Rapid Screening for 15 Sulfonamide Residues in Foods of Animal Origin by High-Performance Liquid Chromatography–UV Method

Rapid Screening for 15 Sulfonamide Residues in Foods of Animal Origin by High-Performance Liquid... Abstract A rapid and simple high-performance liquid chromatography–ultraviolet method was developed for the separation and quantification of 15 sulfonamides (SAs) in foods of animal origin without the need of clean-up procedure. A mixture of acetonitrile–formic acid–ammonium acetate–water was used as the mobile phase to separate 15 SAs on a C18 column with gradient. The selected SAs were separated completely from the matrix mixture based on different retention behaviors at different concentration of acetonitrile. The effects of the additive of formic acid and ammonium acetate in mobile phases on the separation of SAs were also investigated. The additive can greatly improve the resolution between SAs and impurities, so that the SAs can be quantified directly under the optimized chromatographic condition the sample preparation which does not need extra sample clean-up procedure. Complete baseline separation of 15 SAs was achieved in <40 min, the linear range is 0.01–130 μg/mL with a correlation coefficient R2-value > 0.999. Excellent method reproducibility was found by intra- and inter-day precisions with the relative standard deviation <9.5%. The detection limit was <11.0 ng/mL and it can be used for routine screening of the SA residues in foods of animal origin. Introduction Sulfonamides (SAs) represent a class of antibacterial compounds. They were widely used in the prophylaxis and treatment of bacterial diseases in veterinary clinic (1). Excessive or uncontrolled dosages to animals before sales to consumers can cause residual problems in food of animal origin. Regular consumption of food containing antibiotics can cause allergic reactions and antibiotic resistance in humans (2, 3). To limit these possible adverse consequences, each country has set limits for antibiotic residues in original animal food; both the European Union and China set a maximum residue limit (MRL) of 100 μg/kg for SA. In order to meet the needs of daily large number of routine determination of antibiotic residue, a simply and rapid screening method for the quantification of SAs is necessary. A survey found that the levels of residues of SA have decreased over the years due to optimization of administration schemes and application of co-medication to avoid detection. Many laboratories now use a combination of screening and confirmatory methods to cope with relatively large numbers of samples and to increase the reliability of the final result. Confirmatory methods only include liquid chromatography/tandem mass spectrometry (4–6) and gas chromatography/tandem mass spectrometry (7). These confirmatory methods based on chromatographic approaches can provide both qualitative and quantitative detection results with satisfactory sensitivity and reproducibility. However, they require expensive instrumentation and more expense, and thus are not suitable for routine inspection. As the alternative of confirmatory methods, various routine screening methods for the determination of SA residuals in animal tissue and body fluids have been developed. They include high-performance liquid chromatography (HPLC) coupled with fluorescence (8) or ultraviolet (UV) (9–13), capillary electrophoresis (14), electrochemiluminescence (15), molecularly imprinted polymer (16–18) and the enzyme linked immunosorbent assay (19). Among them, derivatization of SAs with fluorescamine can separate and detect several SAs, but introduces another variability and may complicate the methodology in a screening procedure (20, 21), and high-performance liquid chromatography–ultraviolet (HPLC–UV) is one of most widely used methods in all the inspection centers and labs, but the current established methods need sample clean-up steps or were only able to determine a few SA residuals in one chromatographic run. This work was to develop a simply HPLC–UV analytical method for simultaneous determination of 15 SAs with simplified sample extraction for routine analysis, which was used as a screening method before be identified by confirmatory methods. The linearity of calibration curve, recovery, precision and lower limit of detection (LLD) were studied to evaluate the developed method. The method was successfully used to determine the content of SA residues in pork, beef, mutton and milk. Methods Chemicals, standards solutions and materials The 15 sulfonamide standards, including sulfasulfacetamide (SA), sulfadiazine (SDZ), sulfathiazole (STZ), sulfapyridine (SP), sulfamerazine (SMR), and sulfamethazine (SMZ), sulfamethoxypyridazine (SMP), sulfameter (SM), sulfamonomethoxine (SMM), sulfachloropyridazine (SCP), sulfadoxine (SDX), sulfamethoxazole (SMX), sulfisoxazole (SIZ), sulfadimethoxine (SDM) and sulfaphenazole (SPP), (Table I), were purchased from the laboratories of Dr Ehrenstorfer (Augsburg, Germany). Stock solutions of the 15 SAs were prepared by dissolving 10 mg of each compound in 100 mL methanol. Pork, beef and mutton were obtained from the supermarket and preserved at − 20°C until use. Fresh whole milk was purchased from supermarket and stored at 4°C before use. Acetonitrile, isopropanol and methanol were obtained from Fisher (HPLC grade, New Jersey, USA). Formic acid and ammonium acetate were obtained from Tianjin Chemical Reagents Company (Tianjin, China). Deionized water was prepared by using a Milli-Q water filtration system (EMD Millipore Corp., Billerica, MA, USA). All solvents for HPLC were filtered through 0.45-mm filters (Millipore) and degassed in an ultrasonic bath. Table I. Structures and molecular weights of SAs samples Antibiotics  Symbol  MW  Structure  Sulfacetamide  SA  214.2    Sulfadiazine  SDZ  250.3    Sulfathiazole  STZ  255.3    Sulfapyridine  SP  249.3    Sulfamerazine  SMR  264.3    Sulfamethazine  SMZ  278.3    Sulfamethoxypyridazine  SMP  280.3    Sulfameter  SM  280.3    Sulfamonomethoxine  SMM  280.3    Sulfachloropyridazine  SCP  284.7    Sulfadoxine  SDX  310.3    Sulfamethoxazole  SMX  253.3    Sulfisoxazole  SIZ  267.3    Sulfadimethoxine  SDM  310.3    Sulfaphenazole  SPP  314.4    Antibiotics  Symbol  MW  Structure  Sulfacetamide  SA  214.2    Sulfadiazine  SDZ  250.3    Sulfathiazole  STZ  255.3    Sulfapyridine  SP  249.3    Sulfamerazine  SMR  264.3    Sulfamethazine  SMZ  278.3    Sulfamethoxypyridazine  SMP  280.3    Sulfameter  SM  280.3    Sulfamonomethoxine  SMM  280.3    Sulfachloropyridazine  SCP  284.7    Sulfadoxine  SDX  310.3    Sulfamethoxazole  SMX  253.3    Sulfisoxazole  SIZ  267.3    Sulfadimethoxine  SDM  310.3    Sulfaphenazole  SPP  314.4    Table I. Structures and molecular weights of SAs samples Antibiotics  Symbol  MW  Structure  Sulfacetamide  SA  214.2    Sulfadiazine  SDZ  250.3    Sulfathiazole  STZ  255.3    Sulfapyridine  SP  249.3    Sulfamerazine  SMR  264.3    Sulfamethazine  SMZ  278.3    Sulfamethoxypyridazine  SMP  280.3    Sulfameter  SM  280.3    Sulfamonomethoxine  SMM  280.3    Sulfachloropyridazine  SCP  284.7    Sulfadoxine  SDX  310.3    Sulfamethoxazole  SMX  253.3    Sulfisoxazole  SIZ  267.3    Sulfadimethoxine  SDM  310.3    Sulfaphenazole  SPP  314.4    Antibiotics  Symbol  MW  Structure  Sulfacetamide  SA  214.2    Sulfadiazine  SDZ  250.3    Sulfathiazole  STZ  255.3    Sulfapyridine  SP  249.3    Sulfamerazine  SMR  264.3    Sulfamethazine  SMZ  278.3    Sulfamethoxypyridazine  SMP  280.3    Sulfameter  SM  280.3    Sulfamonomethoxine  SMM  280.3    Sulfachloropyridazine  SCP  284.7    Sulfadoxine  SDX  310.3    Sulfamethoxazole  SMX  253.3    Sulfisoxazole  SIZ  267.3    Sulfadimethoxine  SDM  310.3    Sulfaphenazole  SPP  314.4    Sample preparation from tissue and milk SAs in pork, beef and mutton tissues were extracted using a previously reported method with minor modification (10). Briefly, 5 g of pork, beef or mutton were finely homogenized in 20 mL acetonitrile in a homogenizer (Xinzhi DY89-1, Ningbo, China). The homogenate was then centrifuged at 10,000 × g for 5 min, and the supernatant was recovered and transferred to a 50-mL flask. The tissue residue was washed with 10 mL acetonitrile, repeated twice, and concentrated in an evaporator at 35–42°C under reduced pressure. The evaporated residue was then dissolved in 0.5 mL acetonitrile and transferred to a 5-mL vial. The solubilization of residue with acetonitrile was repeated five more times to maximize the recovery. The acetonitrile solvent was then evaporated under a gentle stream of nitrogen. The final residue was dissolved in 1.0 mL of acetonitrile and filtered through a 0.22-μm organic filter to remove insoluble materials before HPLC analysis. SAs in milk were extracted using a modified method (22). Briefly, 0.5 mL trichloroacetic acid was added into 4.5-mL milk sample and vibrated for a few seconds on a vortex mixer, and then centrifuged 114 at 10,000 × g for 5 min. Finally, the supernatant was collected with a volume adjusted to 5.0 mL using acetonitrile followed by filtering through 0.22-μm organic filters for HPLC analysis. SAs analysis by HPLC The analyses were carried using an Agilent Technologies HP1200 series HPLC system equipped with a quaternary gradient pump, a diode array detector, and ChemStation Data-Analysis System (Agilent, Palo Alto, CA, USA). Chromatographic separation was performed at ambient temperature on Inertsil ODS-3 (250 mm × 4.6 mm i.d., 5 μm particle) analytical columns (Shimadzu, Japan) at a flow rate of 0.8 mL/min via a ternary gradient. Eluent A consisted of formic acid–water, eluent B is formic acid in acetonitrile solution and eluent C is ammonium acetate aqueous solution. Separation was performed with a gradient shown in Table II. SAs were monitored with UV detector at a wavelength of 270 nm. Table II. Gradients used for liquid chromatography Time (min)  Eluent A (%) formic acid aqueous solution (25 mM)  Eluent B (%) formic acid in acetonitrile solution (25 mM)  Eluent C (%) ammonium acetate aqueous solution (10 mM)  0.01  62  8  30  3.00  62  8  30  16.00  45  25  30  32.00  35  35  30  40.00  30  40  30  Time (min)  Eluent A (%) formic acid aqueous solution (25 mM)  Eluent B (%) formic acid in acetonitrile solution (25 mM)  Eluent C (%) ammonium acetate aqueous solution (10 mM)  0.01  62  8  30  3.00  62  8  30  16.00  45  25  30  32.00  35  35  30  40.00  30  40  30  Table II. Gradients used for liquid chromatography Time (min)  Eluent A (%) formic acid aqueous solution (25 mM)  Eluent B (%) formic acid in acetonitrile solution (25 mM)  Eluent C (%) ammonium acetate aqueous solution (10 mM)  0.01  62  8  30  3.00  62  8  30  16.00  45  25  30  32.00  35  35  30  40.00  30  40  30  Time (min)  Eluent A (%) formic acid aqueous solution (25 mM)  Eluent B (%) formic acid in acetonitrile solution (25 mM)  Eluent C (%) ammonium acetate aqueous solution (10 mM)  0.01  62  8  30  3.00  62  8  30  16.00  45  25  30  32.00  35  35  30  40.00  30  40  30  Linear range and limit of detection The linear correlation and dynamic range of SAs detection were determined from calibration curves generated by serial analysis of SAs standards. A calibration curve for each compound was calculated based on peak areas obtained by serially injecting 20 μL of acetonitrile–diluted solutions of SA standards. Stock solutions of individual SAs were serially diluted and spiked into matrix solution extracted from the pork. The lowest detection limits were determined as concentration corresponding to three times of noise (S/N = 3). Precision The precision of the method was evaluated by using SAs-spiked samples. Five extractions of same SAs-spiked tissue sample were tested consecutively to determine intra-day relative standard deviations (RSDs). For inter-day precision, five extractions of same SAs-spiked tissue sample were tested in three sequences over 5 days. Recovery The recovery of the assay was determined by spiking known amounts of standards into test samples. Six SAs-spiked samples and six control samples were compared. For pork or beef sample, 2.5 or 0.5 μg of the 15 SAs antibiotics were directly spiked into 5.0 g tissues. For milk sample, 2.5 or 0.5 μg of the 15 SAs antibiotics were directly spiked into 5.0 mL milk. The extraction of SAs was performed as described in Sample preparation from tissue and milk. The control samples were subjected to the same procedure as test samples. Results Optimization of chromatographic conditions SAs vary substantially in their hydrophobicity. Numerous protonated and unprotonated forms of SAs are presented due to their amphoteric properties and wide pKa ranges between 1.85 and 7.4. Therefore, chromatographic separation of SAs is very difficult in a short LC run. In this study, mixtures of acetonitrile–water–formic acid–ammonium acetate were evaluated for their potential to separate SAs in complex matrix. A mobile phase with a high initial concentration of acetonitrile (~23%) was unable to separate most of SA standards added into the pork (Figure 1A), several peaks of SAs were severely trailing and overlapped. With decreasing the initial concentration of acetonitrile (~8%), the retention times of SAs were extended, and the SAs can be separated from the main interferences (Figure 1B). In order to further improve the resolution and sensitivity of peaks, formic acid was added into the mobile phase. The retention times of SAs were remarkably shortened and the resolution and sensitivity of peaks were improved dramatically when 2 mM of formic acid was used. Continue to increase the acidity of mobile phase, the SAs peaks were completely separated except for SDZ and STZ when the concentration of formic acid reached 25 mM (Figure 1D). The separation of SAs was very sensitive to the acidity of mobile phase due to stronger interaction between protonated forms of SAs and RP stationary phases. To better separate SDZ and STZ, ammonium acetate was also added into the mobile phase. With the increase of concentration, the resolution of SDZ and STZ became higher and higher (Figures 1E and F). When the concentration of ammonium acetate reached 10 mM, all SAs-spiked into the pork were baseline separated (Figure 1F). The optimized chromatographic conditions are shown in Table II. Figure 1. View largeDownload slide Chromatographic separation of the sample extracted from pork spiked with SAs standard solution (5 mg/ L each) with different mobile phase: (A) H2O (77%) + acetonitrile (23%); (B) H2O (92%) + acetonitrile (8%); (C) 2 mM formic acid (92%) + 2 mM formic acid in acetonitrile (8%). (D) 25 mM formic acid (92%) + 25 mM formic acid in acetonitrile (8%); (E) 25 mM formic acid (62%) + 25 mM formic acid in acetonitrile (8%) + 5 mM ammonium acetate (30%); (F) 25 mM formic acid (62%) + 25 mM formic acid in acetonitrile (8%) + 10 mM ammonium acetate (30%); in (A) chromatographic programs, the ratio of acetonitrile become from 23% to 40% within 40 min, and in all other chromatographic programs, the ratio of acetonitrile or formic acid in acetonitrile become from 8% to 40% within 40 min, and the ratio of ammonium acetate aqueous solution was kept unchanged. Peak 1: SA; 2: SDZ; 3: STZ; 4: SP; 5: SMR; 6: SMZ; 7: SMP; 8: SM; 9: SMM; 10: SCP; 11: SDX; 12: SMX; 13: SIZ; 14: SDM; 15: SPP. Figure 1. View largeDownload slide Chromatographic separation of the sample extracted from pork spiked with SAs standard solution (5 mg/ L each) with different mobile phase: (A) H2O (77%) + acetonitrile (23%); (B) H2O (92%) + acetonitrile (8%); (C) 2 mM formic acid (92%) + 2 mM formic acid in acetonitrile (8%). (D) 25 mM formic acid (92%) + 25 mM formic acid in acetonitrile (8%); (E) 25 mM formic acid (62%) + 25 mM formic acid in acetonitrile (8%) + 5 mM ammonium acetate (30%); (F) 25 mM formic acid (62%) + 25 mM formic acid in acetonitrile (8%) + 10 mM ammonium acetate (30%); in (A) chromatographic programs, the ratio of acetonitrile become from 23% to 40% within 40 min, and in all other chromatographic programs, the ratio of acetonitrile or formic acid in acetonitrile become from 8% to 40% within 40 min, and the ratio of ammonium acetate aqueous solution was kept unchanged. Peak 1: SA; 2: SDZ; 3: STZ; 4: SP; 5: SMR; 6: SMZ; 7: SMP; 8: SM; 9: SMM; 10: SCP; 11: SDX; 12: SMX; 13: SIZ; 14: SDM; 15: SPP. Linear range, limit of detection Under the optimized conditions, the linear ranges for SA, SDZ, STZ SP, SMR, SMZ, SMP, SM, SMM were 0.01–100 μg/mL, for SCP, SDX, SMX, SIZ being 0.02–110 μg/mL and for SDM and SPP being 0.025–130 μg/mL. The correlation coefficients of calibration curves were >0.999. The LLD were calculated based on a signal-to-noise ratio of 3, and the values varied from 6.5 to 11.0 ng/mL (Table III). The method provides sensitive detection with wide linear range. Table III. Linear correlation, ranges, and lower limit of detection of SAs selected Antibiotics  Regression equation  Coefficient (R2)  Linear range (μg/mL)  Limita (ng/mL)  SA  y = 586.94x−97.625  0.9997  0.01–100  6.5  SDZ  y = 539.84x + 111.71  0.9994  0.01–100  7.0  STZ  y = 414.37x−27.44  0.9999  0.01–100  7.5  SP  y = 509.54x−26.576  0.9999  0.01–100  8.0  SMR  y = 545.04x−20.619  0.9997  0.01–100  8.5  SMZ  y = 575.51x−98.697  0.9992  0.01–100  9.0  SMP  y = 497.41x−0.0923  0.9998  0.01–100  10  SM  y = 465.42x + 69.798  0.9993  0.01–100  10  SMM  y = 488.8x + 20.422  0.9997  0.01–100  11  SCP  y = 464.41x−21.589  0.9997  0.02–110  10  SDX  y = 523.5x−96.603  0.9993  0.02–110  9.5  SMX  y = 558.04x + 13.708  0.9993  0.02–110  9.5  SIZ  y = 552.62x−5.6391  0.9994  0.02–110  10  SDM  y = 511.39x−14.526  0.9992  0.025–130  10  SPP  y = 418.7x−60.568  0.9994  0.025–130  11  Antibiotics  Regression equation  Coefficient (R2)  Linear range (μg/mL)  Limita (ng/mL)  SA  y = 586.94x−97.625  0.9997  0.01–100  6.5  SDZ  y = 539.84x + 111.71  0.9994  0.01–100  7.0  STZ  y = 414.37x−27.44  0.9999  0.01–100  7.5  SP  y = 509.54x−26.576  0.9999  0.01–100  8.0  SMR  y = 545.04x−20.619  0.9997  0.01–100  8.5  SMZ  y = 575.51x−98.697  0.9992  0.01–100  9.0  SMP  y = 497.41x−0.0923  0.9998  0.01–100  10  SM  y = 465.42x + 69.798  0.9993  0.01–100  10  SMM  y = 488.8x + 20.422  0.9997  0.01–100  11  SCP  y = 464.41x−21.589  0.9997  0.02–110  10  SDX  y = 523.5x−96.603  0.9993  0.02–110  9.5  SMX  y = 558.04x + 13.708  0.9993  0.02–110  9.5  SIZ  y = 552.62x−5.6391  0.9994  0.02–110  10  SDM  y = 511.39x−14.526  0.9992  0.025–130  10  SPP  y = 418.7x−60.568  0.9994  0.025–130  11  aS/N = 3. Table III. Linear correlation, ranges, and lower limit of detection of SAs selected Antibiotics  Regression equation  Coefficient (R2)  Linear range (μg/mL)  Limita (ng/mL)  SA  y = 586.94x−97.625  0.9997  0.01–100  6.5  SDZ  y = 539.84x + 111.71  0.9994  0.01–100  7.0  STZ  y = 414.37x−27.44  0.9999  0.01–100  7.5  SP  y = 509.54x−26.576  0.9999  0.01–100  8.0  SMR  y = 545.04x−20.619  0.9997  0.01–100  8.5  SMZ  y = 575.51x−98.697  0.9992  0.01–100  9.0  SMP  y = 497.41x−0.0923  0.9998  0.01–100  10  SM  y = 465.42x + 69.798  0.9993  0.01–100  10  SMM  y = 488.8x + 20.422  0.9997  0.01–100  11  SCP  y = 464.41x−21.589  0.9997  0.02–110  10  SDX  y = 523.5x−96.603  0.9993  0.02–110  9.5  SMX  y = 558.04x + 13.708  0.9993  0.02–110  9.5  SIZ  y = 552.62x−5.6391  0.9994  0.02–110  10  SDM  y = 511.39x−14.526  0.9992  0.025–130  10  SPP  y = 418.7x−60.568  0.9994  0.025–130  11  Antibiotics  Regression equation  Coefficient (R2)  Linear range (μg/mL)  Limita (ng/mL)  SA  y = 586.94x−97.625  0.9997  0.01–100  6.5  SDZ  y = 539.84x + 111.71  0.9994  0.01–100  7.0  STZ  y = 414.37x−27.44  0.9999  0.01–100  7.5  SP  y = 509.54x−26.576  0.9999  0.01–100  8.0  SMR  y = 545.04x−20.619  0.9997  0.01–100  8.5  SMZ  y = 575.51x−98.697  0.9992  0.01–100  9.0  SMP  y = 497.41x−0.0923  0.9998  0.01–100  10  SM  y = 465.42x + 69.798  0.9993  0.01–100  10  SMM  y = 488.8x + 20.422  0.9997  0.01–100  11  SCP  y = 464.41x−21.589  0.9997  0.02–110  10  SDX  y = 523.5x−96.603  0.9993  0.02–110  9.5  SMX  y = 558.04x + 13.708  0.9993  0.02–110  9.5  SIZ  y = 552.62x−5.6391  0.9994  0.02–110  10  SDM  y = 511.39x−14.526  0.9992  0.025–130  10  SPP  y = 418.7x−60.568  0.9994  0.025–130  11  aS/N = 3. Precision and reproducibility Five samples extracted from same SAs-spiked tissue (0.1 μg/kg) or milk samples (0.1 μg/mL) were analyzed consecutively and also in different days (d = 5, n = 5). The precision was high for both intra-day (RSD values of 0.78–7.35%) and inter-day assay (RSD values of 1.52–9.87%), respectively. It is worth noting that the milk samples showed the lowest intra and inter-day RSD, which is probably due to less sample extracted. The results are shown in Table IV. Both inter- and intra-day RSDs are <10%, indicating a high degree of assay precision. Long-term reproducibility was also verified. After 14 months, we used the same method to separate the standards, compared with the chromatogram made 14 months before, the variation of retention time is not <0.1 min, and thus the reproducibility is good. Table IV. Analysis precisionsa for each SA compound Standard  Intra-day RSD (%, n = 5)  Inter-day RSD (%, n = 5)  Pork  Beef  Mutton  Milk  Pork  Beef  Mutton  Milk  SA  2.64  2.45  2.85  1.70  5.56  4.48  3.69  2.55  SDZ  3.87  5.24  3.31  0.89  6.36  7.31  4.54  1.63  STZ  3.64  6.86  3.29  0.78  6.39  6.83  4.95  1.52  SP  2.49  7.35  2.67  0.93  4.38  7.91  4.85  3.88  SMR  5.68  5.43  4.82  1.22  8.40  8.16  5.67  2.34  SMZ  6.57  4.10  6.12  2.53  9.87  6.28  6.98  2.02  SMP  4.82  3.22  4.19  3.31  7.52  8.24  5.58  2.49  SM  4.27  2.83  5.32  2.64  5.03  7.88  5.89  3.67  SMM  6.50  5.50  4.25  1.57  6.64  8.01  5.67  4.70  SCP  7.13  4.79  7.20  2.64  9.20  5.97  7.75  5.29  SDX  5.92  5.52  4.37  1.52  8.31  5.89  5.51  3.56  SMX  4.61  6.80  4.83  1.54  7.22  8.75  5.62  4.51  SIZ  2.71  3.25  5.27  1.87  5.28  6.06  5.99  5.83  SDM  3.83  4.79  4.91  3.04  4.63  5.60  6.01  3.16  SPP  4.65  4.31  4.88  2.72  5.90  4.46  6.12  5.97  Standard  Intra-day RSD (%, n = 5)  Inter-day RSD (%, n = 5)  Pork  Beef  Mutton  Milk  Pork  Beef  Mutton  Milk  SA  2.64  2.45  2.85  1.70  5.56  4.48  3.69  2.55  SDZ  3.87  5.24  3.31  0.89  6.36  7.31  4.54  1.63  STZ  3.64  6.86  3.29  0.78  6.39  6.83  4.95  1.52  SP  2.49  7.35  2.67  0.93  4.38  7.91  4.85  3.88  SMR  5.68  5.43  4.82  1.22  8.40  8.16  5.67  2.34  SMZ  6.57  4.10  6.12  2.53  9.87  6.28  6.98  2.02  SMP  4.82  3.22  4.19  3.31  7.52  8.24  5.58  2.49  SM  4.27  2.83  5.32  2.64  5.03  7.88  5.89  3.67  SMM  6.50  5.50  4.25  1.57  6.64  8.01  5.67  4.70  SCP  7.13  4.79  7.20  2.64  9.20  5.97  7.75  5.29  SDX  5.92  5.52  4.37  1.52  8.31  5.89  5.51  3.56  SMX  4.61  6.80  4.83  1.54  7.22  8.75  5.62  4.51  SIZ  2.71  3.25  5.27  1.87  5.28  6.06  5.99  5.83  SDM  3.83  4.79  4.91  3.04  4.63  5.60  6.01  3.16  SPP  4.65  4.31  4.88  2.72  5.90  4.46  6.12  5.97  aSpiking 0.1 μg/kg in the tissue sample and 0.1 μg/mL in the milk. Table IV. Analysis precisionsa for each SA compound Standard  Intra-day RSD (%, n = 5)  Inter-day RSD (%, n = 5)  Pork  Beef  Mutton  Milk  Pork  Beef  Mutton  Milk  SA  2.64  2.45  2.85  1.70  5.56  4.48  3.69  2.55  SDZ  3.87  5.24  3.31  0.89  6.36  7.31  4.54  1.63  STZ  3.64  6.86  3.29  0.78  6.39  6.83  4.95  1.52  SP  2.49  7.35  2.67  0.93  4.38  7.91  4.85  3.88  SMR  5.68  5.43  4.82  1.22  8.40  8.16  5.67  2.34  SMZ  6.57  4.10  6.12  2.53  9.87  6.28  6.98  2.02  SMP  4.82  3.22  4.19  3.31  7.52  8.24  5.58  2.49  SM  4.27  2.83  5.32  2.64  5.03  7.88  5.89  3.67  SMM  6.50  5.50  4.25  1.57  6.64  8.01  5.67  4.70  SCP  7.13  4.79  7.20  2.64  9.20  5.97  7.75  5.29  SDX  5.92  5.52  4.37  1.52  8.31  5.89  5.51  3.56  SMX  4.61  6.80  4.83  1.54  7.22  8.75  5.62  4.51  SIZ  2.71  3.25  5.27  1.87  5.28  6.06  5.99  5.83  SDM  3.83  4.79  4.91  3.04  4.63  5.60  6.01  3.16  SPP  4.65  4.31  4.88  2.72  5.90  4.46  6.12  5.97  Standard  Intra-day RSD (%, n = 5)  Inter-day RSD (%, n = 5)  Pork  Beef  Mutton  Milk  Pork  Beef  Mutton  Milk  SA  2.64  2.45  2.85  1.70  5.56  4.48  3.69  2.55  SDZ  3.87  5.24  3.31  0.89  6.36  7.31  4.54  1.63  STZ  3.64  6.86  3.29  0.78  6.39  6.83  4.95  1.52  SP  2.49  7.35  2.67  0.93  4.38  7.91  4.85  3.88  SMR  5.68  5.43  4.82  1.22  8.40  8.16  5.67  2.34  SMZ  6.57  4.10  6.12  2.53  9.87  6.28  6.98  2.02  SMP  4.82  3.22  4.19  3.31  7.52  8.24  5.58  2.49  SM  4.27  2.83  5.32  2.64  5.03  7.88  5.89  3.67  SMM  6.50  5.50  4.25  1.57  6.64  8.01  5.67  4.70  SCP  7.13  4.79  7.20  2.64  9.20  5.97  7.75  5.29  SDX  5.92  5.52  4.37  1.52  8.31  5.89  5.51  3.56  SMX  4.61  6.80  4.83  1.54  7.22  8.75  5.62  4.51  SIZ  2.71  3.25  5.27  1.87  5.28  6.06  5.99  5.83  SDM  3.83  4.79  4.91  3.04  4.63  5.60  6.01  3.16  SPP  4.65  4.31  4.88  2.72  5.90  4.46  6.12  5.97  aSpiking 0.1 μg/kg in the tissue sample and 0.1 μg/mL in the milk. Recovery Different samples including pork, beef, mutton and milk were selected for maximum variability with respect to provenance and matrix. Spiked samples at two levels: 0.5 μg/kg or 0.1 μg/kg for tissue and 0.5 μg/L or 0.1 μg/L for milk, were analyzed to evaluate the recovery (Table V). Excellent recoveries of 85–95% for high concentration samples and less satisfactory recoveries of 81–93% for low concentration samples were obtained. Table V. Recovery of residual SAs from pork, beef, mutton and milk matrices Standard  % Recovery  Pork  Beef  Mutton  Milk  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/mL  Spiking 0.1 μg/mL  SA  88.6 ± 5.3  86.9 ± 6.1  90.2 ± 6.6  84.8 ± 6.7  86.1 ± 4.2  90.4 ± 4.4  91.9 ± 3.9  90.7 ± 2.0  SDZ  84.6 ± 4.1  85.0 ± 4.5  89.3 ± 5.8  82.6 ± 5.8  85.4 ± 5.5  89.9 ± 5.7  92.2 ± 4.3  93.6 ± 4.2  STZ  91.3 ± 7.5  90.3 ± 5.9  89.7 ± 8.4  86.5 ± 4.2  88.6 ± 6.4  92.1 ± 5.3  94.8 ± 5.5  91.5 ± 3.6  SP  92.7 ± 4.8  87.6 ± 6.8  93.6 ± 6.1  87.8 ± 7.9  90.3 ± 4.8  86.8 ± 4.9  93.7 ± 5.0  93.3 ± 4.4  SMR  90.5 ± 6.7  85.8 ± 4.7  92.0 ± 5.7  91.6 ± 8.5  91.1 ± 5.5  91.5 ± 5.8  93.0 ± 4.4  91.9 ± 5.7  SMZ  87.8 ± 5.9  82.7 ± 5.2  88.9 ± 4.2  85.0 ± 6.4  89.0 ± 5.8  92.4 ± 6.4  90.6 ± 5.2  91.8 ± 5.3  SMP  90.4 ± 7.0  85.2 ± 6.0  91.0 ± 5.9  87.3 ± 6.7  92.7 ± 6.2  91.9 ± 6.1  92.7 ± 5.7  90.5 ± 4.7  SM  92.4 ± 5.7  88.6 ± 7.4  90.5 ± 6.7  86.0 ± 7.5  93.5 ± 7.1  89.8 ± 5.5  95.3 ± 4.9  93.1 ± 3.3  SMM  93.0 ± 4.4  90.4 ± 8.8  87.7 ± 7.4  82.7 ± 6.0  87.9 ± 6.3  90.8 ± 5.9  92.3 ± 3.6  92.2 ± 2.7  SCP  89.2 ± 7.2  85.2 ± 7.5  89.4 ± 7.3  88.9 ± 5.3  90.2 ± 4.5  91.4 ± 4.8  94.1 ± 2.4  90.3 ± 2.5  SDX  86.9 ± 5.8  81.9 ± 5.0  94.2 ± 8.0  85.1 ± 6.6  85.9 ± 5.4  92.5 ± 5.1  89.7 ± 4.7  91.0 ± 4.8  SMX  85.1 ± 4.7  84.7 ± 5.7  87.4 ± 6.3  88.3 ± 7.4  89.4 ± 4.7  91.8 ± 6.1  89.5 ± 2.5  87.6 ± 5.8  SIZ  89.6 ± 8.8  88.6 ± 6.9  83.7 ± 5.7  84.2 ± 6.2  87.7 ± 6.6  90.6 ± 5.7  88.6 ± 4.5  86.4 ± 4.2  SDM  85.6 ± 6.3  81.8 ± 5.6  85.8 ± 6.8  81.6 ± 5.8  91.6 ± 5.7  89.8 ± 6.6  86.9 ± 5.3  87.2 ± 4.9  SPP  86.5 ± 5.9  81.5 ± 6.0  87.9 ± 7.2  83.4 ± 5.5  92.3 ± 6.7  90.9 ± 6.0  87.3 ± 4.8  85.5 ± 5.2  Standard  % Recovery  Pork  Beef  Mutton  Milk  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/mL  Spiking 0.1 μg/mL  SA  88.6 ± 5.3  86.9 ± 6.1  90.2 ± 6.6  84.8 ± 6.7  86.1 ± 4.2  90.4 ± 4.4  91.9 ± 3.9  90.7 ± 2.0  SDZ  84.6 ± 4.1  85.0 ± 4.5  89.3 ± 5.8  82.6 ± 5.8  85.4 ± 5.5  89.9 ± 5.7  92.2 ± 4.3  93.6 ± 4.2  STZ  91.3 ± 7.5  90.3 ± 5.9  89.7 ± 8.4  86.5 ± 4.2  88.6 ± 6.4  92.1 ± 5.3  94.8 ± 5.5  91.5 ± 3.6  SP  92.7 ± 4.8  87.6 ± 6.8  93.6 ± 6.1  87.8 ± 7.9  90.3 ± 4.8  86.8 ± 4.9  93.7 ± 5.0  93.3 ± 4.4  SMR  90.5 ± 6.7  85.8 ± 4.7  92.0 ± 5.7  91.6 ± 8.5  91.1 ± 5.5  91.5 ± 5.8  93.0 ± 4.4  91.9 ± 5.7  SMZ  87.8 ± 5.9  82.7 ± 5.2  88.9 ± 4.2  85.0 ± 6.4  89.0 ± 5.8  92.4 ± 6.4  90.6 ± 5.2  91.8 ± 5.3  SMP  90.4 ± 7.0  85.2 ± 6.0  91.0 ± 5.9  87.3 ± 6.7  92.7 ± 6.2  91.9 ± 6.1  92.7 ± 5.7  90.5 ± 4.7  SM  92.4 ± 5.7  88.6 ± 7.4  90.5 ± 6.7  86.0 ± 7.5  93.5 ± 7.1  89.8 ± 5.5  95.3 ± 4.9  93.1 ± 3.3  SMM  93.0 ± 4.4  90.4 ± 8.8  87.7 ± 7.4  82.7 ± 6.0  87.9 ± 6.3  90.8 ± 5.9  92.3 ± 3.6  92.2 ± 2.7  SCP  89.2 ± 7.2  85.2 ± 7.5  89.4 ± 7.3  88.9 ± 5.3  90.2 ± 4.5  91.4 ± 4.8  94.1 ± 2.4  90.3 ± 2.5  SDX  86.9 ± 5.8  81.9 ± 5.0  94.2 ± 8.0  85.1 ± 6.6  85.9 ± 5.4  92.5 ± 5.1  89.7 ± 4.7  91.0 ± 4.8  SMX  85.1 ± 4.7  84.7 ± 5.7  87.4 ± 6.3  88.3 ± 7.4  89.4 ± 4.7  91.8 ± 6.1  89.5 ± 2.5  87.6 ± 5.8  SIZ  89.6 ± 8.8  88.6 ± 6.9  83.7 ± 5.7  84.2 ± 6.2  87.7 ± 6.6  90.6 ± 5.7  88.6 ± 4.5  86.4 ± 4.2  SDM  85.6 ± 6.3  81.8 ± 5.6  85.8 ± 6.8  81.6 ± 5.8  91.6 ± 5.7  89.8 ± 6.6  86.9 ± 5.3  87.2 ± 4.9  SPP  86.5 ± 5.9  81.5 ± 6.0  87.9 ± 7.2  83.4 ± 5.5  92.3 ± 6.7  90.9 ± 6.0  87.3 ± 4.8  85.5 ± 5.2  Table V. Recovery of residual SAs from pork, beef, mutton and milk matrices Standard  % Recovery  Pork  Beef  Mutton  Milk  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/mL  Spiking 0.1 μg/mL  SA  88.6 ± 5.3  86.9 ± 6.1  90.2 ± 6.6  84.8 ± 6.7  86.1 ± 4.2  90.4 ± 4.4  91.9 ± 3.9  90.7 ± 2.0  SDZ  84.6 ± 4.1  85.0 ± 4.5  89.3 ± 5.8  82.6 ± 5.8  85.4 ± 5.5  89.9 ± 5.7  92.2 ± 4.3  93.6 ± 4.2  STZ  91.3 ± 7.5  90.3 ± 5.9  89.7 ± 8.4  86.5 ± 4.2  88.6 ± 6.4  92.1 ± 5.3  94.8 ± 5.5  91.5 ± 3.6  SP  92.7 ± 4.8  87.6 ± 6.8  93.6 ± 6.1  87.8 ± 7.9  90.3 ± 4.8  86.8 ± 4.9  93.7 ± 5.0  93.3 ± 4.4  SMR  90.5 ± 6.7  85.8 ± 4.7  92.0 ± 5.7  91.6 ± 8.5  91.1 ± 5.5  91.5 ± 5.8  93.0 ± 4.4  91.9 ± 5.7  SMZ  87.8 ± 5.9  82.7 ± 5.2  88.9 ± 4.2  85.0 ± 6.4  89.0 ± 5.8  92.4 ± 6.4  90.6 ± 5.2  91.8 ± 5.3  SMP  90.4 ± 7.0  85.2 ± 6.0  91.0 ± 5.9  87.3 ± 6.7  92.7 ± 6.2  91.9 ± 6.1  92.7 ± 5.7  90.5 ± 4.7  SM  92.4 ± 5.7  88.6 ± 7.4  90.5 ± 6.7  86.0 ± 7.5  93.5 ± 7.1  89.8 ± 5.5  95.3 ± 4.9  93.1 ± 3.3  SMM  93.0 ± 4.4  90.4 ± 8.8  87.7 ± 7.4  82.7 ± 6.0  87.9 ± 6.3  90.8 ± 5.9  92.3 ± 3.6  92.2 ± 2.7  SCP  89.2 ± 7.2  85.2 ± 7.5  89.4 ± 7.3  88.9 ± 5.3  90.2 ± 4.5  91.4 ± 4.8  94.1 ± 2.4  90.3 ± 2.5  SDX  86.9 ± 5.8  81.9 ± 5.0  94.2 ± 8.0  85.1 ± 6.6  85.9 ± 5.4  92.5 ± 5.1  89.7 ± 4.7  91.0 ± 4.8  SMX  85.1 ± 4.7  84.7 ± 5.7  87.4 ± 6.3  88.3 ± 7.4  89.4 ± 4.7  91.8 ± 6.1  89.5 ± 2.5  87.6 ± 5.8  SIZ  89.6 ± 8.8  88.6 ± 6.9  83.7 ± 5.7  84.2 ± 6.2  87.7 ± 6.6  90.6 ± 5.7  88.6 ± 4.5  86.4 ± 4.2  SDM  85.6 ± 6.3  81.8 ± 5.6  85.8 ± 6.8  81.6 ± 5.8  91.6 ± 5.7  89.8 ± 6.6  86.9 ± 5.3  87.2 ± 4.9  SPP  86.5 ± 5.9  81.5 ± 6.0  87.9 ± 7.2  83.4 ± 5.5  92.3 ± 6.7  90.9 ± 6.0  87.3 ± 4.8  85.5 ± 5.2  Standard  % Recovery  Pork  Beef  Mutton  Milk  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/mL  Spiking 0.1 μg/mL  SA  88.6 ± 5.3  86.9 ± 6.1  90.2 ± 6.6  84.8 ± 6.7  86.1 ± 4.2  90.4 ± 4.4  91.9 ± 3.9  90.7 ± 2.0  SDZ  84.6 ± 4.1  85.0 ± 4.5  89.3 ± 5.8  82.6 ± 5.8  85.4 ± 5.5  89.9 ± 5.7  92.2 ± 4.3  93.6 ± 4.2  STZ  91.3 ± 7.5  90.3 ± 5.9  89.7 ± 8.4  86.5 ± 4.2  88.6 ± 6.4  92.1 ± 5.3  94.8 ± 5.5  91.5 ± 3.6  SP  92.7 ± 4.8  87.6 ± 6.8  93.6 ± 6.1  87.8 ± 7.9  90.3 ± 4.8  86.8 ± 4.9  93.7 ± 5.0  93.3 ± 4.4  SMR  90.5 ± 6.7  85.8 ± 4.7  92.0 ± 5.7  91.6 ± 8.5  91.1 ± 5.5  91.5 ± 5.8  93.0 ± 4.4  91.9 ± 5.7  SMZ  87.8 ± 5.9  82.7 ± 5.2  88.9 ± 4.2  85.0 ± 6.4  89.0 ± 5.8  92.4 ± 6.4  90.6 ± 5.2  91.8 ± 5.3  SMP  90.4 ± 7.0  85.2 ± 6.0  91.0 ± 5.9  87.3 ± 6.7  92.7 ± 6.2  91.9 ± 6.1  92.7 ± 5.7  90.5 ± 4.7  SM  92.4 ± 5.7  88.6 ± 7.4  90.5 ± 6.7  86.0 ± 7.5  93.5 ± 7.1  89.8 ± 5.5  95.3 ± 4.9  93.1 ± 3.3  SMM  93.0 ± 4.4  90.4 ± 8.8  87.7 ± 7.4  82.7 ± 6.0  87.9 ± 6.3  90.8 ± 5.9  92.3 ± 3.6  92.2 ± 2.7  SCP  89.2 ± 7.2  85.2 ± 7.5  89.4 ± 7.3  88.9 ± 5.3  90.2 ± 4.5  91.4 ± 4.8  94.1 ± 2.4  90.3 ± 2.5  SDX  86.9 ± 5.8  81.9 ± 5.0  94.2 ± 8.0  85.1 ± 6.6  85.9 ± 5.4  92.5 ± 5.1  89.7 ± 4.7  91.0 ± 4.8  SMX  85.1 ± 4.7  84.7 ± 5.7  87.4 ± 6.3  88.3 ± 7.4  89.4 ± 4.7  91.8 ± 6.1  89.5 ± 2.5  87.6 ± 5.8  SIZ  89.6 ± 8.8  88.6 ± 6.9  83.7 ± 5.7  84.2 ± 6.2  87.7 ± 6.6  90.6 ± 5.7  88.6 ± 4.5  86.4 ± 4.2  SDM  85.6 ± 6.3  81.8 ± 5.6  85.8 ± 6.8  81.6 ± 5.8  91.6 ± 5.7  89.8 ± 6.6  86.9 ± 5.3  87.2 ± 4.9  SPP  86.5 ± 5.9  81.5 ± 6.0  87.9 ± 7.2  83.4 ± 5.5  92.3 ± 6.7  90.9 ± 6.0  87.3 ± 4.8  85.5 ± 5.2  Validation procedure This method was validated with other three different tested samples, including beef, mutton and milk. Typical chromatograms of the blank and spiked samples are shown in Figure 2. SAs were not detected in blank samples and there were no significant interference peaks observed at the retention time of SA standard. All of the spiked SA standards were well separated and detected with low abundance peaks from matrix partially overlapped with the SAs. The quantitative results were still satisfactory because the interference from the overlapped peaks were minimal. Figure 2. View largeDownload slide Typical chromatograms for three spiked (A) and blank samples (B). A: beef; B: mutton; C: milk. The concentrations of spiked analytes are 0.1 μg/kg or 0.1 μg/L. The chromatographic program were the same as Table II. Peaks 1: SA; 2: SDZ; 3: STZ; 4: SP; 5: SMR; 6: SMZ; 7: SMP; 8: SM; 9: SMM; 10: SCP; 11: SDX; 12: SMX; 13: SIZ; 14: SDM; 15: SPP. Figure 2. View largeDownload slide Typical chromatograms for three spiked (A) and blank samples (B). A: beef; B: mutton; C: milk. The concentrations of spiked analytes are 0.1 μg/kg or 0.1 μg/L. The chromatographic program were the same as Table II. Peaks 1: SA; 2: SDZ; 3: STZ; 4: SP; 5: SMR; 6: SMZ; 7: SMP; 8: SM; 9: SMM; 10: SCP; 11: SDX; 12: SMX; 13: SIZ; 14: SDM; 15: SPP. Application to samples The method was used to analyze 142 pork samples from the local Food and Drug Administration. The samples were randomly obtained from the supermarket, free market and street vendors of whole Shaanxi province in China according to the local government monitoring plan. Twenty-five samples containing SAs residues were detected. Positive milk samples containing 3, 2 or 1 SAs were 1.5%, 7.4% and 8.7%, respectively. The frequent SAs residues detected in pork samples were STZ, SMR, SMP SM and SDX. The SAs content of the positive samples ranged from 6 μg/kg to 163 μg/kg. Eight samples were detected with the levels of SAs residues higher than the established MRL. The results were consistent with that of the local Food and Drug Administration and showed that this method was able to screen samples for SAs routinely before performing confirmatory tests. Discussion The quantification of SAs can be severely interfered by the impurity from complex matrix (23). Therefore, extensive sample preparation are necessary to remove interferences, such as liquid–liquid extraction (24), dispersive liquid–liquid microextraction (22, 25, 26), matrix solid-phase dispersion (27), solid-phase extraction (28–30) and solid-phase microextraction (21, 31, 32). But the clean-up steps are labor-intensive and time-consuming leading to low recoveries and low throughput. Numerous efforts have been reported to emphasize these shortcomings. Complete elimination of the clean-up step was developed for determination of the SAs content of fish (22, 33), calf and pig tissue (10) by HPLC–UV, but these simplified methods can be only able to simultaneous evaluate fewer SAs in one chromatographic run. The extraction procedure for SAs we developed didn’t need extra sample clean-up step, which reduced the sample preparation and boring physical labor. In the reported HPLC methods for SAs separation, most of that used inorganic acids or salts as an additive to improve the peaks’ resolution or shape. However, when the proportion of water in the mobile phase is low, inorganic acids or salts easily precipitate from the mobile phase, these very tiny precipitate particles can damage the pump head of HPLC instrument (34). In this method, we use the formic acid and ammonium acetate as the mobile phase additive, which are easily soluble in organic solvents. Since the isolated sulfonamides have both an acid residue and a base residue, it is an amphoteric molecule and, if directly separated, exhibits a variety of properties, making the separation more complex. In order to decrease this complexity, we add formic acid in the mobile phase and reduce the pH of the mobile phase, so the basic group will be protonated and acidic groups will not ionize. Then we add salt, utilizing the effect of its ion pair to make sulfonamides only show different hydrophobicity so that they are easier to separate. In addition, organic additives were compatible with mass spectrum and can be apply to the LC-MS or GC-MS of confirmatory method. Therefore, using of organic additives soluble in solvents is very important. In general, C18 stationary phase can be tolerated in a pH range of 2–8. Originally, we wanted to use the pH determination of flow meter pH, but due to the interference caused by the mobile phase containing acetonitrile, the pH of the mobile phase is very difficult to accurately determine. However, we can roughly estimate the pH of the mobile phase according to its pKa: The final concentration of formic acid in mobile phase is 0.025 M × 0.7 = 0.0175 M, and its pKa = 3.75; In the mobile phase [H+]≈Ka∙C=10−3.75×0.0175≈0.00176, pH = 2.75. Therefore, the pH of the mobile phase is within the tolerable range of the C18 stationary phase, demonstrating that the acidic mobile phase has not degraded it. We also examined the separation of sulfonamides from different chromatographic columns. In a contrasting test, we used the Shimadzu Inertsil ODS-3 analytical columns in comparison with Agilent Eclipse XBD C18 analytical column of the same chromatographic conditions to separate sulfonamides with the results as shown below in the sulfonamides standard chromatogram made by Inertsil ODS-3 analytical columns in Figure 3A, and made by Agilent Eclipse XBD C18 analytical column in Figure 3B. From comparing the two figures, we see the retention behavior of the two different brands of columns is slightly different so the retention time and resolution varies, it is normal and acceptable. The separation of various solutes in different columns may need further optimization. Figure 3. View largeDownload slide The chromatograms of SA standards separated by Shimadzu Inertsil ODS-3 (250 mm × 4.6 mm i.d., 5 μm particle) analytical columns (A) and Agilent Eclipse XBD C18 (250 mm × 4.6 mm i.d., 5 μm particle) analytical column. The other chromatographic condition is the same as Section SAs analysis by HPLC. Peaks 1: SA; 2: SDZ; 3: STZ; 4: SP; 5: SMR; 6: SMZ; 7: SMP; 8: SM; 9: SMM; 10: SCP; 11: SDX; 12: SMX; 13: SIZ; 14: SDM; 15: SPP. Figure 3. View largeDownload slide The chromatograms of SA standards separated by Shimadzu Inertsil ODS-3 (250 mm × 4.6 mm i.d., 5 μm particle) analytical columns (A) and Agilent Eclipse XBD C18 (250 mm × 4.6 mm i.d., 5 μm particle) analytical column. The other chromatographic condition is the same as Section SAs analysis by HPLC. Peaks 1: SA; 2: SDZ; 3: STZ; 4: SP; 5: SMR; 6: SMZ; 7: SMP; 8: SM; 9: SMM; 10: SCP; 11: SDX; 12: SMX; 13: SIZ; 14: SDM; 15: SPP. In conclusion, the additive can greatly improve the resolution and sensitivity. The 15 SAs were successfully separated and quantified in pork, beef, mutton and milk without extra sample clean-up steps. Quantitative data showed good precision, linear Range and limit of detection. 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Rapid Screening for 15 Sulfonamide Residues in Foods of Animal Origin by High-Performance Liquid Chromatography–UV Method

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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1945-239X
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10.1093/chromsci/bmy037
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Abstract

Abstract A rapid and simple high-performance liquid chromatography–ultraviolet method was developed for the separation and quantification of 15 sulfonamides (SAs) in foods of animal origin without the need of clean-up procedure. A mixture of acetonitrile–formic acid–ammonium acetate–water was used as the mobile phase to separate 15 SAs on a C18 column with gradient. The selected SAs were separated completely from the matrix mixture based on different retention behaviors at different concentration of acetonitrile. The effects of the additive of formic acid and ammonium acetate in mobile phases on the separation of SAs were also investigated. The additive can greatly improve the resolution between SAs and impurities, so that the SAs can be quantified directly under the optimized chromatographic condition the sample preparation which does not need extra sample clean-up procedure. Complete baseline separation of 15 SAs was achieved in <40 min, the linear range is 0.01–130 μg/mL with a correlation coefficient R2-value > 0.999. Excellent method reproducibility was found by intra- and inter-day precisions with the relative standard deviation <9.5%. The detection limit was <11.0 ng/mL and it can be used for routine screening of the SA residues in foods of animal origin. Introduction Sulfonamides (SAs) represent a class of antibacterial compounds. They were widely used in the prophylaxis and treatment of bacterial diseases in veterinary clinic (1). Excessive or uncontrolled dosages to animals before sales to consumers can cause residual problems in food of animal origin. Regular consumption of food containing antibiotics can cause allergic reactions and antibiotic resistance in humans (2, 3). To limit these possible adverse consequences, each country has set limits for antibiotic residues in original animal food; both the European Union and China set a maximum residue limit (MRL) of 100 μg/kg for SA. In order to meet the needs of daily large number of routine determination of antibiotic residue, a simply and rapid screening method for the quantification of SAs is necessary. A survey found that the levels of residues of SA have decreased over the years due to optimization of administration schemes and application of co-medication to avoid detection. Many laboratories now use a combination of screening and confirmatory methods to cope with relatively large numbers of samples and to increase the reliability of the final result. Confirmatory methods only include liquid chromatography/tandem mass spectrometry (4–6) and gas chromatography/tandem mass spectrometry (7). These confirmatory methods based on chromatographic approaches can provide both qualitative and quantitative detection results with satisfactory sensitivity and reproducibility. However, they require expensive instrumentation and more expense, and thus are not suitable for routine inspection. As the alternative of confirmatory methods, various routine screening methods for the determination of SA residuals in animal tissue and body fluids have been developed. They include high-performance liquid chromatography (HPLC) coupled with fluorescence (8) or ultraviolet (UV) (9–13), capillary electrophoresis (14), electrochemiluminescence (15), molecularly imprinted polymer (16–18) and the enzyme linked immunosorbent assay (19). Among them, derivatization of SAs with fluorescamine can separate and detect several SAs, but introduces another variability and may complicate the methodology in a screening procedure (20, 21), and high-performance liquid chromatography–ultraviolet (HPLC–UV) is one of most widely used methods in all the inspection centers and labs, but the current established methods need sample clean-up steps or were only able to determine a few SA residuals in one chromatographic run. This work was to develop a simply HPLC–UV analytical method for simultaneous determination of 15 SAs with simplified sample extraction for routine analysis, which was used as a screening method before be identified by confirmatory methods. The linearity of calibration curve, recovery, precision and lower limit of detection (LLD) were studied to evaluate the developed method. The method was successfully used to determine the content of SA residues in pork, beef, mutton and milk. Methods Chemicals, standards solutions and materials The 15 sulfonamide standards, including sulfasulfacetamide (SA), sulfadiazine (SDZ), sulfathiazole (STZ), sulfapyridine (SP), sulfamerazine (SMR), and sulfamethazine (SMZ), sulfamethoxypyridazine (SMP), sulfameter (SM), sulfamonomethoxine (SMM), sulfachloropyridazine (SCP), sulfadoxine (SDX), sulfamethoxazole (SMX), sulfisoxazole (SIZ), sulfadimethoxine (SDM) and sulfaphenazole (SPP), (Table I), were purchased from the laboratories of Dr Ehrenstorfer (Augsburg, Germany). Stock solutions of the 15 SAs were prepared by dissolving 10 mg of each compound in 100 mL methanol. Pork, beef and mutton were obtained from the supermarket and preserved at − 20°C until use. Fresh whole milk was purchased from supermarket and stored at 4°C before use. Acetonitrile, isopropanol and methanol were obtained from Fisher (HPLC grade, New Jersey, USA). Formic acid and ammonium acetate were obtained from Tianjin Chemical Reagents Company (Tianjin, China). Deionized water was prepared by using a Milli-Q water filtration system (EMD Millipore Corp., Billerica, MA, USA). All solvents for HPLC were filtered through 0.45-mm filters (Millipore) and degassed in an ultrasonic bath. Table I. Structures and molecular weights of SAs samples Antibiotics  Symbol  MW  Structure  Sulfacetamide  SA  214.2    Sulfadiazine  SDZ  250.3    Sulfathiazole  STZ  255.3    Sulfapyridine  SP  249.3    Sulfamerazine  SMR  264.3    Sulfamethazine  SMZ  278.3    Sulfamethoxypyridazine  SMP  280.3    Sulfameter  SM  280.3    Sulfamonomethoxine  SMM  280.3    Sulfachloropyridazine  SCP  284.7    Sulfadoxine  SDX  310.3    Sulfamethoxazole  SMX  253.3    Sulfisoxazole  SIZ  267.3    Sulfadimethoxine  SDM  310.3    Sulfaphenazole  SPP  314.4    Antibiotics  Symbol  MW  Structure  Sulfacetamide  SA  214.2    Sulfadiazine  SDZ  250.3    Sulfathiazole  STZ  255.3    Sulfapyridine  SP  249.3    Sulfamerazine  SMR  264.3    Sulfamethazine  SMZ  278.3    Sulfamethoxypyridazine  SMP  280.3    Sulfameter  SM  280.3    Sulfamonomethoxine  SMM  280.3    Sulfachloropyridazine  SCP  284.7    Sulfadoxine  SDX  310.3    Sulfamethoxazole  SMX  253.3    Sulfisoxazole  SIZ  267.3    Sulfadimethoxine  SDM  310.3    Sulfaphenazole  SPP  314.4    Table I. Structures and molecular weights of SAs samples Antibiotics  Symbol  MW  Structure  Sulfacetamide  SA  214.2    Sulfadiazine  SDZ  250.3    Sulfathiazole  STZ  255.3    Sulfapyridine  SP  249.3    Sulfamerazine  SMR  264.3    Sulfamethazine  SMZ  278.3    Sulfamethoxypyridazine  SMP  280.3    Sulfameter  SM  280.3    Sulfamonomethoxine  SMM  280.3    Sulfachloropyridazine  SCP  284.7    Sulfadoxine  SDX  310.3    Sulfamethoxazole  SMX  253.3    Sulfisoxazole  SIZ  267.3    Sulfadimethoxine  SDM  310.3    Sulfaphenazole  SPP  314.4    Antibiotics  Symbol  MW  Structure  Sulfacetamide  SA  214.2    Sulfadiazine  SDZ  250.3    Sulfathiazole  STZ  255.3    Sulfapyridine  SP  249.3    Sulfamerazine  SMR  264.3    Sulfamethazine  SMZ  278.3    Sulfamethoxypyridazine  SMP  280.3    Sulfameter  SM  280.3    Sulfamonomethoxine  SMM  280.3    Sulfachloropyridazine  SCP  284.7    Sulfadoxine  SDX  310.3    Sulfamethoxazole  SMX  253.3    Sulfisoxazole  SIZ  267.3    Sulfadimethoxine  SDM  310.3    Sulfaphenazole  SPP  314.4    Sample preparation from tissue and milk SAs in pork, beef and mutton tissues were extracted using a previously reported method with minor modification (10). Briefly, 5 g of pork, beef or mutton were finely homogenized in 20 mL acetonitrile in a homogenizer (Xinzhi DY89-1, Ningbo, China). The homogenate was then centrifuged at 10,000 × g for 5 min, and the supernatant was recovered and transferred to a 50-mL flask. The tissue residue was washed with 10 mL acetonitrile, repeated twice, and concentrated in an evaporator at 35–42°C under reduced pressure. The evaporated residue was then dissolved in 0.5 mL acetonitrile and transferred to a 5-mL vial. The solubilization of residue with acetonitrile was repeated five more times to maximize the recovery. The acetonitrile solvent was then evaporated under a gentle stream of nitrogen. The final residue was dissolved in 1.0 mL of acetonitrile and filtered through a 0.22-μm organic filter to remove insoluble materials before HPLC analysis. SAs in milk were extracted using a modified method (22). Briefly, 0.5 mL trichloroacetic acid was added into 4.5-mL milk sample and vibrated for a few seconds on a vortex mixer, and then centrifuged 114 at 10,000 × g for 5 min. Finally, the supernatant was collected with a volume adjusted to 5.0 mL using acetonitrile followed by filtering through 0.22-μm organic filters for HPLC analysis. SAs analysis by HPLC The analyses were carried using an Agilent Technologies HP1200 series HPLC system equipped with a quaternary gradient pump, a diode array detector, and ChemStation Data-Analysis System (Agilent, Palo Alto, CA, USA). Chromatographic separation was performed at ambient temperature on Inertsil ODS-3 (250 mm × 4.6 mm i.d., 5 μm particle) analytical columns (Shimadzu, Japan) at a flow rate of 0.8 mL/min via a ternary gradient. Eluent A consisted of formic acid–water, eluent B is formic acid in acetonitrile solution and eluent C is ammonium acetate aqueous solution. Separation was performed with a gradient shown in Table II. SAs were monitored with UV detector at a wavelength of 270 nm. Table II. Gradients used for liquid chromatography Time (min)  Eluent A (%) formic acid aqueous solution (25 mM)  Eluent B (%) formic acid in acetonitrile solution (25 mM)  Eluent C (%) ammonium acetate aqueous solution (10 mM)  0.01  62  8  30  3.00  62  8  30  16.00  45  25  30  32.00  35  35  30  40.00  30  40  30  Time (min)  Eluent A (%) formic acid aqueous solution (25 mM)  Eluent B (%) formic acid in acetonitrile solution (25 mM)  Eluent C (%) ammonium acetate aqueous solution (10 mM)  0.01  62  8  30  3.00  62  8  30  16.00  45  25  30  32.00  35  35  30  40.00  30  40  30  Table II. Gradients used for liquid chromatography Time (min)  Eluent A (%) formic acid aqueous solution (25 mM)  Eluent B (%) formic acid in acetonitrile solution (25 mM)  Eluent C (%) ammonium acetate aqueous solution (10 mM)  0.01  62  8  30  3.00  62  8  30  16.00  45  25  30  32.00  35  35  30  40.00  30  40  30  Time (min)  Eluent A (%) formic acid aqueous solution (25 mM)  Eluent B (%) formic acid in acetonitrile solution (25 mM)  Eluent C (%) ammonium acetate aqueous solution (10 mM)  0.01  62  8  30  3.00  62  8  30  16.00  45  25  30  32.00  35  35  30  40.00  30  40  30  Linear range and limit of detection The linear correlation and dynamic range of SAs detection were determined from calibration curves generated by serial analysis of SAs standards. A calibration curve for each compound was calculated based on peak areas obtained by serially injecting 20 μL of acetonitrile–diluted solutions of SA standards. Stock solutions of individual SAs were serially diluted and spiked into matrix solution extracted from the pork. The lowest detection limits were determined as concentration corresponding to three times of noise (S/N = 3). Precision The precision of the method was evaluated by using SAs-spiked samples. Five extractions of same SAs-spiked tissue sample were tested consecutively to determine intra-day relative standard deviations (RSDs). For inter-day precision, five extractions of same SAs-spiked tissue sample were tested in three sequences over 5 days. Recovery The recovery of the assay was determined by spiking known amounts of standards into test samples. Six SAs-spiked samples and six control samples were compared. For pork or beef sample, 2.5 or 0.5 μg of the 15 SAs antibiotics were directly spiked into 5.0 g tissues. For milk sample, 2.5 or 0.5 μg of the 15 SAs antibiotics were directly spiked into 5.0 mL milk. The extraction of SAs was performed as described in Sample preparation from tissue and milk. The control samples were subjected to the same procedure as test samples. Results Optimization of chromatographic conditions SAs vary substantially in their hydrophobicity. Numerous protonated and unprotonated forms of SAs are presented due to their amphoteric properties and wide pKa ranges between 1.85 and 7.4. Therefore, chromatographic separation of SAs is very difficult in a short LC run. In this study, mixtures of acetonitrile–water–formic acid–ammonium acetate were evaluated for their potential to separate SAs in complex matrix. A mobile phase with a high initial concentration of acetonitrile (~23%) was unable to separate most of SA standards added into the pork (Figure 1A), several peaks of SAs were severely trailing and overlapped. With decreasing the initial concentration of acetonitrile (~8%), the retention times of SAs were extended, and the SAs can be separated from the main interferences (Figure 1B). In order to further improve the resolution and sensitivity of peaks, formic acid was added into the mobile phase. The retention times of SAs were remarkably shortened and the resolution and sensitivity of peaks were improved dramatically when 2 mM of formic acid was used. Continue to increase the acidity of mobile phase, the SAs peaks were completely separated except for SDZ and STZ when the concentration of formic acid reached 25 mM (Figure 1D). The separation of SAs was very sensitive to the acidity of mobile phase due to stronger interaction between protonated forms of SAs and RP stationary phases. To better separate SDZ and STZ, ammonium acetate was also added into the mobile phase. With the increase of concentration, the resolution of SDZ and STZ became higher and higher (Figures 1E and F). When the concentration of ammonium acetate reached 10 mM, all SAs-spiked into the pork were baseline separated (Figure 1F). The optimized chromatographic conditions are shown in Table II. Figure 1. View largeDownload slide Chromatographic separation of the sample extracted from pork spiked with SAs standard solution (5 mg/ L each) with different mobile phase: (A) H2O (77%) + acetonitrile (23%); (B) H2O (92%) + acetonitrile (8%); (C) 2 mM formic acid (92%) + 2 mM formic acid in acetonitrile (8%). (D) 25 mM formic acid (92%) + 25 mM formic acid in acetonitrile (8%); (E) 25 mM formic acid (62%) + 25 mM formic acid in acetonitrile (8%) + 5 mM ammonium acetate (30%); (F) 25 mM formic acid (62%) + 25 mM formic acid in acetonitrile (8%) + 10 mM ammonium acetate (30%); in (A) chromatographic programs, the ratio of acetonitrile become from 23% to 40% within 40 min, and in all other chromatographic programs, the ratio of acetonitrile or formic acid in acetonitrile become from 8% to 40% within 40 min, and the ratio of ammonium acetate aqueous solution was kept unchanged. Peak 1: SA; 2: SDZ; 3: STZ; 4: SP; 5: SMR; 6: SMZ; 7: SMP; 8: SM; 9: SMM; 10: SCP; 11: SDX; 12: SMX; 13: SIZ; 14: SDM; 15: SPP. Figure 1. View largeDownload slide Chromatographic separation of the sample extracted from pork spiked with SAs standard solution (5 mg/ L each) with different mobile phase: (A) H2O (77%) + acetonitrile (23%); (B) H2O (92%) + acetonitrile (8%); (C) 2 mM formic acid (92%) + 2 mM formic acid in acetonitrile (8%). (D) 25 mM formic acid (92%) + 25 mM formic acid in acetonitrile (8%); (E) 25 mM formic acid (62%) + 25 mM formic acid in acetonitrile (8%) + 5 mM ammonium acetate (30%); (F) 25 mM formic acid (62%) + 25 mM formic acid in acetonitrile (8%) + 10 mM ammonium acetate (30%); in (A) chromatographic programs, the ratio of acetonitrile become from 23% to 40% within 40 min, and in all other chromatographic programs, the ratio of acetonitrile or formic acid in acetonitrile become from 8% to 40% within 40 min, and the ratio of ammonium acetate aqueous solution was kept unchanged. Peak 1: SA; 2: SDZ; 3: STZ; 4: SP; 5: SMR; 6: SMZ; 7: SMP; 8: SM; 9: SMM; 10: SCP; 11: SDX; 12: SMX; 13: SIZ; 14: SDM; 15: SPP. Linear range, limit of detection Under the optimized conditions, the linear ranges for SA, SDZ, STZ SP, SMR, SMZ, SMP, SM, SMM were 0.01–100 μg/mL, for SCP, SDX, SMX, SIZ being 0.02–110 μg/mL and for SDM and SPP being 0.025–130 μg/mL. The correlation coefficients of calibration curves were >0.999. The LLD were calculated based on a signal-to-noise ratio of 3, and the values varied from 6.5 to 11.0 ng/mL (Table III). The method provides sensitive detection with wide linear range. Table III. Linear correlation, ranges, and lower limit of detection of SAs selected Antibiotics  Regression equation  Coefficient (R2)  Linear range (μg/mL)  Limita (ng/mL)  SA  y = 586.94x−97.625  0.9997  0.01–100  6.5  SDZ  y = 539.84x + 111.71  0.9994  0.01–100  7.0  STZ  y = 414.37x−27.44  0.9999  0.01–100  7.5  SP  y = 509.54x−26.576  0.9999  0.01–100  8.0  SMR  y = 545.04x−20.619  0.9997  0.01–100  8.5  SMZ  y = 575.51x−98.697  0.9992  0.01–100  9.0  SMP  y = 497.41x−0.0923  0.9998  0.01–100  10  SM  y = 465.42x + 69.798  0.9993  0.01–100  10  SMM  y = 488.8x + 20.422  0.9997  0.01–100  11  SCP  y = 464.41x−21.589  0.9997  0.02–110  10  SDX  y = 523.5x−96.603  0.9993  0.02–110  9.5  SMX  y = 558.04x + 13.708  0.9993  0.02–110  9.5  SIZ  y = 552.62x−5.6391  0.9994  0.02–110  10  SDM  y = 511.39x−14.526  0.9992  0.025–130  10  SPP  y = 418.7x−60.568  0.9994  0.025–130  11  Antibiotics  Regression equation  Coefficient (R2)  Linear range (μg/mL)  Limita (ng/mL)  SA  y = 586.94x−97.625  0.9997  0.01–100  6.5  SDZ  y = 539.84x + 111.71  0.9994  0.01–100  7.0  STZ  y = 414.37x−27.44  0.9999  0.01–100  7.5  SP  y = 509.54x−26.576  0.9999  0.01–100  8.0  SMR  y = 545.04x−20.619  0.9997  0.01–100  8.5  SMZ  y = 575.51x−98.697  0.9992  0.01–100  9.0  SMP  y = 497.41x−0.0923  0.9998  0.01–100  10  SM  y = 465.42x + 69.798  0.9993  0.01–100  10  SMM  y = 488.8x + 20.422  0.9997  0.01–100  11  SCP  y = 464.41x−21.589  0.9997  0.02–110  10  SDX  y = 523.5x−96.603  0.9993  0.02–110  9.5  SMX  y = 558.04x + 13.708  0.9993  0.02–110  9.5  SIZ  y = 552.62x−5.6391  0.9994  0.02–110  10  SDM  y = 511.39x−14.526  0.9992  0.025–130  10  SPP  y = 418.7x−60.568  0.9994  0.025–130  11  aS/N = 3. Table III. Linear correlation, ranges, and lower limit of detection of SAs selected Antibiotics  Regression equation  Coefficient (R2)  Linear range (μg/mL)  Limita (ng/mL)  SA  y = 586.94x−97.625  0.9997  0.01–100  6.5  SDZ  y = 539.84x + 111.71  0.9994  0.01–100  7.0  STZ  y = 414.37x−27.44  0.9999  0.01–100  7.5  SP  y = 509.54x−26.576  0.9999  0.01–100  8.0  SMR  y = 545.04x−20.619  0.9997  0.01–100  8.5  SMZ  y = 575.51x−98.697  0.9992  0.01–100  9.0  SMP  y = 497.41x−0.0923  0.9998  0.01–100  10  SM  y = 465.42x + 69.798  0.9993  0.01–100  10  SMM  y = 488.8x + 20.422  0.9997  0.01–100  11  SCP  y = 464.41x−21.589  0.9997  0.02–110  10  SDX  y = 523.5x−96.603  0.9993  0.02–110  9.5  SMX  y = 558.04x + 13.708  0.9993  0.02–110  9.5  SIZ  y = 552.62x−5.6391  0.9994  0.02–110  10  SDM  y = 511.39x−14.526  0.9992  0.025–130  10  SPP  y = 418.7x−60.568  0.9994  0.025–130  11  Antibiotics  Regression equation  Coefficient (R2)  Linear range (μg/mL)  Limita (ng/mL)  SA  y = 586.94x−97.625  0.9997  0.01–100  6.5  SDZ  y = 539.84x + 111.71  0.9994  0.01–100  7.0  STZ  y = 414.37x−27.44  0.9999  0.01–100  7.5  SP  y = 509.54x−26.576  0.9999  0.01–100  8.0  SMR  y = 545.04x−20.619  0.9997  0.01–100  8.5  SMZ  y = 575.51x−98.697  0.9992  0.01–100  9.0  SMP  y = 497.41x−0.0923  0.9998  0.01–100  10  SM  y = 465.42x + 69.798  0.9993  0.01–100  10  SMM  y = 488.8x + 20.422  0.9997  0.01–100  11  SCP  y = 464.41x−21.589  0.9997  0.02–110  10  SDX  y = 523.5x−96.603  0.9993  0.02–110  9.5  SMX  y = 558.04x + 13.708  0.9993  0.02–110  9.5  SIZ  y = 552.62x−5.6391  0.9994  0.02–110  10  SDM  y = 511.39x−14.526  0.9992  0.025–130  10  SPP  y = 418.7x−60.568  0.9994  0.025–130  11  aS/N = 3. Precision and reproducibility Five samples extracted from same SAs-spiked tissue (0.1 μg/kg) or milk samples (0.1 μg/mL) were analyzed consecutively and also in different days (d = 5, n = 5). The precision was high for both intra-day (RSD values of 0.78–7.35%) and inter-day assay (RSD values of 1.52–9.87%), respectively. It is worth noting that the milk samples showed the lowest intra and inter-day RSD, which is probably due to less sample extracted. The results are shown in Table IV. Both inter- and intra-day RSDs are <10%, indicating a high degree of assay precision. Long-term reproducibility was also verified. After 14 months, we used the same method to separate the standards, compared with the chromatogram made 14 months before, the variation of retention time is not <0.1 min, and thus the reproducibility is good. Table IV. Analysis precisionsa for each SA compound Standard  Intra-day RSD (%, n = 5)  Inter-day RSD (%, n = 5)  Pork  Beef  Mutton  Milk  Pork  Beef  Mutton  Milk  SA  2.64  2.45  2.85  1.70  5.56  4.48  3.69  2.55  SDZ  3.87  5.24  3.31  0.89  6.36  7.31  4.54  1.63  STZ  3.64  6.86  3.29  0.78  6.39  6.83  4.95  1.52  SP  2.49  7.35  2.67  0.93  4.38  7.91  4.85  3.88  SMR  5.68  5.43  4.82  1.22  8.40  8.16  5.67  2.34  SMZ  6.57  4.10  6.12  2.53  9.87  6.28  6.98  2.02  SMP  4.82  3.22  4.19  3.31  7.52  8.24  5.58  2.49  SM  4.27  2.83  5.32  2.64  5.03  7.88  5.89  3.67  SMM  6.50  5.50  4.25  1.57  6.64  8.01  5.67  4.70  SCP  7.13  4.79  7.20  2.64  9.20  5.97  7.75  5.29  SDX  5.92  5.52  4.37  1.52  8.31  5.89  5.51  3.56  SMX  4.61  6.80  4.83  1.54  7.22  8.75  5.62  4.51  SIZ  2.71  3.25  5.27  1.87  5.28  6.06  5.99  5.83  SDM  3.83  4.79  4.91  3.04  4.63  5.60  6.01  3.16  SPP  4.65  4.31  4.88  2.72  5.90  4.46  6.12  5.97  Standard  Intra-day RSD (%, n = 5)  Inter-day RSD (%, n = 5)  Pork  Beef  Mutton  Milk  Pork  Beef  Mutton  Milk  SA  2.64  2.45  2.85  1.70  5.56  4.48  3.69  2.55  SDZ  3.87  5.24  3.31  0.89  6.36  7.31  4.54  1.63  STZ  3.64  6.86  3.29  0.78  6.39  6.83  4.95  1.52  SP  2.49  7.35  2.67  0.93  4.38  7.91  4.85  3.88  SMR  5.68  5.43  4.82  1.22  8.40  8.16  5.67  2.34  SMZ  6.57  4.10  6.12  2.53  9.87  6.28  6.98  2.02  SMP  4.82  3.22  4.19  3.31  7.52  8.24  5.58  2.49  SM  4.27  2.83  5.32  2.64  5.03  7.88  5.89  3.67  SMM  6.50  5.50  4.25  1.57  6.64  8.01  5.67  4.70  SCP  7.13  4.79  7.20  2.64  9.20  5.97  7.75  5.29  SDX  5.92  5.52  4.37  1.52  8.31  5.89  5.51  3.56  SMX  4.61  6.80  4.83  1.54  7.22  8.75  5.62  4.51  SIZ  2.71  3.25  5.27  1.87  5.28  6.06  5.99  5.83  SDM  3.83  4.79  4.91  3.04  4.63  5.60  6.01  3.16  SPP  4.65  4.31  4.88  2.72  5.90  4.46  6.12  5.97  aSpiking 0.1 μg/kg in the tissue sample and 0.1 μg/mL in the milk. Table IV. Analysis precisionsa for each SA compound Standard  Intra-day RSD (%, n = 5)  Inter-day RSD (%, n = 5)  Pork  Beef  Mutton  Milk  Pork  Beef  Mutton  Milk  SA  2.64  2.45  2.85  1.70  5.56  4.48  3.69  2.55  SDZ  3.87  5.24  3.31  0.89  6.36  7.31  4.54  1.63  STZ  3.64  6.86  3.29  0.78  6.39  6.83  4.95  1.52  SP  2.49  7.35  2.67  0.93  4.38  7.91  4.85  3.88  SMR  5.68  5.43  4.82  1.22  8.40  8.16  5.67  2.34  SMZ  6.57  4.10  6.12  2.53  9.87  6.28  6.98  2.02  SMP  4.82  3.22  4.19  3.31  7.52  8.24  5.58  2.49  SM  4.27  2.83  5.32  2.64  5.03  7.88  5.89  3.67  SMM  6.50  5.50  4.25  1.57  6.64  8.01  5.67  4.70  SCP  7.13  4.79  7.20  2.64  9.20  5.97  7.75  5.29  SDX  5.92  5.52  4.37  1.52  8.31  5.89  5.51  3.56  SMX  4.61  6.80  4.83  1.54  7.22  8.75  5.62  4.51  SIZ  2.71  3.25  5.27  1.87  5.28  6.06  5.99  5.83  SDM  3.83  4.79  4.91  3.04  4.63  5.60  6.01  3.16  SPP  4.65  4.31  4.88  2.72  5.90  4.46  6.12  5.97  Standard  Intra-day RSD (%, n = 5)  Inter-day RSD (%, n = 5)  Pork  Beef  Mutton  Milk  Pork  Beef  Mutton  Milk  SA  2.64  2.45  2.85  1.70  5.56  4.48  3.69  2.55  SDZ  3.87  5.24  3.31  0.89  6.36  7.31  4.54  1.63  STZ  3.64  6.86  3.29  0.78  6.39  6.83  4.95  1.52  SP  2.49  7.35  2.67  0.93  4.38  7.91  4.85  3.88  SMR  5.68  5.43  4.82  1.22  8.40  8.16  5.67  2.34  SMZ  6.57  4.10  6.12  2.53  9.87  6.28  6.98  2.02  SMP  4.82  3.22  4.19  3.31  7.52  8.24  5.58  2.49  SM  4.27  2.83  5.32  2.64  5.03  7.88  5.89  3.67  SMM  6.50  5.50  4.25  1.57  6.64  8.01  5.67  4.70  SCP  7.13  4.79  7.20  2.64  9.20  5.97  7.75  5.29  SDX  5.92  5.52  4.37  1.52  8.31  5.89  5.51  3.56  SMX  4.61  6.80  4.83  1.54  7.22  8.75  5.62  4.51  SIZ  2.71  3.25  5.27  1.87  5.28  6.06  5.99  5.83  SDM  3.83  4.79  4.91  3.04  4.63  5.60  6.01  3.16  SPP  4.65  4.31  4.88  2.72  5.90  4.46  6.12  5.97  aSpiking 0.1 μg/kg in the tissue sample and 0.1 μg/mL in the milk. Recovery Different samples including pork, beef, mutton and milk were selected for maximum variability with respect to provenance and matrix. Spiked samples at two levels: 0.5 μg/kg or 0.1 μg/kg for tissue and 0.5 μg/L or 0.1 μg/L for milk, were analyzed to evaluate the recovery (Table V). Excellent recoveries of 85–95% for high concentration samples and less satisfactory recoveries of 81–93% for low concentration samples were obtained. Table V. Recovery of residual SAs from pork, beef, mutton and milk matrices Standard  % Recovery  Pork  Beef  Mutton  Milk  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/mL  Spiking 0.1 μg/mL  SA  88.6 ± 5.3  86.9 ± 6.1  90.2 ± 6.6  84.8 ± 6.7  86.1 ± 4.2  90.4 ± 4.4  91.9 ± 3.9  90.7 ± 2.0  SDZ  84.6 ± 4.1  85.0 ± 4.5  89.3 ± 5.8  82.6 ± 5.8  85.4 ± 5.5  89.9 ± 5.7  92.2 ± 4.3  93.6 ± 4.2  STZ  91.3 ± 7.5  90.3 ± 5.9  89.7 ± 8.4  86.5 ± 4.2  88.6 ± 6.4  92.1 ± 5.3  94.8 ± 5.5  91.5 ± 3.6  SP  92.7 ± 4.8  87.6 ± 6.8  93.6 ± 6.1  87.8 ± 7.9  90.3 ± 4.8  86.8 ± 4.9  93.7 ± 5.0  93.3 ± 4.4  SMR  90.5 ± 6.7  85.8 ± 4.7  92.0 ± 5.7  91.6 ± 8.5  91.1 ± 5.5  91.5 ± 5.8  93.0 ± 4.4  91.9 ± 5.7  SMZ  87.8 ± 5.9  82.7 ± 5.2  88.9 ± 4.2  85.0 ± 6.4  89.0 ± 5.8  92.4 ± 6.4  90.6 ± 5.2  91.8 ± 5.3  SMP  90.4 ± 7.0  85.2 ± 6.0  91.0 ± 5.9  87.3 ± 6.7  92.7 ± 6.2  91.9 ± 6.1  92.7 ± 5.7  90.5 ± 4.7  SM  92.4 ± 5.7  88.6 ± 7.4  90.5 ± 6.7  86.0 ± 7.5  93.5 ± 7.1  89.8 ± 5.5  95.3 ± 4.9  93.1 ± 3.3  SMM  93.0 ± 4.4  90.4 ± 8.8  87.7 ± 7.4  82.7 ± 6.0  87.9 ± 6.3  90.8 ± 5.9  92.3 ± 3.6  92.2 ± 2.7  SCP  89.2 ± 7.2  85.2 ± 7.5  89.4 ± 7.3  88.9 ± 5.3  90.2 ± 4.5  91.4 ± 4.8  94.1 ± 2.4  90.3 ± 2.5  SDX  86.9 ± 5.8  81.9 ± 5.0  94.2 ± 8.0  85.1 ± 6.6  85.9 ± 5.4  92.5 ± 5.1  89.7 ± 4.7  91.0 ± 4.8  SMX  85.1 ± 4.7  84.7 ± 5.7  87.4 ± 6.3  88.3 ± 7.4  89.4 ± 4.7  91.8 ± 6.1  89.5 ± 2.5  87.6 ± 5.8  SIZ  89.6 ± 8.8  88.6 ± 6.9  83.7 ± 5.7  84.2 ± 6.2  87.7 ± 6.6  90.6 ± 5.7  88.6 ± 4.5  86.4 ± 4.2  SDM  85.6 ± 6.3  81.8 ± 5.6  85.8 ± 6.8  81.6 ± 5.8  91.6 ± 5.7  89.8 ± 6.6  86.9 ± 5.3  87.2 ± 4.9  SPP  86.5 ± 5.9  81.5 ± 6.0  87.9 ± 7.2  83.4 ± 5.5  92.3 ± 6.7  90.9 ± 6.0  87.3 ± 4.8  85.5 ± 5.2  Standard  % Recovery  Pork  Beef  Mutton  Milk  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/mL  Spiking 0.1 μg/mL  SA  88.6 ± 5.3  86.9 ± 6.1  90.2 ± 6.6  84.8 ± 6.7  86.1 ± 4.2  90.4 ± 4.4  91.9 ± 3.9  90.7 ± 2.0  SDZ  84.6 ± 4.1  85.0 ± 4.5  89.3 ± 5.8  82.6 ± 5.8  85.4 ± 5.5  89.9 ± 5.7  92.2 ± 4.3  93.6 ± 4.2  STZ  91.3 ± 7.5  90.3 ± 5.9  89.7 ± 8.4  86.5 ± 4.2  88.6 ± 6.4  92.1 ± 5.3  94.8 ± 5.5  91.5 ± 3.6  SP  92.7 ± 4.8  87.6 ± 6.8  93.6 ± 6.1  87.8 ± 7.9  90.3 ± 4.8  86.8 ± 4.9  93.7 ± 5.0  93.3 ± 4.4  SMR  90.5 ± 6.7  85.8 ± 4.7  92.0 ± 5.7  91.6 ± 8.5  91.1 ± 5.5  91.5 ± 5.8  93.0 ± 4.4  91.9 ± 5.7  SMZ  87.8 ± 5.9  82.7 ± 5.2  88.9 ± 4.2  85.0 ± 6.4  89.0 ± 5.8  92.4 ± 6.4  90.6 ± 5.2  91.8 ± 5.3  SMP  90.4 ± 7.0  85.2 ± 6.0  91.0 ± 5.9  87.3 ± 6.7  92.7 ± 6.2  91.9 ± 6.1  92.7 ± 5.7  90.5 ± 4.7  SM  92.4 ± 5.7  88.6 ± 7.4  90.5 ± 6.7  86.0 ± 7.5  93.5 ± 7.1  89.8 ± 5.5  95.3 ± 4.9  93.1 ± 3.3  SMM  93.0 ± 4.4  90.4 ± 8.8  87.7 ± 7.4  82.7 ± 6.0  87.9 ± 6.3  90.8 ± 5.9  92.3 ± 3.6  92.2 ± 2.7  SCP  89.2 ± 7.2  85.2 ± 7.5  89.4 ± 7.3  88.9 ± 5.3  90.2 ± 4.5  91.4 ± 4.8  94.1 ± 2.4  90.3 ± 2.5  SDX  86.9 ± 5.8  81.9 ± 5.0  94.2 ± 8.0  85.1 ± 6.6  85.9 ± 5.4  92.5 ± 5.1  89.7 ± 4.7  91.0 ± 4.8  SMX  85.1 ± 4.7  84.7 ± 5.7  87.4 ± 6.3  88.3 ± 7.4  89.4 ± 4.7  91.8 ± 6.1  89.5 ± 2.5  87.6 ± 5.8  SIZ  89.6 ± 8.8  88.6 ± 6.9  83.7 ± 5.7  84.2 ± 6.2  87.7 ± 6.6  90.6 ± 5.7  88.6 ± 4.5  86.4 ± 4.2  SDM  85.6 ± 6.3  81.8 ± 5.6  85.8 ± 6.8  81.6 ± 5.8  91.6 ± 5.7  89.8 ± 6.6  86.9 ± 5.3  87.2 ± 4.9  SPP  86.5 ± 5.9  81.5 ± 6.0  87.9 ± 7.2  83.4 ± 5.5  92.3 ± 6.7  90.9 ± 6.0  87.3 ± 4.8  85.5 ± 5.2  Table V. Recovery of residual SAs from pork, beef, mutton and milk matrices Standard  % Recovery  Pork  Beef  Mutton  Milk  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/mL  Spiking 0.1 μg/mL  SA  88.6 ± 5.3  86.9 ± 6.1  90.2 ± 6.6  84.8 ± 6.7  86.1 ± 4.2  90.4 ± 4.4  91.9 ± 3.9  90.7 ± 2.0  SDZ  84.6 ± 4.1  85.0 ± 4.5  89.3 ± 5.8  82.6 ± 5.8  85.4 ± 5.5  89.9 ± 5.7  92.2 ± 4.3  93.6 ± 4.2  STZ  91.3 ± 7.5  90.3 ± 5.9  89.7 ± 8.4  86.5 ± 4.2  88.6 ± 6.4  92.1 ± 5.3  94.8 ± 5.5  91.5 ± 3.6  SP  92.7 ± 4.8  87.6 ± 6.8  93.6 ± 6.1  87.8 ± 7.9  90.3 ± 4.8  86.8 ± 4.9  93.7 ± 5.0  93.3 ± 4.4  SMR  90.5 ± 6.7  85.8 ± 4.7  92.0 ± 5.7  91.6 ± 8.5  91.1 ± 5.5  91.5 ± 5.8  93.0 ± 4.4  91.9 ± 5.7  SMZ  87.8 ± 5.9  82.7 ± 5.2  88.9 ± 4.2  85.0 ± 6.4  89.0 ± 5.8  92.4 ± 6.4  90.6 ± 5.2  91.8 ± 5.3  SMP  90.4 ± 7.0  85.2 ± 6.0  91.0 ± 5.9  87.3 ± 6.7  92.7 ± 6.2  91.9 ± 6.1  92.7 ± 5.7  90.5 ± 4.7  SM  92.4 ± 5.7  88.6 ± 7.4  90.5 ± 6.7  86.0 ± 7.5  93.5 ± 7.1  89.8 ± 5.5  95.3 ± 4.9  93.1 ± 3.3  SMM  93.0 ± 4.4  90.4 ± 8.8  87.7 ± 7.4  82.7 ± 6.0  87.9 ± 6.3  90.8 ± 5.9  92.3 ± 3.6  92.2 ± 2.7  SCP  89.2 ± 7.2  85.2 ± 7.5  89.4 ± 7.3  88.9 ± 5.3  90.2 ± 4.5  91.4 ± 4.8  94.1 ± 2.4  90.3 ± 2.5  SDX  86.9 ± 5.8  81.9 ± 5.0  94.2 ± 8.0  85.1 ± 6.6  85.9 ± 5.4  92.5 ± 5.1  89.7 ± 4.7  91.0 ± 4.8  SMX  85.1 ± 4.7  84.7 ± 5.7  87.4 ± 6.3  88.3 ± 7.4  89.4 ± 4.7  91.8 ± 6.1  89.5 ± 2.5  87.6 ± 5.8  SIZ  89.6 ± 8.8  88.6 ± 6.9  83.7 ± 5.7  84.2 ± 6.2  87.7 ± 6.6  90.6 ± 5.7  88.6 ± 4.5  86.4 ± 4.2  SDM  85.6 ± 6.3  81.8 ± 5.6  85.8 ± 6.8  81.6 ± 5.8  91.6 ± 5.7  89.8 ± 6.6  86.9 ± 5.3  87.2 ± 4.9  SPP  86.5 ± 5.9  81.5 ± 6.0  87.9 ± 7.2  83.4 ± 5.5  92.3 ± 6.7  90.9 ± 6.0  87.3 ± 4.8  85.5 ± 5.2  Standard  % Recovery  Pork  Beef  Mutton  Milk  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/g  Spiking 0.1 μg/g  Spiking 0.5 μg/mL  Spiking 0.1 μg/mL  SA  88.6 ± 5.3  86.9 ± 6.1  90.2 ± 6.6  84.8 ± 6.7  86.1 ± 4.2  90.4 ± 4.4  91.9 ± 3.9  90.7 ± 2.0  SDZ  84.6 ± 4.1  85.0 ± 4.5  89.3 ± 5.8  82.6 ± 5.8  85.4 ± 5.5  89.9 ± 5.7  92.2 ± 4.3  93.6 ± 4.2  STZ  91.3 ± 7.5  90.3 ± 5.9  89.7 ± 8.4  86.5 ± 4.2  88.6 ± 6.4  92.1 ± 5.3  94.8 ± 5.5  91.5 ± 3.6  SP  92.7 ± 4.8  87.6 ± 6.8  93.6 ± 6.1  87.8 ± 7.9  90.3 ± 4.8  86.8 ± 4.9  93.7 ± 5.0  93.3 ± 4.4  SMR  90.5 ± 6.7  85.8 ± 4.7  92.0 ± 5.7  91.6 ± 8.5  91.1 ± 5.5  91.5 ± 5.8  93.0 ± 4.4  91.9 ± 5.7  SMZ  87.8 ± 5.9  82.7 ± 5.2  88.9 ± 4.2  85.0 ± 6.4  89.0 ± 5.8  92.4 ± 6.4  90.6 ± 5.2  91.8 ± 5.3  SMP  90.4 ± 7.0  85.2 ± 6.0  91.0 ± 5.9  87.3 ± 6.7  92.7 ± 6.2  91.9 ± 6.1  92.7 ± 5.7  90.5 ± 4.7  SM  92.4 ± 5.7  88.6 ± 7.4  90.5 ± 6.7  86.0 ± 7.5  93.5 ± 7.1  89.8 ± 5.5  95.3 ± 4.9  93.1 ± 3.3  SMM  93.0 ± 4.4  90.4 ± 8.8  87.7 ± 7.4  82.7 ± 6.0  87.9 ± 6.3  90.8 ± 5.9  92.3 ± 3.6  92.2 ± 2.7  SCP  89.2 ± 7.2  85.2 ± 7.5  89.4 ± 7.3  88.9 ± 5.3  90.2 ± 4.5  91.4 ± 4.8  94.1 ± 2.4  90.3 ± 2.5  SDX  86.9 ± 5.8  81.9 ± 5.0  94.2 ± 8.0  85.1 ± 6.6  85.9 ± 5.4  92.5 ± 5.1  89.7 ± 4.7  91.0 ± 4.8  SMX  85.1 ± 4.7  84.7 ± 5.7  87.4 ± 6.3  88.3 ± 7.4  89.4 ± 4.7  91.8 ± 6.1  89.5 ± 2.5  87.6 ± 5.8  SIZ  89.6 ± 8.8  88.6 ± 6.9  83.7 ± 5.7  84.2 ± 6.2  87.7 ± 6.6  90.6 ± 5.7  88.6 ± 4.5  86.4 ± 4.2  SDM  85.6 ± 6.3  81.8 ± 5.6  85.8 ± 6.8  81.6 ± 5.8  91.6 ± 5.7  89.8 ± 6.6  86.9 ± 5.3  87.2 ± 4.9  SPP  86.5 ± 5.9  81.5 ± 6.0  87.9 ± 7.2  83.4 ± 5.5  92.3 ± 6.7  90.9 ± 6.0  87.3 ± 4.8  85.5 ± 5.2  Validation procedure This method was validated with other three different tested samples, including beef, mutton and milk. Typical chromatograms of the blank and spiked samples are shown in Figure 2. SAs were not detected in blank samples and there were no significant interference peaks observed at the retention time of SA standard. All of the spiked SA standards were well separated and detected with low abundance peaks from matrix partially overlapped with the SAs. The quantitative results were still satisfactory because the interference from the overlapped peaks were minimal. Figure 2. View largeDownload slide Typical chromatograms for three spiked (A) and blank samples (B). A: beef; B: mutton; C: milk. The concentrations of spiked analytes are 0.1 μg/kg or 0.1 μg/L. The chromatographic program were the same as Table II. Peaks 1: SA; 2: SDZ; 3: STZ; 4: SP; 5: SMR; 6: SMZ; 7: SMP; 8: SM; 9: SMM; 10: SCP; 11: SDX; 12: SMX; 13: SIZ; 14: SDM; 15: SPP. Figure 2. View largeDownload slide Typical chromatograms for three spiked (A) and blank samples (B). A: beef; B: mutton; C: milk. The concentrations of spiked analytes are 0.1 μg/kg or 0.1 μg/L. The chromatographic program were the same as Table II. Peaks 1: SA; 2: SDZ; 3: STZ; 4: SP; 5: SMR; 6: SMZ; 7: SMP; 8: SM; 9: SMM; 10: SCP; 11: SDX; 12: SMX; 13: SIZ; 14: SDM; 15: SPP. Application to samples The method was used to analyze 142 pork samples from the local Food and Drug Administration. The samples were randomly obtained from the supermarket, free market and street vendors of whole Shaanxi province in China according to the local government monitoring plan. Twenty-five samples containing SAs residues were detected. Positive milk samples containing 3, 2 or 1 SAs were 1.5%, 7.4% and 8.7%, respectively. The frequent SAs residues detected in pork samples were STZ, SMR, SMP SM and SDX. The SAs content of the positive samples ranged from 6 μg/kg to 163 μg/kg. Eight samples were detected with the levels of SAs residues higher than the established MRL. The results were consistent with that of the local Food and Drug Administration and showed that this method was able to screen samples for SAs routinely before performing confirmatory tests. Discussion The quantification of SAs can be severely interfered by the impurity from complex matrix (23). Therefore, extensive sample preparation are necessary to remove interferences, such as liquid–liquid extraction (24), dispersive liquid–liquid microextraction (22, 25, 26), matrix solid-phase dispersion (27), solid-phase extraction (28–30) and solid-phase microextraction (21, 31, 32). But the clean-up steps are labor-intensive and time-consuming leading to low recoveries and low throughput. Numerous efforts have been reported to emphasize these shortcomings. Complete elimination of the clean-up step was developed for determination of the SAs content of fish (22, 33), calf and pig tissue (10) by HPLC–UV, but these simplified methods can be only able to simultaneous evaluate fewer SAs in one chromatographic run. The extraction procedure for SAs we developed didn’t need extra sample clean-up step, which reduced the sample preparation and boring physical labor. In the reported HPLC methods for SAs separation, most of that used inorganic acids or salts as an additive to improve the peaks’ resolution or shape. However, when the proportion of water in the mobile phase is low, inorganic acids or salts easily precipitate from the mobile phase, these very tiny precipitate particles can damage the pump head of HPLC instrument (34). In this method, we use the formic acid and ammonium acetate as the mobile phase additive, which are easily soluble in organic solvents. Since the isolated sulfonamides have both an acid residue and a base residue, it is an amphoteric molecule and, if directly separated, exhibits a variety of properties, making the separation more complex. In order to decrease this complexity, we add formic acid in the mobile phase and reduce the pH of the mobile phase, so the basic group will be protonated and acidic groups will not ionize. Then we add salt, utilizing the effect of its ion pair to make sulfonamides only show different hydrophobicity so that they are easier to separate. In addition, organic additives were compatible with mass spectrum and can be apply to the LC-MS or GC-MS of confirmatory method. Therefore, using of organic additives soluble in solvents is very important. In general, C18 stationary phase can be tolerated in a pH range of 2–8. Originally, we wanted to use the pH determination of flow meter pH, but due to the interference caused by the mobile phase containing acetonitrile, the pH of the mobile phase is very difficult to accurately determine. However, we can roughly estimate the pH of the mobile phase according to its pKa: The final concentration of formic acid in mobile phase is 0.025 M × 0.7 = 0.0175 M, and its pKa = 3.75; In the mobile phase [H+]≈Ka∙C=10−3.75×0.0175≈0.00176, pH = 2.75. Therefore, the pH of the mobile phase is within the tolerable range of the C18 stationary phase, demonstrating that the acidic mobile phase has not degraded it. We also examined the separation of sulfonamides from different chromatographic columns. In a contrasting test, we used the Shimadzu Inertsil ODS-3 analytical columns in comparison with Agilent Eclipse XBD C18 analytical column of the same chromatographic conditions to separate sulfonamides with the results as shown below in the sulfonamides standard chromatogram made by Inertsil ODS-3 analytical columns in Figure 3A, and made by Agilent Eclipse XBD C18 analytical column in Figure 3B. From comparing the two figures, we see the retention behavior of the two different brands of columns is slightly different so the retention time and resolution varies, it is normal and acceptable. The separation of various solutes in different columns may need further optimization. Figure 3. View largeDownload slide The chromatograms of SA standards separated by Shimadzu Inertsil ODS-3 (250 mm × 4.6 mm i.d., 5 μm particle) analytical columns (A) and Agilent Eclipse XBD C18 (250 mm × 4.6 mm i.d., 5 μm particle) analytical column. The other chromatographic condition is the same as Section SAs analysis by HPLC. Peaks 1: SA; 2: SDZ; 3: STZ; 4: SP; 5: SMR; 6: SMZ; 7: SMP; 8: SM; 9: SMM; 10: SCP; 11: SDX; 12: SMX; 13: SIZ; 14: SDM; 15: SPP. Figure 3. View largeDownload slide The chromatograms of SA standards separated by Shimadzu Inertsil ODS-3 (250 mm × 4.6 mm i.d., 5 μm particle) analytical columns (A) and Agilent Eclipse XBD C18 (250 mm × 4.6 mm i.d., 5 μm particle) analytical column. The other chromatographic condition is the same as Section SAs analysis by HPLC. Peaks 1: SA; 2: SDZ; 3: STZ; 4: SP; 5: SMR; 6: SMZ; 7: SMP; 8: SM; 9: SMM; 10: SCP; 11: SDX; 12: SMX; 13: SIZ; 14: SDM; 15: SPP. In conclusion, the additive can greatly improve the resolution and sensitivity. The 15 SAs were successfully separated and quantified in pork, beef, mutton and milk without extra sample clean-up steps. Quantitative data showed good precision, linear Range and limit of detection. 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Journal of Chromatographic ScienceOxford University Press

Published: Apr 26, 2018

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