Preservatives are added to many food products such as soda, juices, and sauces to prevent decompositions resulting from microbial and alteration caused by chemical changes. The most commonly used global preservatives in food are SA, BA and their corresponding salts as well as parabens (properties of these preservatives are shown in Table 1)   . Preservatives are added to extend the shelf life of unstable food products preventing the alteration in texture, flavour and color, as well as, to protect the health of consumers. However, adding high amount of preservatives could cause alteration in taste. Whereas, less than optimal amount will have little effect on microbial growth resulting in failure of preservative properties of the substance. Maximum levels for preservatives application in food products are subjected to international food law regulations     . Therefore, an effective and efficient analytical method is required to assure that the amount of the added preservative is within the permitted levels. Several analytical methods have been applied in research studies to determine preservatives in foods include high performance liquid chromatography (HPLC)   , headspace gas chromatography-flame ionization detector (HS GC-FID)  , gas chromatography-mass spectrometry  , and miniature ion trap mass spectrometer  .
In a previous study we optimized and validated a reliable method based on derivatisation and dispersive liquid-liquid microextraction method, followed by gas chromatography-mass spectrometry (GC-MS). Fast automated Scan/SIM Type (FASST) acquisition was applied to simultaneously determine (qualitative and quantitative analysis) two different classes of preservatives, i.e., carboxylic acid and phenolic compounds in representative pharmaceuticals and cosmetics  . FASST is a type of mass acquisition that switches between Scan and SIM mode for a single group during a measurement. As a result, acquisition parameters for both Scan and SIM modes can be specified in the same group. Here we report on the application of the develop method to the analysis of the studied preservatives in two different food products (i.e., soft drink and sauces).
Table 1. Properties of SA, SB, MP and PP.
2. Materials and Methods
Methylparaben (99%, MP), propylparaben (99%, PP), sorbic acid (98%, SA), and isobutyl chloroformate (99%, iBCF) were acquired from Merck (Germany). Sodium benzoate (99%, SB), sodium hydroxide (98%, NaOH), pyridine (98%, Pyr), and chloroform (99.5%, CHCl3) were provided by BDH (England). Ethanol and acetonitrile were purchased from Sigma-Aldrich (USA). Individual stock solutions of each target analyte at a concentration of 1000 µg/mL were prepared in distilled water and stored at 4˚C. Further dilutions and mixtures were prepared on the day of analysis in distilled water.
2.2. Food Samples
Liquid (i.e., soft drink) and semi-solid (i.e., sauces) food samples by global brands were purchased from local Baghdad stores. An approximately 1.0 gm of sauces sample was diluted to 10 mL with deionized water and then treated with an ultrasonic bath for 10 min to disperse the aliquot. The sauces sample was then filtered to remove undissolved particles prior to analysis. Whereas, 1mL of soft drink was diluted to 10 mL and degassed for 10 min in the ultrasonic bath at room temperature. Samples were spiked with 5 µg/mL of the corresponding mixture of the target analytes at different dilution ratio (i.e., 1:50, 1:100, 1:1000, and 1:2000) to evaluate matrix effect of real samples.
2.3. Derivatization and DLLME Procedures
The applied method for preparing and extracting iBCF derivatives of the target analytes in foods was adopted from our previous study  . Briefly, acylation derivatisation was performed with 30 µL of iBCF, 30 µL of pyridine, and 150 µL ethanol, which was followed by DLLME approach to recover the generated derivatives with 1mL of CHCl3. After centrifugation, 1 µL from the organic phase was injected into the GC-MS. Figure 1 illustrates the applied method.
2.4. GC-MS Analysis
Analyses were performed on a Gas Chromatograph (GC 2010Plus) coupled to a Mass Spectrometer (QPMS 2010Ultra), both instruments were made in Shimadzu/Japan. The detailed setup parameters for the GC-MS system are shown in Table 2. The GC-MS spectra were evaluated by GCMSsolution v.4 software (Shimadzu, Japan). Target analytes were searched in the National Institute of Standards and Technology (NIST) MS search v.8 library browser and verified by comparing with the authentic standards.
3. Results and Discussion
3.1. Targeted GC-MS Analysis
According to the combined derivatisation-extraction method that has been optimized in our pervious study, two families of preservatives (i.e., carboxylic acid
Figure 1. A scheme of the applied method  .
Table 2. GC-MS setup parameters.
and phenolic compounds) were investigated in food samples. As reported before, a tenfold increase in sensitivity has been observed for all studied analytes when compared to those analysed without derivatisation. An improvement in the chromatographic performance, i.e., symmetric peak shape and responses has been observed with the applied method. The two-step derivatisation protocol was performed by applying pH adjustment to simultaneously detect the carboxylic acid and phenolic compounds in the studied samples. Compound detection was based on FASST acquisition where high sensitivity for the quantitative analysis (SIM mode) and mass spectra for unknown identification (Scan mode) can be achieved. Ions with higher abundance (higher S/N ratio) was selected for better sensitivity, whereas, ions with higher m/z ratio were chosen for better selectivity. As a result, ions monitored in FASST mode for all derivatives of target analytes were: m/z (67), 95, 140 for SA; m/z 77, (105), 122, 150 for BA; m/z 57, 121, (152) for MP; and m/z 57, 121, (138) for PP; the quantifier ions being in brackets (Table 3).
During the derivatisation reaction, minor derivatives with a ratio of less than 5% for all analytes and major derivatives for both classes were generated in the reaction medium. The major derivatives of MP and PP were detected as alkoxycarbonly esters, whereas, alkyl esters were superior for SA and BA (Figure 2). Figure 3 shows the extracted ion chromatograms for the major derivatives of target preservatives that were used for the quantification analysis.
The matrix effect (ME) was evaluated accordingly to the dilution ratios mentioned in section (2.2). Percent recoveries of the target analytes from the samples were generally improved for all analytes. Sauce samples showed a higher percentage of recovery when applying 100-fold dilution, while, 1000-dilution was required to analyse soft drink samples (Figure 4).
Table 3. GC-MS analyses of target analytes as iBCF derivatives.
ES: ethyl sorbate; IS: isobutyl sorbate; EB: ethyls benzoate; EMP: ethyl methylparaben carbonate; IMP: isobutyl methylparaben carbonate; EPP: ethyl propylparaben carbonate; IPP: isobutyl propylparaben carbonate. *hydrogen migration and rearrangement; values in bold represent base peak ions; vs: very small ion peak; m/z: mass to charge ratio; ND: not detected. [M+•]: molecular ion; [M-15]: loss of methyl group (-CH3); [M-28]: loss of ethylene group (-C2H4); [M-31]: loss of methoxy group (-OCH3); [M-56]: loss of isobutyl group (-C4H8); [M-59]: loss of propoxy group (-OC3H7); [M-72]: loss of ethoxycarbonyl group (C3H4O2); [M-100]: loss of isobutoxycarbonyl group (C5H8O2).
Figure 2. Overall scheme of iBCF derivatisation of four target compounds at two pH medium aqueous system: (1) MP; (2) PP; (3) SA; (4) BA.
Figure 3. GC-MS (FASST) ion chromatograms of derivatised: SA (a); BA (b) characterized as ethyl esters; MP (c); and PP (d) characterized as isobutoxycarbonyl esters.
Figure 4. Recovery of target preservatives (SA, BA, MP, and PP) spiked into food samples after dilution of 1:100 and 1:1000.
3.2. Application to Real Samples
The applicability of the method to real samples was evaluated by the determination of the target analytes to accurately quantify the target preservatives in the selected samples (i.e., soft drink and sauce). The target analytes were determined according to the standard addition method where real samples spiked at three concentration levels (0.5, 1.0, and 5.0 µg/mL). Each sample was analyzed in triplicate, and the mean values of the concentration of SA were 210 µg/mL and 1000 µg/mL in soft drink and sauce samples, respectively. On the other hand, BA (226 µg/mL) was only detected in soft drink, whereas, parabens were not found in any sample collected from the local stores. The added preservatives that are labelled on the sample must not exceed the maximum allowed limit. As reported in food survey of benzoates and sorbates in soft drinks, 150 µg/mL is the maximum permitted level for BA, whereas, 300 µg/mL for SA (as a single additive) and 250 µg/mL (as a combined additive with BA) are the maximum allowed level  . Benzoate is largely used in beverage industry because of the high concentration of fructose corn syrup in soft drinks including water-based, flavoured, and carbonated drinks  . As a result, benzoate containing soft drinks account for the highest human consumption in the world. Also, chemical changes can occur to BA due to the presence of other chemical ingredients in a sample resulting in the generation of carcinogenic benzene. Therefore, food control systems are essential for imported and exported foods to ensure health and safety of domestic consumers, and to maintain consumer confidence in the food system.
3.3. Non-Targeted GC-MS Analysis
In addition to the main objective targeted analysis of the commonly used preservatives in food, non-targeted approach of the applied samples was achieved by only a single run. In another word, the key benefit of this multi-analysis approach was the non-targeted analysis that allowed screening of unknown substances in the samples without authentic standards. The chromatogram of Figure 5 verifies that the used method can be applied for quantitative and monitoring analysis. The ingredients (citric acid, malic acid) found in soft drinks were putatively characterised as ethyl derivatives (Figure 6).
Figure 5. Resultant ion chromatogram from the GC-MS (FASST) analysis of soft drink for the added ingredients.
Figure 6. Mass spectra for three ingredients found in soft drink samples that putatively identified as (a) diethyl malate, (b) triethyl citrate, and (c) tetraethyl citrate at RT of 12.87 min, 15.72 min, and 16.31 min, respectively.
In comparison to previous studies, the applied method demonstrated the convenient usage of isobutyl chloroformate derivatisation to qualitative and quantitative analysis of various ingredient compounds present in different food matrices by GC-MS in FASST mode. FASST acquisition allows improved sensitivety and selectivity of analytes as well as minizes sample matrix interferences. The application of this method along with appropriate standard addition with sample dilution approach proved that matrix effects were strongly demininshed for two classes of preservatives in two different matrices of real samples, and therefore will find a wide-range application for routine analysis in food control laboratories.
The method based on derivatization-DLLME followed by GC-MS FASST for the simultaneous determination of the commonly used preservatives (i.e., SA, BA, MP and PP) in food samples has been applied. The present study demonstrated the capability of performing this multicomponent analytical method conveniently in laboratory-based quality control.
The authors gratefully thank to the Mass Spectrometry Service Laboratory at the University of Al-Mustansiriya/College of Science/Department of Chemistry, for the permission to conduct the GC-MS experiments and would also like to thank Dr Unber Shafique for proofreading the manuscript.
 Martin, Y.C. (1996) Exploring QSAR: Hydrophobic, Electronic, and Steric Constants C. Hansch, A. Leo, and D. Hoekman. American Chemical Society, Washington, DC. 1995. Xix + 348 pp. 22 × 28.5 cm. Exploring QSAR: Fundamentals and Applications in Chemistry and Biology. C. Hansch and A. Leo. American Chemical Society, Washington, DC. 1995. Xvii + 557 pp. 18.5 × 26 cm. ISBN 0-8412-2993-7 (set). $99.95 (set). Journal of Medicinal Chemistry, 39, 1189-1190.
 Angelov, T., Vlasenko, A. and Tashkov, W. (2007) HPLC Determination of pKa of Parabens and Investigation on their Lipophilicity Parameters. Journal of Liquid Chromatography & Related Technologies, 31, 188-197.
 Yang, P., Ren, H., Qiu, H., Liu, X. and Jiang, S. (2011) Determination of Four Trace Preservatives in Street Food by Ionic Liquid-Based Dispersive Liquid-Liquid Micro-Extraction. Chemical Papers, 65, 747-753.
 Sugiura, J. and Nakajima, M. (2017) Simultaneous Determination of Nine Preservatives in Food by Liquid Chromatography with the Aid of Coagulant in the Clean-Up Process. Food Additives & Contaminants: Part A, 1-10.
 Dong, C., Mei, Y. and Chen, L. (2006) Simultaneous Determination of Sorbic and Benzoic Acids in Food Dressing by Headspace Solid-Phase Microextraction and Gas Chromatography. Journal of Chromatography A, 1117, 109-114.
 Djatmika, R., et al. (2016) Determination of Paraben Preservatives in Seafood Using Matrix Solid-Phase Dispersion and On-Line Acetylation Gas Chromatography- Mass Spectrometry. Journal of Chromatography B, 1036-1037, 93-99.
 Ma, Q., et al. (2016) Direct Identification of Prohibited Substances in Cosmetics and Foodstuffs Using Ambient Ionization on a Miniature Mass Spectrometry System. Analytica Chimica Acta, 912, 65-73.
 (2008) Food Standards Agency, Food Surveys, Survey of Benzoates and Sorbates in Soft Drinks.