Journal of Pharmaceutical and Biomedical Analysis 107 (2015) 526–534
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Ultra-trace analysis of 12 ˇ2 -agonists in pork, beef, mutton and chicken by ultrahigh-performance liquid-chromatography–quadrupole-orbitrap tandem mass spectrometry Changchuan Guo a , Feng Shi a,∗ , Liping Gong a , Huijie Tan a , Defu Hu a , Jinling Zhang b,∗∗ a b
Shandong Institute for Food and Drug Control, Jinan, Shandong 250101, China Cancer Center, Linyi’s People Hospital Affiliated to Shandong University, Linyi, Shandong 276000, China
a r t i c l e
i n f o
Article history: Received 23 November 2014 Received in revised form 23 January 2015 Accepted 24 January 2015 Available online 31 January 2015 Keywords: Orbitrap ˇ2 -Agonist Food animal muscles Residues Detection
a b s t r a c t This paper presents an application of ultrahigh-performance liquid-chromatography – quadrupole – orbitrap high resolution mass spectrometry (UHPLC–Q–Orbitrap HRMS) for the ultra-trace analysis of 12 ˇ2 -agonists in pork, beef, mutton and chicken meat. The mass spectrometer was operated in Full MS/dd-MS2 (data-dependent MS2 ) mode, under which a Full MS scan was followed by a dd-MS2 scan with a fragmentation energy. The quantification was achieved using matrix-matched standard calibration curves with salbutamol-d3 and clenbuterol-d9 as the internal standards. The method validation included assessment of selectivity, sensitivity, calibration curve, accuracy, precision, recovery, matrix effect and stability. The results show an exceptional linear relationship with the concentrations of the analytes over wide concentration ranges (e.g., 0.01–50 g/kg for clenbuterol) as all the fitting coefficients of determination r2 are >0.9986. The detection limits (LODs) were in the range of 0.0033–0.01 g/kg, which was much lower than the current reported methods. The recoveries were able to reach 73.0–88.7%, while the matrix effects ranged from 83.7% to 92.8%. Analysis of 400 pork, beef, mutton and chicken samples reveal that only 4.25% samples were positive for ˇ2 -agonists. The detected ˇ2 -agonists involved salbutamol, clenbuterol, ractopamine and clorprenaline. Overall, the novel Q-Orbitrap technique was demonstrated to have great performance for the screening, identification and quantification of ultra-trace ˇ2 -agonists used in food animal muscles, which helps to ensure food safety and public health. © 2015 Elsevier B.V. All rights reserved.
1. Introduction ˇ2 -Agonists (structures see Fig. 1) are a class of synthetic drugs commonly used in the clinical treatments for acute symptoms of asthma owing to their bronchodilator activities [1]. These compounds are efficient partitioning agents to reduce the body fat and enhance growth of cattle, sheep and swine when used in high dosage [2]. Therefore, in the past several decades, ˇ2 -agonists, especially salbutamol and clenbuterol, have been widely used as growth promoters in the animal breeding [3]. Although ˇ2 -agonists have been prohibited for use in meat production in China and Europe [4,5], a certain number of animal-feeding manufacturers
∗ Corresponding author. Tel.: +86 531 81216563; fax: +86 531 81216563. ∗∗ Corresponding author. Tel.: +86 13697818180; fax: +86 539 8216254. E-mail addresses: [email protected] (F. Shi), jinlingzhang [email protected] (J. Zhang). http://dx.doi.org/10.1016/j.jpba.2015.01.048 0731-7085/© 2015 Elsevier B.V. All rights reserved.
and farmers still use these drugs as fodder additives illegally. The abuse of this banned substance has led to serious cases of intoxication in patients after the intake of contaminated beef or pig meat [6–8]. The analytical methods hitherto used for the detection of ˇ2 agonists include hapten microarray [9], capillary electrophoresis (CE) [10,11], gas chromatography mass spectrometry (GC–MS) [12] and liquid chromatography–mass spectrometry (LC–MS) [13–18]. However, there are limitations with these methods. First, the sensitivity was moderate but not high enough. All the detection limits (LODs) of these methods are over the range of 0.05–0.12 g/kg or 0.08–100 ng/mL. Compared with these methods, the Q-Orbitrap HRMS method could provide a much higher sensitivity. The LOD of Q-Orbitrap HRMS can reach 0.0033 g/kg (viz. 0.0165 ng/mL), which is an extremely low detecting level. Second, in most of the current LC–MS methods the liquid chromatography is used together with low resolution mass spectrometry (LRMS) analyzer such as ion trap (IT) or triple-quadrupole (QqQ). Different from
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LRMS, the high resolution mass spectrometry (HRMS) such as Orbitrap MS could provide much higher mass accuracy, which benefits the high-resolution identification of residues in biological matrices. Orbitrap is the newest member of HRMS analyzers. It combines high speed with excellent quantification properties and is favored in plenty of analytical applications [19]. Experiments have been carried out on a modified Orbitrap EliteTM instrument and the Orbitrap technology is demonstrated to successfully resolve peaks in excess of 1,000,000 FWHM, offering an incomparable mass accuracy [20]. As a new generation of the Orbitrap instruments, the Q-Orbitrap (Q-ExactiveTM , hybrid quadrupole-orbitrap mass spectrometer) combines high-performance quadrupole precursor selection with high resolution and accurate mass (HR/AM) Orbitrap detection. The incorporation of non-targeted full scan screening
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and targeted MS/MS analysis makes the Q-Orbitrap an ideal tool for fast and easy screening. All the present ions are monitored without any pre-selection and identified based on the analytes’ exact mass. The Orbitrap acquires product ion spectra with accurate mass measurements, which are essential for unequivocal identification of detailed structural information. The Orbitrap technique has been reported to be applied to both qualitative and quantitative analysis in various fields such as doping control, drugs of abuse, pesticide residues, etc. [21–23]. The aim of the present study was to develop a rapid and effective multi-analyte method coupling ultrahigh-performance liquid chromatography (UHPLC) to Q-Orbitrap HRMS for the detection of 12 ˇ2 -agonists. This method was successfully applied to the screening, identification and quantification of ˇ2 -agonists
Fig. 1. Chemical structures of studied ˇ2 -agonists.
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multi-residues in pork, beef, mutton and chicken samples. To the best of our knowledge, this is the first report on the application of Q-Orbitrap HRMS to the simultaneous qualitative and quantitative analysis of ˇ2 -agonists in food animal muscles. 2. Materials and methods 2.1. Chemicals and reagents Salbutamol (99.5%), terbutaline (98.5%), ractopamine (97.0%), clenbuterol (98.5%) and brombuterol (98.5%) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Cimaterol (99.8%), cimbuterol (99.6%), mapenterol (99.0%), bromchlorbuterol (99.3%), salbutamol-d3 (internal standard, ISTD, 98.3%) and clenbuterol-d9 (ISTD, 98.0%) were obtained from Witega (Berlin, Germany). Isoxsuprine (99.0%) and mabuterol (98.0%) were supplied by Fluka (Bucks, Switzerland) and TRC (Toronto, Canada), respectively. Clorprenaline (98.0%) was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Their chemical structures are showed in Fig. 1. HPLC grade methanol, acetonitrile and formic acid (FA, ≥99.0%) were purchased from TEDIA Inc. (USA). Analytical grade acetic acid (≥99.5%), sodium acetate (≥99.0%), perchloric acid (70.0–72.0%), sodium hydroxide (≥96.0%), sodium chloride (≥99.5%), isopropanol (≥99.7%), ethyl acetate (≥99.5%) and ammonium hydroxide (25.0–28.0%) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). ˇ-Glucuronidase/arylsulfatase from Helix pomatia was purchased from Roche Diagnostica GmbH (Mannheim, Germany). Bond Elut Plexa PCX solid phase extraction (SPE) cartridges were supplied by Agilent (USA). Ultrapure water (18.2 M) was obtained from a Milli-Q Advantage A10 ultrapure water purification system. 2.2. Instrumentation The UHPLC–Q-Orbitrap HRMS system is composed of an Accela 1250 LC pump and an Accela open autosampler coupled with a Q ExactiveTM HR mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). XCalibur 2.2 software (Thermo Fisher Scientific, San Jose, CA, USA) was used for instrument control and data processing, while Q Exactive 2.1 (for tune application) software (Thermo Fisher Scientific) was used to control the mass spectrometer. Chromatographic separation was achieved on a Hypersil GOLD aQ C18 chromatographic column (100 mm × 2.1 mm, 1.9 m) (Thermo Fisher Scientific).
2.4. Sample pretreatment An improved sample preparation procedure was proposed referring to several methods that have been reported [13,14]. An aliquot of 2.00 g homogenized food animal muscles and 8 mL sodium acetate (0.2 mol/L, pH 5.2) were added to a 50 mL polypropylene centrifuge tube and mixed thoroughly. The mixture was then incubated with 50 L ˇ-glucuronidase/arylsulfatase for 12 h at 37 ◦ C in water bath. After being cooled down to room temperature, 100 L mixed ISTD solution (100 ng/mL) was added to the enzyme digested sample. After horizontal oscillation for 15 min and centrifugation for 10 min (9391 × g), 4 mL extract was transferred into another centrifuge tube. Afterward, 5 mL of 0.1 mol/L perchloric acid was added and the pH was adjusted to 1.0 with 1.0 mol/L perchloric acid. The mixture was centrifuged again at 9391 × g for 10 min and the pH of the extract was adjusted to 11.0 with 10 mol/L NaOH solution. 10 mL saturated sodium chloride solution and 10 mL isopropanol–ethyl acetate (6:4, v/v) was added, followed by vortex for 1 min and centrifugation at 9391 × g for 10 min. 5 mL extract was collected and evaporated to dryness with nitrogen evaporator at 40 ◦ C in water bath. The residue was dissolved in 5 mL sodium acetate (0.2 mol/L, pH 5.2), followed by sonication for 5 min. Whereafter, the residue solution was subjected to SPE. The Bond Elut Plexa PCX (3 mL, 60 mg) cartridge was sequentially conditioned with 3 mL methanol and 3 mL water. After the sample was loaded, the cartridge was washed with 2 mL H2 O, 2 mL 2% FA, 2 mL methanol and dried with vacuum. Finally, the analytes were eluted with 2 mL of methanol containing 5% ammonia. The eluent was evaporated with a gentle N2 stream at 40 ◦ C in water bath and reconstituted to 200 L with initial mobile phase. After vortex for 1 min and centrifugation at 9391 × g for 10 min, the extract solution was injected into chromatographic column for the Q-Orbitrap HRMS analysis. The sample equivalent in the final extract was 2.5 g/mL. 2.5. Chromatographic conditions Chromatographic separation was performed on a Hypersil Gold aQ C18 column (100 mm × 2.1 mm, 1.9 m). The autosampler tray temperature, column oven temperature, flow rate and injection volume were set at 20 ◦ C, 30 ◦ C, 300 L/min and 5 L, respectively. The mobile phase consisted of water (A) and acetonitrile (B), both containing 0.1% FA. The gradient used for eluting analytes with mobile phase is as follows: 0–0.5 min, 4% B; 0.5–2.0 min, 4% B–25% B; 2.0–7.0 min, 25% B–65% B; 7.0–7.5 min, 65% B–95% B; 7.5–9.5 min, 95% B; 9.6–12.0 min, 4% B.
2.3. Standard solutions 2.6. Mass spectrometry conditions Individual stock standard solutions were prepared in methanol at concentration of 1 mg/mL and stored at −20 ◦ C. The mixed working standard solutions were prepared daily by proportional dilution of the stock standard solutions. The salbutamol-d3 and clenbuterol-d9 stock ISTD solutions were prepared in methanol at 100 g/mL and stored at −20 ◦ C. The mixed working ISTD solutions were prepared daily by dilution of the stock ISTD solutions. To quantificationally analyze the 12 ˇ2 -agonists in food animal muscles, the matrix-matched calibration curves were constructed. Seven multi-component standard solutions were prepared by spiking standard mixture into blank mixed matrices at different concentrations. All animal muscle food samples including pork, beef, mutton and chicken were collected by Shandong Food and Drug Administration. Samples were received frozen and kept at −20 ◦ C until analysis.
The Q-Orbitrap HRMS was equipped with a HESI-II ion source and the positive ionization mode was applied. The spray voltage, capillary temperature, vaporizer temperature and were set to 3.0 kV, 350 ◦ C and 250 ◦ C, respectively. The sheath gas, auxiliary gas, sweep gas and S-lens RF level were set to 40, 15, 0 (arbitrary units) and 50 V, respectively. The analysis was executed in Full MS/ddMS2 (data-dependent MS2 ) mode, under which a Full MS scan event (SE1) was followed by a data-dependent scan applied with a fragmentation energy (SE2). The mass spectrometer acquired a Full MS scan at the resolution of 70,000 (FWHM at 200 m/z). The automatic gain control (AGC) target (the number of ions to fill C-Trap) was set to 1.0e6 with a maximum injection time (IT) of 100 ms. The Full MS scan range was from 150 to 400 m/z. For the dd-MS2 scan, the mass resolution was 35,000 FWHM with AGC target at 2.0e5 , maximum IT 50 ms, isolation window 0.4 m/z, underfill ratio 1.0%,
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intensity threshold 4.0e4 , apex trigger 3–5 s, exclude isotopes “on”, and dynamic exclusion 10.0 s. 2.7. Method validation Method validation was carried out with ˇ2 -agonists assay in herbal medicines and dietary supplements referring to the US Food and Drug Administration (USFDA) guidelines [24]. The validation parameters included selectivity, sensitivity, calibration curve, accuracy and precision, recovery, matrix effect and stability. The homogenized blank food animal muscles (pork, beef, mutton and chicken) were used as blank matrices in the following method validation. 2.7.1. Selectivity The selectivity of the analyzing method should be assayed using a minimum of six independent sources of the same matrix. The selectivity should be ensured at the lowest calibrated level (LCL). 2.7.2. Calibration curve For quantificational analysis, salbutamol-d3 was used as the ISTD of salbutamol, terbutaline and ractopamine, while clenbuterol-d9 was used as the ISTD of other ˇ2 -agonists. A matrixmatched calibration curve was constructed by linear regression of the ratios of chromatographic peak areas of the standards and the ISTD and the linearity was assessed by the coefficient of determination (r2 ). The LOD was the lowest concentration that can be detected giving a signal-to-noise ratio of at least 3-fold (S/N ≥ 3). The LCL was defined as the lowest concentration of the calibration curve, giving a signal-to-noise ratio of at least 10–fold (S/N ≥ 10), acceptable accuracy (80–120%) and sufficient precision (within 20%). 2.7.3. Accuracy and precision The accuracy was calculated from the ratio of the mean values of the detected concentration (Cdet ) and the nominal concentration (Cnom ) as following: (Cdet /Cnom ) × 100. The precision was expressed as relative standard deviation (RSD), which was calculated as RSD% = [standard deviation (SD)/Cdet ] × 100. 2.7.4. Recovery The recoveries of ˇ2 -agonists from matrices were evaluated by comparing the response of ˇ2 -agonists after extraction from matrices with the response of the same concentration analytes spiked into the eluent from blank matrices. Recoveries of 12 ˇ2 -agonists were determined at three levels (low, medium and high).
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2.7.5. Matrix effect The matrix effect was evaluated by analyzing the response of analytes prepared in solvent and in extracted blank matrix at the same concentration. The value of matrix effect can be calculated as (Eq. (1)): ME (%) = B/A × 100
(1)
A refers to the peak areas obtained from neat solution standards, while B refers to the corresponding peak areas of standards spiked after extraction from matrix [25,26]. 2.7.6. Stability The post-preparative stability was measured by repeatedly determining the processed QC samples which were kept in the autosampler (20 ◦ C) for 24 h. The concentrations of the samples were calculated according to original calibration standards. 2.8. Calculation The chromatograms were processed using XCalibur 2.2 software developed by Thermo Fisher Scientific. All calculations were completed in Microsoft Excel 2010 (Microsoft Co., Redmond, USA) or OriginPro 8.0 (OriginLab Co., Northampton, MA, USA). 3. Results and discussion 3.1. Development of the UHPLC–Q-Orbitrap HRMS method As discussed above, all analytes were measured in positive mode and the precursor ion selected was [M+H]+ in all cases. For each analyte, the experimentally determined mass of the corresponding ion was evaluated according to the theoretical mass calculated by Xcalibur 2.1. Mass deviations were defined as 106 × [(measured mass − theoretical mass)/theoretical mass] and expressed in ppm. All the MS parameters were optimized and set to the optimal values in order to provide the best response of the analytes. The optimal parameter values of the HESI source were summarized in Section 2.6. The simultaneous qualitative and quantitative analysis of ˇ2 -agonists was performed in Full MS/dd-MS2 mode, which includes a full scan followed by MS/MS scan of precursors in the inclusion list (Table 1). The inclusion list with the monoisotopic mass and specific NCEs of the precursors was utilized to generate sensitive products since precursors are selected in the quadrupole. For the selection of the mass resolution of the Full MS scan, compromise has to be made between selectivity and sensitivity. A higher mass resolution gives rise to a better mass accuracy and thereby better selectivity. However, an extremely high resolution (such as
Table 1 Retention time, accurate mass, NCEs, correlation coefficients, linear ranges and LCLs of 12 ˇ2 -agonists. Analyte
Retention time (min)
Salbutamol Salbutamol-d3 Terbutaline Cimaterol Cimbuterol Ractopamine Clorprenaline Clenbuterol-d9 Clenbuterol Bromchlorbuterol Brombuterol Isoxsuprine Mabuterol Mapenterol
3.98 3.96 3.98 4.11 4.54 5.19 5.26 5.65 5.68 5.84 6.01 6.16 6.34 6.89
Protonated ion mass [M+H]+
Theoretical
Experimental
240.1594 243.1783 226.1438 202.1339 234.1601 302.1751 214.0993 286.1434 277.0869 323.0343 366.9838 302.1751 311.1133 325.1289
240.1590 243.1778 226.1434 202.1336 234.1597 302.1750 214.0990 286.1429 277.0866 323.0337 366.9833 302.1750 311.1128 325.1282
Mass error (ppm)
Normalized collision energy (NCE)
Correlation coefficient (r2 )
−1.7 −2.1 −1.8 −1.5 −1.7 −0.3 −1.4 −1.7 −1.1 −1.9 −1.4 −0.3 −1.6 −2.2
22 40 58 48 53 32 29 34 12 42 16 32 16 42
0.9992 0.9990 0.9987 0.9991 0.9994 0.9988 0.9995 0.9988 0.9991 0.9996 0.9986 0.9987 0.9992 0.9990
Linear range (g/kg)
0.01–50 0.01–50 0.03–50 0.01–50 0.01–50 0.03–50 0.01–50 0.03–50 0.01–50 0.01–50 0.01–50 0.01–50 0.01–50 0.01–50
LCL (g/kg)
0.01 0.01 0.03 0.01 0.01 0.03 0.01 0.03 0.01 0.01 0.01 0.01 0.01 0.01
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Fig. 2. Extracted ion chromatograms of ˇ2 -agonists in (A) a blank pork matrix and (B) a pork matrix spiked with 1 g/kg ˇ2 -agonists standards.
140,000 FWHM) would significantly affect the sensitivity due to the reduced scanning speed, given the same experimental time. To strike a balance between the selectivity and the sensitivity with Full MS, a mass resolution of 70,000 FWHM was selected and this
turned out to be optimal for majority of the analytes. For the ddMS2 scan, 35,000 FWHM was used for time-saving and to ensure sufficient scan points of the Full MS. Under such conditions, the fragment ions can be used for further identification of the target
Fig. 3. Suggested fragmentation profiles of ˇ2 -agonists.
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Table 2 Comparison of methods for the detection of ˇ2 -agonists in food animal muscle matrices. Method
The number of ˇ2 -agonists
LOD (case with clenbuterol)
Linear range (case with clenbuterol)
Application
References
CE CE LC–MS LC–MS LC–MS HRMS
5 4 7 16 3 12
0.10 g/mL 22.45 ng/mL 0.05 g/kg 0.06 g/kg 0.12 g/kg 0.0033 g/kg (viz. 0.0165 ng/mL)
0.20–100 g/mL 0.069–2.77 g/mL / 0.1–10 g/kg 0.5–20 g/kg 0.01–50 g/kg (viz. 0.025–125 ng/mL)
Pig feed, pig urine and pig liver Pig feed and urine Animal liver and urine Pig liver, kidney and muscle Pork Food animal muscles
[10] [11] [13] [14] [15] Present paper
analytes. The data-dependent approach employed here should better be described as exact-mass-dependent acquisition as it merely depends on the exact mass in the inclusion list to trigger dd-MS/MS and the fragmentation is carried out with the NCEs specified in the inclusion list. In addition, the selected mass tolerance window was also considered as a crucial parameter regarding selectivity. The optimal value for this parameter (in ppm) is compound-specific and dependent on the detection capability, signal intensity and occurrence of matrix interferences [27]. In the case of ˇ2 -agonists, all mass spectra were filtered through a mass tolerance window of 5 ppm to reconstruct extracted ion chromatograms (XICs) and to acquire adequate selectivity. The mass window of 5 ppm was sufficient to eliminate the background from the complex matrix and to generate a sharp and resolved peak, which represents the specific compound without interference. The peak shape and the chromatographic resolution were the main criteria of evaluation during optimization of the UHPLC method. The Hypersil GOLD aQ C18 chromatographic column, which was used throughout this study, provides sharp absorbance peaks and excellent chromatographic resolution. Since all the analytes contain basic groups, 0.1% FA was added to the mobile phase to enhance the protonation and sensitivity. Accordingly, 0.1% FA H2 O–0.1% FA acetonitrile was selected as the binary mobile solvents system. The elution gradient procedure enabled chromatographic separation of 12 ˇ2 -agonists within 12 min. A typical extracted ion chromatogram of matrices spiked with 1 g/kg ˇ2 -agonists standards is shown in Fig. 2B. The result shows that the retention time (RT) ranged from 3.98 min (salbutamol) to 6.89 min (mapenterol). The theoretical and experimental masses for each analyte are given in Table 1. The mass of all the precursor ions corresponded to that of protonated molecules ([M+H]+ ). As shown in Table 1, mass measurement accuracy was less than 2.2 ppm, indicating a high level of confidence between the theoretical values and experimental masses measured by Orbitrap HRMS for all analytes. Furthermore, the MS2 product ions of analytes (showed in Fig. 3) were utilized as diagnostic ions for each compound by accurate mass measurements and would be considered for further identification.
3.2. Method validation 3.2.1. Selectivity The selectivity of the analyzing method was assayed using six independent sources of the same matrix. Fig. 2A shows the representative chromatograms of a blank pork matrix, while a pork matrix spiked with 1 g/kg ˇ2 -agonists is shown in Fig. 2B. The reconstruction of XICs with a narrow mass tolerance window (5 ppm) provided sufficient resolving power to distinguish analytes from isobaric co-eluting sample matrix compounds. No interfering peaks were detected at RTs of all analytes.
3.2.2. Calibration curve The LCLs and LODs were determined as the lowest analyte concentrations that produced signal-to-noise (S/N) ratios of at least 3 and 10, respectively, via the analysis of the target compounds in blank matrix. All LOD and LOQ values were in ranges of 0.0033–0.01 and 0.01–0.03 g/kg, respectively. Series of matrix-matched calibration curves were constructed by linear regression of the ratios of chromatographic peak areas of the standards and the deuterated ISTD versus nominal concentrations. The linearity of calibration curve was assessed by the coefficients of determination (r2 ). As presented in Table 1, the calibration curves showed great linearity as the coefficients of determination (r2 ) are higher than 0.9986 in all the measurements. In comparison with other methods reported, the present Orbitrap HRMS method offers higher sensitivity and broader linear ranges (shown in Table 2). Besides, the number of the analytes that can be measured simultaneously (12) was more than most of these methods. 3.2.3. Accuracy and precision Table 3 gives an overview of the values of the intra-day and inter-day accuracy and precision at three spiking levels (low, medium and high). The intra-day and inter-day accuracy of ˇ2 agonists ranged from 96.2% to 104.1% and 94.5% to 106.3%, respectively. The intra- and inter-day precision were in the range of 0.4–7.6% and 0.7–8.7%, respectively. These results indicated that the method was reliable for accurate and precise analysis. 3.2.4. Recovery and matrix effect The analyte recovery of this procedure was evaluated by spiked blank samples at three concentrations (low, medium and high), with each condition performed in five replicates. As shown in Table 3, the mean recovery of each compound was in the range of 73.0–88.7%. The results of matrix effects evaluation are shown in Table 3. Signal suppression or enhancement effect was considered tolerable if the matrix effect value was between 80% and 120%. As shown in Table 3, the matrix effects of analytes ranged from 83.7% to 92.8% with SDs less than 5.5%. This suggests that the food animal muscles matrix have tolerable low suppression on the MS response of ˇ2 agonists. With such low level of matrix effects, this method would be reliable for analysis in food animal muscles matrix. 3.2.5. Stability As summarized in Table 3, the post-preparative stability was expressed as a percentage of the initial concentration (first analyzed batch) of the analytes in QC samples. The values of all analytes stabilities in matrices were in the range of 96.9–103.6%, indicating that all 12 ˇ2 -agonists were stable in auto sampler at 20 ◦ C for 24 h. The results suggested that the established method was reliable and suitable for large-scale sample screening.
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Table 3 Accuracy, precision, matrix effect, recovery and stability for the determination of 12 ˇ2 -agonists in food animal muscle matrices (n = 5). Analyte
Salbutamol
Terbutaline
Cimaterol
Cimbuterol
Ractopamine
Clorprenaline
Clenbuterol
Bromchlorbuterol
Brombuterol
Isoxsuprine
Mabuterol
Mapenterol
QC concentration (g/kg)
0.012 0.8 40 0.012 0.8 40 0.04 1.0 40 0.012 0.8 40 0.012 0.8 40 0.04 1.0 40 0.012 0.8 40 0.04 1.0 40 0.012 0.8 40 0.012 0.8 40 0.012 0.8 40 0.012 0.8 40
Accuracy (%)
Precision (RSD, %)
Intra-day
Inter-day
Intra-day
Inter-day
102.6 102.1 100.7 102.0 101.8 97.9 98.4 98.1 101.5 102.5 99.2 98.4 96.8 103.0 98.4 96.9 102.3 103.2 97.6 100.8 98.2 96.3 97.1 101.0 98.5 104.1 99.3 96.6 97.0 101.6 102.9 103.8 98.4 103.5 96.2 101.6
105.1 103.4 102.5 102.9 104.1 96.8 97.6 96.9 102.9 105.3 96.6 95.8 96.1 104.8 99.0 94.5 103.7 104.4 97.2 103.4 97.7 97.6 96.4 98.7 97.2 106.3 96.7 95.7 96.2 103.8 104.2 105.5 97.5 105.0 95.3 103.7
4.9 3.8 1.2 5.7 1.1 1.4 3.4 6.8 2.5 7.6 1.3 2.3 1.9 4.8 0.6 7.2 1.1 0.9 4.4 4.6 0.4 0.9 3.2 1.3 5.7 3.2 0.7 3.2 5.3 0.8 5.1 6.1 1.1 6.7 2.5 1.2
6.7 4.4 2.5 7.0 1.8 1.7 4.0 8.6 3.4 8.7 2.5 3.0 2.7 6.3 1.1 8.2 1.9 1.3 5.5 4.9 0.7 2.1 4.6 1.6 6.5 3.4 1.0 4.0 6.5 2.3 5.7 6.7 1.6 8.2 4.0 1.7
3.3. Application of the Orbitrap HRMS method to real samples The Orbitrap HRMS method was applied to the simultaneous qualitative and quantitative analysis of ˇ2 -agonists in 400 batches of pork, beef, mutton and chicken meat, which were collected by Shandong Food and Drug Administration. These food animal muscles samples were most representative because the sampling locations covered slaughterhouses, farm product markets, supermarkets and restaurants all over the Shandong Province. Considering the fact that ˇ2 -agonists have been frequently detected in food animal muscles in China for the past few years, the significance of a novel screening assay with high sensitivity and selectivity in this field is obvious. The identification of the target compounds was based on the RT windows (RTWs) and exact mass. RTWs were defined as the average RT plus or minus 3-fold SD value of the RT (RT ± 3 × SD). If there was no signal at all or the signal was below the LOD level within the RTW, as well as the exact mass error was beyond 5 ppm, the sample was considered negative. On the other hand, if a signal was detected showing an exact mass error
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Ultra-trace analysis of 12 ˇ2 -agonists in pork, beef, mutton and chicken by ultrahigh-performance liquid-chromatography–quadrupole-orbitrap tandem mass spectrometry Changchuan Guo a , Feng Shi a,∗ , Liping Gong a , Huijie Tan a , Defu Hu a , Jinling Zhang b,∗∗ a b
Shandong Institute for Food and Drug Control, Jinan, Shandong 250101, China Cancer Center, Linyi’s People Hospital Affiliated to Shandong University, Linyi, Shandong 276000, China
a r t i c l e
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Article history: Received 23 November 2014 Received in revised form 23 January 2015 Accepted 24 January 2015 Available online 31 January 2015 Keywords: Orbitrap ˇ2 -Agonist Food animal muscles Residues Detection
a b s t r a c t This paper presents an application of ultrahigh-performance liquid-chromatography – quadrupole – orbitrap high resolution mass spectrometry (UHPLC–Q–Orbitrap HRMS) for the ultra-trace analysis of 12 ˇ2 -agonists in pork, beef, mutton and chicken meat. The mass spectrometer was operated in Full MS/dd-MS2 (data-dependent MS2 ) mode, under which a Full MS scan was followed by a dd-MS2 scan with a fragmentation energy. The quantification was achieved using matrix-matched standard calibration curves with salbutamol-d3 and clenbuterol-d9 as the internal standards. The method validation included assessment of selectivity, sensitivity, calibration curve, accuracy, precision, recovery, matrix effect and stability. The results show an exceptional linear relationship with the concentrations of the analytes over wide concentration ranges (e.g., 0.01–50 g/kg for clenbuterol) as all the fitting coefficients of determination r2 are >0.9986. The detection limits (LODs) were in the range of 0.0033–0.01 g/kg, which was much lower than the current reported methods. The recoveries were able to reach 73.0–88.7%, while the matrix effects ranged from 83.7% to 92.8%. Analysis of 400 pork, beef, mutton and chicken samples reveal that only 4.25% samples were positive for ˇ2 -agonists. The detected ˇ2 -agonists involved salbutamol, clenbuterol, ractopamine and clorprenaline. Overall, the novel Q-Orbitrap technique was demonstrated to have great performance for the screening, identification and quantification of ultra-trace ˇ2 -agonists used in food animal muscles, which helps to ensure food safety and public health. © 2015 Elsevier B.V. All rights reserved.
1. Introduction ˇ2 -Agonists (structures see Fig. 1) are a class of synthetic drugs commonly used in the clinical treatments for acute symptoms of asthma owing to their bronchodilator activities [1]. These compounds are efficient partitioning agents to reduce the body fat and enhance growth of cattle, sheep and swine when used in high dosage [2]. Therefore, in the past several decades, ˇ2 -agonists, especially salbutamol and clenbuterol, have been widely used as growth promoters in the animal breeding [3]. Although ˇ2 -agonists have been prohibited for use in meat production in China and Europe [4,5], a certain number of animal-feeding manufacturers
∗ Corresponding author. Tel.: +86 531 81216563; fax: +86 531 81216563. ∗∗ Corresponding author. Tel.: +86 13697818180; fax: +86 539 8216254. E-mail addresses: [email protected] (F. Shi), jinlingzhang [email protected] (J. Zhang). http://dx.doi.org/10.1016/j.jpba.2015.01.048 0731-7085/© 2015 Elsevier B.V. All rights reserved.
and farmers still use these drugs as fodder additives illegally. The abuse of this banned substance has led to serious cases of intoxication in patients after the intake of contaminated beef or pig meat [6–8]. The analytical methods hitherto used for the detection of ˇ2 agonists include hapten microarray [9], capillary electrophoresis (CE) [10,11], gas chromatography mass spectrometry (GC–MS) [12] and liquid chromatography–mass spectrometry (LC–MS) [13–18]. However, there are limitations with these methods. First, the sensitivity was moderate but not high enough. All the detection limits (LODs) of these methods are over the range of 0.05–0.12 g/kg or 0.08–100 ng/mL. Compared with these methods, the Q-Orbitrap HRMS method could provide a much higher sensitivity. The LOD of Q-Orbitrap HRMS can reach 0.0033 g/kg (viz. 0.0165 ng/mL), which is an extremely low detecting level. Second, in most of the current LC–MS methods the liquid chromatography is used together with low resolution mass spectrometry (LRMS) analyzer such as ion trap (IT) or triple-quadrupole (QqQ). Different from
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LRMS, the high resolution mass spectrometry (HRMS) such as Orbitrap MS could provide much higher mass accuracy, which benefits the high-resolution identification of residues in biological matrices. Orbitrap is the newest member of HRMS analyzers. It combines high speed with excellent quantification properties and is favored in plenty of analytical applications [19]. Experiments have been carried out on a modified Orbitrap EliteTM instrument and the Orbitrap technology is demonstrated to successfully resolve peaks in excess of 1,000,000 FWHM, offering an incomparable mass accuracy [20]. As a new generation of the Orbitrap instruments, the Q-Orbitrap (Q-ExactiveTM , hybrid quadrupole-orbitrap mass spectrometer) combines high-performance quadrupole precursor selection with high resolution and accurate mass (HR/AM) Orbitrap detection. The incorporation of non-targeted full scan screening
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and targeted MS/MS analysis makes the Q-Orbitrap an ideal tool for fast and easy screening. All the present ions are monitored without any pre-selection and identified based on the analytes’ exact mass. The Orbitrap acquires product ion spectra with accurate mass measurements, which are essential for unequivocal identification of detailed structural information. The Orbitrap technique has been reported to be applied to both qualitative and quantitative analysis in various fields such as doping control, drugs of abuse, pesticide residues, etc. [21–23]. The aim of the present study was to develop a rapid and effective multi-analyte method coupling ultrahigh-performance liquid chromatography (UHPLC) to Q-Orbitrap HRMS for the detection of 12 ˇ2 -agonists. This method was successfully applied to the screening, identification and quantification of ˇ2 -agonists
Fig. 1. Chemical structures of studied ˇ2 -agonists.
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multi-residues in pork, beef, mutton and chicken samples. To the best of our knowledge, this is the first report on the application of Q-Orbitrap HRMS to the simultaneous qualitative and quantitative analysis of ˇ2 -agonists in food animal muscles. 2. Materials and methods 2.1. Chemicals and reagents Salbutamol (99.5%), terbutaline (98.5%), ractopamine (97.0%), clenbuterol (98.5%) and brombuterol (98.5%) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Cimaterol (99.8%), cimbuterol (99.6%), mapenterol (99.0%), bromchlorbuterol (99.3%), salbutamol-d3 (internal standard, ISTD, 98.3%) and clenbuterol-d9 (ISTD, 98.0%) were obtained from Witega (Berlin, Germany). Isoxsuprine (99.0%) and mabuterol (98.0%) were supplied by Fluka (Bucks, Switzerland) and TRC (Toronto, Canada), respectively. Clorprenaline (98.0%) was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Their chemical structures are showed in Fig. 1. HPLC grade methanol, acetonitrile and formic acid (FA, ≥99.0%) were purchased from TEDIA Inc. (USA). Analytical grade acetic acid (≥99.5%), sodium acetate (≥99.0%), perchloric acid (70.0–72.0%), sodium hydroxide (≥96.0%), sodium chloride (≥99.5%), isopropanol (≥99.7%), ethyl acetate (≥99.5%) and ammonium hydroxide (25.0–28.0%) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). ˇ-Glucuronidase/arylsulfatase from Helix pomatia was purchased from Roche Diagnostica GmbH (Mannheim, Germany). Bond Elut Plexa PCX solid phase extraction (SPE) cartridges were supplied by Agilent (USA). Ultrapure water (18.2 M) was obtained from a Milli-Q Advantage A10 ultrapure water purification system. 2.2. Instrumentation The UHPLC–Q-Orbitrap HRMS system is composed of an Accela 1250 LC pump and an Accela open autosampler coupled with a Q ExactiveTM HR mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). XCalibur 2.2 software (Thermo Fisher Scientific, San Jose, CA, USA) was used for instrument control and data processing, while Q Exactive 2.1 (for tune application) software (Thermo Fisher Scientific) was used to control the mass spectrometer. Chromatographic separation was achieved on a Hypersil GOLD aQ C18 chromatographic column (100 mm × 2.1 mm, 1.9 m) (Thermo Fisher Scientific).
2.4. Sample pretreatment An improved sample preparation procedure was proposed referring to several methods that have been reported [13,14]. An aliquot of 2.00 g homogenized food animal muscles and 8 mL sodium acetate (0.2 mol/L, pH 5.2) were added to a 50 mL polypropylene centrifuge tube and mixed thoroughly. The mixture was then incubated with 50 L ˇ-glucuronidase/arylsulfatase for 12 h at 37 ◦ C in water bath. After being cooled down to room temperature, 100 L mixed ISTD solution (100 ng/mL) was added to the enzyme digested sample. After horizontal oscillation for 15 min and centrifugation for 10 min (9391 × g), 4 mL extract was transferred into another centrifuge tube. Afterward, 5 mL of 0.1 mol/L perchloric acid was added and the pH was adjusted to 1.0 with 1.0 mol/L perchloric acid. The mixture was centrifuged again at 9391 × g for 10 min and the pH of the extract was adjusted to 11.0 with 10 mol/L NaOH solution. 10 mL saturated sodium chloride solution and 10 mL isopropanol–ethyl acetate (6:4, v/v) was added, followed by vortex for 1 min and centrifugation at 9391 × g for 10 min. 5 mL extract was collected and evaporated to dryness with nitrogen evaporator at 40 ◦ C in water bath. The residue was dissolved in 5 mL sodium acetate (0.2 mol/L, pH 5.2), followed by sonication for 5 min. Whereafter, the residue solution was subjected to SPE. The Bond Elut Plexa PCX (3 mL, 60 mg) cartridge was sequentially conditioned with 3 mL methanol and 3 mL water. After the sample was loaded, the cartridge was washed with 2 mL H2 O, 2 mL 2% FA, 2 mL methanol and dried with vacuum. Finally, the analytes were eluted with 2 mL of methanol containing 5% ammonia. The eluent was evaporated with a gentle N2 stream at 40 ◦ C in water bath and reconstituted to 200 L with initial mobile phase. After vortex for 1 min and centrifugation at 9391 × g for 10 min, the extract solution was injected into chromatographic column for the Q-Orbitrap HRMS analysis. The sample equivalent in the final extract was 2.5 g/mL. 2.5. Chromatographic conditions Chromatographic separation was performed on a Hypersil Gold aQ C18 column (100 mm × 2.1 mm, 1.9 m). The autosampler tray temperature, column oven temperature, flow rate and injection volume were set at 20 ◦ C, 30 ◦ C, 300 L/min and 5 L, respectively. The mobile phase consisted of water (A) and acetonitrile (B), both containing 0.1% FA. The gradient used for eluting analytes with mobile phase is as follows: 0–0.5 min, 4% B; 0.5–2.0 min, 4% B–25% B; 2.0–7.0 min, 25% B–65% B; 7.0–7.5 min, 65% B–95% B; 7.5–9.5 min, 95% B; 9.6–12.0 min, 4% B.
2.3. Standard solutions 2.6. Mass spectrometry conditions Individual stock standard solutions were prepared in methanol at concentration of 1 mg/mL and stored at −20 ◦ C. The mixed working standard solutions were prepared daily by proportional dilution of the stock standard solutions. The salbutamol-d3 and clenbuterol-d9 stock ISTD solutions were prepared in methanol at 100 g/mL and stored at −20 ◦ C. The mixed working ISTD solutions were prepared daily by dilution of the stock ISTD solutions. To quantificationally analyze the 12 ˇ2 -agonists in food animal muscles, the matrix-matched calibration curves were constructed. Seven multi-component standard solutions were prepared by spiking standard mixture into blank mixed matrices at different concentrations. All animal muscle food samples including pork, beef, mutton and chicken were collected by Shandong Food and Drug Administration. Samples were received frozen and kept at −20 ◦ C until analysis.
The Q-Orbitrap HRMS was equipped with a HESI-II ion source and the positive ionization mode was applied. The spray voltage, capillary temperature, vaporizer temperature and were set to 3.0 kV, 350 ◦ C and 250 ◦ C, respectively. The sheath gas, auxiliary gas, sweep gas and S-lens RF level were set to 40, 15, 0 (arbitrary units) and 50 V, respectively. The analysis was executed in Full MS/ddMS2 (data-dependent MS2 ) mode, under which a Full MS scan event (SE1) was followed by a data-dependent scan applied with a fragmentation energy (SE2). The mass spectrometer acquired a Full MS scan at the resolution of 70,000 (FWHM at 200 m/z). The automatic gain control (AGC) target (the number of ions to fill C-Trap) was set to 1.0e6 with a maximum injection time (IT) of 100 ms. The Full MS scan range was from 150 to 400 m/z. For the dd-MS2 scan, the mass resolution was 35,000 FWHM with AGC target at 2.0e5 , maximum IT 50 ms, isolation window 0.4 m/z, underfill ratio 1.0%,
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intensity threshold 4.0e4 , apex trigger 3–5 s, exclude isotopes “on”, and dynamic exclusion 10.0 s. 2.7. Method validation Method validation was carried out with ˇ2 -agonists assay in herbal medicines and dietary supplements referring to the US Food and Drug Administration (USFDA) guidelines [24]. The validation parameters included selectivity, sensitivity, calibration curve, accuracy and precision, recovery, matrix effect and stability. The homogenized blank food animal muscles (pork, beef, mutton and chicken) were used as blank matrices in the following method validation. 2.7.1. Selectivity The selectivity of the analyzing method should be assayed using a minimum of six independent sources of the same matrix. The selectivity should be ensured at the lowest calibrated level (LCL). 2.7.2. Calibration curve For quantificational analysis, salbutamol-d3 was used as the ISTD of salbutamol, terbutaline and ractopamine, while clenbuterol-d9 was used as the ISTD of other ˇ2 -agonists. A matrixmatched calibration curve was constructed by linear regression of the ratios of chromatographic peak areas of the standards and the ISTD and the linearity was assessed by the coefficient of determination (r2 ). The LOD was the lowest concentration that can be detected giving a signal-to-noise ratio of at least 3-fold (S/N ≥ 3). The LCL was defined as the lowest concentration of the calibration curve, giving a signal-to-noise ratio of at least 10–fold (S/N ≥ 10), acceptable accuracy (80–120%) and sufficient precision (within 20%). 2.7.3. Accuracy and precision The accuracy was calculated from the ratio of the mean values of the detected concentration (Cdet ) and the nominal concentration (Cnom ) as following: (Cdet /Cnom ) × 100. The precision was expressed as relative standard deviation (RSD), which was calculated as RSD% = [standard deviation (SD)/Cdet ] × 100. 2.7.4. Recovery The recoveries of ˇ2 -agonists from matrices were evaluated by comparing the response of ˇ2 -agonists after extraction from matrices with the response of the same concentration analytes spiked into the eluent from blank matrices. Recoveries of 12 ˇ2 -agonists were determined at three levels (low, medium and high).
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2.7.5. Matrix effect The matrix effect was evaluated by analyzing the response of analytes prepared in solvent and in extracted blank matrix at the same concentration. The value of matrix effect can be calculated as (Eq. (1)): ME (%) = B/A × 100
(1)
A refers to the peak areas obtained from neat solution standards, while B refers to the corresponding peak areas of standards spiked after extraction from matrix [25,26]. 2.7.6. Stability The post-preparative stability was measured by repeatedly determining the processed QC samples which were kept in the autosampler (20 ◦ C) for 24 h. The concentrations of the samples were calculated according to original calibration standards. 2.8. Calculation The chromatograms were processed using XCalibur 2.2 software developed by Thermo Fisher Scientific. All calculations were completed in Microsoft Excel 2010 (Microsoft Co., Redmond, USA) or OriginPro 8.0 (OriginLab Co., Northampton, MA, USA). 3. Results and discussion 3.1. Development of the UHPLC–Q-Orbitrap HRMS method As discussed above, all analytes were measured in positive mode and the precursor ion selected was [M+H]+ in all cases. For each analyte, the experimentally determined mass of the corresponding ion was evaluated according to the theoretical mass calculated by Xcalibur 2.1. Mass deviations were defined as 106 × [(measured mass − theoretical mass)/theoretical mass] and expressed in ppm. All the MS parameters were optimized and set to the optimal values in order to provide the best response of the analytes. The optimal parameter values of the HESI source were summarized in Section 2.6. The simultaneous qualitative and quantitative analysis of ˇ2 -agonists was performed in Full MS/dd-MS2 mode, which includes a full scan followed by MS/MS scan of precursors in the inclusion list (Table 1). The inclusion list with the monoisotopic mass and specific NCEs of the precursors was utilized to generate sensitive products since precursors are selected in the quadrupole. For the selection of the mass resolution of the Full MS scan, compromise has to be made between selectivity and sensitivity. A higher mass resolution gives rise to a better mass accuracy and thereby better selectivity. However, an extremely high resolution (such as
Table 1 Retention time, accurate mass, NCEs, correlation coefficients, linear ranges and LCLs of 12 ˇ2 -agonists. Analyte
Retention time (min)
Salbutamol Salbutamol-d3 Terbutaline Cimaterol Cimbuterol Ractopamine Clorprenaline Clenbuterol-d9 Clenbuterol Bromchlorbuterol Brombuterol Isoxsuprine Mabuterol Mapenterol
3.98 3.96 3.98 4.11 4.54 5.19 5.26 5.65 5.68 5.84 6.01 6.16 6.34 6.89
Protonated ion mass [M+H]+
Theoretical
Experimental
240.1594 243.1783 226.1438 202.1339 234.1601 302.1751 214.0993 286.1434 277.0869 323.0343 366.9838 302.1751 311.1133 325.1289
240.1590 243.1778 226.1434 202.1336 234.1597 302.1750 214.0990 286.1429 277.0866 323.0337 366.9833 302.1750 311.1128 325.1282
Mass error (ppm)
Normalized collision energy (NCE)
Correlation coefficient (r2 )
−1.7 −2.1 −1.8 −1.5 −1.7 −0.3 −1.4 −1.7 −1.1 −1.9 −1.4 −0.3 −1.6 −2.2
22 40 58 48 53 32 29 34 12 42 16 32 16 42
0.9992 0.9990 0.9987 0.9991 0.9994 0.9988 0.9995 0.9988 0.9991 0.9996 0.9986 0.9987 0.9992 0.9990
Linear range (g/kg)
0.01–50 0.01–50 0.03–50 0.01–50 0.01–50 0.03–50 0.01–50 0.03–50 0.01–50 0.01–50 0.01–50 0.01–50 0.01–50 0.01–50
LCL (g/kg)
0.01 0.01 0.03 0.01 0.01 0.03 0.01 0.03 0.01 0.01 0.01 0.01 0.01 0.01
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Fig. 2. Extracted ion chromatograms of ˇ2 -agonists in (A) a blank pork matrix and (B) a pork matrix spiked with 1 g/kg ˇ2 -agonists standards.
140,000 FWHM) would significantly affect the sensitivity due to the reduced scanning speed, given the same experimental time. To strike a balance between the selectivity and the sensitivity with Full MS, a mass resolution of 70,000 FWHM was selected and this
turned out to be optimal for majority of the analytes. For the ddMS2 scan, 35,000 FWHM was used for time-saving and to ensure sufficient scan points of the Full MS. Under such conditions, the fragment ions can be used for further identification of the target
Fig. 3. Suggested fragmentation profiles of ˇ2 -agonists.
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Table 2 Comparison of methods for the detection of ˇ2 -agonists in food animal muscle matrices. Method
The number of ˇ2 -agonists
LOD (case with clenbuterol)
Linear range (case with clenbuterol)
Application
References
CE CE LC–MS LC–MS LC–MS HRMS
5 4 7 16 3 12
0.10 g/mL 22.45 ng/mL 0.05 g/kg 0.06 g/kg 0.12 g/kg 0.0033 g/kg (viz. 0.0165 ng/mL)
0.20–100 g/mL 0.069–2.77 g/mL / 0.1–10 g/kg 0.5–20 g/kg 0.01–50 g/kg (viz. 0.025–125 ng/mL)
Pig feed, pig urine and pig liver Pig feed and urine Animal liver and urine Pig liver, kidney and muscle Pork Food animal muscles
[10] [11] [13] [14] [15] Present paper
analytes. The data-dependent approach employed here should better be described as exact-mass-dependent acquisition as it merely depends on the exact mass in the inclusion list to trigger dd-MS/MS and the fragmentation is carried out with the NCEs specified in the inclusion list. In addition, the selected mass tolerance window was also considered as a crucial parameter regarding selectivity. The optimal value for this parameter (in ppm) is compound-specific and dependent on the detection capability, signal intensity and occurrence of matrix interferences [27]. In the case of ˇ2 -agonists, all mass spectra were filtered through a mass tolerance window of 5 ppm to reconstruct extracted ion chromatograms (XICs) and to acquire adequate selectivity. The mass window of 5 ppm was sufficient to eliminate the background from the complex matrix and to generate a sharp and resolved peak, which represents the specific compound without interference. The peak shape and the chromatographic resolution were the main criteria of evaluation during optimization of the UHPLC method. The Hypersil GOLD aQ C18 chromatographic column, which was used throughout this study, provides sharp absorbance peaks and excellent chromatographic resolution. Since all the analytes contain basic groups, 0.1% FA was added to the mobile phase to enhance the protonation and sensitivity. Accordingly, 0.1% FA H2 O–0.1% FA acetonitrile was selected as the binary mobile solvents system. The elution gradient procedure enabled chromatographic separation of 12 ˇ2 -agonists within 12 min. A typical extracted ion chromatogram of matrices spiked with 1 g/kg ˇ2 -agonists standards is shown in Fig. 2B. The result shows that the retention time (RT) ranged from 3.98 min (salbutamol) to 6.89 min (mapenterol). The theoretical and experimental masses for each analyte are given in Table 1. The mass of all the precursor ions corresponded to that of protonated molecules ([M+H]+ ). As shown in Table 1, mass measurement accuracy was less than 2.2 ppm, indicating a high level of confidence between the theoretical values and experimental masses measured by Orbitrap HRMS for all analytes. Furthermore, the MS2 product ions of analytes (showed in Fig. 3) were utilized as diagnostic ions for each compound by accurate mass measurements and would be considered for further identification.
3.2. Method validation 3.2.1. Selectivity The selectivity of the analyzing method was assayed using six independent sources of the same matrix. Fig. 2A shows the representative chromatograms of a blank pork matrix, while a pork matrix spiked with 1 g/kg ˇ2 -agonists is shown in Fig. 2B. The reconstruction of XICs with a narrow mass tolerance window (5 ppm) provided sufficient resolving power to distinguish analytes from isobaric co-eluting sample matrix compounds. No interfering peaks were detected at RTs of all analytes.
3.2.2. Calibration curve The LCLs and LODs were determined as the lowest analyte concentrations that produced signal-to-noise (S/N) ratios of at least 3 and 10, respectively, via the analysis of the target compounds in blank matrix. All LOD and LOQ values were in ranges of 0.0033–0.01 and 0.01–0.03 g/kg, respectively. Series of matrix-matched calibration curves were constructed by linear regression of the ratios of chromatographic peak areas of the standards and the deuterated ISTD versus nominal concentrations. The linearity of calibration curve was assessed by the coefficients of determination (r2 ). As presented in Table 1, the calibration curves showed great linearity as the coefficients of determination (r2 ) are higher than 0.9986 in all the measurements. In comparison with other methods reported, the present Orbitrap HRMS method offers higher sensitivity and broader linear ranges (shown in Table 2). Besides, the number of the analytes that can be measured simultaneously (12) was more than most of these methods. 3.2.3. Accuracy and precision Table 3 gives an overview of the values of the intra-day and inter-day accuracy and precision at three spiking levels (low, medium and high). The intra-day and inter-day accuracy of ˇ2 agonists ranged from 96.2% to 104.1% and 94.5% to 106.3%, respectively. The intra- and inter-day precision were in the range of 0.4–7.6% and 0.7–8.7%, respectively. These results indicated that the method was reliable for accurate and precise analysis. 3.2.4. Recovery and matrix effect The analyte recovery of this procedure was evaluated by spiked blank samples at three concentrations (low, medium and high), with each condition performed in five replicates. As shown in Table 3, the mean recovery of each compound was in the range of 73.0–88.7%. The results of matrix effects evaluation are shown in Table 3. Signal suppression or enhancement effect was considered tolerable if the matrix effect value was between 80% and 120%. As shown in Table 3, the matrix effects of analytes ranged from 83.7% to 92.8% with SDs less than 5.5%. This suggests that the food animal muscles matrix have tolerable low suppression on the MS response of ˇ2 agonists. With such low level of matrix effects, this method would be reliable for analysis in food animal muscles matrix. 3.2.5. Stability As summarized in Table 3, the post-preparative stability was expressed as a percentage of the initial concentration (first analyzed batch) of the analytes in QC samples. The values of all analytes stabilities in matrices were in the range of 96.9–103.6%, indicating that all 12 ˇ2 -agonists were stable in auto sampler at 20 ◦ C for 24 h. The results suggested that the established method was reliable and suitable for large-scale sample screening.
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Table 3 Accuracy, precision, matrix effect, recovery and stability for the determination of 12 ˇ2 -agonists in food animal muscle matrices (n = 5). Analyte
Salbutamol
Terbutaline
Cimaterol
Cimbuterol
Ractopamine
Clorprenaline
Clenbuterol
Bromchlorbuterol
Brombuterol
Isoxsuprine
Mabuterol
Mapenterol
QC concentration (g/kg)
0.012 0.8 40 0.012 0.8 40 0.04 1.0 40 0.012 0.8 40 0.012 0.8 40 0.04 1.0 40 0.012 0.8 40 0.04 1.0 40 0.012 0.8 40 0.012 0.8 40 0.012 0.8 40 0.012 0.8 40
Accuracy (%)
Precision (RSD, %)
Intra-day
Inter-day
Intra-day
Inter-day
102.6 102.1 100.7 102.0 101.8 97.9 98.4 98.1 101.5 102.5 99.2 98.4 96.8 103.0 98.4 96.9 102.3 103.2 97.6 100.8 98.2 96.3 97.1 101.0 98.5 104.1 99.3 96.6 97.0 101.6 102.9 103.8 98.4 103.5 96.2 101.6
105.1 103.4 102.5 102.9 104.1 96.8 97.6 96.9 102.9 105.3 96.6 95.8 96.1 104.8 99.0 94.5 103.7 104.4 97.2 103.4 97.7 97.6 96.4 98.7 97.2 106.3 96.7 95.7 96.2 103.8 104.2 105.5 97.5 105.0 95.3 103.7
4.9 3.8 1.2 5.7 1.1 1.4 3.4 6.8 2.5 7.6 1.3 2.3 1.9 4.8 0.6 7.2 1.1 0.9 4.4 4.6 0.4 0.9 3.2 1.3 5.7 3.2 0.7 3.2 5.3 0.8 5.1 6.1 1.1 6.7 2.5 1.2
6.7 4.4 2.5 7.0 1.8 1.7 4.0 8.6 3.4 8.7 2.5 3.0 2.7 6.3 1.1 8.2 1.9 1.3 5.5 4.9 0.7 2.1 4.6 1.6 6.5 3.4 1.0 4.0 6.5 2.3 5.7 6.7 1.6 8.2 4.0 1.7
3.3. Application of the Orbitrap HRMS method to real samples The Orbitrap HRMS method was applied to the simultaneous qualitative and quantitative analysis of ˇ2 -agonists in 400 batches of pork, beef, mutton and chicken meat, which were collected by Shandong Food and Drug Administration. These food animal muscles samples were most representative because the sampling locations covered slaughterhouses, farm product markets, supermarkets and restaurants all over the Shandong Province. Considering the fact that ˇ2 -agonists have been frequently detected in food animal muscles in China for the past few years, the significance of a novel screening assay with high sensitivity and selectivity in this field is obvious. The identification of the target compounds was based on the RT windows (RTWs) and exact mass. RTWs were defined as the average RT plus or minus 3-fold SD value of the RT (RT ± 3 × SD). If there was no signal at all or the signal was below the LOD level within the RTW, as well as the exact mass error was beyond 5 ppm, the sample was considered negative. On the other hand, if a signal was detected showing an exact mass error
