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Determination of Sulfonamides in Chicken Muscle by Pulsed Direct Current Electrospray Ionization Tandem Mass Spectrometry Xian Fu, Hengxing Liang, Bing Xia, Chunyan Huang, Baocheng Ji, and Yan Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03803 • Publication Date (Web): 31 Aug 2017 Downloaded from http://pubs.acs.org on September 7, 2017

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Journal of Agricultural and Food Chemistry

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Determination of Sulfonamides in Chicken Muscle by Pulsed Direct Current

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Electrospray Ionization Tandem Mass Spectrometry

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Xian Fu†, ‡, Hengxing Liang§, Bing Xia†, *, Chunyan Huang§, Baocheng Ji†, ‡,

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Yan Zhou†,*

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†

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Road, Gaoxin Distinct, Chengdu 610041, P. R. China

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‡

University of Chinese Academy of Sciences, Beijing 100049, P. R. China

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§

Chengdu Institute for Food and Drug Control, Chengdu 610045, P. R. China

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*Corresponding author (Tel: 82890810; E-mail: [email protected])

Chengdu Institute of Biology, Chinese Academy of Sciences, No. 93 South Keyuan

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ACS Paragon Plus Environment


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ABSTRACT

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A simple and rapid approach for the simultaneous detection of trace amounts of six

12

sulfonamides in chicken muscle was developed using pulsed direct current electrospray

13

ionization tandem mass spectrometry (pulsed-dc ESI-MS/MS). The pretreatment of

14

chicken muscle samples consisted of two steps, acetonitrile extraction and n-hexane

15

delipidation. Sulfonamides do not need to be derivatized or chromatographed prior to

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pulsed-dc ESI-MS/MS. The factors affecting the performance of pulsed-dc

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ESI-MS/MS were studied. Under optimum conditions, the quantitative performance of

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pulsed-dc ESI-MS/MS was validated according to European Union Decision

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2002/657/EC, and the sensitivity of pulsed-dc ESI-MS/MS was three times higher than

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that of ultra-high-performance liquid chromatography-tandem mass spectrometry

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(UPLC-MS/MS). The limits of detection obtained by pulsed-dc ESI-MS/MS were in

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the range of 0.07-0.11µg/kg. The proposed method was simple, rapid, and sensitive,

23

and was successfully used for quantitation and rapid screening of sulfonamides in real

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chicken muscle samples.

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KEYWORDS: pulsed-dc ESI-MS/MS, UPLC-MS/MS, sulfonamides, chicken muscle,

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European Union Decision 2002/657/EC, quantitation, rapid screening

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INTRODUCTION

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Sulfonamides are widely used in stockbreeding as a cheap but effective antibiotic.

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Due to excessive use, sulfonamide residues in agricultural products are also

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increasingly common, which is harmful to food security and to public health.1 The

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European Union has set a maximum residue limit (MRL) for sulfonamides in food at

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the level of 100 µg/kg.2 The current methods of detecting sulfonamide residues in food

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include

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chromatography,7-8

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(LC-MS/MS).2, 9-12 The sensitivity of microbial assay is relatively low and can produce

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inaccurate results. Employing immunoassays is tedious, and the detection is too

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specific to be used for multi-analytes analysis. In addition, sulfonamides need to be

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derivatized prior to fluorometric, spectrophotometric, and gas chromatographic

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detection, which increases the workload. When using LC and LC-MS/MS, the

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development of LC method is a prerequisite.

microbiological and

methods,3 liquid

immunoassays,4-5

chromatography-tandem

spectrophotometry,6 mass

spectrometry

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To overcome the deficiencies of above mentioned methods, ambient ionization mass

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spectrometry methods have been developed in recent years. Starting from desorption

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electrospray ionization,13 over forty ambient ionization methods have been presented.14

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Ambient ionization methods have the advantages of operating in situ,15 in real-time,16

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on-line,17 with high sensitivity18 and high throughput.19 Among them, constant voltage

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pulsed direct current electrospray ionization (pulsed-dc ESI),20 as a typical

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representative of high sensitivity ionization techniques, was first reported in 2015. 3



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Pulsed electrospray is generated at a capillary emitter tip when direct current voltage is

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applied to the electrode positioned close to, but not touching, a sample solution.

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Pulsed-dc ESI produces long signal durations without losing detection sensitivity. At

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present, there are few reports about the application of ambient ionization methods in the

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quantitative analysis of compounds. Studies on ambient ionization are mainly focused

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on qualitative analysis and the development of new ionization methods. So far, the

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application of pulsed-dc ESI for quantitative analysis has not been reported.

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The objective of this study was to develop a sensitive method for rapid screening and

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quantitation of sulfonamides in food samples without the use of chromatographic

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separation or derivatization. UPLC-MS/MS and pulsed-dc ESI-MS/MS were

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compared. Sulfonamide residues in real chicken muscle samples were quantitated using

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this new presented technique.

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MATERIALS AND METHODS

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Chemicals and materials

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The standards of sulfadimethoxine, 1, sulfameter, 2, sulfamethazine, 3, sulfathiazole,

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4, sulfamethoxazole, 5, and sulfadiazine, 6, were obtained from Dr. Ehrenstorfer

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(Augsburg, Germany). Their structures are shown in Figure 1. n-Hexane of analytical

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grade was purchased from Kelong Chemical Reagent Factory (Chengdu, China).

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Chromatographic grade methanol and acetonitrile were purchased from Fisher

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(Pittsburgh, PA). Ultrapure water was obtained from a Milli-Q water system (Millipore, 4


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Billerica, MA). The borosilicate glass capillaries (0.6 mm i.d., 1.0 mm o.d., without

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filament) were purchased from Narishige Co. Ltd. (Tokyo, Japan). The steel electrode

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(0.3 mm o.d.) was bought from a local hardware store (Chengdu, China). Stock

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solutions of sulfonamides at a concentration of 1.0 mg/mL were prepared monthly in

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methanol and stored at -18 °C.

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Apparatus

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UPLC-MS/MS was performed on an Acquity UPLC system combined with an

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electrospray ionization source (ESI) on a Xevo TQ triple quadrupole mass spectrometer

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(Waters, Milford, MA). The chromatographic column used was a 100 mm x 2.1 mm i.d.,

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1.6 µm, Cortecs UPLC C18 (Waters, Milford, MA).

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Sample

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A total of about 80 samples from different traders were employed. Blank samples

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were confirmed to be free of sulfonamides by pulsed-dc ESI-MS/MS and

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UPLC-MS/MS applying the following procedures. Standard solution of sulfonamides

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was spiked into the blank samples for method validation.

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Sample treatment

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Sample treatment was basically in accordance with the national standards of P. R.

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China (GB/T 21316-2007) (2007).21 5 g of homogenized chicken muscle samples 5



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were accurately weighed and transferred into a round-bottomed flask followed by 20

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mL of acetonitrile to extract the analytes. The liquid was sucked out after being

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vortexed for 2 min and sonicated for 5 min; the precipitate was extracted with

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additional of 20 mL of acetonitrile. Then the liquid was combined and concentrated to 5

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mL by a rotary evaporator at 40 °C. 4 mL of acetonitrile saturated n-hexane was added

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to the supernatant to remove fat by 2 min sonication. The n-hexane layer was discarded

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after 10,000 rpm centrifuge for 3 min, and the acetonitrile layer was evaporated to

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dryness. Additional acetonitrile (20 mL) was added to dissolve the sulfonamides by

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manual shaking and stirring for 2 min, in such case the fat and protein on the flask

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wall would not be dissolved. The solution was transferred to a new round-bottomed

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flask prior to repeat evaporation and dissolution by 10 mL of acetonitrile twice.

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Finally, there was no fat and protein on the flask wall. For UPLC-MS/MS analysis,

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the residues were dissolved with 2 mL of methanol/water (1:9, v/v) and filtered

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through a 0.22 µm filter membrane, and then 10 µL of the solution was injected into

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the UPLC system. Prior to pulsed-dc ESI-MS/MS detection, the remaining residues

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were dissolved in 2 mL of methanol. After filtration through a 0.22 µm filter

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membrane, 0.6 µL of the solution was loaded onto the capillary emitter tip.

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UPLC-MS/MS conditions

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The mobile phase was composed of A (0.3% aqueous formic acid) and B

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(acetonitrile) at a flow rate of 0.3 mL/min.22 The column temperature was kept at 6


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40 °C, and the following gradient procedure was applied: linear change from 5-40% B

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during the first 6 min, 40-95% B over 1 min, held at 95% B for 1 min, then decreased

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to 5% B in 1 min. Multiple-reaction monitoring (MRM) mode was used for mass

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spectrometry data acquisition under positive ionization mode. In addition, the

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capillary voltage was 2.5 kV and desolvation temperature was 500 °C. The

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desolvation and cone gas flow rate were set at 500 L/h and 2 L/h, respectively. Under

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such condition, the retention times were found to be 2.23, 2.51, 3.24, 3.41, 4.35 and

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5.32 min for 6, 4, 3, 2, 5, 1, respectively.

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Determination of sulfonamides by pulsed-dc ESI under MRM mode

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Borosilicate glass capillaries (0.6 mm i.d., 1.0 mm o.d.) were pulled by PC-10 puller

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(Narishige, Tokyo, Japan) to fabricate spray emitters. The internal diameter of the tip

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was 20 µm. Based on the size of the emitter, a self-made 3D-printed insulating holder

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was built to mount the emitter. 0.6 µL of solution was loaded onto the emitter tip by

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micropipettor. A steel electrode with a diameter of 0.3 mm was inserted straight into the

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emitter from its rear portion. The distance from the tip of electrode to sample solution

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was 1 mm and the emitter tip to mass spectrometer inlet was 5 mm. A direct current of

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4.0 kV was applied to the electrode to produce electric field for electrospray generation.

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Spray solvent was methanol. Schematic of the pulsed-dc ESI system is shown in Figure

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2. All manipulations of the pulsed-dc ESI were performed on a three-dimensional

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mobile platform, which increased the repeatability and stability of the experiment 7



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operation. Taking into account the accuracy of the results, 3 was selected as the internal

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standard for quantitation of other five sulfonamides. The MRM parameters were

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consistent with the UPLC-MS/MS condition. Since pulsed-dc ESI is an ambient

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ionization technique, the capillary voltage and desolvation gas flow were zero. Other

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instrument settings were not changed.

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Validation study

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Method validation was performed according to the European Union regulation

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2002/657/EC.23 The following parameters were evaluated: selectivity, linearity, limit

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of detection (LOD), limit of quantitation (LOQ), decision limit (CCα), detection

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capability (CCβ), precision, accuracy, stability, and robustness. Linearity was assessed

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by matrix-matched calibration curve. CCα and CCβ were calculated by the standard

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deviations of the within-laboratory reproducibility. Precision and accuracy were

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investigated at three concentrations (0.5, 1.0 and 1.5 MRL).

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sulfonamides in chicken muscle investigated at 4 °C and -18 °C. In additional, seven

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small changes was deliberately introduced into experiment process to evaluated

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robustness.

The stability of

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RESULTS AND DISCUSSION

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The primary objective and challenge in method development was to find a suitable

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set of conditions that could generate stable signal while achieving optimal sensitivity. 8


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The optimized parameters were the diameter of the emitter tip, the solvent system, the

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distance from the electrode to the solution, and the spray voltage. A 0.6 µL aliquot of a

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standard sulfonamide solution at a concentration of 10 ng/mL was used to optimize the

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parameters. All data was collected in MRM mode.

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Inner diameter of emitter tip

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In the ionization process, pulsed-dc ESI has a similar ionization mechanism to ESI.

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After droplets experienced Taylor cone formation and coulomb explosion, gas ions

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were emitted from the emitter tip.24 It has been proven that better sensitivity could be

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achieved by reducing the size of the emitter tip.25 The inner diameter of the capillary tip

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was 1-3 µm, which could produce a flow rate of 20 to 30 nL/min. Droplet diameters

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decreased as the spray flow rate decreased. Smaller droplets were more desirable

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because of their larger charge-to-volume ratios, which facilitated the formation of gas

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ions. However, small diameter nozzle had a drawback of clogging. We found that even

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the presence of bubbles during the sample loading process might prevent the generation

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of spray, disturb continuous signal, or block the nozzle, which was not conducive to

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obtaining stable data. As the tip diameter decreased, the chances of the tip being

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blocked were multiplied. Hence, after balancing the operational feasibility, stability and

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detection sensitivity, a spray emitter tip with a diameter of 20 µm was chosen, which

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was also the common size of nanoelectrospray tip.26

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Spray solvent

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It has been demonstrated that droplet generation and fragmentation are strongly

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influenced by solvent surface tension. It is noteworthy that reducing the surface tension

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does not necessarily enhance the response of the sample in the mass spectrometer,

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because the solution surface tension can simultaneously affect the charge density on the

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surface of the droplet. For this reason, the influence of solvents on sulfonamide signal

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intensity was investigated in detail. Tests were performed by varying the ratio of

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methanol to water as follows: 0:10, 3:7, 5:5, 7:3, 9:1, and 10:0. As presented in Figure

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3A, larger methanol ratios increased the ion signal intensity. When the spray solvent

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was pure water, the signal durations were much longer than that of pure methanol,

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while the signal intensity was much weaker. This is because the surface tension of water

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is too large to be conducive to forming gas ions. In theory, the sulfonamides are

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derivatives of p-aminobenzene sulfonamide, thus adding formic acid would improve

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ionization efficiency. However, in our experiments, no obvious change in MRM signal

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intensity was observed after adding 0-0.3% (v/v) formic acid to the methanol.

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In addition, mixtures of 50% methanol and three organic solvents for electrospray

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were tested, including acetonitrile,27 dichloromethane,28 and isopropanol29 (Figure 3B).

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Because the viscosity (η(cp)) of isopropanol (2.437) is much greater than methanol

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(0.597), dichloromethane (0.43) and acetonitrile (0.360), which is not conducive to

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spraying, the lowest signal intensity was obtained in methanol/isopropanol (5:5, v/v).

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Since dichloromethane and acetonitrile were aprotic solvents that could not provide 10


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protons to charge the compounds, the signal intensity in pure methanol was higher than

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that in dichloromethane/methanol (5:5, v/v) and acetonitrile/methanol (5:5, v/v).

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Therefore, pure methanol was used for the following experiments. In addition, due to

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the low boiling point of dichloromethane, in the process of dichloromethane

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gasification, water vapor in the nearby air was more likely to be condensed and

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involved in the ionization process;30 the signal in dichloromethane/methanol (5:5, v/v)

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was merely 10% lower than that in methanol.

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Distance from electrode to solution

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The distance from electrode to solution played an important part in this work. In

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consideration of ionization efficiency and operational controllability, the distance

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between the electrode and solution was varied from 5 to 1 mm to avoid coming into

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contact with the solution. As the electrode approached the solution, the signal strength

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also increased accordingly (Figure 3C). It is worth noting that shortening the distance

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did not lead to shorter signal durations. Thus, 1 mm was selected as the experimental

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distance. For comparison, inserting the electrode of 0.3 mm diameter directly into the

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solution was also evaluated. However, the sample solution either adhered to the

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electrode or squeezed out from the capillary tip, leading to severe sample loss; the

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advantages of using pulsed-dc ESI without contacting the solution were noted by the

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absence of these problems.

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Spray voltage

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Studies had shown that increasing the voltage within a certain range could promote

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droplet formation and fragmentation. The droplet radius decreased as the spray voltage

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increased, making gas phase ions more likely to be generated. We therefore conducted a

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series of investigations varying spray voltages from 1.0 to 4.5 kV (Figure 3D). When

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the voltage was lower than 2.0 kV, the ionization signal was rather weak; however, 2.0

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kV was enough to ionize analytes in common nanospray methods.31 This phenomenon

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might be caused by two reasons. Firstly, the electrode was 1 mm away from the solution,

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and consequently the electric field actually applied to the solution was reduced.

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Another reason was that the diameter of the nozzle tip (20 µm) was larger than the

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diameter of the capillary tip (1-3 µm). Theoretically, the required spray voltage

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increases as the nozzle diameter increases. In our experiment, when the applied voltage

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increased from 2.0 to 3.0 kV, there was a noticeable improvement in signal intensity.

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Further increasing the voltage caused slight signal enhancement. The signal intensity of

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4.0 kV was merely 1.5% stronger than that of 3.5 kV. The signal strength dropped

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slightly as higher voltages were applied, which indicated that voltage had a limit to its

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effect on promoting the ionization process. To achieve maximum detection sensitivity,

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4.0 kV was chosen as the optimal spray voltage.

236 237 238

Method validation Both methods were validated in accordance with the European Commission 12


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Decision 2002/657/EC in terms of selectivity, linearity, LOD, LOQ, CCα, CCβ,

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repeatability, inter-day reproducibility, accuracy, stability and robustness.

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Specificity means the ability of a method to measure the analytes accurately in the

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presence of matrix interferences. 20 blank chicken muscle samples from different

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traders were analyzed by both methods; due to the efficient clean-up step and high

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selectivity provided by MRM, no matrix interference was found. However, the

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undetected matrix components may enhance or suppress the signals of analytes. In

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order to evaluate the matrix effect, calibration curves of solvent and chicken muscle

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extract were constructed in the range of 5-1000 ng/mL, respectively. By comparing

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the slope of the standard curve of solvent and chicken muscle extracts, the matrix

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suppression effect for UPLC-MS/MS and pulsed-dc ESI-MS/MS were 2.5-9.1% and

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7.9-13.7%, respectively. When the matrix effect was in the range of -20% to 20%, the

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effect was considered small.32 The small matrix effect may be caused by the efficient

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clean-up step and insensitivity of sulfonamides to matrix.2, 22, 33 To quantitate more

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accurately, the matrix-matched calibration curve was employed.

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Calibration curves were constructed using spiked chicken muscle samples with

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least-squares linear regression model. For UPLC-MS/MS, the calibration curves for

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six sulfonamides were established by plotting the peak areas versus the concentrations

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of analytes, and the spiked level were 0, 2, 4, 20, 40, 80, 200, and 400 µg/kg. For

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pulsed-dc ESI-MS/MS, 3 was used as the internal standard, and the concentration was

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40 µg/kg. The calibration curves for five sulfonamides were established by plotting 13



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the peak area ratio of analytes to internal standard versus the concentrations of

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analytes. Satisfactory linearity was obtained, with correlation coefficients greater than

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0.9966 (Table 1). In the European Commission Decision 2002/657/EC, LOD and

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LOQ were replaced with CCα and CCβ. Therefore, LOD and LOQ were determined

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according to the International Conference on Harmonisation of Technical

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Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines.34

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LOD was the lowest concentration that could be reliably detected but not required to

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be quantitated, with the signal-to-noise ratio over 3:1; LOQ was the lowest level that

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could be reliably quantitated with relative standard deviation (RSD) less than 20%,

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and the signal-to-noise ratio over 10:1. The results in Table 1 showed that the LODs

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of UPLC-MS/MS and pulsed-dc ESI-MS/MS for sulfonamides were in the range of

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0.20-0.32 µg/kg and 0.07-0.11 µg/kg, respectively, which were far below the MRL set

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by European Union. Representative MRM chromatograms of spiked chicken muscle

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samples are shown in Figure 4. The sensitivity of pulsed-dc ESI-MS/MS was about

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three times higher than that of UPLC-MS/MS. There might be three reasons why

275

pulsed-dc ESI-MS/MS was more sensitive than UPLC-MS/MS. First, the inner

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diameter of capillary tip of pulsed-dc ESI was smaller than that of commercial ESI;

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ionization efficiency increased as the diameter of the tip decreased. Second, the spray

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tip of commercial ESI was perpendicular to the mass spectrometer inlet; while the

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spray tip of pulsed-dc ESI was horizontally aligned with the mass spectrometer inlet,

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improving the ion transport efficiency. In addition, the spray tip of pulsed-dc ESI was 14


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only 5 mm away from the 150 °C inlet，which was conducive to the vaporization of

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charged droplets.

283

Decision limit (CCα) and detection capability (CCβ) are parameters used to

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determine whether the sample is compliant. If the content in a sample is at and above

285

CCα, the sample can be regarded as non-compliant with an error of α. 20 blank

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samples spiked with permitted limit concentration (100 µg/kg) were used for CCα

287

measurement. It was calculated as the permitted limit concentration plus 1.64 times

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the corresponding standard deviations of the within-laboratory reproducibility (α =

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5%). If the content in a sample is at CCβ, the sample can be detected and quantitated

290

with an error of β. CCβ was calculated as CCα plus 1.64 times the corresponding

291

standard deviations of 20 samples spiked at CCα (β = 5%). The CCα obtained by

292

both methods was close to MRL value (Table 1), indicating the detection error α was

293

small.

294

Precision experiments were performed at three concentrations (0.5, 1.0 and 1.5

295

MRL). The precision was evaluated by intra-day and inter-day RSDs. Intra-day RSDs

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were obtained from six replicates within one day. Inter-day precision was performed

297

by six replicates within one day and for three consecutive days. As shown in Table 2,

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all RSDs obtained by both methods were lower than 15%, indicating the repeatability

299

and stability of pulsed-dc ESI-MS/MS was comparable to that of UPLC-MS/MS.

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However, the RSD value of pulsed-dc ESI-MS/MS was a little higher than that of

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UPLC-MS/MS. Because any detection technique used for quantitative analysis was 15



302

very demanding and the electrospray ionization was susceptible to environment, the

303

stability of closed ionization was slightly better than that of ambient ionization.

304

Accuracy was assessed by the recovery of three concentrations (0.5, 1.0 and 1.5

305

MRL) and six replicates were performed at each level. The recovery was calculated

306

by the ratio of measured mass to spiked mass. The mean recoveries obtained by

307

UPLC-MS/MS and pulsed-dc ESI-MS/MS were in the range of range of 87.4-101.3%

308

and 88.4-99.0%，respectively (Table 2), which fully fulfilled the criteria of -20% to 10%

309

set in the European Commission Decision 2002/657/EC.

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Spiked chicken muscle samples at the concentration of 100 µg/kg were used for

311

stability test. The results showed that sulfonamides could be saved in 4 °C for 24 h or

312

-18 °C for three days. No sulfonamides were stable for a week, which may be because

313

sulfonamides were degraded by the microbes that bred from chicken muscle.

314

Robustness refers to the susceptibility of a method to the minor differences in the

315

experiment. For pulsed-dc ESI-MS/MS, the variables from sample treatment to

316

detection were evaluated. Seven parameters were vortex time (1.8, 2.2 min),

317

sonication time for extraction (4.5, 5.5 min), volume of acetonitrile for extraction (19,

318

21 mL), volume of n-hexane (3.5, 4.5 mL), brand of methanol (Fisher, Merck), spray

319

voltage (3.95, 4.05 kV) and distance from electrode to solution (0.9, 1.1 cm). The

320

results showed that those parameters had no significant effect on robustness. However,

321

we found that the morphology and inner diameter of the emitter tip had influence on

322

the results. This may be due to the fact that there was a process of ion migration 16


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during spraying, and the morphologies of the spray tip affected the migration of ions

324

differently. In addition, the diameter of the spray tip had different effects on the

325

ionization efficiency of each compound. Therefore, the spray condition of each

326

detection should be controlled.

327 328

Comparison with other detection methods

329

The most important advantage of the proposed method was simplicity, rapidity, and

330

sensitivity. The detection could be performed within 3 min, much faster than

331

spectrophotometry, enzyme-linked immunosorbent assay (ELISA), and liquid

332

chromatography. For example, 10 min was needed for the derivation of sulfonamides

333

with p-dimethylaminocinnamaldehyde before spectrophotometric determination,6, 35 at

334

least 2 h was needed for ELISA,4 and the detection time of HPLC (/UPLC) ranged

335

from 10-40 min.2, 36-37 The LOD of the proposed method (0.07-0.11 µg/kg) was lower

336

than other reported methods, such as enzyme-linked immunosorbent assay

337

(0.04-0.2 µg/g),4 HPLC with fluorescence detection (1.15-2.73 µg/kg),38 and

338

LC-MS/MS (0.43-1.22 µg/kg).33 In addition, satisfactory linearity, precision, accuracy,

339

CCα and CCβ were obtained, indicating pulsed-dc ESI-MS/MS had sufficient

340

performance to be a new candidate for sulfonamide rapid screening and quantitation.

341 342 343

Analysis of real samples To verify the practicability of the proposed method, sulfonamide residues in six 17



344

chicken muscle samples from three sales environments were determined by

345

UPLC-MS/MS and pulsed-dc ESI-MS/MS. Two of the samples were positive;

346

detection rate was 33.3%. The results of the two methods were similar. Detection

347

deviations of the two methods were less than 12%, indicating the quantitative

348

performance of pulsed-dc ESI-MS/MS was reliable. The residues did not exceed MRL

349

(100 µg/kg). MRM chromatograms of a positive sample are shown in Figure 5. 1 and 2

350

were detected in both samples, probably because 1 is widely used for the prevention

351

and treatment chicken coccidiosis, infectious rhinitis, avian cholera, Kaposi's disease,

352

and 2 is also a broad-spectrum antibacterial agent.22 Interestingly, the two positive

353

samples were purchased in a worse environment than the other four samples. Perhaps,

354

the quality of a sample from a better sales environment is more assured.

355

In this work, pulsed-dc ESI, as a new kind of quantitative ambient ionization

356

method, was applied to the rapid screening of sulfonamide residues in chicken muscle

357

samples. Sulfonamides did not need to be derivatized or chromatographed prior to

358

pulsed-dc ESI-MS/MS. The operation of pulsed-dc ESI was simple, and rapid. When

359

pulsed-dc ESI-MS/MS was used to detect sulfonamides in chicken muscle samples,

360

the results fulfilled the criteria in Commission Decision 2002/657/EC. In addition, the

361

sensitivity of pulsed-dc ESI-MS/MS were three times higher than that of

362

UPLC-MS/MS, which was of great significance for the detection of sulfonamide

363

residues in food. Overall, pulsed-dc ESI-MS/MS could be regarded as an option for

364

sensitive, rapid screening and quantitative analysis of sulfonamides in food samples. 18


Page 18 of 36

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365

ASSOCIATED CONTENT

366

Supporting Information

367

Determination of sulfonamide residues in milk samples with pulsed-dc ESI-MS/MS.

368

Table S1: Mass spectrometer characteristics of six sulfonamides. Table S2: Detection

369

results of sulfonamide residues in chicken muscle samples. Figure S1: MRM

370

chromatograms of a blank sample of (A) UPLC-MS/MS and (B) pulsed-dc

371

ESI-MS/MS. Figure S2: Matrix effects of sulfonamides in chicken muscle. Figure S3:

372

The sulfonamide MS/MS fragmentation patterns of the [M+H]+. Figure S4: The

373

MS/MS mass spectra of sulfonamides.

374

This material is available free of charge via the Internet at http://pubs.acs.org.

375 376

AUTHOR INFORMATION

377

Corresponding authors s

378

*E-mail address: [email protected]; [email protected]

379 380

Notes

381

The authors declare no competing financial interest.

382 383

ABBREVIATIONS USED

384

pulsed-dc ESI-MS/MS, pulsed direct current electrospray ionization tandem mass

385

spectrometry; UPLC-MS/MS, ultra-high-performance liquid chromatography-tandem 19



386

mass spectrometry; MRL, maximum residue limit; LOD, limit of detection; LOQ

387

limit of quantitation; CCα, decision limit; CCβ, detection capability.

388 389

ACKNOWLEDGMENTS

390

Dr. Yan Zhou received funding from the National Natural Sciences Foundation of

391

China (21572221), and Dr. Bing Xia received funding from the National Natural

392

Sciences Foundation of China (21672206).

20


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393

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515

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516

FIGURE CAPTIONS:

517

Figure 1. Structures of the sulfonamides studied: sulfadimethoxine, 1; sulfameter, 2;

518

sulfamethazine, 3; sulfathiazole, 4; sulfamethoxazole, 5; sulfadiazine, 6.

519

Figure 2. Schematic of the pulsed-dc ESI.

520

Figure 3. Optimization of the pulsed-dc ESI. (A) and (B) Solvent composition.

521

Distance from electrode to sample solution, 2 mm; Spray voltage, 2.8 kV. (C) Distance

522

from electrode to solution. Spray solvent, methanol; Spray voltage, 2.8 kV. (D) Spray

523

voltage. Distance from electrode to solution, 1 mm; Spray solvent, methanol.

524

Figure 4. (A) UPLC-MS/MS MRM chromatograms of the spiked sample (400 µg/kg),

525

(B) Pulsed-dc ESI-MS/MS MRM chromatograms of the spiked sample at the

526

concentration of 40 µg/kg.

527

Figure 5. MRM chromatograms of a real sample by (A) UPLC-MS/MS and (B)

528

pulsed-dc ESI-MS/MS.

27



Page 28 of 36

Table 1. Calibration Curves, LOD, LOQ, CCα and CCβ for Sulfonamides. analyte

linear range (µg/kg)

regression equation

correlation coefficient

LOD (µg/kg)

LOQ (µg/kg)

CCα (µg/kg)

CCβ (µg/kg)

107.6

UPLC-MS/MS a

1

0-400

y = 409.10x + 1156.7

0.9991

0.20

0.60

103.7

2

0-400

y = 136.84x – 98.56

0.9985

0.32

1.00

103.7

106.4

3

0-400

y = 276.94x – 731.66

0.9973

0.24

0.72

104.2

106.8

4

0-400

y = 215.33x + 25.226

0.9988

0.26

0.84

103.5

107.8

5

0-400

y = 121.89x + 13.271

0.9989

0.32

1.00

104.6

108.1

6

0-400

y = 194.54x + 472.84

0.9984

0.26

0.84

104.3

107.6

1

0-400

yb = 0.0414x + 0.1971

0.9989

0.07

0.21

104.8

108.8

2

0-400

y = 0.0122x + 0.0702

0.9976

0.09

0.28

102.8

106.5

4

0-400

y = 0.0130x + 0.0578

0.9987

0.09

0.28

103.4

107.0

5

0-400

y = 0.0081x + 0.0517

0.9983

0.11

0.33

101.4

105.6

6

0-400

y = 0.0127x + 0.1003

0.9966

0.09

0.28

104.5

107.5

pulsed-dc ESI-MS/MS

a

b

y, the peak areas of analytes; y, the peak area ratio of analytes to internal standard; x, the concentration of analytes (µg/kg).

28


Page 29 of 36


Table 2. Results of Recovery and Precision for Sulfonamides. 0.5 MRL analyte recovery (%) RSDra (%)

RSDRb (%)

1.0 MRL recovery (%) RSDr (%) RSDR (%) UPLC-MS/MS (n=6)

recovery (%)

1.5 MRL RSDr (%)

RSDR (%)

1

92.3

10.1

6.9

95.1

8.0

6.7

88.1

8.1

7.2

2

98.6

9.5

7.0

90.9

6.2

7.7

87.4

6.8

9.0

3

95.3

5.4

5.6

94.6

7.6

8.9

93.1

7.4

8.8

4

96.9

5.8

6.9

91.7

6.7

7.9

92.7

7.0

7.2

5

93.8

6.3

5.4

101.3

6.0

9.9

90.3

7.4

9.8

6

92.6

8.0

8.3

90.6

5.3

6.1

91.7

6.9

8.8

pulsed-dc ESI-MS/MS (n=6) 1

92.8

9.1

8.4

92.3

8.5

9.6

88.4

8.9

8.3

2

99.0

8.0

10.3

93.3

10.0

10.5

89.3.

10.8

9.7

4

92.3

9.2

8.6

93.6

7.6

8.7

91.5

9.1

9.0

5

98.9

10.7

10.9

98.8

9.1

11.7

92.8

7.4

10.1

6

95.6

9.8

11.4

91.8

8.8

10.1

92.6

9.5

10.8

a

b

RSDr, the RSD of repeatability; RSDR, the RSD of inter-day reproducibility.

29



Figure 1

30


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Figure 2

31



Figure 3

32


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Figure 3 (continued)

33



Figure 4

34


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Figure 5

35



Table of Contents Graphic

36


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Determination of Sulfonamides in Chicken Muscle ... - ACS Publications

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