Simultaneous stereoselective determination of seven β-agonists in pork meat samples by ultra-performance liquid chromatography-tandem mass spectrometry

Microchemical Journal 150 (2019) 104082

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Simultaneous stereoselective determination of seven β-agonists in pork meat samples by ultra-performance liquid chromatography-tandem mass spectrometry

T

Bolin Zhu, Liangzhao Cai, Zhen Jiang, Qing Li , Xingjie Guo ⁎

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School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road Shenhe District, 110016 Shenyang, Liaoning Province, PR China

ARTICLE INFO

ABSTRACT

Keywords: β-Agonist Chiral separation Enantiomeric determination Pork muscles UPLC-MS/MS

This paper presents the stereoselective multi-residue analysis of seven β-agonists including salbutamol, clorprenaline, terbutaline, tulobuterol, metaproterenol, cimaterol and formoterol in pork muscles by chiral UPLCMS/MS. The simultaneous enantioseparation of seven analytes was accomplished on a teicoplanin aglyconebased Astec Chirobiotic TAG column under isocratic condition at a flow rate of 0.6 mL min−1. The effects of the mobile phase composition, the type and content of the organic modifier, and the ratio and content of the additive on the retention and resolution of the enantiomers were investigated and optimized. To obtain the best extraction yields, the analytes were extracted from meat muscles by enzymatic hydrolysis, and then cleaned up using Cleanert PCX solid phase extraction cartridge. Then, the proposed method was validated in terms of selectivity, sensitivity, linearity, accuracy and precision, matrix effect, stability. Quantification of the analytes was achieved using matrix-matched standard calibration curve, providing a liner correlation over the concentration range of 0.1–50 ng g−1 for each enantiomer. The limits of detection (LODs) and quantification (LOQs) were in the range of 0.014–0.033 ng g−1 and 0.035–0.070 ng g−1, respectively. The method recoveries were within 84.9–106.9%, while the matrix effects were in the range of 84.3–108.3%. The intra- and inter-precision (expressed as RSD%) were found to be < 11.2%, which confirmed the precision of this method. Taking into consideration obtained values of validation parameters, the method seems to be suitable for routine analysis of the target seven β-agonists in pork meats by UPLC-MS/MS.

1. Introduction β-Agonists (β-adrenomimetics) are therapeutically administered to farm animals as bronchodilatants and tocolitiic agents to relax the smooth muscle cells. Apart from the clinical use and therapeutic value, β-agonists are also well-known as efficient partitioning agents illicitly used in veterinary framework as growth promoters [1]. β-Agonists administrated in amounts above the recommended therapeutic dose would be capable of reducing the body fat and enhancing growth of cattle, swine and sheep. Nevertheless, these compounds are easily left behind in animal bodies, which are a potential danger to consumer. The consumption of contaminated beef or pork meats has led to serious human poisoning cases. As a result, both China and European Union (EU) have banned the use of β-agonists as feed additives for growth promotion. As reported by the EU, residues of these prohibited β-agonists in food animal products constitute a risk for consumer health at any level of concentration, and consequently these substances do not

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require maximum residue limits (MRL) values [2]. Although the use of β-agonists in food-producing animals is banned, some feed manufactures and farmers still use these compounds for more profit. To protect consumers, there is a growing demand for the simultaneous identification and quantification of multiple β-agonists in a single run. For determination of β-agonists in biological and environmental matrices, many liquid chromatography (LC) [3], gas chromatography–mass spectrometry (GC–MS) [4,5], liquid chromatographymass spectrometry (LC-MS and LC-MS/MS) [6–13], and capillary electrophoresis (CE) [14] methods have been developed. Among them, LC-MS/MS has become the main technique for identifying β-agonists because of its high sensitivity, greater selectivity and its lack of timeconsuming derivatisation procedure (compared to GC–MS/MS-based techniques). Many papers based on LC-MS/MS method have been published for the determination of β-agonists in different samples, including urine [6,8,10], feeds [13], sewage [11], plasma [15,16] and animal tissue [7,8,10,12,17]. These studies focused their attentions on

Corresponding authors. E-mail addresses: [email protected] (Q. Li), [email protected] (X. Guo).

https://doi.org/10.1016/j.microc.2019.104082 Received 10 April 2019; Received in revised form 11 June 2019; Accepted 8 July 2019 Available online 09 July 2019 0026-265X/ © 2019 Elsevier B.V. All rights reserved.

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the determination of racemic β-agonists, but to data, only a few articles described the stereoselective determination of the enantiomers of βagonists in different matrices [3,11]. More important, no literature has reported the stereoselective determination of chiral β-agonists in pork meat, which is of great significance in risk assessment and ensuring customer safety. Chemically, β-agonists are hydroxylamine-containing compounds whose amino groups are always secondary; and their molecules contain at least one aromatic ring. The majority of β-agonists are chiral compounds, and presently administrated as racemic mixtures. Typically, the (R)-forms are often 50 to 10,000-fold more effective than their (S)-enantiomers, and the (S)-isomers do not seem to contribute to the pharmacological effects [3, 18, 19]. The different pharmacological and toxicological effects of the enantiomers of β-agonists have aroused intense research on the chiral separation of such compounds. In HPLC, the most common approach for enantioseparation involves the use of chiral stationary phases (CSPs). Macrocyclic glycopeptide-based CSPs are superior than other CSPs as they can be used under multiple modes (normal phase, reversed phase, polar organic and polar ionic modes). The antibiotic-based CSPs were proven to be efficient for the enantioseparation of β-agonists in different literature reports [3, 11, 16, 19-22]. These studies provided the idea that the enantioseparation of βagonists would be achieved on antibiotic-based CSPs. Therefore, Chirobiotic TAG column was herein adopted for the enantiomeric separation and stereoselective determination of seven β-agonists in pork meats. The developed method could be applied to control the enantiomer occurrence and quantify the chiral contaminants in animal meat products. The aim of this study was to develop a stereoselective multi-residue method for simultaneous identification and quantification of seven βagonists, salbutamol, clorprenaline, terbutaline, tulobuterol, metaproterenol, cimaterol and formoterol in pork meats using chiral UPLC-MS/ MS. For chromatographic separation, the macrocyclic glycopeptidebased Chirobiotic TAG column was used. The chromatographic conditions, including the type and content of organic modifier, the concentration and ratio of acid/base, were investigated. Prior to their determination by chiral UPLC-MS/MS, meat samples were prepared by enzymatic hydrolysis and subsequent solid phase extraction (SPE) using PCX cartridge. To estimate the performance of the method, the validation was undertaken in compliance with CD 2002/657/EC. To the best of our knowledge, this is the first report on the simultaneous enantiomeric quantitative analysis of β-agonists in food animal muscles. Moreover, the use of Chirobiotic TAG column for the simultaneous enantioseparation of seven β-agonists is also the first time.

Germany) was purchased from Merck (Germany). The cartridge used for SPE was Cleanert PCX from Agela Technologies (Tianjin, China). 2.2. Instrumentation The chromatographic analysis was carried out using a Waters Acquity™ UPLC instrument (Waters Co., Milford, MA, USA), consisting of a binary delivery system, an autosampler and a thermostatic column compartment. A Micromass Quattro micro™ API mass spectrometer (Waters Co., Milford, MA, USA) equipped with an electrospray ionization interface was used for detection. The Masslynx 4.11 software (Waters, USA) was used to control the UPLC-MS/MS system. The macrocyclic glycopeptide-based stationary phase used for analytical separation was teicoplanin aglycone-containing Chirobiotic TAG™ column, 150 mm × 4.6 mm I.D., 5 μm particle size (Supelco, PA, USA). 2.3. Standard preparation Individual stock standard solutions of seven β-agonists were prepared in methanol at a concentration of 1.0 mg mL−1 and stored at −20 °C prior to the analysis. Seven individual standard solutions at a concentration of 100 μg mL−1 were prepared by diluting each stock standard solution with methanol. One mixed working standard solutions (10 μg mL−1) was daily prepared by dilution of the stock standard solutions with methanol and subsequently kept at 4 °C. Six serial working standard solutions (at concentrations of 2.0, 10.0, 20.0, 100.0, 200.0 and 1000.0 ng mL−1 for all analytes) were obtained by diluting the mixed working solution (10 μg mL−1) with methanol. To construct the spiked calibration curves, 2.0 g of the blank pork sample were mixed with 100 μL of the serial working standard solutions with the aid of vortex mixed (at concentrations of 0.1, 0.5, 1.0, 5.0, 10.0 and 50 ng g−1 for all analytes). After the addition of the working solutions into the blank meat samples, all spiked samples were left at room temperature for 2 h, after which they were kept at 4 °C for 12 h in the dark. Then, these samples were processed according to the following procedure. 2.4. Sample preparation The sample preparation procedure was proposed according to several methods that have been reported [7,9]. After purchasing, the pork fillets were homogenized using a Tissuemiser (Fisher Scientific Power Gen 123) set to a rotate at 30000 rpm. 2.0 g of the homogenized pork muscle (spiked or non-spiked samples) and 8.0 mL of sodium acetate (0.2 mol L−1, pH 5.2) were added to a 50 mL of polypropylene centrifuge tube and then mixed thoroughly. Whereafter, 30 μL of β-glucoronidase/arysulfatase was added, and enzymatic hydrolysis was performed by incubating the mixture in a thermoregulated bath at 37 °C for 12 h. After being cooled down to room temperature, the mixture was centrifuged for 10 min at 5000 rpm, and 4.0 mL of supernatant were transferred to another centrifuge tube. 5.0 mL of 0.1 mol L−1 perchloric acid was added and the pH was adjusted to 1.0 ± 0.3 using 1.0 mol L−1 perchloric acid. The mixture was vortexed for 1.0 min and centrifuged again for 10 min at 5000 rpm. The supernatant was adjusted to a pH of 11.0 using sodium hydroxide (10 mol L−1). 10.0 mL of saturated sodium chloride solution and 10.0 mL of isopropanol-ethyl acetate (6:4, v/v) solution were added to this mixture, which was then allowed to vortex for 1.0 min and centrifuge for 10 min at 5000 rpm. The organic phase was collected and evaporated to dryness with N2 at 50 °C. Finally, the residue was dissolved in 5 mL of sodium acetate (0.2 mol L−1, pH 5.2) for the following SPE process. For the purpose of preconcentration, SPE was performed using Cleanert PCX, the mixed-mode cartridge. The Cleanert PCX (3 mL, 60 mg) cartridge was sequentially conditioned with 3 mL of methanol and 3 mL of water. After the condition step, the sample was loaded

2. Experimental 2.1. Reagents and materials Salbutamol (99.3%), clorprenaline (hydrochloride, 99.9%), tulobuterol (hydrochloride, 97.0%), terbutaline (sulfate, 99.8%), formoterol (fumarate, 98.0%) were purchased from National Institute for food and Drug Control (Beijing, China). Metaproterenol (hemisulfate, 98.0%) and cimaterol (99.3%) were obtained from Toronto Research Chemicals (Toronto, Canada). All standards were supplied as racemates with minimum purity of 97.0%. The chemical structures of the seven βagonists are shown in Table 1. Ultrapure water was obtained from Jilin Wahaha Foods Co., Ltd. (Jilin, China) and was used in all experiments. Methanol and acetonitrile (both MS grade) were provided by SigmaAldrich (Beijing, China). Ammonium hydroxide (25.0–28.0%), glacial acetic acid (≥99.55%), hydrochloric acid (36.5%), perchloric acid (70.0–72.0%), sodium hydroxide (≥96.0%), sodium chloride (≥99.5%), sodium acetate (≥99.0%) (all analytical grade) were purchased from Tianjin Heng Xing Chemical Reagent Co., Ltd. (Tianjin, China). β-Glucuronidase/arylsulfatase (from Helix pomatia, Merck, 2

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Table 1 Retention time, ion pairs, cone voltage and collision energy of MRM for the optimized LC-MS/MS. Structure

a b c d

Precursor ion

Retention time (t1/t2, min)a

Resolution (Rs)

MRM transitionsb

CV (V)c

CE (eV)d

Salbutamol

[M + H]+

26.10/28.63

1.7

240.05 > 147.87 240.05 > 221.91

25

16 8

Terbutaline

[M + H]+

24.13/27.29

2.5

225.88 > 151.87 225.88 > 124.89

25

10 20

Tulobuterol

[M + H]+

21.45/23.55

2.1

227.84 > 153.87 227.84 > 171.87

34

10 8

Clorprenaline

[M + H]+

23.09/24.83

1.6

213.78 > 153.87 213.78 > 195.91

25

14 8

Formoterol

[M + H]+

38.63/42.24

1.5

345.29 > 148.90 345.29 > 120.92

19

18 26

Cimaterol

[M + H]+

35.03/38.13

1.9

219.75 > 201.96 219.75 > 159.92

8

6 14

Metaproterenol

[M + H]+

26.19/28.56

1.7

212.02 > 193.90 212.02 > 151.95

5

10 14

Compound

t1 and t2 means the retention time of the first and second eluted enantiomers. The quantification ion transitions are underlined. CV means cone voltage; CE means collision energy.

through the cartridge. Then, the cartridge was washed with 2 mL of water, 2 mL of 0.1 N hydrochloric acid, 2 mL of methanol. The cartridge was dried under vacuum for 5 min to remove excess of water. Finally, elution was carried out with 3 mL of 5% ammonium hydroxide in methanol. The effluent was evaporated with N2 at 50 °C and then reconstituted with 200 μL of the mobile phase. The resultant solution was filtered through a 0.22 μm membrane filter and used for UPLC-MS/MS analysis.

2.6. Method validation Method validation was performed referring to the European Commission Decision 2002/657/EC [23]. The validation parameters included selectivity, sensitivity, calibration curve, accuracy and precision, matrix effect and stability. The homogenized blank pork muscles were used as blank matrices in the method validation. These samples were prescreened to confirm that they did not contain the seven target β-agonists and thus used for the method validation. In order to ascertain the absence of interference substances near the retention of target analytes, 20 blank pork meat samples were analyzed. The limits of detection (LODs) are defined as the analyte concentration that could produce the signal-noise ratio (S/N) of three, and determined by spiking serially diluted analytes standards into blank meat samples. The limits of quantitation (LOQs) are the lowest concentration, which could give the S/N of at least ten. The CCɑ (decision limit) is defined as the limit, above which the sample is non-compliant with an error probability of ɑ. The CCβ (detection capability) is defined as the lowest content that may be detected, identified and/or quantified with an error probability of β [23]. According to European Commission Decision 2002/657/EC [23], 20 blank samples were analyzed, and the signal-to-noise at the retention time of target analyte multiplied by three was used as the CCɑ. The CCβ was calculated by analyzing 20 blank pork samples spiked with the seven β-agonists at 0.5 ng g−1. 1.64 times the standard deviation of the within-laboratory reproducibility of the measured content plus CCɑ equaled CCβ. To simultaneously determine seven chiral β-agonists in pork meats, the matrix-matched calibration curves were conducted by spiking mixed standard solutions into blank matrices over the concentration of

2.5. Chromatographic and mass spectrometry conditions The simultaneous chiral separation of seven analytes was performed on a Chirobiotic TAG column (150 mm × 4.6 mm, 5 μm) (Supelco, PA, USA). The autosampler tray temperature, column temperature, and injection volume were set at 4 °C, 20 °C, and 10 μL, respectively. An isocratic mobile phase of methanol- acetonitrile-glacial acetic acidammonium hydroxide (80:20:0.15:0.05, v/v/v/v) was applied at a flow rate of 0.6 mL min−1. The Waters Xevo™ TQ MS triple-quadrupole mass spectrometer, equipped with an ESI source, was operated in positive ionization mode (ESI+). The following ESI parameters were optimal: capillary voltage, 3 kV; ion source temperature, 150 °C; desolvation gas flow rate, 1000 L h−1; desolvation temperature, 500 °C. Detection was carried out in multiple reactions monitoring (MRM). Cone voltage (CV) and collision energy (CE) were optimized for each protonated molecular ions [M + H]+ and different product ions, respectively. In Table 1 are summarized [M + H]+, product ions and β-agonists special MS/MS parameters. 3

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0.1–50 ng g−1. After extraction under the optimal conditions, the calibration curve of each enantiomer was made by plotting the peak area against the nominal spiked concentration. The linearity of the calibration curve was determined by linear regression analysis and evaluated by the correlation coefficient (R2). The lowest spiked concentration for the target analytes was selected as 0.5 ng g−1 according to the No. 1025 Bulletin of the Ministry of Agriculture of the People's Republic of China [24]. Accuracy and precision were estimated by analysis of spiked pork samples at three concentrations of 0.5, 0.75 and 1.0 ng g−1 (n = 6 replicates per concentration) over three consecutive days. Relative recovery was calculated by comparing the measured concentration to the nominal concentration. Repeatability (or intra-day precision) was based on the coefficient of variation (intra-day CVs) of the determined responses of 6 replicated spiked samples at three concentrations. Intermediate precision (or inter-day CVs) was measured using 18 spiked muscle samples (n = 6 replicates per concentration), which were analyzed in three consecutive days and also expressed as CVs. The within-laboratory reproducibility was determined using the 18 spiked samples (n = 6 replicated at the lowest spiked concentration of 0.5 ng g−1 and analyzed at three occasions with three different operations). The matrix effect was calculated by analyzing the responses of βagonists dissolved in extracted blank matrix (A) and in pure solvent (B) at the same concentration. The matrix effect can be defined as A/ B × 100%. A value of 100% means the absence of matrix effect. A value above 100% indicates the presence of ionization enhancement, while a value below 100% indicates ionization suppression [7]. The stability of this method was determined in solvent (stock solvent) and in matrix (15 blank pork samples spiked at 0.1 ng g−1).

retention of all analytes, but did not produce any significant difference in the separation. Addition of 10 mM ammonium formate or ammonium acetate to 100% methanol gave lower enantioselectivity compared with that obtained with glacial acetic acid-ammonium hydroxide. From the preliminary examination, it was concluded that the use of glacial acetic acid-ammonium hydroxide as the additive proved to be most effective for the separation of chiral β-agonists. Subsequently, the type and content of the organic modifier, the ratio and concentration of glacial acetic acid-ammonium hydroxide were optimized to establish the best separation condition. Using macrocyclic antibiotic CSPs, methanol is the dominant organic solvent, and therefore chosen as the main solvent of mobile phase in the present study. The use of methanol in combination with acetonitrile is useful solvent combination for chiral separation on macrocyclic antibiotic-based CSPs [21, 25]. Based on this, enantiomeric separation of the seven β-agonists was investigated on Chirobiotic TAG with the mobile phase composed of methanol-acetonitrile-glacial acetic acid-ammonium hydroxide (80:20:0.1:0.1, v/v/v/v). When compared with methanol-glacial acetic acid-ammonium hydroxide (100:0.1:0.1, v/v/v), addition of 20% acetonitrile to the mobile phase led to shorter retention time as well as higher enantioselectivity. In particular, the baseline separation of enantiomers of formoterol, metaproterenol and cimaterol was obtained while only partial separation for the same pairs of enantiomers was observed using mobile phase without acetonitrile. Fig. 1 shows the effects of acetonitrile proportion in the mobile phase on retention and resolution of seven analytes. As illustrated in Fig. 1A, the increase of acetonitrile proportion from 5% to 20% led to a shortening of retention time, and 20%–50% proportion of acetonitrile did not change the retention significantly. As regarding the chiral resolution (Fig. 1B), there was no optimum acetonitrile content at which chiral resolution reached the maximum values for all analytes. For instance, separation of the enantiomers of metaproterenol improved significantly with an increase of acetonitrile content in the mobile phase. But for enantiomers of salbutamol, the addition of acetonitrile to the mobile phase caused the decrease of enantiomeric resolution. Thus, 20% acetonitrile was finally chosen, due to the comprehensive enantioseparation as well as shorter retention obtained for seven analytes. At the ratio of 1:1 of glacial acetic acid: ammonium hydroxide, the effects of the content of additive in the mobile phase on retention and resolution were studied. It was found that decrease of the additive content from 0.1% to 0.01% would prolong the retention, but the enantioselectivity was not significantly affected. Then, the mobile phase composition was optimized by changing the ratio of acid-base and keeping the total content of additive as constant (0.2%). The chiral resolution was found to be improved when increasing the ratio of glacial acetic acid, and the ratio of 3:1 of glacial acetic acid-ammonium hydroxide brought the sufficient chiral separation (Rs > 1.5) for target analytes. Finally, the mobile phase of methanol-acetonitrile-glacial acetic acid-ammonium hydroxide (80:20:0.15:0.05, v/v/v/v) was applied in most cases and enantiomeric separation was achieved for all

3. Results and discussion 3.1. Optimization of enantioseparation The crucial step to achieve the enantioseparation is the choice of chiral selector. A thorough literature research revealed that macrocyclic antibiotic-based CSPs were suitable for enantioseparation of β-agonists [3, 11, 16, 19-22]. In view of these facts, attempts were made to separate the enantiomers of seven β-agonists on the Chirobiotic TAG column. The reversed phase, polar organic and the polar ionic modes were widely used for chromatographic separation because of their utility in LC-MS applications, and thus we were focused on using these three phases for the enantiomeric separation of the target analytes. Reversed mobile phase composed of 90% methanol and 10% 10 mM aq. ammonium acetate buffer, pH 4.0, was first tested, but there was no elution till 50 min. When 100% methanol (polar organic solvent) was used as the mobile phase, the analytes were eluted within 40 min. However, no separation of the seven β-agonists was observed with polar organic mobile phase eluent. The use of just polar alcohol is useful for chiral resolution of only neutral molecules [25]. The seven β-agonists have ionizable groups present in their structures, and the need to add small amounts of acid and base or volatile salt to the mobile phase (polar ionic mode) is imperative. In the present study, the addition of glacial acetic acid and ammonium hydroxide to 100% methanol (methanol-glacial acetic acid-ammonium hydroxide, 100:0.1:0.1, v/v/v) would commonly lead to the improvement of enantioresolution, decrease of retention, and better peak shapes. All seven studied analytes were separated into enantiomers. Especially, three out of the seven analytes (salbutamol, tulobuterol and terbutaline) achieved baseline separation (Rs > 1.5). These results demonstrated that polar ionic mode was suitable for enantioseparation of β-agonists. The use of acidbase additive may induce the ionization of the target analytes and thus intensify their interaction with the ionizable groups on the macrocyclic glycopeptide [25]. To obtain more efficient enantioseparation, different additives (acid-base or buffer) in 100% methanol were tested. The mixture of formic acid-ammonium hydroxide significantly reduced the

Fig. 1. The effect of acetonitrile proportion in mobile phase on the retention (A) and resolutions (B) of target β-agonists on Chirobiotic TAG column. Chromatographic conditions: mobile phase, methanol/acetonitrile containing 0.1% glacial acetic acid and 0.1% ammonium hydroxide; flow rate, 0.6 mL min−1. 4

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Fig. 2. MRM chromatograms: blank pork sample (A); spiked pork sample at 0.5 ng g−1.

0.033 ng g−1 and 0.035–0.070 ng g−1, respectively. The CCɑ values of the method fall into a range of 0.035–0.078 ng g−1, and the CCβ values fall within a range of 0.054–0.150 ng g−1. These results demonstrate the high sensitivity of the present method.

seven β-agonists. The retention and separation data are summarized in Table 1. The representative chromatograms are showed in Fig. 2. 3.2. Sample preparation It has be widely reported that, not all the β-agonists are present in the form of parent compounds in animal tissues, and the biotransformation of most β-agonists includes conjugation with sulfate or glucuronic acid. Thus, the use of glucuronidase, arylsulfatase or both is necessary to release their conjugates in biological materials [6,7,9,17]. In the present study, 30 μL of β-glucoronidase/arysulfatase (containing 15 U mL−1 and 40 U mL−1 of glucuronidase and arylsulfatase) was chosen to hydrolyse the conjugates, which provided better extraction recovery. SPE with mixed-mode sorbents has been the most widely used technique for the purification and extraction of β-agonists [17]. In this work, Cleanert PCX cartridge was used for the clean-up of extract. As it has been widely reported in the literature, Cleanert PCX cartridge would provide both reversed-phase retention and strong-anion exchange mechanisms, and thus allowed great specificity and retention of basic drugs under acidic conditions [7, 13, 26]. Additionally, other parameters, including the pH of the sodium acetate buffer, the washing solution after the loading, and the type and content of the eluent were also optimized. The optimal extraction conditions (see “Experimental” section) gave satisfactory recoveries (≥70%) for all β-agonists as well as the absence of the possible matrix interferences (Fig. 2).

3.3.3. Calibration curve Series of matrix-matched calibration curves were constructed by linear regression of the peak area of each enantiomer against the nominal concentration over the range of 0.1–50 ng g−1. The results are shown in Table 2. It was found that the enantiomers of all β-agonists showed good linear regression in the range of 0.1–50 ng g−1, and the corresponding correlation coefficients (R2) of the calibration curves were > 0.9910. 3.3.4. Accuracy and precision Accuracy and precision of the present method were evaluated by analyzing the spiked pork samples at three concentrations of 0.5, 0.75 and 1.0 ng g−1 (n = 6 replicates per concentration) over three consecutive days. The results are summarized in Table 3. The mean recoveries, intra- and inter-CVs varied from 84.9 to 106.9%, from 2.0 to 11.2%, from 4.3 to 11.1%, respectively. The CVs of the within-laboratory reproducibility were found to be lower than 11.7%. The European Commission Decision 2002/657/EC indicated that, the accuracy should be in the range of 50–120%, and the precision should be below 23% if the fortified concentration was lower than 1.0 ng g−1 [23]. In our study, the accuracy and precision were well within the acceptable limits proposed by European Commission Decision 2002/657/EC.

3.3. Method validation 3.3.1. Selectivity The selectivity of the developed method was evaluated by analyzing 20 blank pork meat samples. Two typical chromatograms of blank pork muscle (Fig. 2A) and the blank sample fortified with 0.5 ng g−1 of seven β-agonists (Fig. 2B) are depicted in Fig. 2. It can be concluded that the present method is specific for the target analytes, as there is no interfering peak in the retention of the analytes.

3.3.5. Matrix effect The results of matrix effect are listed in Table 2. As shown, the matrix effects of target analytes were in the range of 84.3–108.3%. In pork matrix, the MS responses of most β-agonists were found to be suppressed, except for metaproterenol. The enantiomers of metaproterenol showed signal-enhancement with matrix effects above 100%. The matrix effect (suppression or enhancement) can be considered to be ignored if the matrix effect ranges from 80% to 120% [6]. In our study, the pork matrix has tolerable signal-suppression (signal enhancement for metaproterenol) on the responses of target β-agonists.

3.3.2. Sensitivity The results of LODs, LOQs, CCɑ and CCβ are presented in Table 2. The LODs and LOQs for the target analytes ranged from 0.014 to 5

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Table 2 Correlation coefficient (R2), linearity range, LOQ and LOD, CCɑ and CCβ of seven β-agonists in pork muscle samples. Analyte Salbutamol E1a Salbutamol E2b Clorprenaline E1 Clorprenaline E2 Tulobuterol E1 Tulobuterol E2 Terbutaline E1 Terbutaline E2 Formoterol E1 Formoterol E2 Metaproterenol E1 Metaproterenol E2 Cimaterol E1 Cimaterol E2 a b

Linearity range (ng g−1) 0.1–50 0.1–50 0.1–50 0.1–50 0.1–50 0.1–50 0.1–50 0.1–50 0.1–50 0.1–50 0.1–50 0.1–50 0.1–50 0.1–50

R2 0.9955 0.9947 0.9938 0.9964 0.9984 0.9935 0.9967 0.9954 0.9927 0.9910 0.9986 0.9974 0.9969 0.9943

LOD (ng g−1)

LOQ (ng g−1)

CCɑ (ng g−1)

CCβ (ng g−1)

Matrix effects (%)

0.014 0.016 0.020 0.022 0.025 0.019 0.033 0.029 0.019 0.020 0.023 0.021 0.031 0.030

0.050 0.046 0.035 0.038 0.055 0.048 0.056 0.050 0.039 0.040 0.045 0.042 0.066 0.070

0.047 0.054 0.038 0.042 0.064 0.058 0.044 0.049 0.035 0.042 0.064 0.078 0.043 0.054

0.087 0.120 0.054 0.078 0.098 0.110 0.078 0.100 0.060 0.095 0.126 0.150 0.088 0.110

90.5 92.3 88.9 90.1 86.2 84.3 98.2 97.4 91.1 88.4 105.8 108.3 96.5 93.2

E1 means the first eluted peak. E2 means the second eluted peak.

Table 3 Recovery and CVs of seven β-agonists enantiomer in spiked pork muscle samples. Analyte

Salbutamol E1a Salbutamol E2b Terbutaline E1 Terbutaline E2 Tulobuterol E1 Tulobuterol E2 Clorprenaline E1 Clorprenaline E2 Formoterol E1 Formoterol E2 Cimaterol E1 Cimaterol E2 Metaproterenol E1 Metaproterenol E2

a b

Spiked level (ng g−1)

0.5 0.75 1.0 0.5 0.75 1.0 0.5 0.75 1.0 0.5 0.75 1.0 0.5 0.75 1.0 0.5 0.75 1.0 0.5 0.75 1.0 0.5 0.75 1.0 0.5 0.75 1.0 0.5 0.75 1.0 0.5 0.75 1.0 0.5 0.75 1.0 0.5 0.75 1.0 0.5 0.75 1.0

Mean recovery (%, n = 18)

92.0 97.4 103.9 98.2 96.4 91.0 102.3 95.0 96.6 104.9 89.4 91.7 90.8 94.9 102.2 102.8 94.9 89.5 93.6 105.8 103.7 95.7 92.9 104.7 86.4 88.0 93.2 94.0 104.4 91.2 95.0 104.9 101.3 96.8 84.9 90.9 96.0 103.6 91.2 88.8 91.6 106.9

Intra-day CVs (%, n = 6) Day 1

Day 2

Day 3

7.8 5.9 10.7 3.4 4.2 6.0 3.0 7.4 3.9 5.7 6.0 4.1 6.7 3.5 4.4 6.9 2.0 5.9 6.3 3.9 8.0 5.3 5.7 4.0 5.2 3.9 5.1 4.7 4.4 5.6 7.2 10.2 8.8 4.2 5.0 4.3 3.9 6.0 8.1 4.8 5.9 3.4

6.2 6.6 5.1 4.2 6.1 3.0 3.9 4.3 4.7 5.9 4.0 10.9 5.9 4.0 4.8 6.4 3.9 3.7 5.3 8.1 4.6 7.2 5.0 7.1 4.5 5.2 4.8 6.6 8.4 6.9 4.8 5.3 7.1 3.8 4.2 3.0 5.5 9.4 7.5 5.0 4.7 4.1

4.4 4.7 6.4 5.8 3.9 7.4 8.0 6.4 5.0 4.5 7.3 5.9 8.8 6.2 6.4 4.3 5.4 5.8 8.0 11.2 6.0 4.2 6.7 3.4 3.9 4.8 5.5 4.1 5.2 5.1 4.4 6.1 5.0 4.9 7.0 5.8 5.3 4.6 5.4 3.8 4.6 4.9

E1 means the first eluted peak. E2 means the second eluted peak.

6

Inter-day CVs (%, n = 18)

Within-laboratory reproducibility (%, n = 18)

5.8 8.4 9.2 6.8 7.7 6.9 10.2 8.2 5.9 5.8 8.4 11.1 9.0 6.9 7.0 8.8 5.7 5.3 9.3 10.2 8.8 7.9 6.0 5.6 6.1 5.7 6.5 8.4 6.4 7.5 8.5 9.7 7.6 6.8 7.7 6.0 4.3 9.0 7.2 4.9 6.6 5.3

6.0 – – 9.8 – – 8.0 – – 5.0 – – 10.6 – – 7.3 – – 9.2 – – 7.7 – – 9.4 – – 11.7 – – 10.0 – – 6.9 – – 9.4 – – 7.5 – –

Microchemical Journal 150 (2019) 104082

B. Zhu, et al.

3.3.6. Stability The standard solutions prepared in methanol were stored at −20 °C for three months. These solutions were analyzed every month, and the corresponding peak areas were compared with the responses obtained by freshly prepared standard solutions. The values of all analytes were in the range of 97.7%–103.4%, indicating the stability of seven βagonists in pure solvent. Spiked blank samples at the lowest concentration (0.1 ng g−1 for each enantiomer) were also stored at −20 °C, and then analyzed after 3, 7 and 14 days. The relative recovery of each enantiomer was calculated by comparing the measured concentration to the nominal concentration. The recoveries of all enantiomers were in the range of 82.1–109.4 with RSD lower than 8.3%, revealing the stability of seven β-agonists in pork matrix.

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3.4. Application to real samples The present method was successfully applied to the analysis of real pork samples purchased from different local markets in Benxi/China. The samples were ground, divided into 2.0 g and stored at −20 °C in dark until analysis. Among the 30 pork samples, no β-agonists was detected. The use of β-agonists in animal breeding for human consumption is strictly prohibited in China [27]. 4. Conclusions The enantiomers of seven β-agonists were simultaneously separated on the Chirobiotic TAG column with the mobile phase composition of methanol-acetonitrile-glacial acetic acid-ammonium hydroxide (80:20:0.15:0.05, v/v/v/v). The mobile phase used in this study is polar ionic phase containing volatile additives suited for LC-MS application. Then, a sensitive chiral UPLC-MS/MS method was developed for the enantioselective multi-residue analysis of seven banned β-agonists in pork muscle samples. The present method was validated and demonstrated good linearity and accuracy. The limits of detection of each enantiomer in pork muscle samples (0.1 ng g−1) were satisfying for the trace-analysis of β-agonists. To the best of our knowledge, this is the first report on the stereoselective determination of seven chiral β-agonists in animal muscles. Declaration of Competing Interest None. Acknowledgements This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References [1] M. Salem, H. Levesque, T.W. Moon, C.E. Rexroad, J. Yao, Anabolic effects of feeding β2-adrenergic agonists on rainbow trout muscle proteases and proteins, Comp. Biochem. Phys. A 144 (2006) 145–154. [2] Council of the European Union, Commission Regulation 2010/37/EC of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin, Off. J. Eur. Communities L15 (2010) 1–72. [3] N. Rosales-Conrado, M.E.D. León-González, L.M. Polo-Díez, Development and validation of analytical method for clenbuterol chiral determination in animal feed by direct liquid chromatography, Food Anal. Methods 8 (2015) 2647–2659.

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