 60.8 117.0
82.8 127.9 141.9 65.7 149.5 71.6 134.0
692
L. Jank et al. / Talanta 144 (2015) 686–695
values inside that range, ME was considered negligible. Comparison was performed trough the evaluation of calibration curves slope ratio (RS¼1.070.1). To muscle, 3 distinct species were validated (bovine, swine and poultry). To evaluate a possible matrix effect provoked by differences between tissues, a matrix-matched calibration curve plus blank samples (n ¼6) for each species were spiked at MRL level [45,48]. Calculated concentrations for each sample set were compared trough variance analysis.
sensitivity. As stationary phase, two columns were tested: a Zorbax Eclipse XDB-C18 (Agilent, 150 mm 4.6 mm, 5 μm) and an Agella Durashell RP (100 mm 2.1 mm, 5 μm), which generated the best result, once it presented a better separation with less time of analysis. Final chromatographic method has a total time of 12 min, which is interesting for routine analysis, since more samples can be analyzed in a short period. Chromatograms of a sample of bovine milk fortified in MRL level concentration for each analyte are shown in Fig. 2.
3. Results and discussion 3.3. Validation procedure 3.1. Sample extraction To promote a more reproducible milk protein precipitation, the chosen extraction solvent (ACN) has been added in 3 aliquots, corresponding to 2.0, 1.0 and 1.0 mL, and the samples have been shake between each ACN addition. This simple procedure was much more efficient to promote protein precipitation than the addition of the whole solvent volume in just one or two aliquots. In the case of muscle samples, a mechanical homogenizer (Ultra Turrax) was employed to promote an efficient tissue disruption. In addition to liquid–liquid extraction (LLE), many authors adopted a clean-up step such as solid phase extraction (SPE) [37– 39] to remove other impurities that are also extracted by organic solvent. To reduce costs and time of analysis, the present method was developed aiming the use of LLE without additional clean-up steps. Notwithstanding, the introduction of contaminants from sample into chromatographic system could damage chromatographic column and contaminate mass spectrometry system, interfering in chromatographic response. To avoid the excessive presence of sample co-extractives using just LLE several strategies can be applied. Herein, the use of small injection volumes has been optimized. The injection of small volumes of sample is a cheap and simple technique that could minimize matrix effects and co-extractives interference [52]. In order to obtain a compromise between the better response of analytes and the minimum matrix effects, 3 distinct injection volumes were evaluated: 2.0, 5.0 and 10.0 mL. The coefficient of variation (CV) for different volumes, analytes and matrices were determined (n ¼7). With exception of TLM, which presented a slightly better result with 5.0 mL of sample injection, all compounds presented the minor CV and a satisfactory response when 2.0 mL of extract was injected, as can be observed in Fig. 1. After LLE followed by centrifugation, the obtained organic extract was very limpid and adequate to be directly injected in LC– MS/MS system. The sample preparation method has showed high efficiency to extract the target compounds with the advantages of be fast and environment-friendly, once generate less solvent residues than other methods that use SPE. The sample preparation method was successfully applied to muscle and milk, generating sufficiently clear extract to MS analysis, without the need of very frequently reported analytical steps as filtration, evaporation, fat removal, or more complex techniques as SPE [33,35].
Validation procedure was carried out according to EU Commission Decision 2002/657/EC requirements and Brazilian validation guidelines [43,44]. Parameters considered more significant are described as follows. All parameters are summarized in Table 4. 3.4. Limit of detection (LOD) and limit of quantification (LOQ) Determination of lowest concentration detectable (LOD), as required by guidelines for implementation of EU Decision and Brazil legislation [43,44], were performed constructing calibration curves with lower concentrations than those used in previous tests. These lower concentrations corresponded to 0.06, 0.12 and 0.18 MRL. All samples have been correctly detected even at lower point evaluated. LOQ was defined as the lower point of a “normal” calibration curve, i.e. 25% of the MRL for each compound in each matrix. LOD and LOQ values are presented in Table 4. 3.5. Decision limit (CCα) and detection capability (CCβ) The decision limit (CCα) and the detection capability (CCβ) were calculated plotting all data obtained from the precision determination and applying the calibration curves approach as described in Commission Decision 2002/657/EC and also in conformity with the ISO 11843 [44]. Briefly, the signal was plotted against the added concentration and the corresponding concentration at the y intercept plus 1.64 times the standard deviation of the within-laboratory reproducibility gave the CCα values.CCβ was calculated by summing of the concentration at the CCα and 1.64 times the standard deviation of the within-reproducibility of the mean measured content at the MRL concentration level. Tables 4, 5 and 6 show CCα and CCβ values for all matrices. Although these parameters do not present criteria for upper limits, determined values were considered satisfactory. 3.6. Specificity Blank samples (n¼ 20) of each matrix (bovine milk; bovine, swine and poultry muscle) have been analyzed to evaluate presence of interference. None significant interference has been observed for any analyte. Thus, the method was considered as specific for the target compounds.
3.2. LC–MS/MS
3.7. Accuracy, linearity, repeatability and reproducibility
Injection of individual standard solutions and mixtures of the standards were performed for optimization of various MS/MS conditions. Conditions for fragmentation of the monitored ions were obtained at an ESI source temperature of 300 °C and at flow injection of 10 μL min 1. The six compounds investigated showed response in the positive mode (ESI þ). To perform simultaneous analysis of all compounds, positive ESI mode was chosen. Formic acid 0.1% was the chosen additive to enhance peak resolution and
Accuracy was determined by the comparison between calculated concentrations for spiked samples at MRL level (n ¼21) and the assigned value. To the evaluated levels, accuracy values are acceptable inside the range 80–110%. The only exception was the accuracy value for LNC in bovine muscle (65%). Accuracy values are also included in Tables 4 and 5. To evaluate the method linearity, our laboratory adopted an internal criteria specific for calibration curves that was prepared in
L. Jank et al. / Talanta 144 (2015) 686–695
matrix (r2 4 0.95). All assays for linearity shows values of r2 above 0.98 and adequate linearity inside the range corresponding to 0.25 MRL–3.0 MRL for all analytes. When non-compliant samples containing analytes which the adopted validation level was lower than the MRL, matrix-matched calibration curves must be prepared taking in account the dilution factor adequate for fit the concentration levels inside that linearity range. Repeatability was evaluated in terms of intra-day and inter-day precision. Intra-day precision was determined by the comparison of calculated concentration for spiked samples (n ¼7) in 3 distinct levels (0.5, 1.0 and 1.5 MRL) for each matrix and each species. By variance analysis (ANOVA) of each dataset, coefficients of variation (CV) were obtained. To the evaluated levels, these coefficients must be lower than 16%. As all CV values were below then criteria, method repeatability was considered as adequate. For inter-day precision determination, each dataset was repeated in 3 different days, obtaining a total of 21 measurements for each analyte (7 3). Repeatability levels for the concentration level corresponding to MRL for any analyte in the matrices milk and bovine muscle are showed in Tables 4 and 5. Reproducibility was calculated by the analysis of more 2 complementary and independent datasets, performed by different analysts, in 2 different days. The method was considered reproducible for the matrices milk and muscle since all CV values were below 23%, the upper limit (for the MRL level) which was calculated using Horwittz equation. Results are given in Tables 4 and 5.
693
two lincosamides antibiotics in bovine milk and bovine, swine and chicken muscle. Almost all currently methodologies for macrolides and lincosamides antibiotics residues analysis are multiclass methods and generally need a SPE as clean-up procedure followed in some cases for additional clean-up steps (defatting, filtration, etc.). The development of a LLE sample preparation method, avoiding the use of SPE cartridges, is of great value, since it becomes cheaper and faster, factors that play a major role on deciding what method should be adopted for routine analysis. Moreover, this extraction method can be applied at low complexity laboratories. Validation was performed observing regulatory criteria and demonstrates satisfactory results for all parameters. The present method is able to use in Brazilian National Residue Control Plan as a versatile analytical tool to monitoring and determine the occurrence of lincosamides and macrolides residues in food matrices.
Acknowledgments The authors would like to thank the Coordination for the Improvement of Higher Education Personnel (CAPES) for the fellowships provided for Louise Jank and Juliana Bazzan Arsand, and to National Council for Scientific and Technological Development (CNPq) for the fellowship provided to Magda Targa Martins.
References 3.8. Matrix effects As described above, the slopes ratio (RS) was used to estimate the presence of matrix effects wherein RS ¼1.0 70.1 was adopted as criteria [48–51]. To milk analysis, ERT and TYL showed RS values nearby 1.0, but all other analytes expressed a significant difference. In the case of muscle, the matrix effect evaluation was carried out using bovine muscle as study case: TYL, LNC and AZT demonstrate a complete overlapping of calibration curves. However, for SPR, ERT, TLM and CLN the presence of matrix effects was detected by RS values out of range. Generally, the overall cause for ME is the occurrence of unexpected compounds that co-elute in the chromatographic separation and affect the analytes ionization. Frequently, these interfering species are originated from the sample itself, and are coextracted in the sample preparation techniques [54]. In the present study, considering the low complexity of the sample extraction procedure, has been expected the presence of endogenous compounds in the final extract. Thus, for both muscle and milk samples, the conclusion of the matrix effect evaluation is that the use of calibration curves prepared in matrix is mandatory. To evaluate the matrix effect in poultry and swine muscle, batches of blank samples spiked at MRL or VL level (n ¼ 6) were analyzed and calculated using a matrix-matched calibration curve prepared using a blank bovine muscle sample. By variance analysis, the coefficient of variation was determined for each analyte. The upper limit of 23% for CV was adopted as criteria for acceptance. All CV values were below than the upper limit. Thus, as poultry and swine muscle samples could be correctly analyzed, i.e. with CV values equal or lower than those observed in reproducibility study, a calibration curve prepared using one species can be used to analyze samples from other species.
4. Conclusion The developed method showed high sensitivity, satisfactory results for identification and quantification of five macrolides and
[1] Y. Tao, G. Yu, D. Chen, Y. Pan, Z. Liu, H. Wei, et al., Determination of 17 macrolide antibiotics and avermectins residues in meat with accelerated solvent extraction by liquid chromatography-tandem mass spectrometry, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 897 (2012) 64–71, http://dx.doi.org/ 10.1016/j.jchromb.2012.04.011. [2] M. Sismotto, J.A.R. Paschoal, F.G.R. Reyes, Analytical and regulatory aspects for determination of macrolide residues in animal-derived foods by liquid chromatography associated with mass spectrometry, Quim. Nova 36 (2013) 449–461, http://dx.doi.org/10.1590/S0100-40422013000300016. [3] W.-R. Chen, Y. Ding, C.T. Johnston, B.J. Teppen, S.A. Boyd, H. Li, Reaction of lincosamide antibiotics with manganese oxide in aqueous solution, Environ. Sci. Technol. 44 (2010) 4486–4492, http://dx.doi.org/10.1021/es1000598. [4] S. Pyörälä, K.E. Baptiste, B. Catry, E. van Duijkeren, C. Greko, M.A. Moreno, et al., Macrolides and lincosamides in cattle and pigs: use and development of antimicrobial resistance, Vet. J. 200 (2014) 230–239, http://dx.doi.org/10.1016/ j.tvjl.2014.02.028. [5] P. Struciński, K. Góralczyk, K. Czaja, A. Hernik, W. Korcz, J.K. Ludwicki, Consumer risk assessment in case of maximum residue levels (MRLs) violations in food, Rocz. Państw. Zakładu Hig. 58 (2007) 377–388. [6] I. Pecorelli, R. Bibi, L. Fioroni, A. Piersanti, R. Galarini, Sulfonamides residues analysis: evaluation of results dispersion at maximum residual limit by the expanded uncertainty and 2002/657/EC decision limit approaches, Anal. Chim. Acta 529 (2005) 15–20, http://dx.doi.org/10.1016/j.aca.2004.08.039. [7] European Commission, Commission regulation (EU) No. 37/2010 of 22 December 2009: on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal orig, Off. J. Eur. Union (2010) 1–72. [8] Ministério da Agricultura, Pecuária e Abastecimento, Instrução Normativa SDA n° 11, Diário Of. União, 2014, p. 5. [9] A. de Queiroz Mauricio, E.S. Lins, The National Agricultural Laboratories of Brazil and the control of residues and contaminants in food, Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 29 (2012) 482–489, http://dx.doi.org/10.1080/19440049.2011.620987. [10] A. d Q. Mauricio, E.S. Lins, M.B. Alvarenga, A National Residue Control Plan from the analytical perspective – the Brazilian case, Anal. Chim. Acta 637 (2009) 333–336, http://dx.doi.org/10.1016/j.aca.2008.09.061. [11] K. Bousova, H. Senyuva, K. Mittendorf, Multiresidue automated turbulent flow online LC–MS/MS method for the determination of antibiotics in milk, Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 29 (2012) 1901–1912, http://dx.doi.org/10.1080/19440049.2012.726744. [12] S.B. Turnipseed, J.M. Storey, S.B. Clark, K.E. Miller, Analysis of veterinary drugs and metabolites in milk using quadrupole time-of-flight liquid chromatography–mass spectrometry, J. Agric. Food Chem. 59 (2011) 7569–7581, http: //dx.doi.org/10.1021/jf103808t. [13] D.A. Bohm, C.S. Stachel, P. Gowik, Multi-method for the determination of antibiotics of different substance groups in milk and validation in accordance with Commission Decision 2002/657/EC, J. Chromatogr. A 1216 (2009) 8217–8223, http://dx.doi.org/10.1016/j.chroma.2009.06.058.
694
L. Jank et al. / Talanta 144 (2015) 686–695
[14] M. Gaugain-Juhel, B. Delépine, S. Gautier, M.P. Fourmond, V. Gaudin, D. Hurtaud-Pessel, et al., Validation of a liquid chromatography–tandem mass spectrometry screening method to monitor 58 antibiotics in milk: a qualitative approach, Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 26 (2009) 1459–1471, http://dx.doi.org/10.1080/02652030903150575. [15] D. Hurtaud-Pessel, T. Jagadeshwar-Reddy, E. Verdon, Development of a new screening method for the detection of antibiotic residues in muscle tissues using liquid chromatography and high resolution mass spectrometry with a LC-LTQ-Orbitrap instrument, Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 28 (2011) 1340–1351, http://dx.doi.org/10.1080/ 19440049.2011.605772. [16] H. Berrada, J.C. Moltó, J. Mañes, G. Font, Determination of aminoglycoside and macrolide antibiotics in meat by pressurized liquid extraction and LC–ESI–MS, J. Sep. Sci. 33 (2010) 522–529, http://dx.doi.org/10.1002/jssc.200900682. [17] K. Granelli, C. Elgerud, A. Lundström, A. Ohlsson, P. Sjöberg, Rapid multi-residue analysis of antibiotics in muscle by liquid chromatography–tandem mass spectrometry, Anal. Chim. Acta 637 (2009) 87–91, http://dx.doi.org/10.1016/j. aca.2008.08.025. [18] R.P. Lopes, D.V. Augusti, A.G.M. Oliveira, F.A.S. Oliveira, E.A. Vargas, R. Augusti, Development and validation of a methodology to qualitatively screening veterinary drugs in porcine muscle via an innovative extraction/clean-up procedure and LC-MS/MS analysis, Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 28 (2011) 1667–1676, http://dx.doi.org/10.1080/ 19440049.2011.609136. [19] S. Biselli, U. Schwalb, A. Meyer, L. Hartig, A multi-class, multi-analyte method for routine analysis of 84 veterinary drugs in chicken muscle using simple extraction and LC–MS/MS, Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 30 (2013) 921–939, http://dx.doi.org/10.1080/ 19440049.2013.777976. [20] V. Carretero, C. Blasco, Y. Picó, Multi-class determination of antimicrobials in meat by pressurized liquid extraction and liquid chromatography–tandem mass spectrometry, J. Chromatogr. A 1209 (2008) 162–173, http://dx.doi.org/ 10.1016/j.chroma.2008.09.011. [21] Z.-F. Yue, X.-X. Chen, L.-Q. Xie, C.-N. Ji, H.-H. Hua, Determination of residues of lincosamides and macrolides in animal tissue by high performance liquid chromatography tandem electrospray mass spectrometry, Fenxi Huaxue Chin. J. Anal. Chem. 35 (2007) 1290–1294. [22] D.A. Bohm, C.S. Stachel, P. Gowik, Validation of a multi-residue method for the determination of several antibiotic groups in honey by LC–MS/MS, Anal. Bioanal. Chem. 403 (2012) 2943–2953, http://dx.doi.org/10.1007/ s00216-012-5868-z. [23] M.I. Lopez, J.S. Pettis, I.B. Smith, P.-S. Chu, Multiclass determination and confirmation of antibiotic residues in honey using LC–MS/MS, J. Agric. Food Chem. 56 (2008) 1553–1559, http://dx.doi.org/10.1021/jf073236w. [24] B.F. Spisso, R.G. Ferreira, M.U. Pereira, M.A. Monteiro, T.T. Cruz, R.P. Da Costa, et al., Simultaneous determination of polyether ionophores, macrolides and lincosamides in hen eggs by liquid chromatography–electrospray ionization tandem mass spectrometry using a simple solvent extraction, Anal. Chim. Acta 682 (2010) 82–92, http://dx.doi.org/10.1016/j.aca.2010.09.047. [25] M. Gbylik, A. Posyniak, K. Mitrowska, T. Bladek, J. Zmudzki, Multi-residue determination of antibiotics in fish by liquid chromatography-tandem mass spectrometry, Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 30 (2013) 940–948, http://dx.doi.org/10.1080/19440049.2013.780210. [26] C.P. Rezende, L.F. Souza, M.P. Almeida, P.G. Dias, M.H. Diniz, J.C. Garcia, Optimisation and validation of a quantitative and confirmatory method for residues of macrolide antibiotics and lincomycin in kidney by liquid chromatography coupled to mass spectrometry, Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 29 (2012) 587–595, http://dx.doi.org/10.1080/ 19440049.2011.652196. [27] L.C. Dickson, C.I. O’Byrne, W. Chan, A quantitative method for residues of macrolide antibiotics in porcine kidney by liquid chromatography/Tandem mass spectrometry, J. AOAC Int. 95 (2012) 567–575, http://dx.doi.org/10.5740/ jaoacint.11-139. [28] W. Xie, C. Han, J. Hou, F. Wang, Y. Qian, J. Xi, Simultaneous determination of multiveterinary drug residues in pork meat by liquid chromatography–tandem mass spectrometry combined with solid phase extraction, J. Sep. Sci. 35 (2012) 3447–3454, http://dx.doi.org/10.1002/jssc.201200407. [29] S. Smith, C. Gieseker, R. Reimschuessel, C.-S. Decker, M.C. Carson, Simultaneous screening and confirmation of multiple classes of drug residues in fish by liquid chromatography-ion trap mass spectrometry, J. Chromatogr. A 1216 (2009) 8224–8232, http://dx.doi.org/10.1016/j.chroma.2009.06.077. [30] A. Boscher, C. Guignard, T. Pellet, L. Hoffmann, T. Bohn, Development of a multi-class method for the quantification of veterinary drug residues in feedingstuffs by liquid chromatography–tandem mass spectrometry, J. Chromatogr. A 1217 (2010) 6394–6404, http://dx.doi.org/10.1016/j. chroma.2010.08.024. [31] C. Chiaochan, U. Koesukwiwat, S. Yudthavorasit, N. Leepipatpiboon, Efficient hydrophilic interaction liquid chromatography–tandem mass spectrometry for the multiclass analysis of veterinary drugs in chicken muscle, Anal. Chim. Acta 682 (2010) 117–129, http://dx.doi.org/10.1016/j.aca.2010.09.048. [32] P.A. Martos, S.J. Lehotay, B. Shurmer, Ultratrace analysis of nine macrolides, including tulathromycin A (Draxxin), in edible animal tissues with minicolumn liquid chromatography tandem mass spectrometry, J. Agric. Food Chem. 56 (2008) 8844–8850, http://dx.doi.org/10.1021/jf801747u. [33] H. Berrada, F. Borrull, G. Font, J.C. Moltó, R.M. Marcé, Validation of a confirmatory method for the determination of macrolides in liver and kidney
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43] [44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
animal tissues in accordance with the European Union regulation 2002/657/ EC, J. Chromatogr. A 1157 (2007) 281–288, http://dx.doi.org/10.1016/j. chroma.2007.05.021. A. Freitas, J. Barbosa, F. Ramos, Development and validation of a multi-residue and multiclass ultra-high-pressure liquid chromatography–tandem mass spectrometry screening of antibiotics in milk, Int. Dairy J. 33 (2013) 38–43, http://dx.doi.org/10.1016/j.idairyj.2013.05.019. A. Freitas, J. Barbosa, F. Ramos, Multi-residue and multi-class method for the determination of antibiotics in bovine muscle by ultra-high-performance liquid chromatography tandem mass spectrometry, Meat Sci. 98 (2014) 58–64, http://dx.doi.org/10.1016/j.meatsci.2014.04.003. L.C. Dickson, Performance characterization of a quantitative liquid chromatography–tandem mass spectrometric method for 12 macrolide and lincosamide antibiotics in salmon, shrimp and tilapia, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 967 (2014) 203–210, http://dx.doi.org/10.1016/j. jchromb.2014.07.031. J. Wang, D. Leung, F. Butterworth, Determination of five macrolide antibiotic residues in eggs using liquid chromatography/electrospray ionization tandem mass spectrometry, J. Agric. Food Chem. 53 (2005) 1857–1865, http://dx.doi. org/10.1021/jf048414p. J. Wang, D. Leung, S.P. Lenz, Determination of five macrolide antibiotic residues in raw milk using liquid chromatography–electrospray ionization tandem mass spectrometry, J. Agric. Food Chem. 54 (2006) 2873–2880, http://dx. doi.org/10.1021/jf060068j. J. Wang, D. Leung, Analyses of macrolide antibiotic residues in eggs, raw milk, and honey using both ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry and high-performance liquid chromatography/tandem mass spectrometry, Rapid Commun. Mass Spectrom. 21 (2007) 3213–3222, http://dx.doi.org/10.1002/rcm.3207. M. Gbylik-Sikorska, A. Posyniak, T. Sniegocki, J. Zmudzki, Liquid chromatography–tandem mass spectrometry multiclass method for the determination of antibiotics residues in water samples from water supply systems in foodproducing animal farms, Chemosphere 119 (2015) 8–15, http://dx.doi.org/ 10.1016/j.chemosphere.2014.04.105. K. Bousova, H. Senyuva, K. Mittendorf, Quantitative multi-residue method for determination antibiotics in chicken meat using turbulent flow chromatography coupled to liquid chromatography–tandem mass spectrometry, J. Chromatogr. A 1274 (2013) 19–27, http://dx.doi.org/10.1016/j. chroma.2012.11.067. European Commission, 2014/891/EU: Commission implementing decision of 8 December 2014 amending decision 2011/163/EU on the approval of plans submitted by third countries in accordance with article 29 of council directive 96/23/EC, Off. J. Eur. Union (2014) 17–22. Brasil. Ministério da Agricultura Pecuária e Abastecimento, Manual de garantia da qualidade analítica, 1st ed., MAPA/ACS, Brasília, 2011. European Commission, Decision (2002/657/EC) of 12 August 2002 implementing council directive 96/23/EC concerning the performance of analytical methods and interpretation of results, Off. J. Eur. Communities (2002) 8–36. R.B. Hoff, F. Barreto, J. Melo, M.T. Martins, T.M. Pizzolato, M. do Carmo Ruaro Peralba, Scope extension validation protocol: inclusion of analytes and matrices in an LC–MS/MS sulfonamide residues method, Food Addit Contam Part A Chem Anal Control Expo Risk Assess 31 (2014) 39–47, http://dx.doi.org/ 10.1080/19440049.2013.861082 (131106185253008). L. Jank, R.B. Hoff, F.J. da Costa, T.M. Pizzolato, Simultaneous determination of eight antibiotics from distinct classes in surface and wastewater samples by solid-phase extraction and high-performance liquid chromatography-electrospray ionisation mass spectrometry, Int. J. Environ. Anal. Chem. 94 (2014) 1013–1037, http://dx.doi.org/10.1080/03067319.2014.914184. G. Rübensam, F. Barreto, R.B. Hoff, T.M. Pizzolato, Determination of avermectin and milbemycin residues in bovine muscle by liquid chromatography-tandem mass spectrometry and fluorescence detection using solvent extraction and low temperature cleanup, Food Control 29 (2013) 55–60, http://dx.doi.org/ 10.1016/j.foodcont.2012.05.075. R.B. Hoff, G. Rübensam, L. Jank, F. Barreto, M. do, C. Ruaro Peralba, T. M. Pizzolato, et al., Analytical quality assurance in veterinary drug residue analysis methods: matrix effects determination and monitoring for sulfonamides analysis, Talanta 132 (2015) 443–450, http://dx.doi.org/10.1016/j. talanta.2014.08.046. R. Romero-González, A. Garrido Frenich, J.L. Martínez Vidal, O.D. Prestes, S. L. Grio, Simultaneous determination of pesticides, biopesticides and mycotoxins in organic products applying a quick, easy, cheap, effective, rugged and safe extraction procedure and ultra-high performance liquid chromatography–tandem mass spectrometry, J. Chromatogr. A 1218 (2011) 1477–1485, http://dx.doi.org/10.1016/j.chroma.2011.01.034. M. Sulyok, R. Krska, R. Schuhmacher, A liquid chromatography/tandem mass spectrometric multi-mycotoxin method for the quantification of 87 analytes and its application to semi-quantitative screening of moldy food samples, Anal. Bioanal. Chem. 389 (2007) 1505–1523, http://dx.doi.org/10.1007/ s00216-007-1542-2. B.K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng, Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS, Anal. Chem. 75 (2003) 3019–3030, http://dx.doi.org/10.1021/ ac020361s. K. Deventer, O.J. Pozo, A.G. Verstraete, P. Van Eenoo, Dilute-and-shoot-liquid chromatography–mass spectrometry for urine analysis in doping control and
L. Jank et al. / Talanta 144 (2015) 686–695 analytical toxicology, TrAC – Trends Anal. Chem. 55 (2014) 1–13, http://dx.doi. org/10.1016/j.trac.2013.10.012. [53] Commission of the European Communities, Commission Decision 2002/657/ EC, 2002. [54] F. Gosetti, E. Mazzucco, D. Zampieri, M.C. Gennaro, Signal suppression/
enhancement in high-performance liquid chromatography tandem mass spectrometry, J. Chromatogr. A 1217 (2010) 3929–3937, http://dx.doi.org/ 10.1016/j.chroma.2009.11.060.
695
