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Non-targeted Screening and Determination of Sulfonamides: A Dispersive Micro Solid-phase Extraction Approach to the Analysis of Milk and Honey Samples using Liquid Chromatography- High Resolution Mass Spectrometry Shuping Hu, Min Zhao, Yiyuan Xi, Qiqi Mao, Xudong Zhou, Dawei Chen, and Pengcheng Yan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05773 • Publication Date (Web): 17 Feb 2017 Downloaded from http://pubs.acs.org on February 18, 2017

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Journal of Agricultural and Food Chemistry

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Non-targeted Screening and Determination of Sulfonamides: A Dispersive Micro

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Solid-phase Extraction Approach to the Analysis of Milk and Honey Samples using

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Liquid Chromatography- High Resolution Mass Spectrometry

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Shuping Hu,† Min Zhao,† Yiyuan Xi,† Qiqi Mao,† Xudong Zhou,† Dawei Chen,*,‡

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Pengcheng Yan*,†

7

†

Zhejiang 325035, China

8 9 10

School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou,

‡

Key Laboratory of Food Safety Risk Assessment, Ministry of Health; China National

Center for Food Safety Risk Assessment, Beijing 100021, China

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*Corresponding Author: (Tel: +86-10-67779768. Fax: +86-10-67790051. E-mail:

13

[email protected].)

1

ACS Paragon Plus Environment


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Abstract

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A simple, rapid, sensitive, selective, and environmentally friendly method, based on

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dispersive micro solid-phase extraction approach (dispersive micro SPE) coupled with

17

liquid chromatography-high resolution mass spectrometry (LC-HRMS) was

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established for the analysis of sulfonamides in honey and milk. An efficient

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non-targeted screening strategy was designed to discover and identify known and

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unknown sulfonamides in honey and milk using full-MS/all ion fragmentation (AIF)

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mass spectrometry acquisition mode. The experimental parameters and conditions of

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dispersive micro SPE on extraction efficiency were optimized in detail. Under the

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optimized conditions, the dispersive micro SPE method showed a low limit of

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detection (LOD) for the targeted sulfonamides ranging from 0.003-0.2 µg/L in milk

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and 0.01-1 µg/kg in honey with the recoveries of the analytes between 68.8% and

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115.8%. Compared with the reported methods, improvements in convenience, low

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cost, and environmental friendliness were obtained in this study.

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Keywords: dispersive micro solid-phase extraction (dispersive micro SPE),

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sulfonamides, honey, milk, high resolution mass spectrometry (HRMS), all ion

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fragmentation (AIF)

2


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INTRODUCTION

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Sulfonamides (Figure 1) are an important group of antibiotics extensively used in

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human and veterinary medicine due to their high efficiency, inexpensiveness and

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wide-spectrum antimicrobial activity.1 However, the possible presence of sulfonamide

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residues in animal foods has become a public health concern, mainly because

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excessive exposure can increase the risk of drug resistance and cause some side

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effects.2 To protect human health from this potential risk, the European Union (EU)

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has set a maximum residue limit (MRL) for sulfonamides at the total level of 100 ng/g

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in animal foods, such as meat, milk, and eggs.3 Thus, rapid and efficient analytical

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methods are required for monitoring of sulfonamides at trace-residual level.

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Among several reported analytical methods,4-15 liquid chromatography-mass

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spectrometry (LC-MS) is still the most useful detection technique because of its high

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sensitivity, especially in the analysis of trace sulfonamide residues in milk and honey.

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However, one of the main limitations to mass spectrometry is the interference of

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matrix effect, which will affect the trueness and precision of quantitative analysis.16 In

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order to reduce the occurrence of matrix effect, a more exhaustive sample preparation

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method is extremely important prior to the analysis by LC-MS. Several sample

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preparation methods have been described for analysis of sulfonamides in milk and

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honey, such as solid-phase extraction (SPE),4,6,11-14 stir bar sorptive extraction,7

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dispersive liquid-liquid microextraction.9 Among all these methods, SPE is the most

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frequent one used for efficient extraction, however, it requires multiple operation

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steps and consumes a large amount of organic solvents and sorbents. Fortunately, 3



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some new pretreatment methods, such as micro solid-phase extraction (micro SPE)

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and dispersive micro solid-phase extraction (dispersive micro SPE), have been used

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for residue analysis based on the traditional SPE technique.17–22 For example, Ibarra et

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al.10 developed a micro SPE and HPLC method using Fe3O4–SiO2–phenyl modified

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sorbent to determine sulfonamides in milk, but required a relatively long extraction

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time (15 min) and a large amount of sorbent (100 mg) to achieve the adsorption

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equilibration.

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(MIL-101(Cr)@GO) was also used as dispersive micro SPE sorbent for the

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pretreatment of twelve sulfonamides in milk.15 Although the amount of sorbent was

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smaller (5 mg), this method required an additional liquid-liquid extraction procedure

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and a relatively longer extraction time (20 min). Additionally, so far these magnetic

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materials are not readily available in most laboratories. Therefore, it is very important

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to find a kind of sorbent which possesses high adsorption capabilities for

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sulfonamides in a shorter extraction time and also be commercially available. Cation

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exchange material is assumed to be a promising sorbent for analysis of contaminants

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in food matrices because it can rapidly adsorb alkaline compounds with high chemical

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selectivity and has been widely applied in SPE method.23-25

Furthermore,

metal-organic

framework/graphite

oxide

material

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Among the cation exchange materials, polymer cation exchange (PCX) material

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can yield superior adsorption efficiency for alkaline compounds than the silica-based

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cation exchange material because of higher surface area. Unlike other adsorbents

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applied for dispersive micro SPE (carbon nanotubes, graphene and inorganic

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nanoparticles),26 the adsorption mechanism of PCX is not based on the principle of 4


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hydrogen bonds, π-π stacking interactions, electrostatic forces and hydrophobic

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interactions, but the ability of ion exchange. This mechanism can ensure the same

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adsorption capacity in organic solvent media, which also extend the application of this

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material from aqueous samples to other food matrices, such as the animal derived

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foods. In this work, a simple dispersive micro SPE method with PCX material as

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sorbent is present for the extraction and determination of 24 sulfonamides in milk and

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honey aiming at reducing the extraction time. Additionally, the non-targeted screening

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technique is becoming more and more important with the frequent occurrence of

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illegal drugs adulterated in food. This study also presents an efficient non-targeted

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screening strategy to discover and identify known and unknown sulfonamides in milk

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and honey using high resolution mass spectrometry (HRMS).

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MATERIALS AND METHODS

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Chemicals and Materials

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PCX powder (40-60 µm, average particle size) was purchased from Bonna-Agela

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Technologies (Tianjin, China). Ultra-pure water (H2O) was purified by a Milli-Q

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system (Milford, MA). HPLC-grade acetonitrile (MeCN), methanol (MeOH) and

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other chemical reagents were commercially available. 24 sulfonamides (Figure 1)

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including sulfaguanidine, 1, sulfanilamide, 2, sulfacetamide, 3, sulfadiazine, 4,

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sulfisomidine, 5, sulfathiazole, 6, sulfapyridine, 7, sulfamerazine, 8, trimethoprim, 9,

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sulfamoxole,

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sulfamethoxypyridazine, 14, sulfamonomethoxine, 15, sulfachlorpyridazine, 16,

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sulfamethoxazole, 17, sulfadoxine, 18, sulfisoxazole, 19, sulfabenzamide, 20,

10,

sulfamethazine,

11,

sulfamethizole,

5


12,

sulfameter,

13,


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sulfaclozine, 21, sulfadimethoxine, 22, sulfaphenazole, 23, and sulfaquinoxaline, 24

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(>96.5% purity) and 14 isotope internal standards (IS) (13C6-2, 13C6-4, 13C6-6, 13C6-7,

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13

C6-8, D3-9, 13C6-11, D3-14, D4-15, D4-17, D3-18, 13C6-19, 13C6-22, 13C6-24) were all

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obtained from Dr. Ehrenstorfer (Augsburg, Germany) and Sigma-Aldrich (Shanghai,

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China). The individual stock solutions of 24 sulfonamides and ISs (1.0 mg/mL) were

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prepared in MeOH. Mixed working standard solutions (1 mg/L and 10 mg/L) were

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prepared by diluting the stock solutions with MeOH. All standard solutions were

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stored at -20 °C and stable for 60 days.

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Milk samples were purchased from the local markets from Beijing in China and

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collected from different brands and manufactures, and honey samples were collected

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from different botanical origins in China through the food safety risk monitoring plan

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for which the laboratory of China National Center for Food Safety Risk Assessment is

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responsible.

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Dispersive Micro SPE for Samples Preparation

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Dispersive micro SPE procedure was carried out by a 5 mL syringe with a syringe

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filter. The sample solution was placed in a centrifuge tube with 15 mg PCX which had

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been dispersed in 2 mL H2O. The solution was vortexed for 30 s and transferred into a

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5 mL syringe with a syringe filter. Then the solution was passed through the syringe

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and filter manually and discarded. After decantation of the sample solution, the PCX

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sorbent was washed with 1 mL H2O again and eluted with 1 mL of 5% ammonium

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hydroxide in MeCN-H2O (50:50 v/v). The eluate was used for analysis. An overview

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of sample preparation for the milk and honey is shown in Figure 2.

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Apparatus Conditions

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The ultra-high performance liquid chromatography (UHPLC) analysis was carried 6


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out on a Dionex Ultimate 3000 system (Sunnyvale, California) with a Waters HSS T3

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(100 mm × 2.1 mm i.d., 1.8 µm) column (Milford, Massachusetts) and a Thermo AQ

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C18 (150 mm × 2.1 mm i.d., 1.7 µm) column (Bremen, Germany). The column

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temperature was set at 40 °C. A mixture of H2O (A) and MeOH (B) containing 0.1%

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formic acid was used as mobile phase. The separation was accomplished by using a

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gradient elution at 0.3 mL/min (0-2 min 2-20% B, 2-6 min 20-40% B, 6-10 min

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40-100% B, 10-12 min 100-100% B, 12-13 min 100-2% B, 13-16 min, 2-2% B).

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The analysis was conducted with a Q-Exactive HRMS (Thermo Scientific, Bremen,

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Germany) equipped with a heated electrospray ionization (HESI) source. All the data

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were acquired under the full-MS/all ion fragmentation (AIF) mode using positive

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electrospray ionization (ESI+). The detailed ion source parameters were published

132

previously.27 Data were processed with XCalibur and ToxID 2.2 software (Thermo

133

Scientific).

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Method Validation

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The calibration curve was established by analysis of a series of concentrations of

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the standard solution, and was plotted by the peak area ratio (y): the standard to

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respective IS; versus concentration (x) of the analyte. LOD (S/N=3) and LOQ

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(S/N=10) were calculated by the spiked experiments of the lowest concentration. The

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method precision (intra-day and inter-day precision) and accuracy (percentage

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recoveries) were evaluated by five replicate analysis of sulfonamides in spiked milk

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and honey samples at three different concentrations. Intra-day precision (so called

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repeatability, in terms of % RSDr) was determined on the same day. For inter-day

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precision (so called reproducibility, in terms of % RSDR), the spiked samples were

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analyzed on three consequent days. The extraction recoveries were determined by

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comparing the measured values with the expected values. The matrix effect was 7



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estimated by building calibration curves from a matrix solution (milk or honey) and a

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matrix free solution, and was expressed as the signal suppression or enhancement by

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the slope ratio of the analytes in matrix and matrix free solution.

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RESULTS AND DISCUSSION

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Optimization of UHPLC Conditions

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In this experiment, two frequently-used types of analytical columns, including AQ

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C18 and HSS T3 were tested. Both columns are compatible with 100% aqueous mobile

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phase, and suitable to retain and separate some polar compounds. As shown in Table

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1, 13/14/15, 16/21, 18/22 and 10/19 are four sets of isomers with the same molecular

155

weight, even with similar fragment ions. Therefore, it is essential for the four sets of

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isomers to be chromatographically separable. The results showed that 16/21, 18/22

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and 10/19 had good chromatographic separation in both columns, whereas the T3

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column had a better separation effect and superior ionization efficiency than the C18

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column for 13/14/15 (Figures 3A and B). As for the mobile phase, MeCN-H2O and

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MeOH-H2O with 0.1% formic acid were compared. Figure 3A shows that 13/14 did

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not achieve complete separation in MeCN-H2O containing 0.1% formic acid. Finally,

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MeOH-H2O with 0.1% formic acid was selected as the optimum mobile phase (Figure

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3C), by which an adequately good ionization efficiency of the 24 sulfonamides was

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obtained with high sensitivity and good separation resolution.

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Non-targeted Screening of Sulfonamides by Full-MS/AIF Mode

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Full-MS/AIF acquisition mode was applied for the multi-target and non-targeted

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screening approach. The AIF mode provides the fragmentation of all the generated

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precursor ions. Accordingly, the use of full-MS/AIF mode allows to all necessary

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information about the precursor and fragment ions to be obtained in one run.

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Although the mixing of product ion spectra could be caused by the absence of 8


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precursor ion selection in the quadrupole instrument under AIF mode, adequate

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specificity could still be achieved in the screening of sulfonamides using HRMS

173

analysis.

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In this work, an efficient non-targeted screening strategy was developed to discover

175

and identify known and unknown sulfonamides in milk and honey by full-MS/AIF.

176

For the known sulfonamides, a sulfonamides database was created with 72

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sulfonamides including 47 metabolites. Based on the established UHPLC-HRMS

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method, the samples were analyzed in full-MS/AIF mode. In the sulfonamides

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database, compound name, elemental composition, polarity, exact mass (m/z),

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retention time (tR) and characteristic fragment ions were included for each

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sulfonamide. In particular, MS/MS spectra were obtained by stepped fragmentation at

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20%, 35%, and 50% of normalized collision energy (NCE) in higher energy

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collisional dissociation cell for further identification of sulfonamides. Fortunately,

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several specific fragment ions were observed in the analysis of fragment ions for

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different kinds of sulfonamides. All the sulfonamides except for TMP had the same

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fragment ions (m/z 156.01138, 108.04439, and 92.04948) due to the same structural

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skeleton. For the unknown sulfonamides not found in the database, further compound

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discovery and structure elucidation were performed under AIF mode using the

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characteristic fragment ions of sulfonamides.

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An example of this is presented in Figure 4. Firstly, the extractive characteristic

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fragment ions chromatograms from the mixed product ion spectra were obtained by

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AIF mode. Secondly, the retrospective data analysis of so far unknown compounds

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was performed by full-MS to obtain its corresponding precursor ion, and the possible

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precursor ion was extracted from the full-MS. In this case, the ion m/z 215.04843 in

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the chromatogram (Figure 4A) was selected and it was found to have the same tR and 9



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peak shape as the characteristic fragment ions in Figures 4B-D, indicating that it was

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the precursor ion of these fragment ions. Finally, the QualBrowser of XCalibur was

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used to assign molecular formula to each exact mass based on atomic constraints set

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by the parent sulfonamides and the measured isotopic pattern. The atomic settings

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were a minimum of 6xC, 9xH, 2xO, 2xN, 1xS, in accordance with the atomic

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composition of the parent sulfonamide. When the standard deviation was within 5

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ppm, and the measured isotope pattern fit the atomic composition, the molecular

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formula was considered plausible. The result shows that the elemental composition of

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the selected precursor ion m/z 215.04843 [M+H]+ is C8H11O2N2S, indicating a

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standard deviation of 0.1 ppm. By this non-targeted screening strategy, we can obtain

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a glimpse of the unknown sulfonamides, including their metabolites, because the

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standards are relatively scarce in various monitoring laboratories.

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Optimization for Sample Preparation

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In this work, PCX material was selected as the sorbent in dispersive micro SPE

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procedure for the extraction of 24 sulfonamides from milk and honey due to the

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characteristic that a rapid adsorption equilibration can be achieved in 30 s as shown in

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our preliminary study. Additionally, the extraction efficiency is also affected by other

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parameters, including the pH of sample solution, the amount of PCX, the desorption

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solvent and volume.

215

Sulfonamides, as weakly alkaline substances, are protonated under acid conditions

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and the rapid adsorption capacities of PCX material for sulfonamides was mainly

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based on the ion exchange mechanism. Thus, the pH level of sample solution was

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investigated first. However, our results revealed that there was no obvious difference

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in the extraction efficiencies of sulfonamides when the pH of sample solution was in

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the range from 1 to 5 and lack of pH adjustment (pH 6.4). This phenomenon may be 10


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attributed to the fact that the PCX material possesses considerable strong ion

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exchange abilities, and the pH of sample solution (pH 6.4) is adequate to keep the

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sulfonamides positively charged. Moreover, the amounts of PCX ranging from 10-25

224

mg were also carefully studied. It was observed that a maximum extraction efficiency

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for all 24 sulfonamides was achieved at 15 mg, and there was no significant increase

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on extraction efficiency when the amount of sorbent was further increased, which

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meant that 15 mg of PCX was adequate to extract the sulfonamides. A proper

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desorption solvent is a significant factor in the elution of sulfonamides. In this work,

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MeCN-H2O (1:1, v/v) with different ratios of ammonium hydroxide (1-10%) was

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evaluated for the efficiency of releasing the sulfonamides from PCX sorbent.

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Desorption efficiencies gradually increased as the concentration of ammonium

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hydroxide was increased from 1-5% and slightly changed when the concentration was

233

up to 10%. Furthermore, the correlation between desorption efficiency and solvent

234

volume ranging from 0.5-2 mL was also studied. As expected, the desorption

235

efficiency increased with the volume of solvent, and reached a maximum equilibrium

236

above 1 mL. Therefore, the optimum volume of desorption solvent was determined as

237

1 mL.

238

Matrix Effects Evaluation

239

After optimization of the dispersive micro SPE procedure, matrix effects for the

240

sulfonamides in milk and honey were studied by comparing the slope ratios of the

241

analytes in matrix and matrix free solution. Generally, matrix effect was considered

242

tolerable if the ratio was between 0.8 and 1.2. As shown in Table 1, weak matrix

243

effects were observed in honey matrices with slope ratios of 0.87-1.14 for all 24

244

sulfonamides. The proposed method yielded weaker matrix effects than those of

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reported SPE method in honey,8,13 while the sample dilution factors and 11



246

chromatographic conditions were completely different from those reported methods.

247

As for milk matrices, with moderate to high levels of proteins, different matrix effects

248

were observed for the sulfonamides. It can be seen that no apparent matrix effects

249

were observed for the 13 sulfonamides with slope ratios between 0.8 and 1.2, such as

250

5, 8, and so on. However, several sulfonamides exhibited a strong signal suppression

251

effect (0.51-0.74), and a signal enhancement effect (1.23 for 17 and 1.24 for 9) was

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also observed in milk matrices. In order to reduce the influence of matrix effects, the

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matrix-matched calibration curve was frequently used in various studies.7,8,11 In this

254

work, an isotopic dilution technique was used for compensating different matrix

255

effects and the loss of recoveries in milk and honey matrices.

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Analytical Performance

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The analytical performance of sulfonamides based on the dispersive micro SPE

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method is listed in Table 1. Method specificity was evaluated by analyzing the blank

259

samples, and no interfering peak was observed at the tR of the analytes. The

260

correlation coefficients (R2 = 0.9979-1.0000) of all 24 sulfonamides in the studied

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ranges indicated good linearity relationships by the internal standard calibration curve.

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For all 24 sulfonamides, the LODs were in the range of 0.003-1 µg/L (or µg/kg), and

263

LOQs varied between 0.01 and 3 µg/L (or µg/kg) for the milk and honey. The

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precision and accuracy were estimated by analyzing the spiked blank samples at three

265

concentration levels. As shown in Table 2, acceptable results were achieved for all 24

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sulfonamides in two matrices. Mean recoveries for all the sulfonamides fell into the

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range of 74.3-115.8%, along with RSDr range from 1.3-6.4% and RSDR of less than

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12.8% in honey. As for milk, the recoveries were exhibited between 68.8 and 112.6%,

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with RSDr and RSDR range from 1.0-13.2%.

270

Comparison with Previous Analytical Methods 12


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The proposed dispersive micro SPE-UHPLC-HRMS is a new method for the rapid

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monitoring of sulfonamides in milk and honey. The SPE method has been extensively

273

used as a pretreatment step.4,6,11-14 However, the dispersive micro SPE method is

274

superior in convenience, low cost, and environmental friendliness. For example, the

275

lower consumption of organic solvent (only 0.5 mL MeCN) and sorbent material (15

276

mg) was achieved in our dispersive micro SPE procedure. Unlike the disposable use

277

of SPE column, the sorbent material could be reused by washing with 5% ammonium

278

hydroxide in MeOH and H2O respectively after dispersive micro SPE procedure,

279

indicating that this sorbent would be a type of green and sustainable material. For

280

comparison, stir bar sorptive extraction7 required a 10 min of extraction time with less

281

organic solvent (90 µL MeOH), but dispersive liquid-liquid microextraction9

282

consumed more organic solvent (1 mL chloroform as extraction solvent and 1.9 mL

283

MeCN as disperser solvent). For dispersive micro SPE or micro SPE method, the

284

Fe3O4–SiO2–phenyl modified sorbent10 and MIL-101(Cr)@GO15 have also been used

285

for the extraction of sulfonamides. Compared with those methods, our method

286

required a shorter extraction time (approximately 30 s) than MIL-101(Cr)@GO

287

method (20 min) and less amount of sorbent material (15 mg) than

288

Fe3O4–SiO2–phenyl modified sorbent method (100 mg). Additionally, combined with

289

HRMS technique, lower detection levels with acceptable or better precision and

290

recovery were obtained in our work, and the total number of detected sulfonamides

291

was 24, including sulfanilamide, 2, which was rarely involved in previous studies.

292

Therefore,

293

environmentally-friendly and rapid analysis of sulfonamides in milk and honey.

294

Application to Real Samples

295

the

proposed

method

enabled

a

relatively

inexpensive,

The proposed method was applied to the determination of sulfonamides in 18 milk 13



296

and 51 honey samples collected from different commercial brands. Firstly, we tried to

297

use non-targeted screening to identify samples that may contain sulfonamides in the

298

absence of authentic standards. Taking milk as an example, three chromatographic

299

peaks were observed by analyzing the characteristic fragment ions of sulfonamides.

300

Then, through further analysis of parent ions, it was found that those three molecular

301

compositions were C10H11O4N2S, C11H10D3O3N4S and C12H12D3O4N4S, respectively.

302

Among them, the first peak is sulfadiazine, 4 (m/z 251.0603), and the other two peaks

303

are isotope internal standard (D3-14, m/z 284.0897; D3-18, m/z 314.1002) spiked in

304

milk. Secondly, we applied existing standards to validate and accurately quantitate

305

sulfonamides. The results showed that sulfadiazine, 4 (0.3-9.7 µg/L for the three

306

detected samples) and sulfamerazine, 8 (4.9-7.4 µg/L for the two detected samples)

307

were commonly detected in the milk samples. However, sulfamoxole, 10 (3.9 µg/kg)

308

was detected only in a honey sample.

309

In conclusion, we developed an efficient non-targeted screening strategy for the

310

discovery and identification of sulfonamides in honey and milk using UHPLC-HRMS.

311

Sulfonamides database searching by full-MS and the identification of characteristic

312

fragment ions by AIF were performed to accomplish the discovery of the known and

313

unknown sulfonamides. Furthermore, a dispersive micro SPE method based on PCX

314

material was used for the sample pretreatment. The proposed dispersive micro SPE

315

method exhibited following advantages: (a) convenient and timesaving because the

316

traditional evaporation and centrifugation steps can be omitted and a very short

317

extraction time (30 s) can be achieved based on the high adsorption capacity and

318

selectivity of PCX sorbent material; (b) low-cost and environmentally-friendly

319

compared to the traditional SPE method due to the requirement of less amount of

320

sorbent and organic solvent; (c) the relatively fewer matrix effects in honey and milk; 14


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(d) efficient monitoring of trace sulfonamide residues in milk and honey when

322

combined with UHPLC-HRMS analysis. What is more, the ability of ion exchange of

323

PCX material would extend the application of this material to organic solvent media

324

from animal food matrices. The dispersive micro SPE-UHPLC-HRMS method is

325

rapid, efficient, sensitive, low-cost and environmentally friendly, which would be

326

suitable for the risk monitoring of sulfonamides in the field of food safety, and can

327

rapidly identify potential unknown and known sulfonamides in the absence of

328

sulfonamides authentic standards.

329

Abbreviations Used

330

AIF, all ion fragmentation; SPE, solid-phase extraction; HESI, heated electrospray

331

ionization; NCE, normalized collision energy.

332

ASSOCIATED CONTENT

333

Supporting Information

334

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

335

Figure S1 (A) The extraction ions at m/z 156.01138, 108.04439, and 92.04948 by AIF

336

for all 24 sulfonamides; (B) extraction ion at m/z 281.0708 by full scan; (C) the mixed

337

product ion spectrum at the retention time (8.32 min).

338

Figure S2 Effects of: (A) the amount of PCX; (B) the ammonium hydroxide

339

concentration in MeCN; (C) the eluting solution volume on the dispersive micro SPE

340

procedure for the 24 sulfonamides (n = 3).

341

Figure S3 The extraction ions at m/z 156.01138, 108.04439, and 92.04948 by AIF for

342

sulfonamides in milk.

343

Figure S4 Workflow of the non-targeted screening of sulfonamides by

344

UHPLC-HRMS.

345

Table S1 The extraction conditions for optimization of each parameter by analyzing 15



Page 16 of 31

346

spiked samples.

347

Table S2 Information for the internal standards of sulfonamides.

348

Table S3 Comparison of various analytical methods developed for analysis of

349

sulfonamides in milk and honey matrices.

350

Sulfonamides database.

351

AUTHOR INFORMATION

352

Corresponding Authors

353

*(D.

354

[email protected].

355

*(P.

356

[email protected].

357

Funding

358

This research was funded by the National Nature Science of Foundation of China

359

(21607035).

360

Notes

361

The authors declare no competing financial interest.

362

REFERENCES

363

(1) Dmitrienko, S. G.; Kochuk, E. V.; Apyari, V. V.; Tolmacheva, V. V.; Zolotov, Y.

364

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365

detection - A review. Anal. Chim. Acta. 2014, 850, 6-25.

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(2) Shen, Q.; Jin, R.; Xue, J.; Lu, Y.; Dai, Z. Analysis of trace levels of sulfonamides

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in fish tissue using micro-scale pipette tip-matrix solid-phase dispersion and fast

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liquid chromatography tandem mass spectrometry. Food Chem. 2016, 194, 508-515.

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(3) European Union Council Regulation. Directive 675/92/EEC. Official Journal of

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Fax:

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E-mail:

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chromatography-tandem mass spectrometry method for the determination of

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sulfonamides, trimethoprim and dapsone in honey and validation according to

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Commission Decision 2002/657/EC for banned compounds. Talanta. 2012, 97, 32-41.

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(7) Yu, C.; Hu, B. C18-coated stir bar sorptive extraction combined with high

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performance liquid chromatography-electrospray tandem mass spectrometry for the

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analysis of sulfonamides in milk and milk powder. Talanta. 2012, 90, 77-84.

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quantification of nine sulfonamides in bovine milk using HPLC/MS/MS and without

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using SPE. Food Chem. 2013, 141, 2294-2299.

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(9) Arroyo-Manzanares, N.; Gamiz-Gracia, L.; Garcia-Campana, A. M. Alternative

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sample treatments for the determination of sulfonamides in milk by HPLC with

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fluorescence detection. Food Chem. 2014, 143, 459-464.

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(10) Ibarra, I. S.; Miranda, J. M.; Rodriguez, J. A.; Nebot, C.; Cepeda, A. Magnetic

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solid phase extraction followed by high-performance liquid chromatography for the

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determination of sulphonamides in milk samples. Food Chem. 2014, 157, 511-517.

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(11) Meng, Z.; Shi, Z.; Liang S.; Dong, X.; Li, H.; Sun, H. Residues investigation of

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fluoroquinolones and sulphonamides and their metabolites in bovine milk by 17



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determination of 88 veterinary drug residues in milk using automated TurborFlow

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online clean-up mode coupled to liquid chromatography-tandem mass spectrometry.

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Talanta. 2016, 148, 401-411.

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(13) Hou, X. L.; Chen, G.; Zhu, L.; Yang, T.; Zhao, J.; Wang, L.; Wu, Y. L.

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Development and validation of an ultra high performance liquid chromatography

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tandem mass spectrometry method for simultaneous determination of sulfonamides,

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quinolones and benzimidazoles in bovine milk. J. Chromatogr. B. 2014, 962, 20-29.

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(14) Gu, X.; Wu, J. P.; Zhang, X.; Li, D. N.; Yan, F.; Zhou, Y. R. Determination of 14

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sulfonamides residue in milk by on-line solid phase extraction in cation exchange

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mode/liquid chromatography-tandem mass spectrometry. Chinese J. Anal. Chem.

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(15) Jia, X.; Zhao, P.; Ye, X.; Zhang, L.; Wang, T.; Chen, Q.; Hou, X. A novel

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metal-organic framework composite MIL-101(Cr)@GO as an efficient sorbent in

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dispersive micro-solid phase extraction coupling with UHPLC-MS/MS for the

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determination of sulfonamides in milk samples. Talanta. 2016, in press,

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http://dx.doi.org/10.1016/j.talanta.2016.08.086.

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using porous metal-organic framework MIL-101 as sorbent. J. Chromatogr. A. 2015,

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1401, 9-16.

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dispersive micro-solid phase extraction using CTAB-modified zeolite NaY sorbent

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coupled with HPLC for the determination of carbamate insecticides. J. Agric. Food

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solid-phase extraction based on magnetic multiwalled carbon nanotubes for the

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determination of polycyclic aromatic hydrocarbons in edible oils. J. Agric. Food

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Chem. 2011, 59, 12794-12800.

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extraction with nano-TiO2 as adsorbent for the determination of mercury species.

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chlorophenols in human urine by gas chromatography/mass spectrometry. J.

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characterization of dissolved organic matter (DOM) in refinery wastewater by revised

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phase retention and ion-exchange adsorption solid phase extraction followed by ESI

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Ion-exchange solid-phase extraction combined with liquid chromatography-tandem

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mass spectrometry for the determination of veterinary drugs in organic fertilizers. J.

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Miniaturized solid-phase extraction technique. TrAC-Trends Anal. Chem. 2015, 73,

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(27) Chen, D. W.; Miao, H.; Zou, J. H.; Cao, P.; Ma, N.; Zhao, Y. F.; Wu, Y. N.

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457

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458

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20


Page 20 of 31

Page 21 of 31


459

Figure Captions

460

Figure 1 Structures of the sulfonamides studied: sulfaguanidine, 1, sulfanilamide, 2,

461

sulfacetamide, 3, sulfadiazine, 4, sulfisomidine, 5, sulfathiazole, 6, sulfapyridine, 7,

462

sulfamerazine,

463

sulfamethizole, 12, sulfameter, 13, sulfamethoxypyridazine, 14, sulfamonomethoxine,

464

15, sulfachlorpyridazine, 16, sulfamethoxazole, 17, sulfadoxine, 18, sulfisoxazole, 19,

465

sulfabenzamide, 20, sulfaclozine, 21, sulfadimethoxine, 22, sulfaphenazole, 23, and

466

sulfaquinoxaline, 24.

467

Figure 2 An overview of sample preparation for the milk and honey

468

Figure 3 Chromatograms for sulfameter, 13, sulfamethoxypyridazine, 14, and

469

sulfamonomethoxine, 15 under different chromatographic conditions: (A) T3 column

470

with mobile phase MeCN-H2O with 0.1% formic acid; (B) C18 column with mobile

471

phase MeCN-H2O with 0.1% formic acid; (C) T3 column with mobile phase

472

MeOH-H2O with 0.1% formic acid.

473

Figure 4 Chromatograms for sulfacetamide, 3, under different acquisition modes: (A)

474

extraction ion at m/z 215.0490 by full scan; (B) extraction ion at m/z 156.01138 by

475

AIF; (C) extraction ion at m/z 108.04439 by AIF; (D) extraction ion at m/z 92.04948

476

by AIF.

8,

trimethoprim,

9,

sulfamoxole,

10,

21


sulfamethazine,

11,


Page 22 of 31

Table 1 Calibration Curve Equations, Correlation Coefficients (R2), LODs, LOQs and Matrix Effects for the 24 Sulfonamides Honey

Milk

Analytes

tR (min)

[M+H]+ (m/z)

Linear range (µg/L)

Linearity equation

R2

LOD (µg/kg)

LOQ (µg/kg)

Matrix effects

LOD (µg/L)

LOQ (µg/L)

Matrix effects

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

2.59 2.89 5.06 6.08 6.16 6.53 6.84 7.28 7.56 7.86 8.18 7.98 7.92 8.32 8.91 8.66 8.76 9.19 9.13 9.60 10.14 10.39

215.0603 173.0385 215.0490 251.0603 279.0916 256.0214 250.0650 265.0759 291.1457 268.0756 279.0916 271.0323 281.0708 281.0708 281.0708 285.0213 254.0599 311.0814 268.0756 277.0647 285.0213 311.0814

0.01-10 0.5-20 0.2-10 0.02-10 0.05-10 0.01-10 0.01-10 0.01-10 0.01-10 0.01-10 0.05-10 0.02-10 0.005-5 0.005-5 0.01-10 0.01-10 0.005-5 0.01-10 0.01-10 0.02-10 0.01-10 0.01-10

Y = -0.0005+0.7438*X Y = -0.0054+0.0170*X Y = -0.0091+0.4118*X Y = -0.0037+0.4926*X Y = -0.0024+0.7124*X Y = -0.0050+0.7814*X Y = -0.0027+0.8378*X Y = -0.0019+0.8396*X Y = -0.0255+4.843*X Y = -0.0021+0.5579*X Y = -0.0086+0.9011*X Y = -0.0011+0.3714*X Y = 0.0014+0.5980*X Y = 0.0013+0.7043*X Y = 0.0007+3.112*X Y = -0.0024+0.2968*X Y = -0.0013+1.329*X Y = -0.0002+0.8679*X Y = -0.0051+0.8952*X Y = -0.0113+1.079*X Y = -0.0052+0.4315*X Y = 0.0022+0.9524*X

0.9989 0.9979 0.9997 0.9997 0.9999 1.0000 0.9999 1.0000 0.9990 0.9999 1.0000 0.9999 0.9999 0.9999 0.9999 0.9999 1.0000 1.0000 1.0000 0.9998 0.9997 0.9999

0.02 1.0 0.3 0.05 0.06 0.02 0.02 0.02 0.02 0.02 0.1 0.05 0.01 0.01 0.02 0.02 0.01 0.02 0.02 0.05 0.02 0.02

0.06 3.0 1.0 0.15 0.3 0.06 0.06 0.06 0.06 0.06 0.3 0.15 0.03 0.03 0.06 0.06 0.03 0.06 0.06 0.15 0.06 0.06

0.89 1.04 0.87 0.91 1.04 1.03 1.03 1.07 1.14 1.10 1.05 1.07 1.05 1.05 1.08 1.07 1.09 1.02 1.01 1.04 1.07 1.06

0.005 0.2 0.1 0.01 0.02 0.01 0.01 0.005 0.005 0.01 0.02 0.01 0.003 0.003 0.01 0.005 0.003 0.01 0.01 0.01 0.01 0.01

0.02 0.5 0.2 0.03 0.06 0.02 0.02 0.02 0.02 0.02 0.06 0.03 0.01 0.01 0.02 0.02 0.01 0.02 0.02 0.03 0.02 0.02

0.61 0.74 0.72 0.74 0.94 0.71 0.74 1.01 1.24 0.98 0.82 0.88 1.09 0.83 0.60 0.83 1.23 0.83 0.73 0.51 1.01 0.92

22


Page 23 of 31


23 24

9.97 10.66

315.0916 301.0759

0.01-10 0.1-10

Y = -0.0040+1.038*X Y = -0.0055+0.2336*X

0.9999 0.9993

23


0.02 0.2

0.06 0.6

1.06 1.09

0.01 0.1

0.02 0.2

0.94 0.96


Page 24 of 31

Table 2 Recovery and Precision Values of 24 Sulfonamides from Different Spiked Levels by the Proposed Method

Analytes 1

2

3

4

5

6

7

Honey (n = 5)

Fortified level (µg/kg)

R (%)

RSDr (%)

0.06 0.3 1.5 3.0 15 75 1.0 5.0 25 0.15 0.75 4.0 0.3 1.5 7.5 0.06 0.3 1.5 0.06 0.3 1.5

74.3 82.7 83.8 78.5 83.1 97.1 82.8 95.2 89.9 103.6 97.2 105.2 88.3 110.4 108.2 77.5 83.9 103.9 101.4 98.5 114.2

5.8 3.1 2.1 8.3 5.9 3.2 5.7 3.6 4.2 6.5 5.7 4.1 2.1 3.1 2.7 5.5 1.9 3.3 2.4 1.8 1.6

Milk (n = 5)

RSDR (%)

Fortified level (µg/L)

R (%)

RSDr (%)

RSDR (%)

10.7 5.2 7.4 11.2 7.3 3.6 7.9 6.3 4.9 6.8 6.3 5.2 6.4 7.0 4.6 5.9 2.5 4.8 4.3 3.7 6.2

0.02 0.1 0.5 0.5 2.5 12.5 0.2 1.0 5.0 0.03 0.15 0.75 0.06 0.3 1.5 0.02 0.1 0.5 0.02 0.1 0.5

84.1 76.3 85.9 68.8 73.2 79.4 86.2 83.8 85.2 93.1 98.6 99.3 74.2 95.2 107.4 78.6 89.2 99.3 93.2 99.8 106.7

4.9 2.8 1.7 6.2 8.0 5.2 4.4 1.2 2.0 6.2 3.7 4.6 3.7 3.1 2.2 5.6 4.2 1.4 2.2 1.2 1.7

6.3 4.7 3.2 13.2 8.4 11.7 5.2 3.0 4.8 7.3 6.2 5.8 4.4 4.0 3.5 7.4 8.3 4.9 4.2 4.9 3.7

24


Page 25 of 31


8

9

10

11

12

13

14

15

16

0.06 0.3 1.5 0.06 0.3 1.5 0.06 0.3 1.5 0.3 1.5 7.5 0.15 0.75 4.0 0.03 0.15 0.75 0.03 0.15 0.75 0.06 0.3 1.5 0.06 0.3

95.1 90.6 107.7 96.3 102.5 105.3 89.3 94.2 99.3 104.2 94.8 98.6 70.6 93.2 96.6 83.8 92.2 107.1 95.2 87.9 106.3 87.5 90.3 88.5 82.0 86.4

5.3 6.4 3.6 4.7 3.6 3.1 2.7 3.4 2.5 2.9 3.6 2.4 7.6 5.8 2.1 2.5 5.2 1.9 4.2 2.8 2.7 6.4 2.1 4.2 3.8 1.7

8.2 7.2 3.8 5.0 4.1 5.0 4.2 3.9 4.7 4.0 4.6 3.2 12.8 7.2 3.1 4.4 5.8 3.2 6.6 3.9 6.3 8.7 3.7 5.6 7.7 4.3

25


0.02 0.1 0.5 0.02 0.1 0.5 0.02 0.1 0.5 0.06 0.3 1.5 0.03 0.15 0.75 0.01 0.05 0.25 0.01 0.05 0.25 0.02 0.1 0.5 0.02 0.1

85.6 86.1 102.0 87.7 99.1 96.6 88.4 93.7 107.3 97.2 102.7 95.9 79.2 94.3 86.9 79.2 84.2 94.8 85.5 93.6 95.3 96.1 88.8 104.6 92.0 83.5

6.6 4.3 1.6 3.6 2.5 1.2 5.2 3.8 2.3 3.7 2.3 1.4 6.4 4.6 3.2 6.6 6.2 3.0 2.3 3.0 1.7 5.2 2.8 2.3 2.7 3.6

7.9 5.7 3.3 6.3 3.3 3.9 9.3 5.2 4.9 5.2 6.3 2.8 7.7 6.9 3.7 10.2 8.5 7.3 4.4 5.8 3.5 8.2 4.2 3.9 5.3 4.8


17

18

19

20

21

22

23

24

1.5 0.03 0.15 0.75 0.06 0.3 1.5 0.06 0.3 1.5 0.15 0.75 4.0 0.06 0.3 1.5 0.06 0.3 1.5 0.06 0.3 1.5 0.6 3.0 15

92.7 87.4 94.3 97.2 73.8 79.4 83.4 95.2 93.1 103.6 97.5 104.5 111.5 92.1 89.8 94.7 93.0 86.3 96.4 74.9 83.7 98.3 109.4 115.8 113.2

1.6 4.8 3.9 2.8 2.6 5.7 4.4 5.0 3.6 5.7 3.1 2.4 2.6 1.6 1.5 2.3 6.2 1.3 2.2 4.6 2.9 1.4 6.4 5.6 4.1

3.2 7.8 6.1 3.0 5.9 6.0 5.7 8.9 4.3 7.4 5.5 5.1 3.5 2.2 3.6 4.2 8.2 4.2 3.9 5.0 3.7 3.6 9.2 10.4 8.9

26


0.5 0.01 0.05 0.25 0.02 0.1 0.5 0.02 0.1 0.5 0.03 0.15 0.75 0.02 0.1 0.5 0.02 0.1 0.5 0.02 0.1 0.5 0.2 1.0 5.0

Page 26 of 31

87.3 74.4 79.6 83.2 82.4 74.8 87.9 91.4 104.3 104.9 86.9 98.5 107.6 93.6 92.1 108.4 76.7 93.3 95.9 80.2 87.2 89.3 102.8 97.4 112.6

4.1 2.1 3.4 1.7 3.2 3.1 2.8 3.1 2.5 3.9 2.8 1.0 1.7 2.7 1.1 2.6 5.7 3.6 3.2 2.1 2.6 1.5 5.1 3.7 4.5

6.2 5.4 5.5 2.7 6.3 7.9 5.8 4.8 5.1 6.3 5.2 3.9 4.6 3.7 2.9 4.0 12.1 7.2 5.4 4.3 3.8 2.7 7.7 8.6 6.8

Page 27 of 31


Figure 1

27



Honey (2.0 g)

Milk (1.0 mL)

Dissolved and diluted to 10 mL with H2O

1.0 mL extract using dispersive micro SPE with 15 mg PCX as sorbent

Washed with 1 mL of H2O

Eluted by 1.0 mL 5% ammonium hydroxide in MeCN/H2O (50:50 v/v)

UHPLC-HRMS by Full MS-AIF mode Figure 2

28


Page 28 of 31

Page 29 of 31


80

14

13

100

A

tR: 5.88

tR: 6.07

15

60

tR: 6.73

40

Relative Abundance (%)

20 0 100 80

tR: 8.06 13, 14

B

60 40

15 t : 8.43 R

20 0 100

tR: 7.92 13

C 80

tR: 8.32 14 tR: 8.91 15

60 40 20 0 5.0

5.2

5.4

5.6

5.8

6.0

6.2

6.4

6.6

6.8

7.0

7.2

7.4

7.6

7.8

Time (min) Figure 3

29


8.0

8.2

8.4

8.6

8.8

9.0

9.2

9.4

9.6


Page 30 of 31

100 80

tR: 5.06

A

60 40 20

Relative Abundance

Relative Abundance (%)

0 100 80

B

tR: 5.07

C

tR: 5.07

D

tR: 5.07

60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 4.4

4.5

4.6

4.7

4.8

4.9

5.0

5.1

5.2 Time (min)

5.3

Time (min) Figure 4

30


5.4

5.5

5.6

5.7

5.8

5.9

Page 31 of 31


Table of Contents Graphic

31


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