One - factor - at - a - time method combined with ICP MS for determining 11 elements in soy sauce and their migration from the containing glass bottles

: This study was designed to determine the 11 metal elements ( Al, Cr, Mn, Fe, As, Ni, Cu, Zn, Cd, Sb, and Pb ) in soy sauce and their migration from the con taining glass bottles. Inductively coupled plasma mass spectrometry ( ICP - MS ) was applied for the determination of the elements and one - factor - at - a - time method was employed for optimizing the ICP - MS parameters in migra tion experiment and microwave digestion experiment. The developed method was successfully applied to deter mine the content of 11 elements. The results showed that the experiment had excellent correlation and sensitivity. The accuracy of the elements in the migration study and test of soy sauce itself ranging from 84.25% to 118.75% was satis ﬁ ed, and the precision of the method was vali dated and the RSD was no more than 15.5%. The concen tration of all the detected metal elements migrated from the glass bottles were between 0.3450 and 2.398 ng · mL − 1 , and the risk assessment indicated that the metal elements in soy sauce had no risk to the public health. The proposed methodology in this study was successfully applied for the quality control for metal elements in soy sauce and the containing glass bottles for the ﬁ rst time, and a research method suitable for soy sauce consumption process con trol and risk assessment has been established.


Introduction
As one of the popular condiments, soy sauce originated from Asia and spread all over the world. It is mainly made from soybeans and flour via fermentation using microbial enzymes. From the analytical point of view, soy sauce is a complex matrix, and contains about 8% proteins, 5% carbohydrates, 0.8% fiber, 0.6% lipids, 5.5% sodium, and 0.4% potassium [1,2]. However, the undesired pollution in soy sauce may result in high level of metal elements which could be a potential risk to human health [2]. Metal elements such as lead and copper in soy sauce are mainly derived from soil, raw materials, environment, production process, and packaging materials [3,4]. The packaging materials play an important role in food safety and shelf life by protecting the products from external light, microorganisms, and air.
Chemical migration mainly depends on the type of material contacting the food products [5]. Studies showed that packaging materials can contaminate food with some harmful elements [6,7]. Glass is widely used in packaging materials for soy sauce because of its eco-friendly property and low cost. However, it was reported that at least 18 different elements were found in glass (may be as stabilizers), including aluminum, antimony, arsenic, barium, bismuth, boron, calcium, germanium, hafnium, lanthanum, magnesium, niobium, strontium, tantalum, titanium, yttrium, zinc, and zirconium [8][9][10]. Previous research [11,12] reports showed that glass bottles contaminated bottled water with heavy metals. Therefore, the investigation of the unwanted elements which migrate from soy sauce packaging materials is of great importance.
The toxicity of elements is closely related to their exposure concentration. Chemical elements are essential for biological processes and play an important role in normal growth and development of the organism, but can be toxic when taken in excess [13,14]. Nowadays, soy sauce is consumed worldwide, the amount of soy sauce made in China was around ten million tons in 2016 [2]. When studying the migration of elements from the glass bottles, it is also necessary to evaluate the health risk of chemical elements intake from the daily consumed soy sauce. Target hazard quotient (THQ) is an effective tool to evaluate the health risk of different elements, which is calculated based on the ratio of estimated daily intake (EDI) to oral reference dose (RfD) [15] of soy sauce. This method is based on the Region III risk-based concentration table from United States Environmental Protection Agency (USEPA) [16]. Furthermore, RfD refers to Integrated Risk Information System from USEPA [17]. Simultaneously, Food and Agriculture Organization of the United Nations/World Health Organization/the Joint Expert Committee on food additives evaluated the safety of contaminants and toxins in food, and finally revised the concept of provisional tolerable weekly intake (PTWI). Accordingly, risk assessment was carried out for the chemical elements on human health through PTWI and THQ.
Currently, inductively coupled plasma mass spectrometry (ICP-MS) is widely used for the determination of chemical elements [3] at ppt levels and it is recommended by China National Food Safety Standard for the determination of elements in condiments, such as soy sauce [18,19]. International Organization for Standardization (ISO) 7,086-1:2,000 requires that a glass bottle should be briefly washed with a solution containing a nonacidic detergent at a temperature of approximately 40°C, followed by rinsing with tap water, and then distilled water, and finally filled with a 4% aqueous acetic acid (CH 3 COOH) solution.
This study optimized the microwave digestion method for soy sauce sample pretreatment, selected the optimal system, and established an ICP-MS method for the simultaneous determination of 11 metal elements. This research aims to determine the chemical elements in soy sauce and their possible migration from glass bottles. And the health risk assessment on the chemical elements' migration from the glass bottles into soy sauce has been carried out for the first time. This study provided a reference to further control quality of chemical elements in condiments and their packaging materials.

Materials and reagents
All chemicals and reagents were of chromatographic grade. Nitric acid (69% HNO 3 ) was purchased from Beijing Chemical Works (Beijing, China). Ethanol (CH 3 CH 2 OH) was obtained from Tianjin Concord Technology Co., Ltd (Tianjing, China). Acetic acid (CH 3 COOH) was acquired from Sinopharm Group Chemical Reagent Co., Ltd (Shanghai, China). The water used for the experiment came from Shenyang Wahaha Rongtai Food Co., Ltd (Shenyang, China). Hydrogen peroxide (H 2 O 2 ) was obtained from Shenyang Yuwang Chemical Glass Instrument Co., Ltd (Shenyang, China).
The standards of Al(III), Cr(III), Mn(II), Fe(III), Ni(II), Zn(II), As(III), Cd(II), Sb(III), and Pb(II) were purchased from Guobiao Testing & Certification Co., Ltd (Beijing, China) and Cu(II) were purchased from AnPu Experimental Technology Co., Ltd (Shanghai, China). The working standard solutions were prepared daily by diluting each singleelement standard stock solution. All standard solutions need to be sealed and stored below 4°C in dark before analysis.

Apparatus
The constant temperature and humidity incubator, LRHS-300F-II, was purchased from Shanghai Longyue Instrument Equipment Co., Ltd (Shanghai, China). The Multiwave Pro microwave digestor (Anton Paar, Austria) and the BHW-09A45 acid-driven processor (Anton Paar, Austria) were used for sample digestion. All the elements were measured by an Agilent 7800 ICP-MS (Agilent, USA) with a MicroMist nebulizer. The instrumental conditions are set in Table 1.

Sample preparation
In the experiment, seven different brands of soy sauce with different packagings were randomly selected from the local supermarket in Shenyang, Liaoning. The glass bottles of three different brands were studied for the migration of elements.
Before test, all the glass bottles were rinsed with distilled water and soaked in 4% (v/v) CH 3 COOH at 22°C for 24 h. Then, 10 mL of fresh extraction solution was transferred to a microwave digestion tank and heated at 150°C for 60 min in order to digest CH 3 COOH. After cooling down, the remaining solution in the microwave digestion tank was transferred into a 50 mL volumetric flask and diluted to the volume with 2% (v/v) HNO 3 . All the sample solutions were prepared in duplicate.
The purpose of the digestion experiment was to make it easier to extract the chemical elements in soy sauce. To test elements in soy sauce, 1 mL of soy sauce sample, 6 mL of HNO 3 , and 2 mL of H 2 O 2 were added into the microwave digestion tank and digested in a microwave system at 180°C for 15 min. After the completion of digestion, the tank was placed in a hot plate for 30 min to evaporate the solvents. Then, the cooled and dried sample was diluted with 2% (v/v) HNO 3 and transferred to a 50 mL volumetric flask before analyzing using ICP-MS.

Method validation
The linear regression analysis was verified by the correlation coefficient (R 2 ) of the calibration curve for each element. The limit of detection (LOD) of 11 metal elements was estimated to be three times of the standard deviation (SD) of ten blank samples. The limit of quantification   (LOQ) was determined to be ten times of the above SD. To assess recovery, each chemical element was added to the samples at concentrations of 80%, 100%, 120% in triplicate.
To validate the precision and accuracy for migration study and test the elements in the soy sauce itself, the samples were spiked with mixed standards at three levels and then analyzed as described for normal samples in triplicate.

Extraction, migration, and digestion tests
To confirm the effect of extraction time [20], extraction with 4% (v/v) CH 3 COOH for 48 h resulted in a near-logarithmic profile. 10 mL of samples were withdrawn at 1, 2, 4, 8, 24, and 48 h, and heated at 150°C for 60 min to remove the organic solvents. After cooling down to ambient temperature, samples were diluted to 50 mL with 2% (v/v) HNO 3 . In order to study the effect of different acids on the migration, two mixed standards (1.0 and 20.0 ng·mL −1 ) were prepared according to the sample preparation  Different digestion solvents in microwave digestion of soy sauce was evaluated. The recovery of the elements in three different solvents (8 mL of HNO 3 , 7 mL of HNO 3 with 1 mL of H 2 O 2 , and 6 mL of HNO 3 with 2 mL of H 2 O 2 ) was compared. In order to verify whether the acid concentration will affect the results, a blank digestion solution was prepared.
3 Results and discussion 3.1 Optimization of experiment conditions 3

.1.1 Condition optimization for migration experiment
The migration experiment condition of the elements from glass bottles was optimized ( Table 2). Concentration ratios of the metals in different leaching solutions vary significantly from acid to acid [21]. Preliminary data showed that the type of the organic solvent and incubating time were critical for the migration of 11 elements and the concentration of element ions was examined in the eluate after different solvent erosions. The different leaching solutions were evaluated in microwave digestion including 10% (v/v in water) CH 3 CH 2 OH, 20% (v/v in water) CH 3 CH 2 OH, and 4% (v/v in water) CH 3 COOH. The results showed that 4% (v/v in water) CH 3 COOH had the best migration effect and indicated that the presence of H 2 O 2 made the digestion more complete because they could make the digestion system to have stronger oxidation ability [22].
The effect of migration time is shown in Figure 1. After 24 h, the increasing rate of concentration of each element in 4% (v/v) CH 3 COOH started to slow down, indicating that the migration reached stable state.
The effect of different acidity on the migration is shown in Figure 2, and the results were in the range of 0.8-1.2. There was no interference with the organic solvent in the analytical values.

Optimization of digestion condition
In most cases, the decomposition of samples could be affected by a combination of different parameters, such as the microwave power, digestion time, pressure, and temperature in the sealed digestive tube [3,23].  with HNO 3 [24]. H 2 O 2 may increase the solubility of Pb [25]. Therefore, different levels of H 2 O 2 in the digestion solvent were investigated. Results showed that 2 mL of H 2 O 2 with 6 mL of HNO 3 as the digestion solution had the highest recovery ( Figure 3).
The effect of the digestion time was also studied, and the recovery of the elements at different digestion times (5, 10, 15, and 20 min) was compared. According to the results in Figure 4, the highest recovery was found at 15-20 min.
As to the digestion temperature, it was reported that temperature changes in microwave digestion might affect the results [4]. Efficient digestion for all chemical elements is presented in Figure 5. Moreover, the analytical results showed that 180°C gave a statistically higher recovery than the other temperatures.
The mixed standard solutions (5.0 and 100.0 ng·mL −1 ) were diluted using the blank solution with a fixed 2% (v/v) HNO 3 . The results are shown in Table 3. All the concentrations were in the range of 0.8-1.2%, and there was no significant change in the recovery.

Method validation
The analytical method employed for 11 chemical elements detection was validated on linearity, LOD, LOQ, accuracy, and precision. The results from validation studies indicated the method had a satisfactory performance.
The linearity of the method using the least squares method was evaluated by calculating the regression coefficients. The regression equations were derived from statistical tests and the linear range of the migration experiment was within 0.05-200 ng·mL −1 . In this range, the regression model was considered acceptable for most tested elements (R 2 > 0.978). The LOD levels were between 0.006 ng·mL −1 (LOD Cd) and 0.158 ng·mL −1 (LOD Zn). The LOQ levels were between 0.020 ng·mL −1 (LOQ Cd) and 0.528 ng·mL −1 (LOQ Zn). The experiment had excellent correlation and sensitivity.
The instrument precision of this method was evaluated using RSD and the results are shown in Table 4. All the values for the elements migrated from glass ranged from 0.5% to 1.6%. Note:means the measured value is less than the LOQ.  The precision and accuracy of the elements in the migration study and test of soy sauce itself were validated. The accuracy was found to be between 84.25% and 118.75% for 11 chemical elements, and the RSDs were within 0.4-11.1%. For analyzing elements in soy sauce, the accuracy ranged from 93.57% to 114.65%, and the RSDs were all less than 15.5% ( Table 5). The accuracy and precision of the method were reliable and suitable.

Analysis of real samples
Al, Cr, Mn, Fe, Ni, Cu, Zn, As, Cd, Sb, and Pb in seven brands of soy sauce samples were tested using the validated method, and the contents of 11 elements are shown in Table 6.
Samples from three different brands of glass bottles were analyzed by the validated migration method ( Table 7). Several elements with different concentrations were detected in all the samples. The results showed that the content of each element in the soy sauce in glass bottle did not exceed the national standard [26].

Safety evaluation
Risk assessment was conducted separately for essential and toxic chemical elements. THQ and the EDI of 11 elements were analyzed and compared with PTWI and RfD. The daily intake of soy sauce for Chinese residents (9 g per person per day) was obtained from China Health and Nutrition Survey (CHNS, 2002). The THQ of ingested 11 elements was calculated using the following equation: Note:means the element was not detected.
where IR is the daily intake of soy sauce per person (g·day −1 ); C is the content of every element in the soy sauce (g·kg −1 ); and BWa stands for average of body weight (kg).

THQ EDI RfD
where THQ represents the value of risk (if value of THQ is less than 1, it means that this level is unlikely to cause any obvious deleterious effects to the general health). According to the database of the Nutrition and Chronic Diseases of Chinese residents, the average body weight of Chinese residents is 66.2 kg [27]. The density of soy sauce is regarded as 1.15 × 10 3 kg·m −3 .
The THQ value of each chemical element and the maximum allowed levels in the soy sauce were far less than 1 ( Table 8), suggesting that the listed ingested metal elements in soy sauce is within the safe range. It is critical that humans may underestimate or overestimate the health hazard due to the variety of interactions among the mixture components [28]. Chemical elements intake under real conditions will be lower than that resulting from migration into the food simulant.

Conclusion
In this study, a novel method, one-factor-at-a-time method combined with ICP-MS, for determining Al, Cr, Mn, Fe, Ni, Cu, Zn, As, Cd, Sb, and Pb in soy sauce and their migration from the containing glass bottles was developed. By optimizing the migration experiment and digestion experiment conditions, determining the extraction time, the effect of different acids, and different digestion solvents, the ICP-MS methods were formed. The chemicals in the glass migrate to the soy sauce at very low levels. Combined with the risk assessment, it was confirmed that the soy sauce and glass bottles used in this study were safe. The methods for determining the content of 11 chemical elements are accurate and reliable. Accurate and fair results provide theoretical basis for long-term safe storage of soy sauce. This experiment provided a new direction on the quality control of condiments. Author contributions: Xun Gao: writingoriginal draft, writingreview and editing, methodology, and formal analysis; Kexin Chen: writingoriginal draft, formal analysis, and visualization; Yue Zhang: validation and data curation; Miaomiao Chi: investigation and formal analysis.

Conflict of interest: Authors state no conflict of interest.
Data availability statement: All data generated or analyzed during this study are included in this published article. Note:represents the undetected value; *represents the safety evaluation of Pb use the PTWI established by JECFA; EDI means "estimated daily intakes;" THQ means "target hazard quotient." Determination of 11 elements in soy sauce by ICP-MS  19