Rapid analysis of fifteen sulfonamide residues in pork and fish samples by automated on-line solid phase extraction coupled to liquid chromatography–tandem mass spectrometry

Junmei Ma, Sufang Fan, Lei Sun, Liangna He, Yan Zhang,?, Qiang Li,?

aHebei Key Laboratory of Forensic Medicine, College of Forensic Medicine, Hebei Medical University, Shijiazhuang 050017, China

bHebei Food Safety Key Laboratory, Hebei Food Inspection and Research Institute, Shijiazhuang 050227, China

ABSTRACT

The aim of this work was to develop an automated on-line solid phase extraction (SPE) with liquid chromatography-tandem mass spectrometry method for the detection of fifteen sulfonamides in pork and fish samples. Samples were extracted with 0.2% formic acid acetonitrile solution, purified by on-line SPE device with HLB column, then separated by XBridge C18column, using 0.1% formic acid solution and acetonitrile as the mobile phase. Mass spectrometric data was acquired under multiple reaction monitoring (MRM) mode using positive ionization electrospray. Internal standard method was used in the quantification, good linear relationship was got in range of 0.1–100 ng/mL and correlation coefficient was higher than 0.9990. The limits of detection were in the range of 0.125–2.00 μg/kg and the limits of quantitation were in the range of 0.250–5.00 μg/kg. Recoveries of the method were in range of 78.3%–99.3%,relative standard deviation were lower than 10%. The method was simple, sensitivity, and could be used for routine supervision and analysis of fifteen sulfonamides in pork and fish.

Keywords:

Liquid chromatography–tandem mass

spectrometry

On-line solid phase extraction

Sulfonamides

Internal standard quantification

1. Introduction

Sulfonamides are N-substituted derivatives of sulfanilamide, a group of amide of para amino benzene sulfonic acid [1], which are widely used in veterinary medicine for the prevention and therapy of bacterial and protozoal infections due to their broad-spectrum antibacterial, low price, stable properties, and excellent therapeutic effects [2,3]. However, the residues of sulfonamides in food could cause allergic reactions, urination and hematopoietic disorder. Long-term accumulation may deal great harm to human health because it may cause damage to kidney function, drug resistance,carcinogenicity, etc. [4]. To ensure food safety for consumers, EU Commission [5], the U.S. Food and Drug Administration (FDA) [6]and Agriculture Department of China [7] have laid down the maximum residue limit (MRL) on the residues of sulfonamides in food separately (Table 1).

Table 1MRL for sulfonamides in animal-derived foods stipulated by the EU, the U.S. and China.

Table 2Mass spectrometry parameters of analytes.

Various analytical methods have been proposed for detecting sulfonamides, such as capillary electrophoresis [8], biosensor technology [9], immunoassay [10,11], and high performance liquid chromatography [12,13], gas chromatography [14], liquid chromatography-tandem mass spectrometry [15–17], etc.Capillary electrophoresis showed good separation capability for sulfonamides, but suffered from low precision issues. Biosensor technology has good sensitivity, but is prone to false positives.Immunoassay is not suitable for multiresidue and confirmatory analysis. Gas chromatography is relatively difficult because sulfonamides must be volatilized prior to analysis due to the high polarity and low volatility of sulfonamides. High performance liquid chromatography-tandem mass spectrometry is a powerful technique for the analysis of sulfonamides because of its high specificity and sensitivity [18–23]. Animal-derived food have complex matrices, which can easily cause interference to the target and cause pollution to the instrument without purification steps or poor purification methods. Some analytical methods have been developed to determine sulfonamides in various food samples including pork and fish, however most of the reported methods use offline SPE, liquid-liquid extraction purification, or lack of sample purification steps [24–27]. Off-line SPE and liquid-liquid extrac-tion purification methods are complicated to operate, and those purification steps are time-consuming and cost-intensive [28,29].

The on-line SPE technology adopted in this research achieves the combination of sample pretreatment and instrument analysis,which can increase the degree of automation of sample analysis,simplify the pretreatment procedure, improve the work efficiency,and avoid the artificial error in traditional sample preparation process. At present, there are few reports on the application of on-line SPE technology in the detection of sulfonamides in animalderived food [30,31]. The existed on-line SPE technology for the detection of sulfonamides was combined with high performance liquid chromatography, and those methods based only on ultraviolet detector, which are not the proper confirmatory method because of ultraviolet detector’s low sensitivity or specificity. In this study, an on-line SPE device from Spark Holland was combined with liquid chromatography–tandem mass spectrometry in internal standard correction. Efficient purification and separation were achieved through the optimization of on-line SPE peak focus. The proposed method was validated by evaluating recovery, linearity,accuracy, and repeatability, and could be used to monitor the illegal use of sulfonamides.

2. Materials and methods

2.1. Reagents and standards

Acetonitrile, methanol, and formic acid (FA) with HPLC-grade were purchased from Fisher Scientific (Pittsburgh, USA). Deionized water from a Millipore Milli-Q water purification system (Millipore,Bedford, MA, USA) was used for preparation of the chromatographic phase.

Sulfacetamide (SAA), sulfamethizole (SMT), sulfisoxazole(SFZ), sulfachloropyridazine (SCP), sulfadiazine (SDZ), sulfanilamidoisoxazole (SMZ), sulfathiazole (STZ), sulfamonomethoxine(SMM), sulfamerazine(SMR), sulfadimoxine (SDX), sulfapyridine (SPD), sulfamethoxypyridazine (SMP), sulfadimidine(SDM), sulfaphenazolum (SPA), sulfadimethoxine (SDT),13C6-sulfamethoxazole (13C6-STX) were supplied from Dr. Ehrenstorfer(Augsburg, Germany).

Stock solutions of fifteen sulfonamides were prepared in methanol with concentration of 1 mg/mL and stored at ?20°C.Working solutions with appropriate concentrations were prepared before use.

2.2. Instruments

The high-speed refrigerated centrifuge (CR22N, HITACHI,Germany), the vortex mixer (Vortex Genius 3, IKA, Germany), and the ultrasonic cleaner (Elmasonic P300H, Elma, Germany) were used in the procedure of extraction.

Sample pretreatment was performed using an automated SPE system (Symbiosis pico, Spark Holland, Emmen, Netherlands). The on-line SPE unit was connected to a binary LC pumps (Symbiosis SPH 1240, Spark Holland), which was connected to a tandem mass spectrometer (Triple Quad 6500, AB Sciex, USA) equipped with an ESI source. Analyst 1.5.1 was used for data acquisition,and MultiQuant 3.0.1 was used for data processing, while version 1.2.0.0 was used to control the on-line SPE unit. The Oasis HLB cartridge (10 mm × 1 mm, Waters, USA) was used for on-line SPE,and chromatographic separation was performed on an XBridge C18(4.6 mm × 150 mm, 5 μm, Waters, USA) column.

2.3. Samples preparation

Two grams of sample was weighed and transferred to a 50 mL centrifuge tube. Following spiking 50 μL of 100 ng/mL internal standard solutions of13C6-STX, 5 mL of acetonitrile with 0.2% FA,vortex and mix for 1 min, and then sonicate for 30 min, The sample was centrifuged at 8000 r/min for 5 min. After degreasing, the supernatant was filtered and transferred to a sample vial ready for on-line SPE.

2.4. On-line SPE conditions

The on-line SPE cartridge was conditioned with 1000 μL methanol and 1000 μL water at the flow rate of 3500 μL/min, A 100 μL portion of sample extract was then loaded onto the cartridge using 1000 μL water as transfer solvent at 1000 μL/min. The SPE cartridge was then washed successively with 1000 μL water at 3500 μL/min. After the washing procedure was completed, taking place at the left clamp of the automated cartridge exchange,the cartridge was automatically moved onto the right side clamp,where the target analytes were eluted onto the LC column by 200 μL methanol at 100 μL /min. At the same time, a new cartridge was placed on the left side clamp, and the next extraction sequence was initiated.

2.5. Chromatographic separation

The chromatographic separation was performed on a C18column. The column temperature was 35°C, the injection volume was 50 μL, water containing 0.1% (V/V) FA (phase A)and acetonitrile (phase B) were used as mobile phase. Target compounds were separated with a gradient elute, the elution conditions were as follows: 0–0.05 min, 0.6–0.5 mL/min, 98% A;0.05–2.00 min, 0.5 mL/min, 98% A; 2.00–2.05 min, 0.5–0.6 mL/min,98% A; 2.05–10.30 min, 0.6 mL/min 98%–5% A; 10.30–12.30 min,0.6 mL/min, 5% A; 12.30–12.40 min, 0.6 mL/min, 5%–98% A;12.40–15.00 min, 0.6 mL/min, 98% A.

2.6. Mass spectrometry detection

The MS analysis was performed using an ESI source in positive ionization mode. The optimized conditions of mass specptrometry were as follows: the ionization voltage was 5.5 kV, the source temperature was 500°C, pressure of curtain gas was 30 psi, pressure of nebulizer gas was 40 psi, pressure of auxiliary gas was 45 psi, dwell time was 20 ms. Mass spectrometry parameters of analytes were shown in Table 2.

3. Results and discussion

3.1. Optimization of extraction solvents

Sulfonamides all contain polar groups, according to similar compatibility principles, they are easier to dissolve in polar organic solvents. This experiment investigated the extraction effect when acetonitrile and methanol were used as the extraction solvent.When methanol was used as the extraction solvent, the extraction solution was relatively turbid and the recovery was less than that of acetonitrile. Most of the proteins present in pork and fish were precipitated in the presence of acetonitrile. The extraction effect of acetonitrile with various concentrations of formic acid (0, 0.1%,0.2%, 0.5%, 1%) were tested. 0.2% formic acid acetonitrile solution yielded the best reproducibility and recovery of the fifteen sulfonamides. Therefore, 0.2% formic acid acetonitrile solution was selected as the extraction solvent.

3.2. Optimization of on-line SPE conditions

A number of key parameters were still assessed to validate an appropriate on-line SPE procedure. For HLB cartridge, the stronger polarity of the elution solvent, the easier target would be eluted, but it will also wash out impurities. Methanol and acetonitrile were compared as the elution solvent. The result was shown in Fig. 1.Except for SFZ and SMZ, the elution effect of methanol was better than that of acetonitrile. Therefore, methanol was used as elution solvent in this experiment.

Fig. 1. Effect of different elution solvent on the recoveries of fifteen sulfonamides.

Optimization of on-line SPE was aimed to obtain maximum extraction efficiency and sample clean-up. The flow rate for the elution step was an important parameter to be optimized. Under the conditions of injected sample volume (50 μL) and elution volume(200 μL), the elution flow rates of 100, 150, 200 and 250 μL/min were compared. The test was performed to investigate the elution effect based on the response and peak types of fifteen sulfonamides(10 ng/mL). The optimization of elution flow rates was shown in Fig. 2. When the elution rate was 100 μL/min, the peak shapes of fifteen compounds were best and the recoveries were maximum;when the elution flow rate continues to increase, the elution was insufficient, and the peak areas of some compounds were smaller,so the elution flow rate was chosen as 100 μL/min.

Fig. 2. Effect of different elution flow rate on the recoveries of fifteen sulfonamides.

Under the conditions of injected sample volume (50 μL) and elution flow rate (100 μL/min), the elution volume of 50, 100, 150,200, and 250 μL were compared. The optimization of elution flow rates was shown in Fig. 3. The results revealed that when the elution volume was 50 and 100 μL, the target compound could not be effectively eluted. As the elution volume increased, the peak area of the target compound gradually increased. When the elution volume reached 200 μL, the peak area change tended to be stable, so the elution volume was chosen as 200 μL.

Fig. 3. Effect of different elution volume on the recoveries of fifteen sulfonamides.

3.3. Optimization of liquid chromatography conditions

This experiment uses the Symbiosis Pico automatic on-line SPE system, which consists of an on-line SPE device and liquid chromatography. The samples purified by on-line SPE were directly injected into LC column for analysis, which would result in the spread of targets for eluting solution, so the dilution to decrease the percentage of methanol using the mobile phase was necessary to make the peak of target compound focus in the front of LC column. In this study, the total flow rate was fixed at 0.6 mL/min and the percentage of organic in preliminary mobile phase was fixed at 2%. The elution flow rate was 0.1 mL/min, in order to avoid pressure fluctuations caused by the change of total flow rate, which can cause abnormal peaks, the speed of the mobile phase needs to be reduced (from 0.6 mL/min to 0.5 mL/min).

Optimization of the liquid chromatographic conditions was investigated for the optimum separation of all the compounds. Different mobile phase systems acetonitrile-water, acetonitrile-0.1%(V/V) formic acid water, methanol-water and methanol-0.1% (V/V)formic acid water were used. Under ESI+conditions, the addition of formic acid could increase ionization of the compounds, which improved the separation efficiency and the intensity of the mass spectrometry signal. Methanol and acetonitrile as mobile phases had little difference on the signal intensity. When methanol was used as the mobile phase for gradient elution, column pressure varied widely and equilibration was longer. Therefore, acetonitrile-0.1% formic acid aqueous solution was selected as the mobile phase.

3.4. Optimization of mass spectrometry parameters

Syringe injection was used to inject fifteen sulfonamides and the internal standard solutions (100 ng/mL) directly into the mass spectrometer at a rate of 5 μL/min. Precursor ions were found in Q1 MS full scan and two fragments were selected using product ion scan mode. Multiple reaction monitoring (MRM) mode was used to monitor the precursor to product ion transitions. The declustering potential (DP) and collision energy (CE) of the product were optimized. The qualitative and quantitative ion detection parameters of the target compound are shown in Table 2, and extracted ion chromatograms recorded in the MRM acquisition mode for the compounds shown in Fig. 4.

Fig. 4. Extracted ion chromatograms recorded in the MRM acquisition mode for the compounds (a): SAA (7.73 min); (b): SPD (8.25 min); (c): SDZ (8.04 min); (d):SMZ (9.29 min); (e): STZ (8.11 min); (f): SMR (8.42 min); (g): SFZ (9.42 min); (h):SMT (8.60 min); (i): SDM (8.71 min); (j): SMM(8.67 min), SMP (8.93 min); (k): SCP(9.12 min); (l): SDT (9.68 min), SDX (9.26 min); (m): SPA (9.74 min); (n):13C6-STX(9.28 min).

3.5. Method validation

3.5.1. Linearity

Taking into account the losses caused by preparation and matrix effects, the internal standard (13C6-STX) was used to correct the quantitative results. The linearity of the method was determined by constructing calibration curves with different concentrations of fifteen sulfonamides and corresponding isotope internal standards with fixed concentration. As Table 3 showed, the linear range was studied by preparing a calibration curve with a concentration range of 0.1–100 ng/mL for each sulfonamide, and a good linear relationship with correlation coefficients (R2) between 0.9992 and 0.9999 was achieved for fifteen sulfonamides in their respective linear range.

Table 3Linearity, LOD and LOQ of fifteen sulfonamides.

Table 4Recoveries and RSD of fifteen sulfonamides in different matrices (n = 6).

3.5.2. Sensitivity

Limits of detection (LODs) and limits of quantification (LOQs)were evaluated using the spiked samples, and LODs and LOQs of the fifteen sulfonamides in pork and fish samples were calculated by signal-to-noise ratio of 3 and 10 (the ratio between intensity of signal of each compound obtained under MRM conditions and intensity of noise in a spiked sample). The LODs of fifteen sulfonamides were in range of 0.125–2.00 μg/kg, The LOQs of fifteen sulfonamides were in range of 0.25–5.00 μg/kg. LOD and LOQ for the methods on determination of sulfonamides in the pork and fish were shown in Table 4.

3.5.3. Recovery and precision

The recoveries were assessed by spiked blank pork and fish samples at the concentration of 0.25–50 μg/kg, and each level was repeated six times. Precision of the method was expressed by relative standard deviation (RSD). In recovery experiments,fifteen sulfonamides were spiked before extraction and on-line SPE-LC–MS/MS, so statistical results could reveal the loss of analytes during the entire process. The data of recovery and precision were given in Table 4, the average recoveries of fifteen sulfonamides were in range between 78.3% and 99.3% in pork and fish. The RSDs were in range of 0.2%–9.4%.

3.6. Comparison with standard methods

The method established in this study (Method 1), GB/T 20759-2006 (Method 2) [32] and Announcement No. 1025-23-2008 by Ministry of Agriculture (Method 3) [33] were used to detect positive samples. The detection results of the three methods were consistent. However, after the sample was extracted with acetonitrile, the liquid-liquid extraction method was used for purification in Method 2. Comparing to Method 1, Method 2 consumes larger amount of reagent and takes longer time. After the sample was extracted by ethyl acetate, the off-line SPE was used for purification in Method 3, which required activation, equilibration, loading,leaching, elution, nitrogen blowing, and reconstitution. These steps were tedious, and probable to introduce artificial error. The LOD and LOQ in Method 1 were much lower than those in Method 2 and Method 3. Compared with off-line SPE and liquid-liquid extraction, on-line SPE could reduce sample preparation time,increase sample throughput and decrease the risk of sample contamination since the entire SPE process took place in an enclosed circuit. Furthermore, purified samples were transferred directly to the chromatographic column for analysis, without the error-prone steps (e.g. evaporation and reconstitution). Usually, the average time spent to finish the entire off-line SPE process was 2–3 h. It only took 3–5 min to complete the same steps by using on-line SPE. So, the Method 1 is superior to Method 2 and Method 3 in terms of detection cost, measurement efficiency, sensitivity and repeatability.

4. Conclusions

In this experiment, a rapid and sensitive LC–MS/MS method was developed to analyze fifteen sulfonamides based on the on-line SPE technique, and isotope-labeled internal standard was used in the quantitative analysis. The linearity, sensitivity, accuracy and precision of the method were investigated, and the developed method has been successfully used for the detection of fifteen sulfonamides in pork and fish sample.

Declaration of Competing Interest

The authors have declared no conflict of interest.

Acknowledgements

This work was supported by “National Key Research and Development Program of China” (Project No. 2018YFC1603400) and Science and Technology Program of Hebei Province (Project No.19225503D).