Chiang Mai J. Sci. 2019; 46(4) : 703-713http://epg.science.cmu.ac.th/ejournal/Contributed Paper

Gas Chromatography-Mass Spectrometry for Quality Control of Fortified Iodine in Seasoning Powder for Instant NoodlesWarawut Tiyapongpattana* [a], Worawan Malethong [b], Woraphot Wanichalanant [a], Duangjai Nacapricha [b] and Prapin Wilairat [c][a] Department of Chemistry, Faculty of Science and Technology, Thammasat University, Pathum Thani,12120, Thailand.[b] Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand.[c] National Doping Control Centre, Mahidol University, Rama VI Road, Bangkok 10400, Thailand.*Author for correspondence; e-mail: twarawut@gmail.com

Received: 25 November 2018Revised : 12 January 2019

Accepted: 24 January 2019

ABSTRACT In this work, a method of gas chromatography-mass spectrometry (GC-MS) has been

developed for quantitation and identification of iodine (fortified as iodide) content in seasoning powder of instant noodle. Iodide was oxidized to iodine by 2-iodosobenzoate. The generated iodine then derivatized with N,N-dimethylaniline in a phosphate buffer, following by extraction the derivative, 4-iodo- N,N-dimethylaniline, with hexane for further separation and detection by GC-MS. Under the optimal condition, a satisfied validation of data was achieved for linearity, accuracy and precision. A calibration curve (10 – 250 µg I L-1) was obtained from a plot between the area ratio of the derivative to the internal standard, diphenylamine, and the iodide concentration. The coefficient of determination was 0.999. The limit of detection was found to be 3 µg I L-1. The recoveries were obtained in the range of 97% to 101%. This method can be effectively applied to determine fortified iodide in the various flavors of seasoning powder of instant noodle.

Keywords: iodine, iodide, seasoning powder, instant noodle, gas chromatography-mass spectrometry

1. INTRODUCTIONNowadays, Thailand still faces iodine

deficiency disorder (IDD) in many regions of the country. Iodine plays an important role in the body. The thyroid gland needed iodine to produce thyroid hormones that regulate body temperature, metabolic rate, growth and reproduction [1].

Iodine fortification in food might contributes

to the effort of decreasing the iodine deficiency. Because instant noodles are widely and usually consumed, the Ministry of Public Health of Thailand and some industrial foods were motivated and proposed to develop iodine enriched instant noodle. Iodine as potassium iodide was fortified in the seasoning powder because it is packed in an aluminum foil sachet

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inside the pouch of instant noodles to prevent from physical and chemical damage. Moreover, iodide is not exposed to heat and moisture during the noodle processing [2].

Iodine is recommended daily intake (Thai RDI) of 150 µg [3]. The Ministry of Public Health of Thailand established only the iodine content not less than 20 mg kg-1 and not more than 40 mg kg-1 of edible salt [4]. Although a low intake of iodine over a long period of time may cause thyroid gland increases in size in order to trap iodine, so-called “goiter”, excessive iodine intake can cause thyroid problems and should also be avoided. In order to food processing control of iodine addition and mixing for uniform iodine dispersion, and consumer protection to make certain safety in the use of food, the effective analytical method for determination of iodine in food or an ingredient in food was required.

From the literature, many analytical methods have been proposed for the determination of iodide including colorimetric measurement [5, 6], the Sveikina method [7], the Sandell-Kolthoff reaction [8], chemiluminescence [9], X-ray fluorescence (XRF) [10], tandem mass spectrometry (MS/MS) [11], neutron activation analysis (NAA) [12] and high performance liquid chromatography (HPLC) [13]. Inductively coupled plasma (ICP) and ion chromatography (IC) was commonly used for the detection of iodine determination in food [14] including salt and seasoning powder [15].

L. Nunticha et al. [15] proposed an analytical method using the inductively coupled plasma optical emission spectrometry (ICP-OES) for the determination of iodine in different kinds of food products including salt and seasoning powder. However, the sensitivity of ICP-OES method for iodine quantitative analysis is quite low because of iodide UV emission under vacuum condition and the interference from matrices emission. Therefore, many inductively coupled plasma coupled with mass spectrometry

(ICP-MS) methods has been developed for the quantitation of iodine [14, 16, 17]. The problems of memory effects and poor signal stability were reported in ICP-MS method for the iodine detection [18]. An ion chromatography (IC) has been also used for iodide analysis. A limitation of the IC method is inadequate sensitive detection by using conductivity, potentiometry or amperometry detector [18]. The derivatization of iodide overcomes this problem in order to provide the high sensitivity method. The derivatizations of iodide have been proposed by the reaction with ethylene oxide [19], by oxidation in the presence of acetone [20] or by reaction with 3-pentanone [21] followed by gas chromatography coupled with electron capture detector (GC-ECD). Other analytical methods provided use of dimethylphenol as derivatizing agent with subsequent analysis of the derivative via HPLC-UV [22] or GC-MS [23] detection. The disadvantageous of these methods is long reaction time to form the derivative. The oxidation of iodide with 2-iodosobenzoate in the presence of 2,6-dimethylaniline [24] or N,N-dimethylaniline [18, 25] have been also proposed. However, these methods required the sample clean-up and preconcentration step (e.g. solid phase extraction, single drop microextraction) for eliminating matrices and improving the sensitivity in pharmaceuticals, iodized salt, milk powder, vegetables and seawater samples.

As far as we are aware, there have been no report on the chromatographic method for the analysis of iodide in seasoning powder. In order to protect consumers against false labeling, unfair advertising and other types, the effective GC-MS method was developed for quantification and identification to determine the iodide content in seasoning powder sachet in various flavors and manufacturer of instant noodle.

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2. MATERIALS AND METHODS2.1 Reagents and Chemicals

All chemicals and reagents used were of analytical reagent (AR) grade. Organic solvents are GC or pesticide grade. Deionized-distilled water (DI water) (18.2 MW•cm-1), obtained from a Milli-Q-system, was used for the preparation of reagent solutions. Potassium iodide, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and diphenylamine were purchased from MERCK (Germany). 2-Iodosobenzoic acid was purchased from Sigma (USA). N,N-Dimethylaniline was purchased from Fluka (Switzerland). 2,6-Dimethylaniline and 2,6-diisopropylaniline were purchased from Acros organics (USA).

2.2 Instrumentation and Chromatographic Conditions

The iodide derivatives analysis was employed the optimal GC-MS system: Thermo Finnigan TRACE GC 2000 equipped with ion trap mass spectrometry detector, Thermo Finnigan PolarisQ (USA). Separation was carried out on a ZB-5 fused silica capillary column (cross-linked 5% phenylpolysiloxane, 30 m × 0.32 mm I.D., 0.25 µm film thickness, phenomenex). Helium (99.999%, Linde), at a constant flow of 1 mL min-1 was used as the carrier gas. The hexane extractant was injected at 1 µL in the splitless mode. The split/splitless injector was operated at 280 ºC. The GC temperature program was initial temperature at 90 ºC for 3 min, then increasing to 210 ºC at 30 ºC min-1 and held for 3 min. For the MS detector, the ion source and transfer line were set at 250 ºC and 300 ºC, respectively. The identification was performed according to the Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results as criteria for identification [26]. Identification of the target analytes was performed using the mass spectrometer in full scan (50 – 350 m/z). The qualifying ions for identification of

4-iodo-N,N-dimethylaniline were 247, 120 and 77 m/z and diphenylamine were 169, 168 and 77, respectively.

2.3 Standard and Reagent Solutions Preparation

1000 mg I L-1 iodide standard solution was prepared by dissolving 0.1307 g of oven-dried potassium iodide (105 ̊ C overnight) and diluting to 100 mL in a volumetric flask with DI water. The stock solution of KI was kept in a plastic bottle and storage in the refrigerator. The derivatizing reagent, 190 mg L-1 of N,N-dimethylaniline solution was prepared by adding 20 µL of N,N-dimethylaniline (99.5%, d = 0.956) in 100 mL volumetric flask and made up to volume with methanol. 0.26 mol L-1 of phosphate buffer pH 6.4 was made. 20 g each of potassium dihydrogen phosphate and dipotassium hydrogen phosphate were dissolved in 500 mL DI water and adjusted to pH 6.4 with phosphoric acid and sodium hydroxide. In order to prepare 0.015 mol L-1 of sodium-2-iodosobenzoate reagent, 1 g of 2-iodosobenzoic acid was stirred in 20 mL of 0.2 mol L-1 NaOH and then diluted to 250 mL with DI water [25].

2.4 Derivatization and Extraction ProcedureAn appropriate standard solution of

iodide or sample volume was pipetted into a test tube. A 0.5 mL of phosphate buffer pH 6.4, 0.5 mL of 190 mg L-1 N,N-dimethylaniline solution and 0.4 mL of 0.015 mol L-1 sodium-2-iodosobenzoate were added and mixed by vortex for 1 min at room temperature. After that, 40 µL of 60 mg L-1 diphenylamine and 1 mL of n-hexane were added. The solution was extracted into n-hexane layer by horizontal shaking for 10 min and centrifuging at 5,000 rpm for 5 min. 1 µL of n-hexane portion was injected to GC-MS.

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2.5 Sample PreparationVarious flavors and manufacturers of instant

noodle were purchased from a supermarket (Bangkok, Thailand).Ten sachets were weighed and homogenized in a porcelain mortar. An appropriate portion of powder equivalent to the mass of one sachet was accurately weighed and dissolved in 50.0 mL DI water.

After magnetic stirring for 10 min, the solution was filtered through Whatman no.1 filter paper to remove solid matrices. The clear solution was finally filtered through a 0.20 µm nylon syringe filter. Derivatization and extraction were carried out as described in section 2.4 with three replicate analyses. 1 μL aliquot was injected into the GC-MS system. The quantitation was performed using

the standard addition method. The different amounts of the standard were added directly to five aliquots of each analyzed sample.

3. RESULTS AND DISCUSSION3.1 Derivatization of Iodide

In order to analyze iodide by gas chromatography, derivatization step is one of the most important sample preparation. Iodide should be oxidized only to iodine (which undergoes derivatization) and not further to iodate (which escapes derivatization). After oxidation of iodide with 2-iodosobenzoate, only iodine is produced in neutral or feebly acidic solutions [18, 22 – 25]. The generated iodine is subsequently derivatized with a reagent as shown in Figure 1(A) – 1(B).

Figure 1. (A) The oxidation reaction of iodide (B) the expected derivatization reaction of iodine and (C) the derivatization reaction of iodine with N,N-dimethylaniline.

The suitable derivatizing reagent was examined. Criteria for the selection is based on its reactivity to iodine with easy operation and its derivative product must be miscible in an organic extraction solvent.

In the preliminary study, 2,6-diisopropylaniline

was proposed and examined as the derivatizing reagent. 2,6-Diisopropylaniline reacts with iodine to produce 4-iodo-2,6-diisopropylamine. Because of amine and isopropyl groups in the aromatic ring, the amine group is a strong activating substituent, classified as ortho- and

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para-direction but at ortho-position is substituted with isopropyl group, so only the para-position is available for iodine. The result showed that 4-iodo-2,6-diisopropylaniline can be separated and detected by GC-MS. Unfortunately, when this reagent was applied to the sample, the unknown peak was co-eluting with the iodo-derivative.

N,N-Dimethylaniline [18, 25] and 2,6-dimethylaniline [24] have been proposed for derivatization of iodide in iodized table salt and seawater. These two derivatizing reagents

were investigated for its possibility apply to seasoning powder sample.

The derivatization time was the first parameter for comparing the performance of both derivatizing reagents as shown in Figure 2. The reaction time was varied from 1 to 60 min. The derivatization with N,N-dimethylaniline was completed within 1 min at room temperature and kept constant till 60 min (Figure 2(A)). While the formation of 4-Iodo-2,6-dimethylaniline was increased and kept persistent after 10 min at room temperature (Figure 2(B)).

Figure 2. Effect of reaction time on the yield of (A) 4-iodo-N,N-dimethylaniline and (B) 4-Iodo-2,6-dimethylaniline.

The method sensitivity achieved from using these two derivatizing reagents was also compared by using the slope of linear equations. The linear ranges of iodide with N,N-dimethylaniline and 2,6-dimethylaniline reactions were from 10 – 250 µg L-1 with the linear equation of 0.0011x + 0.0023 (r2 = 0.9989) and 0.0012x + 0.0190 (r2 = 0.9980). The result showed that there was no significant difference in sensitivity of two derivatizing reagents.

From the above results, the fast reaction time and high sensitivity were considered. N,N-dimethylaniline was therefore selected for derivatization of iodide through this work. The derivatization reaction using N,N-dimethylaniline was shown in Figure 1(C).

3.2 Effect of Extraction3.2.1 Extraction solvent

A suitable extraction solvent for 4-iodo-

N,N-dimethylaniline should have the following properties: (i) good extraction affinity to ensure high enrichment; (ii) immiscibility in water; (iii) good chromatographic behavior to be separated from the derivative peak in the chromatogram. According to these properties ethyl acetate, methyl tertiary butyl ether (MTBE) and hexane which are the difference in polarity were tested. Due to the derivative is a nonpolar compound as expected, the extraction efficiencies were increased by the extraction with ethyl acetate, MTBE and hexane in accordance with their polarity index of 4.4, 2.4 and 0.1, respectively [27]. The extracted amounts of the 4-iodo-N,N-dimethylaniline obtaining from ethyl acetate and MTBE were 30% and 45% of hexane extractant, respectively. Moreover, the 4-iodo-N,N-dimethylaniline and diphenylamine were clearly separated from the hexane peak. Hexane was therefore chosen as an extraction

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solvent and used in the subsequent experiments.

3.2.2 Extraction volumeThe volume of hexane affected extraction

efficiency and sensitivity. The larger volume of extraction solvent provides higher extraction

efficiency due to a large area for extracting the derivative. In contrast, the sensitivity of the method is decreased because of the dilution effect. In this work, the hexane volumes of 200, 400, 800 and 1000 µL were studied as shown in Figure 3.

Figure 3. Effect of hexane volume on the extraction efficiency of 4-iodo-N,N-dimethylaniline.

The results showed that at lower volumes, the derivative was more preconcentrated. However, the separation and collection of hexane from the aqueous sample was not easily achieved resulting in poorer precision of analysis. For compromise among extraction efficiency, sensitivity and precision, a 1000 µL volume of hexane was selected.

3.2.3 Extraction timeThe last parameter affecting to an extraction

efficiency of 4-iodo-N,N-dimethylaniline was extraction time. In this work, the horizontal shaker speed was fixed at 300 rpm but different shaking times varied from 5 to 30 min. Figure 4. shows the increase of the peak area of 4-iodo-N,N-dimethylaniline when extending shaking time from 5 to 10 min. After 10 min, the peak area kept constant indicating that 4-iodo-N,N-dimethylaniline was reached the equilibrium. Based on the results, the extraction by shaker at 10 min was chosen for compromising high extraction efficiency and short analysis time.

3.3 Method ValidationThe developed GC-MS method was

validated using the Association of Official Analytical Communities (AOAC) requirements for single laboratory validation of chemical methods as a guideline [28]. In addition, an internal standard was used for improving the analytical performance of the method. The internal standard is a compound added to a sample in known concentration to facilitate the quantitative determination of the analytes. The internal standard should be similar in the chemical behavior and analytical response to the analyte. In this work, a fixed volume of diphenylamine using as an internal standard solution was added to the standard and sample solutions. This method corrects for run-to-run-variation in extraction efficiency, chromatographic response and ionization variability [29]. The analytical performances of the method were summarized in Table 1.

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3.3.1 LinearityThe internal standard calibration curve was

carried out using seven standard concentrations with three replicate injections of iodide. When the calibration solutions and sample were analyzed, the peak area of the derivative was

divided by the peak area of diphenylamine to produce a peak area ratio value. The results showed that the working range of the method was linear in the range of 10 – 250 µg I L-1 with the acceptable coefficient of determination (r2) of 0.999.

Figure 4. Effect of extraction time by hexane on the extraction efficiency of 4-iodo-N,N-dimethylaniline.

Table 1. Analytical performance of the proposed GC-MS method.

Parameter Criteria Value

Internal calibration curve- Range (µg I L-1)- r2 r2

> 0.9910 – 250r2

= 0.999

LODa (µg I L-1) 3*

LOQb (µg I L-1) 10*

Precision: Repeatability (n = 6)- Relative retention time

%RSD (HORRATr)c

- Relative peak area%RSD (HORRATr)

c

Accuracy: %Recovery (n = 7)

(0.5 – 2.0)

(0.5 – 2.0)75 – 120

0.51 % (0.17)

4.06 % (1.35)97 – 101

a LOD value in solution based on three signal-to-noise ratio criteria (3S/N)b LOQ value in solution based on ten signal-to-noise ratio criteria (10S/N)c HORRATr = RSDr (found)/RSDr (calculated); RSDr calculated at 1,000 µg mL-1 = 3 % * using quantifying ion according to the guideline of European Commission Decision.

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3.3.2 Limits of detection (LOD) and quantification (LOQ)

The iodide standard solutions were prepared and performed the analysis by the proposed method. Limit of detection (LOD) was defined as a concentration that gives three times detectable signal to noise (3S/N). A concentration that gives detectable signal related to the concentration of iodide at ten times signal to noise (10S/N) was a limit of quantification (LOQ). The limits of detection and quantification were 3 and 10 µg I L-1, respectively.

3.3.3 PrecisionIn order to determine the instrumental

system precision, the analyzing a 1000 µg I mL-1 iodide certified standard solution which is traceable to National Institute of Standard and Technology (NIST) was performed. According to the AOAC requirements, the precision of the method was evaluated using the HORRAT method. An acceptable value of the repeatability is a half and twice times of the calculated value [28]. The result showed that the relative %RSD values of retention time and peak area ratio were less than 4.06 (n = 6), demonstrating good repeatability of the method (RSDr at 1000 µg mL-1 is 3 %). However, the precision of the relative retention time was lower than HORRATr value (0.5) due to the use of internal standard improved the precision of the method.

3.3.4 AccuracyThe accuracy of the method was performed

by analyzing a 1000 µg I mL-1 iodide certified standard solution and the recovery of iodide in seasoning powders.

The certified standard solution was to be found at 966 µg I mL-1 (n = 6). No significant difference between the results obtaining from the GC-MS and the certified value using t-test at 95 % confidence interval (tstat = 2.03, tcritical = 2.57).

Recovery study was carried out by fortification of 100 µg I L-1 in the sample. The results showed that the recoveries of seasoning powder were in the range of 97 – 101%. Therefore, the recovery values established the satisfactory accuracy of the method.

3.4 Application to SampleOnce the analytical performance was

validated, the proposed GC-MS method was applied to determine the iodine content as iodide in seven seasoning powder samples with the difference in flavors. Because of the high varieties of flavors and ingredients of seasoning powder samples, the standard addition was used for eliminating matrix effects from a measurement. All sample were collected and prepared as described in section 2.5. The examples of total ion chromatogram of sample B and mass spectrums of 4-iodo-N,N-dimethylaniline and diphenylamine were presented in Figure 5. The iodide contents in seasoning powders were found ranging from 23 – 148 µg I per sachet (Table 2).

4. CONCLUSIONSA new GC-MS method was successfully

developed for the determination of iodide content in seasoning powder of instant noodle. The method provided the high performance in terms of accuracy, precision and sufficient sensitivity for the analysis of iodide in various matrices (flavor) of seasoning powder. The sensitivity is possible to increase by using a lower volume of extraction solvent if required. The advantage of the method was not only used the simple derivatization but the solid phase extraction for preconcentration was also not required.

ACKNOWLEDGEMENTSThe authors gratefully acknowledge

the financial support provided by Thailand Research Fund (Contract No. MRG5280180).

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Figure 5. (A) The total ion chromatogram (TIC) of sample B and mass spectrums of (B) 4-iodo-N,N-dimethylaniline and (C) diphenylamine. Peak identification: (*) 4-iodo-N,N-dimethylaniline and (#) diphenylamine.

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The authors are also thankful to Department of Chemistry, Faculty of Science, Mahidol University and Department of Chemistry, the Central Scientific Instrument Center (CSIC), the Center of Scientific Equipment for Advanced Research (TU-CSEAR), Thammasat University for facility and instrument support throughout this research.

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Table 2. Determination of iodide content in seasoning powder and its recovery studied.

Sample Iodide Content(µg I per sachet; Mean ± SD)*

% Recovery(Mean ± SD)*

A 30.99 ± 1.35 98.12 ± 1.33B 102.21 ± 4.15 97.49 ± 0.44C 57.80 ± 2.31 100.51 ± 2.01D 38.14 ± 1.56 99.20 ± 2.88E 55.94 ± 2.32 97.00 ± 0.39F 23.14 ± 1.88 98.20 ± 1.13G 148.15 ± 8.83 101.00 ± 0.57

*Three replicate analyses.

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