DOI: https://doi.org/10.21203/rs.3.rs-2201547/v1
Chlorate (ClO3-) is formed as a by-product when using chlorine, chlorine dioxide or hypochlorite for disinfection (European Food Safety Authority (EFSA), 2015). The presence of chlorate in food can arise from the use of chlorinated water for food processing and the disinfection of food-processing equipment (European Food Safety Authority (EFSA), 2015).
Perchlorate (ClO4−) is a chemical pollutant, which is released into the environment from natural and man-made sources (European Food Safety Authority (EFSA), 2014). Perchlorates are mainly derived from the use of natural fertilizers containing perchlorates (Cao et al., 2019; Du et al., 2019), industrial emissions of perchlorates (especially from the use of ammonium perchlorate in rocket and missile solid propellants) (Song et al., 2019; Lumen & George, 2017; Gan et al., 2015; Du et al., 2019) and naturally formed perchlorates in the atmosphere and surface water (European Food Safety Authority (EFSA), 2014). Perchlorate in foods of animal origin mainly come from the intake of food and feed containing perchlorate by animals. Perchlorate in plant-based foods mainly come from soil or irrigation water containing perchlorate. Processed foods could be contaminated with perchlorate during processing.
In 2014, the European Food Safety Authority (EFSA) assessed the public health risks of perchlorate in foods, especially fruits and vegetables, and found that acute exposure to perchlorate had no adverse effect on human health (except for fetal and infant health) (European Food Safety Authority (EFSA), 2014);Chronic exposure could cause long-term inhibition of thyroid uptake of iodine, lead to the development of toxic multinodular goitre and result in hyperthyroidism (Cao et al., 2019; European Food Safety Authority (EFSA), 2014; Pleus et al., 2018); In 2015, the risks to human health related to the presence of chlorate in food were assessed by the EFSA Panel on contaminants in the food chain (European Food Safety Authority (EFSA), 2015). The critical acute effect in humans identified in cases of poisoning is induction of methaemoglobinaemia, followed by lysis of red blood cells that can lead eventually to renal failure (European Food Safety Authority (EFSA), 2015; Al-Otoum et al., 2016). The chronic exposure harm of chlorate was consistent with that of perchlorate, Inhibition of iodine uptake in humans was identified as the critical effect for chronic exposure to chlorate (European Food Safety Authority (EFSA), 2015). A tolerable daily intake (TDI) of 3 µg/kg b.w. was set by read across from a TDI of 0.3 µg/kg b.w. derived for this effect for perchlorate, multiplied by a factor of 10 to account for the lower potency of chlorate (European Food Safety Authority (EFSA), 2015). In 2017, the European Food Safety Agency (EFSA) assessed the dietary exposure of perchlorate in European population, and found that the exposure of perchlorate in all age groups might possibly exceed the daily tolerance intake in Europe (European Food Safety Authority (EFSA), 2017). The maximum levels of chlorate and perchlorate in certain foods was amended by commission regulation (EU) 2020/749 (European Commission 749, 2020) and (EU) 2020/685 (European Commission 685, 2020), respectively.
For detection of chlorate and perchlorate in foods, several methods had been developed using ion chromatography (IC) (Sungur & Sangun., 2011; Canas et al., 2006; Niemann et al., 2006), ion chromatography-tandem mass spectrometry (IC - MS/MS) (Zhang et al.,2007; Aribi et al., 2006; Martinelango et al., 2006;Melton et al., 2019; Yang et al.,2011), liquid chromatography coupled to mass spectrometry (LC - MS) or liquid chromatography-tandem mass spectrometry (LC - MS/MS) (Xian et al., 2017; Constantinou et al., 2016; Zhao et al., 2018). The sensitivity of ion chromatography was low and the anti-interference ability was weak. The sample pretreatment in ion chromatography was complicated. Compared with IC - MS/MS, liquid chromatography coupled to a triple quadrupole mass spectrometry (LC - QqQ - MS/MS) had been widely used for its applicability and high sensitivity.
In this study, the analytical method of chlorate and perchlorate was improved by optimizing the method of instrument and sample pretreatment such as mobile phase, chromatographic column injection volume, extraction, and purification. Then, the improved method was validated and analyzed in drinking water and 16 types of foods. The performance of the method was compared with the published method (Table 1).
| Matrix | Detection method | Chlorate | Perchlorate | References | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| LOD /µg/L(µg/kg) | LOQ/µg/L(µg/kg) | Recovery/% | RSD/% | Retention time/min | LOD /µg/L(µg/kg) | LOQ/µg/L(µg/kg) | Recovery/% | RSD/% | Retention time/min | |||
| Flour | LC-MS/MS | - | - | - | - | - | 0.1 | 2.0 | 84.6–104.9 | 2.9–8.2 | 2.9 | Xian et al. (2017) |
| Drinking water | LC-MS/MS | - | 10 | - | - | - | - | 5.0 | - | - | - | Constantinou et al. (2019) |
| Fruit and vegetables | LC-MS/MS | - | 50 | 86.8–113 | 5.1–24.6 | 4.3 | - | 50 | 87.2–111 | 2.2–14.9 | 5.9 | Constantinou et al. (2019) |
| Baby food | IC-HRMS | - | 2.0 | 85.0-105 | 8.0–11.0 | 12.0 | - | 2.0 | 78.0-108 | 8.0–12.0 | 23.4 | Panseri et al. (2020) |
| Ozonated saline | IC-MS | 0.10 | 0.33 | 82.7–97.4 | 0.81–3.5 | 14.2 | 0.04 | 0.13 | - | 9.0–12.0 | 21.0 | Yin et al. (2020) |
| Tea | UPLC-MS/MS | - | - | - | - | - | 1.0 | 10 | 79.2–105 | 1.3–16.3 | 1.7 | Liu et al. (2019) |
| Drinking water | UPLC-MS/MS | 0.06 | 0.20 | 96.5–109 | 4.8 | 4.6 | 0.015 | 0.05 | 99.3–111 | 2.1 | 2.2 | This paper |
| Fresh food | UPLC-MS/MS | 1.8 | 6.0 | 86.5–103 | 2.5–7.4 | 0.31 | 1.0 | 91.3–111 | 2.6–7.9 | |||
| dry food | UPLC-MS/MS | 5.4 | 18.0 | 0.91 | 3.0 | |||||||
| Note: -: not reported. | ||||||||||||
The 1000 mg/L Chlorate (IC grade) and 1000 mg/L perchlorate (IC grade) were obtained from NSI Lab (USA) and Inorganic Ventures (USA), respectively. The 200 µg/mL chlorate-18O3 and 100 µg/mL perchlorate-18O4 were purchased from EURL-SRM (USA) and Cambridge (USA). The quality control sample of chlorate (S242–5272) and perchlorate (QCI − 108) were acquired from ERA (USA) and NSI Lab (USA). They were stored at 4°C.
Acetonitrile and methanol (HPLC grade) were purchased from Merck (Germany). Ammonium formate (purity ≥ 99.0%, mass spectrometry) was obtained from Sigma-Aldrich (USA). Formic acid (LC - MS grade) was obtained from CNW Technology Co. Ltd. (Germany). Oasis® PRIME HLB (60 mg and 120 mg) Solid phase extraction (SPE) cartridges were purchased from Waters (USA). Supelclean™ ENVI™ − 18 (500 mg) and Supelclean™ ENVI - Carb™ II/PSA (500 mg) SPE cartridges were obtained from Supelco (USA).
The analysis was conducted using an ACQUITY UPLC I CLASS coupled to a Waters Xevo TQ-XS triple - quadrupole mass spectrometer (Waters Corp., Milford, MA, USA). Perchlorate and chlorate were separated using Torus™ DEA(2.1 mm×100 mm, 1.7 µm)maintained at 35°C. The injection volume was 2 µL. The chromatographic separation was carried out employing a binary mobile phase, consisting of 20 mmol/L ammonium formate (A) and acetonitrile (B) at a flow rate of 0.5 mL/min. The gradient elution started at 70% B and increased to 90% B in 3.0 min and held for 2 min. Then, it was decreased to 55% B in 0.2 min and held for 3 min. Finally, the system came back to the initial condition (70% B) for 0.3 min and held for 3.5 min. The total running time was 12 min. All the components to be measured within 5 minutes. The column was rinsed and equilibrated for 7 minutes to maintain good separation performance.
The mass spectrometer was ionized in negative electrospray ionization (ESI) mode, and data was acquired in the multiple reaction monitoring (MRM) mode. The capillary voltage was 0.5 kV, the ion source temperature was 150°C, the desolvation temperature was 500°C, and the desolvation gas flow was 1000 L/Hr, the cone gas flow was 150 L/Hr. The mass spectrum parameters of chlorate, perchlorate and their internal standards are shown in Table 2. The ratios were considered acceptable at 0.32 ± 0.08 for 37Cl : 35Cl.
| Compound | Quantitative ion pair(m/z) | Qualitative ion pair(m/z) | Cone/V | Collision/eV |
|---|---|---|---|---|
| Chlorate | 83.0 > 67.0 | 85.0 > 69.0 | 10 | 14 |
| Perchlorate | 99.0 > 83.0 | 101.0 > 85.0 | 10 | 18 |
| Chlorate-18O3 | 89.0 > 71.0 | - | 10 | 16 |
| Perchlorate-18O4 | 107.0 > 89.0 | - | 10 | 20 |
The sterilized milk and pasteurized milk were contained in liquid milk. The whole milk powder, skim milk powder and formulated milk powder were included in milk powder. The infant formulas contained 0 ~ 6 months, 6 ~ 12 months and 12 ~ 36 months infant formula. The rice, millet, corn, oat and wheat were included in cereals. The citrus fruit, pome fruit, stone fruit, berries and small fruit, miscellaneous fruits with were included in fruits. The vegetables contained root and tuber vegetables, bulb vegetables, fruiting vegetables, brassica vegetables, leaf vegetables, herbs and edible flowers, stem vegetables. The muscles (chicken, duck, cattle, sheep and pig), liver, kidney and edible offal were included in products of animal origin. The tiger spot, moray eel, erythema, gentian spot, green spot, dragon spot and sesame spot were included in marine fish. The spices contained seed spices, fruit spices, root and rhizome spices. The chicken egg, duck egg, geese egg and quail egg were contained in eggs. The mung legumes, soy legumes, red legumes and black legumes were included in legumes. The potatoes and yams were contained in potatoes. The medicinal and food homology included salvia miltiorrhiza, codonopsis, radix pseudostellariae, dendrobium officinale, ganoderma lucidum, and ginger. The spices, dictyophora indusiata and medicine food homology were dry sample. The agaricus blazei murrill were divided into dry samples and fresh samples. The infant supplementary food was divided into infant cereal supplementary food and infant canned supplementary food. All samples (except legumes and potatoes) were collected in circulation by 9 municipal centers for Disease Control and Prevention in Fujian Province. The legumes were provided by Xiamen Zhongjixin Testing Technology Co., LTD and Fujian Guodin Testing Technology Co., LTD. The potatoes were provided by Xiamen Zhongjixin Testing Technology Co., LTD.
An extraction procedure for the determination of chlorate and perchlorate in samples by LC - MS/MS was carried out with modifications of the extraction methods developed previously (Aribi et al, 2006; Liu et al, 2019;). Drinking water: The 0.99 mL sample was accurately removed, 10 µL of mixed internal standard solution was added, vortex mixing was carried out for 10 s, and 0.22 µm polyether sulfone filter was passed through. The fresh foods: 2 g of sample were weighed into a 50 mL polypropylene centrifuge tube, 200 µL of mixed internal standard and 7 mL pure water were added, homogenized and ultrasonicated for 30 min. Next, 13 mL acetonitrile were accurately added, mixed, and centrifuged at 9000 r/min for 10 min. After that, 3 mL supernatant was purified by PRIME HLB 60 mg and 0.22 µm nylon filter membrane. The 1 mL of the primary filtrate was discarded and the continued filtrate was collected for LC - QqQ - MS/MS analysis. The dry foods (e.g., spices, dictyophora indusiata, agaricus blazei mumill, medicine food homology): 1 g of sample were weighed into a 50 mL PTFE centrifuge tube, and then added 300 µL internal standard, 10 mL water and 20 mL acetonitrile. The remaining procedure was mentioned above.
Using pure water as the medium, the mixing standard solution (2.0 mg/L chlorate and 1.0 mg/L perchlorate) and mixing internal standard solution (1000 µg/L chlorate-18O3 and 200 µg/L perchlorate-18O4) were prepared by accurate dilution of each standard solution. The standard working solution with the concentration of chlorate (0, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 40.0, 60.0, 80.0, 120, 160, 200 µg/L) and perchlorate (0, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10.0, 20.0, 30.0, 40.0, 60.0, 80.0, 100 µg/L) was obtained using appropriate dilution of mixed standard solution and dissolving it to the solvent (pure water - acetonitrile (v/v, 1:2)). Each standard working solution was contained 10.0 µg/L chlorate-18O3 and 2.0 µg/L perchlorate-18O4 by appropriate dilution of mixing internal standard solution. All standard solution were stored at 4°C.
The chlorate and perchlorate were inorganic anionic compounds. The retention behavior of chlorate and perchlorate was explored with both Acclaim™ Trinity™ P1 (2.1 mm×100 mm, 3 µm) and Torus™ DEA (2.1mm×100 m, ,1.7 µm). The chlorate and perchlorate could be well separated on Torus™ DEA and Acclaim™ Trinity™ P1 (Fig. 1). The response of chlorate and perchlorate was more than doubled on Torus™ DEA. In foods, the concentration of chlorate and perchlorate was mostly at a low level, so Torus™ DEA was used to seperate.
The effects of mobile phase A (20 mmol/L ammonium formate, 0.2% formic acid and pure water) on the resolution, peak shape, accuracy and sensitivity of perchlorate and chlorate were compared. The results showed that the introduction of formic acid inhibited the response of negative ion mode and affected the sensitivity of the target compound. The introduction of pure water was detrimental to elute of the target compound and competitive ions on Torus™ DEA, which affected the peak shape and accuracy of the target compound. However, 20 mmol/L ammonium formate was favorable to the elution of the target compound and had no inhibitory effect on the negative ion mode. The 20 mmol/L ammonium formate was used as mobile phase A.
The methanol and acetonitrile were compared as mobile phase B. The acetonitrile was better elution capacity, shorter retention time, lower solvent effect and column pressure. The acetonitrile was used as mobile phase B.
The formula milk powder was a complex matrix of proteins, phospholipids, pigments, soluble salts and some artificially added nutrients. After testing multiple formula milk powder continuously with the injection volume of 10 µL, there was retention time drift and strong matrix interference, and the peak shape was broadened and asymmetric (Fig. 2). The retention times of chlorate and perchlorate moved from 4.21 min to 3.80 min and from 2.57 min to 2.32 min, which occurred as competitive anions in the sample take up active sites on the stationary phase and ionization suppression in mass spectrometry. In batch, using small volume injection (2 µL) could make the deviation of retention time less than 2.0%, reduce matrix interference. When the injection volume was reduced by a factor of 5, the peak heights of chlorate (chlorate-18O3) and perchlorate (perchlorate-18O4) are almost 100% and 50% of the original, which met the needs of detection limit. Therefore, in the determination of various foods, the injection volume of 2 µL reached sensitivity requirements, reduced matrix interference and improved the accuracy.
The extraction results of three different extracts (0.1% formic acid - acetonitrile (1:2, v/v), pure water - acetonitrile (1:2, v/v) and 20 mmol/L ammonium formate - acetonitrile (1:2, v/v)) were compared (Fig. 3 and Table 3). The concentrations of chlorate were ranked as follows: 20 mmol/L ammonium formate - acetonitrile > pure water - acetonitrile > 0.1% formic acid - acetonitrile. The concentrations of perchlorate were ranked as follows: 0.1% formic acid - acetonitrile > pure water - acetonitrile > 20 mmol/L ammonium formate - acetonitrile. The RSD of chlorate and perchlorate in three different extraction solutions were 6.25% and 1.96%. Using pure water - acetonitrile (1:2, v/v) was simple, fast and reduce the use of reagents, so it was used as the extraction solution.
| Different extracts | chlorate | perchlorate | ||||
|---|---|---|---|---|---|---|
| Area | IS Area | Concentration(µg/kg) | Area | IS Area | Concentration(µg/kg) | |
| 0.1% formic acid-Acetonitrile (v/v, 1:2) | 1547 | 13168 | 8.83 | 2658 | 16886 | 4.18 |
| Pure water-Acetonitrile (v/v, 1:2) | 1515 | 11265 | 10.1 | 2556 | 16544 | 4.10 |
| 20mmol/L Ammonium formate-Acetonitrile (v/v, 1:2) | 1949 | 14553 | 10.3 | 2452 | 16800 | 3.96 |
| Pure water-Acetonitrile (v/v, 1:4) | 1678 | 13042 | 10.0 | 2350 | 16025 | 4.02 |
| Pure water-Acetonitrile (v/v, 1:1) | 2000 | 16084 | 9.49 | 1481 | 10366 | 3.84 |
| Acetonitrile | 822 | 18484 | 3.65 | 575 | 12511 | 1.21 |
The effects of acetonitrile and methanol as extraction were compared (Fig. 4). The peak heights of chlorate and internal standard was higher when methanol was used for extraction. When using acetonitrile, the peak heights of perchlorate and internal standard was higher. The white crystals deposited at the bottom of the bottle several hours after the milk powder was extracted with methanol for several hours, which was not conducive to mass spectrometry analysis. These crystals were soluble solids, which were completely dissolved after adding water. The acetonitrile was used as the extract without crystallization. This showed that acetonitrile was easier to dissolve inorganic salts. Therefore, acetonitrile was more suitable to extract chlorate and perchlorate from foods.
Different proportions of pure water-acetonitrile were compared (Fig. 5). The protein could not be precipitated When the acetonitrile ratio was 0. The acetonitrile ratio was higher, the effect of protein precipitation was better. The RSD of chlorate and perchlorate extraction with pure water-acetonitrile (v/v, 1:1), pure water-acetonitrile (v/v, 1:2) and pure water-acetonitrile (v/v, 1:4) were 3.32% and 2.92% in fresh food (Table 3), respectively. When acetonitrile was used for extraction, only one third of chlorate and perchlorate were present. This was the sample could not be dispersed without water, and the chlorate and perchlorate could not be extracted effectively (Table 3). Using pure water-acetonitrile (v/v, 1:4) to extract the target, the result of dry food was poor. This was because that dry foods were easy to absorb water and expansion, resulting in not enough water to extract the target, so reduce the gram of dry food to 1 g and increase the volume of extraction to 30 mL. Water-acetonitrile (v/v, 1:2) could extract analyte well, precipitate protein effectively, and reduce the use of organic solvents. The use of pure water-acetonitrile (v/v, 1:2) for different matrix could obtain accurate and reliable determination results and eliminate matrix interference.
The purification effects of four SPE (ENVI™ -18、ENVI-Carb™ II/PSA、PRIME HLB 60 mg and PRIME HLB 120 mg) were compared. The purified solution of four SPE was shallow and transparent. The purified solution of 60 mg and 120 mg of PRIME HLB was divided into 1#, 2# and 3#, which were 1 mL initial filtrate after PRIME HLB purification, 1 mL continuous filtrate after 1 mL initial filtrate was discarded, and 1 mL continuous filtrate after 2 mL initial filtrate was discarded, respectively. The concentrations of 2# and 3# by PRIME HLB 60 mg and PRIME HLB 120 mg were basically the same. The RSD of chlorate and perchlorate purified by ENVI™ -18、ENVI-Carb™ II/PSA、PRIME HLB 60mg (2#、3#)and PRIME HLB 120mg (2#、3#) were 3.72% and 2.69% respectively, which indicated that four SPE could purify the matrix of food. The concentration of perchlorate in 1# of PRIME HLB 60 mg and 120 mg was more than 30% higher than that of 2# and 3#, but the recovery of 1# was 88.3% − 116%, which showed that accuracy could not only be measured by recovery. There were a small amount of chlorate and perchlorate in the PRIME HLB, which caused the high content of 1#. PRIME HLB was not activated, easy to operate, cost-effective, and removed the impurities such as phospholipid, protein and pigment. Therefore, PRIME HLB 60 mg was used to purify foods.
The linear range, calibration curve, correlation coefficient, and quantification (LOQ) of chlorate and perchlorate were confirmed (Table 4). The intraday and interday precision tests were performed after dilution of chlorate (S242-5272) and perchlorate (QCI-108). The intraday mean values (n = 6) of chlorate and perchlorate were 15.0 µg/L and 5.86 µg/L, with RSD of 1.42% and 1.09% respectively. The interday mean values (n = 6) of chlorate and perchlorate were 15.4 µg/L and 6.08 µg/L, with RSD of 4.84% and 2.12% respectively. The interday recovery of chlorate and perchlorate were 101% and 104%. The chemical properties of chlorate and perchlorate were stable. The LOD and LOQ were estimated for a signal-to-noise (S/N) ratio of more than 3 and 10 respectively from the chromatograms of samples spiked at the lowest concentration in food (Chen et al., 2014). The average recovery of chlorate and perchlorate were 86.5–103% and 91.3–111% in every food matrix, with the RSD of 2.53–7.41% and 2.68–7.96% The addition levels of chlorate were 10.0, 40.0 and 80.0 µg/kg, and the addition levels of perchlorate were 5.0, 20.0 and 40.0 µg/kg in fresh food. The dry food was added as twice as fresh food. The chlorate and perchlorate had high accuracy, precision and good repeatability in drinking water and foods by LC - QqQ - MS/MS.
| Compound | Linear range(µg/L) | Regression equation | Correlation coefficient(R2) | LOQ(µg/kg) | ||
|---|---|---|---|---|---|---|
| Drinking water (mg/L) | Fresh food (mg/kg) | Dry food (mg/kg) | ||||
| chlorate | 0.2–200.0 | y = 0.927866x-0.0186662 | 0.9995 | 0.0002 | 0.006 | 0.018 |
| perchlorate | 0.1–100.0 | y = 5.04016x + 0.0837387 | 0.9995 | 0.00005 | 0.001 | 0.003 |
Because milk powder is a typical matrix with high protein, fat and soluble salt, we chose milk powder to investigate the matrix effect as fresh food. The matrix effect of ganoderma lucidum as a representative dried food was investigated. Three related quantitative parameters were obtained by comparing the response of the same concentration target in solvent and milk powder, namely matrix effects (ME), recovery (RE) of the extraction procedure, and process efficiency (PE, including matrix effects and extraction efficiency) (Matuszewski et al., 2003). ME(%) = B/A × 100, RE(%) = C/B × 100, PE(%) = C/A × 100 = (ME × RE)/100, A represents the response value (peak area) of the compound in pure solvent, B represents the response value of the compound added after the extraction of blank sample, C represents the response value of the compound added before the extraction of blank sample (Matuszewski et al., 2003). ME was 79.0–108%, indicating that the LC chromatographic and continuous responses of mass spectrometry could be reproduced by the extracted or purified samples. The RE was 82.9–105%, indicating that the method has achieved acceptable quantitative accuracy, when the standard curve of matrix addition was used. PE was 70.3–110%, indicating that the solvent standard curve was acceptable for quantification.
The results of 18 drinking waters in Fujian Province were much lower than the chlorate limit (700 µg/L) and perchlorate limit (70 µg/L) (Table 5), which were documented in the fourth edition of WHO Drinking Water Guidelines (WHO, 2017). A total of 583 food samples from 16 categories were determined (Table 5). The detection rate of chlorate in 16 kinds of food was ranked: cereals < fruits < medicine food homology < eggs < products of animal origin < spices = dictyophora indusiata < legumes = potatoes < vegetables < liquid milk < marine fish < agaricus blazei murrill < milk powder < infant auxiliary < infant formula. The detection rate of perchlorate in 16 kinds of food was ranked: fruits < potatoes < products of animal origin < cereals = legumes = agaricus blazei murrill < infant auxiliary food, Perchlorate was detected 100% in the other 9 food samples. The average concentration of chlorate in 16 kinds of foods were ranked as: cereal < legumes < eggs < marine fish < infant formula < fruits < liquid milk < medicine food homology < products of animal origin < vegetables < spices < potatoes < dictyophora indusiata Fisch < infant auxiliary food < milk powder < agaricus blazei. The average concentration of perchlorate in 16 kinds of foods were in the order of meats < infant auxiliary food < fruits < infant formula < potatoes < liquid milk < cereals < legumes < agaricus blazei murrill < milk powder < sea fish < dictyophora indusiata fisch < eggs < vegetables < medicine food homology < spices; Although agaricus blazei murrill and dictyophora indusiata fisch were both edible fungi, their chlorate content was very different. The order of chlorate and perchlorate content in each food was different.
| Species | Chlorate | Perchlorate | ||||
|---|---|---|---|---|---|---|
| Detection rate(%) | Content(mg/kg or mg/L) | Mean(mg/kg or mg/L) | Detection rate(%) | Content(mg/kg or mg/L) | Mean(mg/kg or mg/L) | |
| Drinking water(n = 18) | 100 | ND-0.0833 | 0.00759 | 100 | 0.000780–0.00567 | 0.00126 |
| Liquid milk(n = 10) | 40.0 | ND-0.0392 | 0.00654 | 100 | 0.00804–0.0166 | 0.0107 |
| Milk powder(n = 30) | 86.7 | ND -0.248 | 0.0495 | 100 | 0.00354–0.0772 | 0.0288 |
| Infant formula(n = 20) | 100 | 0.0105-0.22 | 0.00554 | 100 | 0.00105–0.0359 | 0.00667 |
| Cereals(n = 30) | 0.00 | ND | ND | 90.0 | ND-0.0536 | 0.0158 |
| Fruits(n = 50) | 2.00 | ND-0.0139 | 0.00616 | 56.0 | ND-0.0488 | 0.0063 |
| Vegetables(n = 60) | 26.7 | ND-0.0765 | 0.0103 | 100 | 0.00204-0.314 | 0.0493 |
| Products of animal origin(n = 109) | 13.8 | ND-0.197 | 0.00903 | 84.4 | ND-0.0411 | 0.00448 |
| Marine fish(n = 25) | 45.0 | ND-0.0111 | 0.00524 | 100 | 0.0026–0.275 | 0.0335 |
| Spices(n = 20) | 15.0 | ND-0.112 | 0.0120 | 100 | 0.0175-4.55 | 1.24 |
| Eggs(n = 59) | 13.3 | ND -0.0204 | 0.00460 | 100 | 0.00404-0.430 | 0.0422 |
| Legumes(n = 20) | 20.0 | ND-0.0131 | 0.00431 | 90.0 | ND-0.223 | 0.0170 |
| Potatoes(n = 20) | 20.0 | ND-0.138 | 0.0122 | 80.0 | ND-0.0452 | 0.00996 |
| Dictyophora indusiata(n = 20) | 15.0 | ND-0.0955 | 0.0150 | 100 | 0.00587-0.139 | 0.0375 |
| Agaricus blazei(n = 20) | 50.0 | ND-2.48 | 0.464 | 90.0 | ND-0.096 | 0.0242 |
| Medicine food homology(n = 50) | 10.0 | ND-0.156 | 0.00868 | 100 | 0.00142-0.537 | 0.0646 |
| Infant auxiliary food(n = 40) | 95.0 | ND-0.274 | 0.0465 | 97.5 | ND-0.0166 | 0.00588 |
| Note: ND : less than the detection limit. | ||||||
The commission regulation (EU) 2020/749 of 4 June 2020 was amended as Annex III to Regulation (EU) No 396/2005 (European Commission 396, 2005) of the European Parliament and of the Council concerning maximum residue levels (MRLs) for chlorate in or on certain products (European Commission 749, 2020). According to (EU) 2020/749, the MRL of chlorate was 0.1 mg/kg in milk, liquid milk did not exceed. Three samples were more than the default MRL of 0.1 mg/kg in milk powder and infant formula respectively, and the over standard rate of milk powder and infant formula were 10.0% and 15.0%; The MRL of chlorate for cereal was 0.05 mg/kg, None of the cereals was detected; none of the fruit exceeded the MRL of 0.05 mg/kg; The vegetables did not exceed the MRL of 0.05 mg/kg; The MRL was 0.05 mg/kg in the products of animal origin, one swine exceeded the MRL, the excess rate was 0.92%; There were two marine fishes slightly higher than default MRL of 0.01 mg/kg, the over standard rate was 8.0%; One zanthoxylum bungeanum maxim exceeded the MRL (0.07 mg/kg) of spices, the exceeded rate was 5.0%; The eggs did not exceed the MRL of 0.05 mg/kg; The MRL of legumes was 0.35 mg/kg, and none of the samples exceeded the MRL; One potato sample exceeded the MRL of 0.05 mg/kg, the exceeded rate was 5.0%; The dictyophora indusiata did not exceed the MRL of cultivated fungi of 0.7 mg/kg; There were four agaricus blazei samples exceeded the MRL of cultivated fungi, and the over-standard rate was 20.0%; one codonopsis pilosula exceeded the MRL (0.05 mg/kg) of homology of medicine and food, and the exceeded rate was 2.0%; There were thirty-two samples of infant auxiliary food exceed the MRL of 0.01 mg/kg, the over-standard rate was 80.0%; The over-standard rate was sorted as: products of animal origin < homology of medicine and food < spices = potatos < marine fish < milk powder < infant formula < agaricus blazei < infant auxiliary food, the remaining seven foods were not exceeded the MRLs.
According to the reference levels for intra-Union trade from 16 March 2015 (European Commission, 2015) and Commission regulation (EU) 2020/685 of 20 may 2020 (European Commission 685, 2020), the MRLs for liquid milk, milk powder, cereals, fruits, vegetables, products of animal origin, marine fishes, eggs, legumes, potatoes, dictyophora indusiata and agaricus blazei were 0.05 mg/kg, the MRLs of infant formula, spices, Medicine food homology and infant supplementary food were 0.01, 0.5, 0.75 and 0.02 mg/kg. According to the above MRLs, seven milk powder samples exceeded the limit, the over-standard rate was 23.3%, and all of them were full cream high calcium milk powder; One sample of infant formula exceeded 0.01 mg/kg, with an excess rate of 15.0%; There were two cereal samples (millet and oat rice) slightly higher than 0.05 mg/kg, and the rate was 6.7%. Eight samples of vegetable were more than 0.05 mg/kg, and the over-standard rate was 25.0%. There were two marine fish samples (all of which were moray eel) exceeding 0.05 mg/kg, and the excess rate was 8.0%. There were ten samples exceeded 0.50 mg/kg in the spices, which were zanthoxylum bungeanum, the over-standard rate was 50.0%. There were seven egg samples (three goose eggs, three duck eggs and one egg) were over 0.05 mg/kg, and the over standard rate was 11.9%. Only one legume sample (black legume) was over 0.05 mg/kg, and the over standard rate was 5.0%. There were five dictyophora indusiata samples were over 0.05 mg/kg, and the rate was 25.0%. Three agaricus blazei samples exceeded 0.05 mg/kg, with a rate of 15.0%. The liquid milk, products of animal origin, fruits and potatos did not exceed 0.05 mg/kg. The homology of medicine and food were herbs with 0.75 mg/kg as the limit, without exceeding the standard. The infant auxiliary food (including cereal auxiliary food and canned auxiliary food) did not exceed 0.02 mg/kg. The over-standard rates of perchlorate were in order of legume < cereal < marine fish < egg < infant formula = agaricus blazei < milk powder < vegetable = dictyophora indusiata < spices, The remaining six categories of food did not exceed the corresponding limits.
A rapid and sensitive LC - QqQ - MS/MS method for the determination of chlorate and perchlorate in foods was established by using solvent standard curve and stable isotope labeling method. The Torus™ DEA column separated chlorate and perchlorate in five minutes, and the sensitivity were good. The ammonium formate (A) and acetonitrile (B) as mobile phase system, chlorate and perchlorate could be eluted without affecting the sensitivity. The small volume injection could reduce matrix interference and retention time drift. The chlorate and perchlorate could be completely extracted from samples with pure water-acetonitrile (v/v, 1:2). The PRIME® HLB 60mg was used to purify the sample matrix. The stable isotope labeling method could avoid matrix effect and accurately determine chlorate and perchlorate in foods. This method was fast, simple, good reproducibility, high sensitivity and accuracy.
In 583 food samples, the highest content of chlorate (2.48 mg/kg) and perchlorate (4.55 mg/kg) were agaricus blazei and zanthoxylum bungeanum maxim respectively. The infant formula had the highest detection rate of chlorate. The detection rate of perchlorate was 100% in liquid milk, infant formula, vegetables, marine fish, spices, eggs, dictyophora indusiata and medicine food homology. The agaricus blazei had highest average concentration of chlorate. The spices had highest average perchlorate concentration of perchlorate. The highest excess rate of chlorate and perchlorate was infant supplementary food (80.0%) and spices (50.0%). According to the above data, the concentration of chlorate and perchlorate in marketed and circulated food had risk of exceeding the EU regulations in Fujian province. The next step will be to performed a human exposure assessment for chlorate and perchlorate in food.
The authors were compliance with ethical standards.
No funding was received for this study.
Wenting Zhang wrote the main manuscript text. Qiuyan Lu，Renjin Zheng，Wenqian Qiu，Yongyou Hua prepared figures 1-5 and tables 1-5. Yuxiang Li reviewed the manuscript.
The authors declared no conflict of interest.