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Abstract ［Objectives］ This study was conducted to explore the application of high performance liquid chromatography to detect sodium salicylate in different milk powder and milk.
［Methods］A high performance liquid chromatographic (HPLC) method was established for determining the sodium salicylate in milk and milk power, with Agilent ZORBAX Eclips XDB C18 column. The recovery and precision of the method were analyzed and discussed.
［Results］ The experimental conditions were determined as mobile phase: methanol+0.02 mol/L ammonium acetate solution, flow rate: 0.8 ml/min, detection wavelength: 300 nm, and column temperature: 30 ℃. The peak area had a good linear relationship with the standard solution in the range of 0.2-10.0 mg/L, and the correlation coefficient (R2) was greater than 0.999. The standard was added into different matrices at four levels. The average recovery of sodium salicylate in milk powder was 90.5%-101.0%, with RSD less than 5.0%, and the average recovery of sodium salicylate in milk was 90.5%-101.4%, with RSD less than 5.0%. The limit of quantification was 1.25 mg/kg in cow milk and 5.00 mg/kg in milk powder.
［Conclusions］The accuracy and precision of sodium salicylate detection in milk powder and milk samples of the experimental method met the requirements. The method is simple, accurate, and reliable, and can meet the needs of actual detection.
Key words Sodium salicylate; Determination; Milk power; Milk; HPLC
Received: June 27, 2021 Accepted: August 28, 2021
Jian CHEN (1979-), male, P. R. China, senior engineer, devoted research about food physical and chemical analysis and testing.
*Corresponding author. E-mail: [email protected].
Salicylic acid, the scientific name of which is o-hydroxybenzoic acid, has a molecular formula of C7H6O3. It is a fat-soluble organic acid that naturally exists in natural willow bark, some vegetables, fruits, and spices［1］. Sodium salicylate, the molecular formula of which is C7H5O3Na, is a salt of salicylic acid, which can be hydrolyzed in an aqueous solution. As an important fine chemical raw material, it can be used to prepare analgesics, rheumatism medicines and other medicines and some analytical reagents, and can also be used in food for disinfection and preservation or as edible synthetic spices. However, sodium salicylate has an irritating effect on the eyes, skin, mucous membranes, and respiratory tracts. After inhalation, it can cause coughing, breathing difficulties and chest pain, nausea, vomiting, headache, dizziness, tinnitus, vision loss, allergic reactions, etc., and large amounts of oral administration can be fatal［2-5］. According to the Announcement No.278 of the Ministry of Agriculture of the Peoples Republic of China, when sodium salicylate is used as a veterinary drug, in accordance with the 65th edition of the veterinary drug specifications, the milk abandonment period shall be at least 48 h, and the milk in the abandonment period shall not be illegally used for commercial purposes［6］. According to the regulations on the use of salicylic acid and sodium salicylate in the "GB 2760-2014 National food safety standard hygienic standard: Hygienic standards for the use of food additives", sodium salicylate should not be added to infant formula foods, sterilized milk or fermented milk［7］. At present, there are still illegal businesses［8］ that artificially add salicylic acid and sodium salicylate to raw milk to prevent premature deterioration of raw milk and promote the extension of the shelf life of raw milk, or illegally use milk that has been abandoned for commercial purposes. However, the detection standards and detection methods for salicylic acid in the food industry in China include "SN/T 4675.15-2016 Determination of salicylic acid, dehydroacetic acid and p-chlorobenzoic acid in wine for export: Liquid chromatography"［9-13］, and the detection of sodium salicylate in food has not yet issued relevant national food safety standards. Therefore, the food industry, especially the dairy industry, urgently needs to develop a fast, universal and efficient method for testing sodium salicylate in milk and cow milk. Since sodium salicylate is a water-soluble salt, which is the opposite of the fat-soluble property of salicylic acid. Therefore, there is no reference to the detection method of salicylic acid in related foods. In this study, sodium salicylate in milk powder and cow milk were determined after extraction with pure water and precipitation of proteins, by high performance liquid chromatography diode array detector. It provides a scientific basis for enterprise risk monitoring, a rapid and accurate method for inspection and testing departments, technical support for the promulgation of relevant national standards, and method reference for official monitoring of illegal applications in the dairy industry.
Materials and Methods
Instruments and reagents
Agilent 1200SL fast liquid chromatograph with DAD detector; Milli-Q System ultrapure water preparation device (Milipor Company, USA); laboratory water: first-grade water, which met the requirements of "GB/T 6682-2008 Water for Analytical Laboratory Use―Specification and Test Methods".
Sodium salicylate standard (purity>99.0%); potassium ferrocyanide, zinc acetate and ammonium acetate, all of which were analytically pure.
Chromatographic conditions
Chromatographic column: Agilent ZORBAX Eclips XDB C18 (250 mm×4.6 mm, 5 μm); mobile phase: methanol+0.02 mol/L ammonium acetate solution=30+70 (V/V); flow rate: 0.8 ml/min; detection wavelength: 300 nm; column temperature: 30 ℃; injection volume: 10 μl.
Preparation of standard stock solution and working solutions
An appropriate amount of sodium salicylate standard was accurately weighed, and dissolved with methanol, obtaining a stock solution with a concentration of 1 000 mg/L. The stock solution was diluted with the mobile phase to standard working solutions of 0.2 mg/L to 10.0 mg/L as required.
Sample preparation pretreatment
A 1 g of milk powder sample (accurate to 0.01 g) was accurately weighed into a 50 ml centrifuge tube, and added with 15 ml of pure water at about 60 ℃, obtaining a mixture which was shaken well to dissolve the sample. The sample liquid was put in an ultrasonic shaker for ultrasonic extraction for 10 min, and then added with 0.25 ml of potassium ferrocyanide solution and 0.25 ml of zinc acetate solution, followed by vortex-mixing. Then, the pretreated sample was transferred to a 25ml volumetric flask and diluted to constant weight. The sample was placed in a refrigerator at 4 ℃ for 10 min to precipitate proteins. The sample solution was centrifuged at 4 500 r/min for 10 min, and the supernatant was filtered with membrane (water phase, 0.2 μm), obtaining a filtrate, which was loaded on the machine for testing. For liquid milk samples, a 4 g of the sample was accurately weighed (accurate to 0.01 g) into a 50 ml centrifuge tube. The subsequent steps were the same as the previous steps for the detection of milk powder samples. Finally, the filtrate was tested on the machine.
Results and Analysis
Standard chromatogram analysis
Under the determined experimental conditions, that is, mobile phase: methanol+0.02 mol/L ammonium acetate solution=30+70 (V/V), flow rate: 0.8 ml/min, detection wavelength: 300 nm, column temperature: at 30 ℃, the detection chromatogram is shown in Fig. 1, and the spectrogram is shown in Fig. 2.
Discussion of experimental results
Standard curve regression equation and correlation coefficient
Under the experimental conditions in this study, a standard curve was drawn with the peak areas and concentrations of the standard solutions. The liquid chromatography showed a good linear relationship in the range of 0.2-10.0 mg/L, and the linear equation was y=16.824x+0.483 (r=0.999 97). The milk powder quantitative limit and liquid milk quantitative limit were 5.0 and 1.25 mg/kg.
Method recovery and precision tests
In this study, milk powder (commercially purchased infant formula milk powder) and liquid milk samples (commercially purchased pure milk) were selected for the recovery test. The concentration levels of sodium salicylate standard added in the milk powder matrix sample were 5.0, 10.0, 20.0 and 100 mg/kg, respectively, and the concentration levels of sodium salicylate standard added in the liquid milk matrix sample were 1.25, 2.50, 5.00 and 25.0 mg/kg. Each supplement level was repeated 6 times. The recovery and precision are shown in Table 1. The milk powder sample chromatogram and quantification limit addition recovery chromatogram are shown in Fig. 3 and Fig. 4, respectively, and the liquid milk sample chromatogram and quantification limit addition recovery chromatogram are shown in Fig. 5 and Fig. 6, respectively.
The results of the tests showed that the average recovery of milk powder in this method was 90.5-101.0%, with relative standard deviations (RSD, n=6) from 2.94 to 4.00%, and the average recovery of liquid milk was 90.5-101.4%, with relative standard deviations (RSD, n=6) from 2.01 to 3.63%, indicating that the method is accurate and reliable.
Agricultural Biotechnology2021
Conclusions and Discussion
The high performance liquid chromatography established in this study could simply and quickly determine the content of sodium salicylate in milk powder and liquid milk. This method showed a good linear relationship in the range of sodium salicylate content from 0.2 to 10 mg/L. The limits of quantification in liquid milk and milk powder were 1.25 and 5.00 mg/kg, respectively. The recovery was between 90.5% and 101.4%, with relative standard deviations from 2.01% to 4.00%. The method is highly efficient and stable, and has high recovery rate and good reproducibility. The method can accurately determine the content of sodium salicylate in milk powder and liquid milk, which provides technical support for enterprises and third-party testing agencies. References
［1］ SHAO RT, DING XY, ZHANG LH, et al. Determination of salicylic acid in raw milk by ultra performance liquid chromatography［J］. Analysis and Testing Technology and Instruments, 2019, 25(2): 114-118. (in Chinese)
［2］ LABIB R, BURY D, BOISLEVE F, et al. A kinetic-based safety assessment of consumer exposure to salicylic acid from cosmetic products demonstrates no evidence of a health risk from developmental toxicity［J］. Regulatory Toxicology and Pharmacology, 2018(94): 245-251.
［3］ HU X, ZHANG Y, ZHANG LJ, et al. Quantitative determination of salicylic acid in milk and dairy products by ultra performance liquid chromatography-tandem mass spectrometry［J］. Journal of Dairy Science and Technology, 2019, 42(30): 30-33. (in Chinese)
［4］ BAENKLER H W. Salicylate intolerance: Pathophysiology, clinical spectrum, diagnosis and treatment［J］. Deutsches rzteblatt International, 2008, 105(8): 137-142.
［5］ MITCHELL J E, SKYPALA I. Aspirin and salicylate in respiratory disease［J］. Rhinology, 2013, 51(3): 195-205.
［6］ Announcement No.278 of the Ministry of Agriculture of the Peoples Republic of China［S］. Beijing: Standards Press of China, 2003. (in Chinese)
［7］ GB 2760-2014 National food safety standard hygienic standard: Hygienic standards for the use of food additives［S］. Beijing: Standards Press of China, 2014. (in Chinese)
［8］ JIN Z, SHEN JW, LIU MR, et al. Hypothetical detection method of salicylic acid and sodium salicylate in raw milk［J］. Silicon Valley, 2013(9): 144-145. (in Chinese)
［9］ SN/T 4675.15-2016 Determination of salicylic acid, dehydroacetic acid and p-chlorobenzoic acid in wine for export: Liquid chromatography［S］. Beijing: Standards Press of China, 2017. (in Chinese)
［10］ Cosmetic safety technical specifications (2015 edition)［S］. State Food and Drug Administration. (in Chinese)
［11］ LI TF, ZHAO FN, ZHANG C, et al. Simultaneous determination of salicylic acid and gibberellic acid in tomato by high performance liquid chromatography tandem mass spectrometry［J］. Food Science, 2016, 37(8): 182-186. (in Chinese)
［12］ TIAN YP, SHU XL, GU LL, et al. Determination of benzoic acid, potassium sorbate, salicylic acid in milk beverage by HPLC-DAD［J］. Chemical World, 2012, 53(1): 24-25. (in Chinese)
［13］ XUE F, LIN YK, MA YL, et al. Research progress in method of determination of salicylic acid in food［J］. Science and Technology of Food Industry 2012, 33(14): 429-431. (in Chinese)
［Methods］A high performance liquid chromatographic (HPLC) method was established for determining the sodium salicylate in milk and milk power, with Agilent ZORBAX Eclips XDB C18 column. The recovery and precision of the method were analyzed and discussed.
［Results］ The experimental conditions were determined as mobile phase: methanol+0.02 mol/L ammonium acetate solution, flow rate: 0.8 ml/min, detection wavelength: 300 nm, and column temperature: 30 ℃. The peak area had a good linear relationship with the standard solution in the range of 0.2-10.0 mg/L, and the correlation coefficient (R2) was greater than 0.999. The standard was added into different matrices at four levels. The average recovery of sodium salicylate in milk powder was 90.5%-101.0%, with RSD less than 5.0%, and the average recovery of sodium salicylate in milk was 90.5%-101.4%, with RSD less than 5.0%. The limit of quantification was 1.25 mg/kg in cow milk and 5.00 mg/kg in milk powder.
［Conclusions］The accuracy and precision of sodium salicylate detection in milk powder and milk samples of the experimental method met the requirements. The method is simple, accurate, and reliable, and can meet the needs of actual detection.
Key words Sodium salicylate; Determination; Milk power; Milk; HPLC
Received: June 27, 2021 Accepted: August 28, 2021
Jian CHEN (1979-), male, P. R. China, senior engineer, devoted research about food physical and chemical analysis and testing.
*Corresponding author. E-mail: [email protected].
Salicylic acid, the scientific name of which is o-hydroxybenzoic acid, has a molecular formula of C7H6O3. It is a fat-soluble organic acid that naturally exists in natural willow bark, some vegetables, fruits, and spices［1］. Sodium salicylate, the molecular formula of which is C7H5O3Na, is a salt of salicylic acid, which can be hydrolyzed in an aqueous solution. As an important fine chemical raw material, it can be used to prepare analgesics, rheumatism medicines and other medicines and some analytical reagents, and can also be used in food for disinfection and preservation or as edible synthetic spices. However, sodium salicylate has an irritating effect on the eyes, skin, mucous membranes, and respiratory tracts. After inhalation, it can cause coughing, breathing difficulties and chest pain, nausea, vomiting, headache, dizziness, tinnitus, vision loss, allergic reactions, etc., and large amounts of oral administration can be fatal［2-5］. According to the Announcement No.278 of the Ministry of Agriculture of the Peoples Republic of China, when sodium salicylate is used as a veterinary drug, in accordance with the 65th edition of the veterinary drug specifications, the milk abandonment period shall be at least 48 h, and the milk in the abandonment period shall not be illegally used for commercial purposes［6］. According to the regulations on the use of salicylic acid and sodium salicylate in the "GB 2760-2014 National food safety standard hygienic standard: Hygienic standards for the use of food additives", sodium salicylate should not be added to infant formula foods, sterilized milk or fermented milk［7］. At present, there are still illegal businesses［8］ that artificially add salicylic acid and sodium salicylate to raw milk to prevent premature deterioration of raw milk and promote the extension of the shelf life of raw milk, or illegally use milk that has been abandoned for commercial purposes. However, the detection standards and detection methods for salicylic acid in the food industry in China include "SN/T 4675.15-2016 Determination of salicylic acid, dehydroacetic acid and p-chlorobenzoic acid in wine for export: Liquid chromatography"［9-13］, and the detection of sodium salicylate in food has not yet issued relevant national food safety standards. Therefore, the food industry, especially the dairy industry, urgently needs to develop a fast, universal and efficient method for testing sodium salicylate in milk and cow milk. Since sodium salicylate is a water-soluble salt, which is the opposite of the fat-soluble property of salicylic acid. Therefore, there is no reference to the detection method of salicylic acid in related foods. In this study, sodium salicylate in milk powder and cow milk were determined after extraction with pure water and precipitation of proteins, by high performance liquid chromatography diode array detector. It provides a scientific basis for enterprise risk monitoring, a rapid and accurate method for inspection and testing departments, technical support for the promulgation of relevant national standards, and method reference for official monitoring of illegal applications in the dairy industry.
Materials and Methods
Instruments and reagents
Agilent 1200SL fast liquid chromatograph with DAD detector; Milli-Q System ultrapure water preparation device (Milipor Company, USA); laboratory water: first-grade water, which met the requirements of "GB/T 6682-2008 Water for Analytical Laboratory Use―Specification and Test Methods".
Sodium salicylate standard (purity>99.0%); potassium ferrocyanide, zinc acetate and ammonium acetate, all of which were analytically pure.
Chromatographic conditions
Chromatographic column: Agilent ZORBAX Eclips XDB C18 (250 mm×4.6 mm, 5 μm); mobile phase: methanol+0.02 mol/L ammonium acetate solution=30+70 (V/V); flow rate: 0.8 ml/min; detection wavelength: 300 nm; column temperature: 30 ℃; injection volume: 10 μl.
Preparation of standard stock solution and working solutions
An appropriate amount of sodium salicylate standard was accurately weighed, and dissolved with methanol, obtaining a stock solution with a concentration of 1 000 mg/L. The stock solution was diluted with the mobile phase to standard working solutions of 0.2 mg/L to 10.0 mg/L as required.
Sample preparation pretreatment
A 1 g of milk powder sample (accurate to 0.01 g) was accurately weighed into a 50 ml centrifuge tube, and added with 15 ml of pure water at about 60 ℃, obtaining a mixture which was shaken well to dissolve the sample. The sample liquid was put in an ultrasonic shaker for ultrasonic extraction for 10 min, and then added with 0.25 ml of potassium ferrocyanide solution and 0.25 ml of zinc acetate solution, followed by vortex-mixing. Then, the pretreated sample was transferred to a 25ml volumetric flask and diluted to constant weight. The sample was placed in a refrigerator at 4 ℃ for 10 min to precipitate proteins. The sample solution was centrifuged at 4 500 r/min for 10 min, and the supernatant was filtered with membrane (water phase, 0.2 μm), obtaining a filtrate, which was loaded on the machine for testing. For liquid milk samples, a 4 g of the sample was accurately weighed (accurate to 0.01 g) into a 50 ml centrifuge tube. The subsequent steps were the same as the previous steps for the detection of milk powder samples. Finally, the filtrate was tested on the machine.
Results and Analysis
Standard chromatogram analysis
Under the determined experimental conditions, that is, mobile phase: methanol+0.02 mol/L ammonium acetate solution=30+70 (V/V), flow rate: 0.8 ml/min, detection wavelength: 300 nm, column temperature: at 30 ℃, the detection chromatogram is shown in Fig. 1, and the spectrogram is shown in Fig. 2.
Discussion of experimental results
Standard curve regression equation and correlation coefficient
Under the experimental conditions in this study, a standard curve was drawn with the peak areas and concentrations of the standard solutions. The liquid chromatography showed a good linear relationship in the range of 0.2-10.0 mg/L, and the linear equation was y=16.824x+0.483 (r=0.999 97). The milk powder quantitative limit and liquid milk quantitative limit were 5.0 and 1.25 mg/kg.
Method recovery and precision tests
In this study, milk powder (commercially purchased infant formula milk powder) and liquid milk samples (commercially purchased pure milk) were selected for the recovery test. The concentration levels of sodium salicylate standard added in the milk powder matrix sample were 5.0, 10.0, 20.0 and 100 mg/kg, respectively, and the concentration levels of sodium salicylate standard added in the liquid milk matrix sample were 1.25, 2.50, 5.00 and 25.0 mg/kg. Each supplement level was repeated 6 times. The recovery and precision are shown in Table 1. The milk powder sample chromatogram and quantification limit addition recovery chromatogram are shown in Fig. 3 and Fig. 4, respectively, and the liquid milk sample chromatogram and quantification limit addition recovery chromatogram are shown in Fig. 5 and Fig. 6, respectively.
The results of the tests showed that the average recovery of milk powder in this method was 90.5-101.0%, with relative standard deviations (RSD, n=6) from 2.94 to 4.00%, and the average recovery of liquid milk was 90.5-101.4%, with relative standard deviations (RSD, n=6) from 2.01 to 3.63%, indicating that the method is accurate and reliable.
Agricultural Biotechnology2021
Conclusions and Discussion
The high performance liquid chromatography established in this study could simply and quickly determine the content of sodium salicylate in milk powder and liquid milk. This method showed a good linear relationship in the range of sodium salicylate content from 0.2 to 10 mg/L. The limits of quantification in liquid milk and milk powder were 1.25 and 5.00 mg/kg, respectively. The recovery was between 90.5% and 101.4%, with relative standard deviations from 2.01% to 4.00%. The method is highly efficient and stable, and has high recovery rate and good reproducibility. The method can accurately determine the content of sodium salicylate in milk powder and liquid milk, which provides technical support for enterprises and third-party testing agencies. References
［1］ SHAO RT, DING XY, ZHANG LH, et al. Determination of salicylic acid in raw milk by ultra performance liquid chromatography［J］. Analysis and Testing Technology and Instruments, 2019, 25(2): 114-118. (in Chinese)
［2］ LABIB R, BURY D, BOISLEVE F, et al. A kinetic-based safety assessment of consumer exposure to salicylic acid from cosmetic products demonstrates no evidence of a health risk from developmental toxicity［J］. Regulatory Toxicology and Pharmacology, 2018(94): 245-251.
［3］ HU X, ZHANG Y, ZHANG LJ, et al. Quantitative determination of salicylic acid in milk and dairy products by ultra performance liquid chromatography-tandem mass spectrometry［J］. Journal of Dairy Science and Technology, 2019, 42(30): 30-33. (in Chinese)
［4］ BAENKLER H W. Salicylate intolerance: Pathophysiology, clinical spectrum, diagnosis and treatment［J］. Deutsches rzteblatt International, 2008, 105(8): 137-142.
［5］ MITCHELL J E, SKYPALA I. Aspirin and salicylate in respiratory disease［J］. Rhinology, 2013, 51(3): 195-205.
［6］ Announcement No.278 of the Ministry of Agriculture of the Peoples Republic of China［S］. Beijing: Standards Press of China, 2003. (in Chinese)
［7］ GB 2760-2014 National food safety standard hygienic standard: Hygienic standards for the use of food additives［S］. Beijing: Standards Press of China, 2014. (in Chinese)
［8］ JIN Z, SHEN JW, LIU MR, et al. Hypothetical detection method of salicylic acid and sodium salicylate in raw milk［J］. Silicon Valley, 2013(9): 144-145. (in Chinese)
［9］ SN/T 4675.15-2016 Determination of salicylic acid, dehydroacetic acid and p-chlorobenzoic acid in wine for export: Liquid chromatography［S］. Beijing: Standards Press of China, 2017. (in Chinese)
［10］ Cosmetic safety technical specifications (2015 edition)［S］. State Food and Drug Administration. (in Chinese)
［11］ LI TF, ZHAO FN, ZHANG C, et al. Simultaneous determination of salicylic acid and gibberellic acid in tomato by high performance liquid chromatography tandem mass spectrometry［J］. Food Science, 2016, 37(8): 182-186. (in Chinese)
［12］ TIAN YP, SHU XL, GU LL, et al. Determination of benzoic acid, potassium sorbate, salicylic acid in milk beverage by HPLC-DAD［J］. Chemical World, 2012, 53(1): 24-25. (in Chinese)
［13］ XUE F, LIN YK, MA YL, et al. Research progress in method of determination of salicylic acid in food［J］. Science and Technology of Food Industry 2012, 33(14): 429-431. (in Chinese)