Simultaneous Quantitative Analysis of Six Isothiazolinones in Water-Based Adhesive Used for Food Contact Materials by High-Performance Liquid Chromatography–Tandem Mass Spectrometry (HPLC–MS/MS)

In this study, a target analytical approach using high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was developed to simultaneously determine six isothiazolinones containing 2-Methylisothiazol-3(2H)-one (MI), 5-Chloro-2-methyl-4-isothiazolin-3-one (CMI), 1,2-benzisothiazolin-3-one (BIT), 2-Octyl-3(2H)-isothiazolinone (OIT), Dichlorooctylisothiazolinone (DCOIT), and 2-methyl-1,2-benzisothiazolin-3-one (MBIT) in water-based adhesive used for food contact materials. The main factors affecting extraction efficiency such as extraction method, extraction time, extraction solvent, and solid–liquid ratio have been evaluated by using real adhesive samples. Multiple-reaction monitoring (MRM) was used for the qualitative and quantitative analyses of targeted isothiazolinones. This method was demonstrated as an effective and reliable technique for detecting multiple isothiazolinones with satisfactory recoveries (81.5~107.3%), and the limits of detection (LOD) and quantification (LOQ) were obtained at a low level. This method was validated and applied to the determination of six isothiazolinones in commercial water-based adhesives. The present results revealed that these adhesives contained a combination of isothiazolinones (BIT, MI, CMI, and MBIT) with the concentration ranging from 2.27 to 123.5 mg/kg. To our knowledge, it is the first time it has been reported that MBIT was detected in water-based adhesives used for food contact materials, which requires a further investigation for its migration to food and the risk to human health.


Optimization of Extraction of Isothiazolinones in Adhesives
Due to very different polarities of these isothiazolinones, sample preparation, and chromatographic separation are crucial for establishing a suitable method to determine multiple compounds. Four samples containing four kinds of isothiazolinones (MI, CMI, BIT, and MBIT) were chosen for the optimization of pretreatment conditions. The main factors affecting extraction efficiency, such as extraction method, extraction time, extraction solvent and solid: liquid ratio were optimized by single-factor experimental design.
Either ultrasonic extraction or vortexes extraction [32] was commonly used for extraction of organic compounds, with the advantage of easy operation and good extraction capability. The extraction efficiency of above methods was studied and compared by following experimental procedure: 0.50 g of water-based adhesive was weighted accurately and subject to extraction with 20 mL methanol by ultrasonic with 300 W power and vortexed at 1600 r/min for 60 min, respectively. It can be seen from Figure 2a that no significant differences were observed between the concentration of isothiazolinones obtained by applying either ultrasound or vortexes method. Given the practical resources in the lab, the vortexes method was selected to perform the extraction.
The extraction time is a key factor affecting the extraction efficiency as longer time usually increase the kinetic energy of the molecules. In this study, the extraction efficiency under different extraction time (15,30,45,60, and 90 min) for adhesive sample 1, 2, 3, and 4 were investigated. 0.50 g of water-based adhesive sample was weighted accurately and subject to vortexes extraction with 20 mL methanol at different extraction time (15,30,45,60, and 90 min). The results are shown in Figure 2b, which revealed that the extraction time actually had key impact on the concentration of the analytes measured. After 60 min's treatment, the concentration of analytes maintained stable for most samples measured. Thus, 60 min was determined as the extraction time in vortexes method. It can be seen from Figure 2b that the concentration of BIT, MI and CMI detected in some samples decline after 90 min extraction, indicating that the degradation of isothiazolinones may occur under long time extraction.
The extraction efficiency for different solvents (methanol, water, acetonitrile and methanol-water (1:1)) was studied and compared by following experimental procedure: 0.50 g of water-based adhesive sample was weighted accurately and subject to vortexes extraction with 20 mL of different solvents (methanol, water and methanol-water (1:1)) for 60 min. The results shown in Figure 2c indicated that either methanol or water has the strong extraction capability. The extraction efficiency on BIT, MI, CMI and MIBT were higher by using methanol as solvent than that using water or methanol-water (1:1) mixture. Therefore, methanol was chose as the solvent to perform the extraction of isothiazolinones from water-based adhesive.
The effects of solid:liquid ratio (1:20, 1:40, 1:60, 1:80 and 1:100) on extraction efficiency were also investigated here. 0.2 g, 0.25 g, 0.33 g, 0.5 g, 1.0 g water-based adhesive was accurately weighted, respectively, and subject to vortexes extraction with 20 mL methanol for 60 min. The results shown in Figure 2d suggesting that the extraction efficiency get boosted with the increasing solid:liquid ratio, it can be explained that the solution easily reaches saturation when the solid:liquid ratio is low, and the targeted compounds cannot be extracted more efficiently. In the case that 1:100 of solid:liquid ratio was applied, a stable concentration of analyte was obtained. Thus, 1:100 of solid:liquid ratio was chose for further extraction process. The optimized extraction condition could be summarized as: 0.2 g water-based adhesive was weighted accurately, and subject to vortexes extraction with 20 mL methanol at 1600 rpm/min for 60 min.

Optimization of Chromatographic and Mass Spectrometric Conditions
Mobile phase system of methanol-water and methanol-water (0.1% formic acid) was evaluated for the selection of mobile phase (See the Supplementary Materials Figure S1). The results showed that the targeted compounds can be completely and effectively separated by using either methanol-water or methanol-0.1% formic acid. Taking into account that the latter mobile phase has a higher response value and the ESI+ mode was used, the component of the molecule ion is more easily ionized under acidic conditions, thus, methanol-0.1% formic acid was chose as the mobile phase for this experiment.
The effects of column temperature on separation of analytes at 30 • C, 40 • C and 50 • C were investigated in this experiment. With the increase of temperature, the retention time for each analyte was expected to be observed earlier. The compound can be separated well under above three column temperatures. The peak area observed was little change and the influence of column temperature on the targeted analytes is not significant. Therefore, the column temperature was set to 30 • C. (Figure 3a).
The total ion chromatograms (TIC) chromatograms of isothiazolinones in the standard solution and water-based adhesive samples obtained under the above optimized experimental conditions was shown in Figure 3. Owing to the presence of alkyl groups, there are more signals response values observed for the fragment ions of OIT and DCOIT than that of other compounds (See Figure 3b). Six targeted analytes were injected into the mass spectrometer to determine the characteristic ions. Six isothiazolinones contain a tertiary nitrogen atom in the molecular structure, and a high response value for [M + H]+ molecular ion peak was obtained in the ESI+ mode. The characteristic secondary fragment ions can be consulted on molecular ion under the action of different collision energy in daughter scan mode. The typical daughter scan mass spectrums and MSscan mass spectrums are shown in Figure 4. It can be seen from Figure 4 Figure 5. Due to isothiazolinones contain a tertiary nitrogen atom in the molecular structure, the fragments were easier to loss the methyl radical, sulfur negative ion, oxygen negative ion, carbonyl group, amino group, and alkyl groups. The multi-reaction monitoring (MRM) mode was applied to collect the secondary fragments produced by the molecular ion in the ESI+ mode. The quantitative ions of six isothiazolinones (MI, CMI, BIT, OIT, DCOIT and MBIT) were selected according to the daughter fragment ions. The mass spectrometry parameters were optimized to determine the highest responding value of each analyte. Mass spectrometry parameters such as characteristic ions, cone voltage, and collision voltage for the targeted six analytes are shown in Table 4. The MRM chromatogram is illustrated in Figure 6.
The data are summarized in Table 1, which demonstrated that the method has satisfactory accuracy, precision, and LOQ. It is well suitable for quantitatively determination of multiple isothiazolinones in the water-based adhesives used for food contact materials.

Accuracy, Precision, and Recovery
Based on the optimized conditions, the accuracy of the method was validated by measuring the recovery of different blank samples spiked with the six isothiazolinones at three distinct levels (0.01 mg/kg,0.5 mg/kg and 10 mg/kg). The precision of the method was validated by the experiment that the samples spiked at each level was detected in six replicated times. The datum is summarized in Table 2. The relative standard deviations (RSD) ranged from 0.9%~5.9%, indicating that the precision and accuracy of the method meet the criteria of methodology.

Test Result for Water-Based Adhesive
The validated method was used to determine the concentration of isothiazolinones in four commercial water-based adhesives used for food contact materials. The results are shown in Table 3. Four isothiazolinones (MI, CMI, BIT and MBIT) were detected with concentration ranging from 2.27 to 23.5 mg/kg, while OIT and DCOIT were not detectable in all samples. MBIT was detected in one water-based adhesive with concentration of 50.17 mg/kg. To our knowledge, it was the first time that MBIT was observed in food contact adhesive. To date, as a new isothiazolinones, MBIT was not authorized by Chinese regulation to be used for food contact adhesive or other food contact materials, therefore no conclusions can be drawn on its potential health risks, and a further investigation will be needed.

Reagents and Standards
Purified water was obtained with a Milli-Q Q-POD ® system (Merck KGaA, Darmstadt, Germany); HPLC-grade methanol was obtained from Thermo Fisher (Waltham, MA, USA); MI, OIT and BIT were purchased from Dr Ehrenstorfer (LGC, London, UK); CMI was purchased from BePure ® ; DCOIT was purchased from Anpel (Shanghai, China); MBIT was purchased from Aikang (Jiangsu, China); Acetic acid and ethanol were purchased from Guangshi (Guangzhou, China).
The standard stock solution (1000 mg/L) for each target analyte was prepared by weighting 10 mg of MI, CMI, BIT, OIT, DCOIT and MBIT (accurate to 0.1 mg) respectively, followed by diluted to 10 mL volume with methanol. The intermediate standard solution (10 mg/L) was prepared by transferring 0.1 mL standard stock solution into 10 mL volumetric flasks, followed by diluted with methanol. Working mix standard solutions contained nine targeted analytes (with concentration of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5 mg/L) were prepared by mixing appropriate volumes of the corresponding intermediate standard solution and dilution with methanol.

Sample for Experimental
For determination of isothiazolinones concentration, four different water-based adhesives (Adhesive sample 1, 2, 3 and 4) were provided by various companies for the study. The details for the name and main characteristics of adhesive collected are confidential and cannot be revealed here.

Sample Preparation
For determination of isothiazolinones concentration, weight 0.2 g adhesive sample (accurately to 0.1 mg) and transferred it to the tube, 20 mL methanol was added and subject to vortex extraction for 60 min, then the fraction collected was filtered through 0.22 µm, followed by LC-MS/MS analysis. Each sample was prepared in duplicate.
The mobile phase through gradient elution was prepared by 0.1% formic acid in water as mobile phase A, and methanol as mobile phase B. The retention time, peak width, and resolution of each solute under different mobile phase conditions and column temperature were studied. A optimized HPLC condition was described as follows: the mobile phase was initially started at 50%(B)(0~1 min), it was gradient up to 95%(B) (1~1.5 min) and maintain for 6min, then it was set to 50% at 6.1min, then 50%(B) was kept at 14 min. The flow rate was kept at 0.3 mL/min. The column temperature was kept at 30 • C and the injection volume was 2 µL. (see the Supplementary Materials Figure S1) Targeted analytes were measured with electrospray ionization (ESI) in the positive mode. Nitrogen was used as a de-solvation gas and was operated at a flow rate of 6L/min at 300 • C. The nebulizer pressure was 45 psi, the capillary was set at 4000 V, the sheath gas temperature was 380 • C, the sheath Gas Flow was operated at 11 L/min; Nozzle voltage was set at 500 V. Mass spectrum parameters on the strength of the molecular ion and fragments ion were studied in MS scan mode and daughter mode (see Figures 3 and 4). The quantitative of analytes were performed in MRM mode. The optimized MRM parameters for six targeted analytes are shown in Table 4.

Conclusions
In this paper, a method for the determination of isothiazolinones in water-based adhesives used for food contact materials by LC-MS/MS was established. The main experimental factors affecting extraction efficiency, such as the extraction method, extraction time, extraction solvent, and solid: liquid ratio have been evaluated and optimized by means of single-factor experimental design, using a real adhesive sample. This method was proved to be an effective and reliable technique, which is suitable for simultaneously detecting multiple isothiazolinones at a low level. A satisfactory linear relationship was obtained in a range of 0.010~1.0 mg/L. The LOD and LOQ was 0.010 mg/L and 0.020 mg/L, respectively. The recoveries ranged from 81.5% to 107.3%, and the relative standard deviation was less than 5.9%. Moreover. This analytical method is also suitable for determination of isothiazolinones used for paints, cosmetics, and paper, etc.
The present results obtained in water-based adhesive samples indicated that these studied adhesives containing a combination of isothiazolinones (BIT, MI, CMI and MBIT) with the concentration ranging from 2.27 to 123.5 mg/kg. MBIT was detected with concentration of 50.17 mg/kg. To our knowledge, it is the first time that MBIT, as a new isothiazolinones derivative, was observed in water-based adhesives. Since MBIT is not a regulated compound used for food contact material in China, a further investigation is needed for its migration to food and the risk to human health.

Conflicts of Interest:
The authors declare no conflict of interest.