Method for measuring content of biotin in composite nutrient supplement

Abstract

The invention relates to the technical field of chemical analysis, in particular to a method for measuring the content of biotin in a compound nutrient supplement. The invention discloses a method for measuring the content of biotin in a compound nutrient supplement. The method comprises the following steps: preparing a sample solution and a standard solution; taking the sample solution and the standard solution, injecting the sample solution and the standard solution into a high performance liquid chromatograph for liquid chromatographic analysis, and determining the biotin content; the conditions of high performance liquid chromatography are as follows: a chromatographic column is a C8 column; the mobile phase A is a monopotassium phosphate solution with the molar concentration of 0.01-0.02 mol / L, and the pH value of the mobile phase A is 2.5; a mobile phase B is acetonitrile; an isocratic elution mode is adopted for elution, and the volume ratio of the mobile phase A to the mobile phase B is (90-95): (10-5); the invention provides a method for determining the content of biotin in a compound nutrient supplement. The method is good in linear relation, high in precision and high in recovery rate.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemical analysis, and in particular to a method for determining the content of biotin in a compound nutrient supplement. BACKGROUND

[0002] Currently, the existing technologies in the field of biotin detection mainly include a microbial method and a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). The microbial method realizes quantitative detection through the dependence of specific microorganisms on biotin, but the detection process is significantly affected by the activity of microorganisms, the composition of culture medium and the fluctuation of culture conditions, resulting in a relatively low accuracy and a relative standard deviation RSD of the detection result generally higher than 5%. The HPLC-MS / MS method needs to rely on a high-performance liquid chromatography-tandem mass spectrometer, which has a high purchase cost and maintenance cost and a high operation complexity, and most conventional laboratories do not have such a special instrument, so it is difficult to popularize and apply.

[0003] Therefore, the existing technologies are limited in the wide application of biotin detection in conventional quality control and primary laboratories due to insufficient detection precision or high equipment threshold and economic cost. Therefore, it is an urgent problem to develop a substitute detection scheme with high precision, low cost and easy operation. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application provides a method for determining the content of biotin in a compound nutrient supplement.

[0005] In a first aspect, the present application provides a method for determining the content of biotin in a compound nutrient supplement, which adopts the following technical scheme: A method for determining the content of biotin in a compound nutrient supplement, comprising the following steps: Preparation of a sample solution and a standard solution; Taking the sample solution and the standard solution, injecting them into a high-performance liquid chromatograph for liquid chromatography analysis to determine the content of biotin.

[0006] Preferably, the conditions for high-performance liquid chromatography analysis include: A chromatographic column: a C8 column with a length of 150 mm, a diameter of 4.6 mm and a filler particle size of 5 μm.

[0007] In a specific implementation, the C8 column is an XB-C8 chromatographic column with a length of 150 mm, a diameter of 4.6 mm and a filler particle size of 5 μm.

[0008] Preferably, the mobile phase A is a 0.01-0.02 mol / L potassium dihydrogen phosphate solution with a pH value of 2.5. Mobile phase B: acetonitrile.

[0009] In a specific embodiment, the mobile phase A is a 0.02 mol / L potassium dihydrogen phosphate solution with a pH value of 2.5; Mobile phase B: acetonitrile.

[0010] Preferably, the flow rate of the mobile phase is 0.5 mL / min to 1.0 mL / min.

[0011] More preferably, the flow rate of the mobile phase is 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min or 1.0 mL / min.

[0012] In a specific embodiment, the flow rate of the mobile phase is 1.0 mL / min.

[0013] Preferably, the injection volume is 20 μL to 50 μL.

[0014] More preferably, the injection volume is 20 μL, 21 μL, 22 μL, 23 μL, 24 μL, 25 μL, 26 μL, 27 μL, 28 μL, 29 μL, 30 μL, 31 μL, 32 μL, 33 μL, 34 μL, 35 μL, 36 μL, 37 μL, 38 μL, 39 μL, 40 μL, 41 μL, 42 μL, 43 μL, 44 μL, 45 μL, 46 μL, 47 μL, 48 μL, 49 μL or 50 μL.

[0015] In a specific embodiment, the injection volume is 50 μL.

[0016] Preferably, the detection wavelength is 205 nm to 210 nm; for example, 205 nm, 206 nm, 207 nm, 208 nm, 209 nm or 210 nm.

[0017] In a specific embodiment, the detection wavelength is 210 nm.

[0018] Preferably, the column temperature is 30 to 35 °C.

[0019] More preferably, the column temperature is 30 °C, 31 °C, 32 °C, 33 °C, 34 °C or 35 °C.

[0020] In a specific embodiment, the column temperature is 35 °C.

[0021] Preferably, the detector is a diode array detector.

[0022] Preferably, the elution is carried out by isocratic elution, and the volume ratio of the mobile phase A to the mobile phase B is (90-95):(10-5).

[0023] More preferably, the volume ratio of the mobile phase A and the mobile phase B is 90:10, 91:9, 92:8, 93:7, 94:6 or 95:5.

[0024] In a specific embodiment, the volume ratio of the mobile phase A and the mobile phase B is 92:8.

[0025] Preferably, the preparation method of the sample solution comprises the following steps: Pre-freezing the composite nutrient supplement sample at -40℃ to -80℃ for 3 to 5 hours, and then vacuum freeze-drying; Taking 0.1 to 0.5 g of the vacuum freeze-dried sample, adding 5 to 6 mL of water with a temperature of 50 to 60℃, and performing ultrasonic hydrolysis for 15 to 25 minutes, and then cooling to 20 to 30℃ and diluting to 10 mL with water, and shaking well; Diluting with water to a solution with a concentration of 1.783 μg / mL to 35.66 μg / mL, shaking well, and filtering to obtain the sample solution.

[0026] Preferably, during the vacuum freeze-drying process, the temperature is 15 to 20℃, the vacuum pressure is 10 to 15 Pa, and the vacuum drying time is 2 to 3 hours. Preferably, during the ultrasonic hydrolysis process, the ultrasonic power is 55 to 65 W.

[0027] Preferably, the composite nutrient supplement sample is any one of a composite vitamin tablet without minerals or a composite vitamin tablet with minerals.

[0028] Preferably, the composite vitamin tablet without minerals comprises vitamin B1, vitamin B2, vitamin B6, vitamin B 12 , biotin, folic acid, nicotinamide, and pantothenic acid.

[0029] Preferably, when the composite nutrient supplement sample is a composite vitamin tablet with minerals, the composite vitamin tablet with minerals is pretreated by mixing 0.5 to 1.5 parts by mass of the composite vitamin tablet with minerals with 0.01 to 0.5 parts by mass of diethylene triamine pentaacetic acid to obtain the pretreated composite vitamin tablet with minerals.

[0030] Preferably, the composite vitamin tablet with minerals comprises vitamin B1, vitamin B2, vitamin B6, vitamin B 12 , biotin, folic acid, nicotinamide, pantothenic acid, calcium, magnesium, manganese, iron, zinc, selenium, and copper.

[0031] Preferably, the preparation method of the standard solution comprises the following steps: Accurately weigh 8.96 mg of biotin standard, dissolve with 60℃ hot water by ultrasonic, and after cooling, dilute to 25 ml to obtain a biotin standard stock solution; Take the standard stock solution, use water as the solvent, and prepare standard working curve solutions with concentrations of 1.783 μg / mL, 3.566 μg / mL, 8.195 μg / mL, 17.830 μg / mL, 26.745 μg / mL, and 35.66 μg / mL.

[0032] By adopting the technical scheme, the sample solution preparation step is improved in the application, and the problems of low biotin extraction efficiency, easy degradation, and matrix interference are solved by vacuum freeze drying and ultrasonic treatment, thereby effectively improving the accuracy and reliability of the detection result.

[0033] The vacuum freeze drying removes water in the sample by low-temperature freezing and vacuum sublimation, on the one hand, reduces the dilution effect of water on biotin and the interference of excipients, and on the other hand, protects the heat-sensitive biotin from thermal degradation in a low-temperature environment throughout the process, and at the same time, forms a porous loose structure inside the tablet by sublimation of ice crystals, which weakens the physical adsorption of biotin and excipients; and then through ultrasonic treatment, the matrix structure of the loose sample after freeze-drying is broken by the cavitation effect of high-frequency sound waves, and the bound biotin is released, which converts the bound biotin into free state, thereby effectively improving the accuracy and reliability of the detection result.

[0034] In summary, the application has at least one of the following beneficial technical effects: The application provides a method for determining the content of biotin in a complex nutrient supplement, which has good linearity, high precision, and high recovery rate. The application provides a method for determining the content of biotin in a complex nutrient supplement, which can separate biotin from the complex nutrient supplement, and the method is simple, fast, safe and reliable, and easy to implement. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 HPLC spectrum of the sample solution in Example 1; Figure 2 HPLC spectrum of the blank solution in Example 1; Figure 3 HPLC spectrum of the biotin standard solution in Example 1; Figure 4 Standard working curve graph in Example 1; Figure 5 HPLC spectrum of the sample solution in Example 2; Figure 6 HPLC spectrum of the blank solution in Example 2; Figure 7This is the HPLC chromatogram of the biotin standard solution in Example 2. Detailed Implementation

[0036] The technical solutions of this application are further illustrated by specific embodiments below. These specific embodiments do not represent a limitation on the scope of protection of this application. Any non-essential modifications and adjustments made by others based on the concept of this application still fall within the scope of protection of this application.

[0037] All raw materials used in this application are commercially available products. The present application will be further described in detail below with reference to embodiments and comparative examples.

[0038] Example 1:

[0039] A method for determining the biotin content in a multivitamin supplement, comprising the following steps: 1. Instruments Electronic balance, ultrasonic cleaner, high performance liquid chromatograph, centrifuge.

[0040] 2. Reagents Potassium dihydrogen phosphate (AR), phosphoric acid (AR), diethylenetriaminepentaacetic acid, DTPA (AR), acetonitrile (chromatographic grade); Biotin standard (Source: China National Institutes for Food and Drug Control, content 99.5%, CAS: 58-85-5).

[0041] Sample of multivitamin supplement: Pankangjian Gu® B-complex vitamin tablets contain 13.76~20mg of vitamin B1, 12.8~20mg of vitamin B2, 6.88~10mg of vitamin B6, and vitamin B1 per 0.5g. 12 6.4~10μg, nicotinamide 34~50mg, folic acid 320~500μg, biotin 60~100μg, pantothenic acid 13.76~20mg.

[0042] 3. Solution preparation: 0.02 mol / L potassium dihydrogen phosphate solution (mobile phase A): Accurately weigh 2.72 g of potassium dihydrogen phosphate, dissolve it in 900 mL of water, adjust the pH to 2.5 with phosphoric acid, dilute to 1000 mL with water, filter, and degas to obtain the solution.

[0043] Acetonitrile (mobile phase B): Take 500 mL of acetonitrile and sonicate for 20 min to obtain the mobile phase B.

[0044] 4. Analytical Methods 4.1 Instrument conditions and parameters Chromatographic conditions: Column: Ultimate® XB-C8, 150mm*4.6mm, 5µm; The detection wavelength was 210 nm; The column temperature was 35℃; The injection volume was 50µL; The flow rate of mobile phase was 1.0ml / min; The detector was a diode array detector; Mobile phase A: 0.02mol / L potassium dihydrogen phosphate solution, pH value was 2.5; Mobile phase B: acetonitrile; The elution was carried out by isocratic elution, and the volume ratio of mobile phase A to mobile phase B was 92:8.

[0045] 4.2, Preparation of sample solution After pre-freezing the composite nutrient supplement sample at-40℃ for 4 hours, vacuum freeze-drying was carried out at a temperature of 18℃ and a vacuum pressure of 13Pa for 2.5 hours; Take 0.25g of the vacuum freeze-dried sample, put it in a 10mL brown volumetric flask, add 6mL of water at a temperature of 55℃, oscillate in an ultrasonic water bath for 20 minutes, the ultrasonic power is 60W; cool to 25℃, then dilute to 10mL with water, shake well; Dilute with water to a concentration of 1.783μg / mL~35.66μg / mL solution, shake well, filter with a 0.45µm filter membrane for standby, get the sample solution.

[0046] 4.3, Preparation of standard solution Precisely weigh 8.96mg of biotin standard in a 25ml brown volumetric flask, ultrasonically dissolve with 60℃ hot water, cool and dilute to the mark, shake well to ensure the solution is completely transparent; store at 2-8℃ for 7 days. As a biotin standard stock solution, the concentration is 356.61μg / ml.

[0047] Take the biotin standard stock solution, use water as solvent, prepare standard working curve solutions with concentrations of 1.783μg / mL, 3.566μg / mL, 8.195μg / mL, 17.830μg / mL, 26.745μg / mL, 35.66μg / mL.

[0048] 4.4, Sample determination Precisely take 20μL of sample solution and standard working curve solution respectively, inject into the liquid chromatograph, determine, i.e. Figure 1 As shown.

[0049] 4.5, Calculation The standard curve gets the regression equation, the peak area corresponding to the sample is brought into the regression equation, and the sample concentration is calculated. The content is calculated according to the following formula: ; Wherein: X - biotin content in the sample, pg / tablet; C - biotin concentration in the sample according to the regression equation, pg / mL; V - dilution volume of the product, mL; m - product specification, g / tablet; W - sample mass, g.

[0050] Methodology validation: The determination method of Example 1 was subjected to methodology validation: 1. Method blank test 1.1. Test method Preparation of blank solution: Without taking sample, the blank solution was prepared according to the sample processing method in Example 1 4.2. Preparation of standard solution: 8.96 mg of biotin standard was precisely weighed into a 25 ml brown volumetric flask, dissolved with 60°C hot water under ultrasonic, and after cooling, the volume was adjusted to the mark, shaken well to ensure complete transparency of the solution; stored at 2-8°C for 7 days. As a biotin standard stock solution, the concentration was 356.61 pg / ml.

[0051] An appropriate amount of biotin standard stock solution was taken and water was used as solvent to prepare standard working curve solutions with concentrations of 1.783 pg / mL, 3.566 pg / mL, 8.195 pg / mL, 17.830 pg / mL, 26.745 pg / mL, and 35.66 pg / mL.

[0052] 20 pL of blank solution and standard working curve solution were precisely taken respectively and injected into the liquid chromatograph for determination. The peak retention time of the blank solution was compared with that of the biotin standard solution.

[0053] 1.2. Test results The HPLC spectrum of the blank solution is shown in Figure 2 ; and the HPLC spectrum of the biotin standard solution is shown in Figure 3 .

[0054] 1.3. Test conclusion According to Figure 2 , Figure 3 , the blank solution has no absorption peak at the biotin peak retention time of the standard solution, indicating that the blank solution has no interference with the determination results.

[0055] 2. Confirmation of linear range 2.1. Test data Respectively, 6 different concentrations of standard working curve solution of 20 μL, injection liquid chromatograph, the test results are shown in Table 1.

[0056] Table 1 Standard curve data table

[0057] 2.2, standard working curve The concentration in Table 1 is taken as the abscissa, and the peak area is taken as the ordinate. The standard working curve is shown in Figure 4 .

[0058] 2.3, linear test conclusion Linear evaluation: the correlation is 0.999, so the method for determining biotin shows good linearity between the concentration of 1.783 μg / mL to 35.66 μg / mL, which meets the requirements of GB / T27417-2017 "Guidelines for Chemical Analysis Method Confirmation and Verification": GB / T27417-2017 requires correlation ≥0.99.

[0059] 3, detection limit and quantitative limit The detection limit DL and the quantitative limit QL of the analysis method are calculated by the signal-to-noise ratio (S / N). DL is defined as the concentration corresponding to S / N=3, and QL is defined as the concentration corresponding to S / N=10.

[0060] 3.1, detection limit When the signal-to-noise ratio (S / N) is 3, the detection limit of biotin is 0.03 μg / mL.

[0061] 3.2, quantitative limit When the signal-to-noise ratio (S / N) is 10, the quantitative limit of biotin is 0.10 μg / mL.

[0062] 4, precision test 4.1, test method 6 samples were weighed, and the sample was treated according to the sample solution preparation method of Example 1 to prepare the sample solution, and the content of the sample was detected, and the RSD (%) was calculated.

[0063] 4.2, test data Table 2 Test data table

[0064] 4.3, test conclusion The RSD of the biotin content of the 6 samples is 1.0%, indicating that the method has good precision, which meets the requirements of GB / T27417-2017 "Guidelines for Chemical Analysis Method Confirmation and Verification": GB / T27417-2017 requires RSD (%) ≤2.0%.

[0065] 5. Recovery rate test 5.1 Test Methods Accurately weigh 8.96 mg of biotin standard into a 25 ml brown volumetric flask, dissolve by sonication in 60 °C hot water, cool, and dilute to the mark. Shake well to ensure the solution is completely transparent. Store at 2–8 °C for 7 days. This serves as a biotin standard stock solution with a concentration of 356.61 μg / ml.

[0066] Spike addition: Accurately weigh 9 portions of sample, divide them into three groups of 3 portions each, and accurately add 0.6 mL, 1.0 mL, and 1.6 mL of biotin standard stock solution with a concentration of 35.66 μg / mL to each group respectively to prepare sample solutions according to the method in Example 1.

[0067] 5.2 The test data are as follows: Table 3 Recovery rate test data

[0068] The amount added = the amount measured in the spiked sample - the amount measured in the whole sample; Recovery rate (%) = Measured amount added / Theoretical amount added × 100%.

[0069] 5.3 Experimental Conclusions The average recovery rate was 96.8%, and the relative standard deviation (RSD) was 1.1%, which meets the requirements of GB / T27417-2017 "Guideline for Conformity Assessment and Validation of Chemical Analysis Methods": the recovery rate is 95~105% when the analyte is >100mg / kg.

[0070] 6. Conclusion The biotin content determination method was tested for linearity, precision, blank, limit of detection, limit of quantitation, and recovery rate. All results met the requirements of GB / T27417-2017 "Guideline for Conformity Assessment and Validation of Chemical Analysis Methods", proving that the content determination method is scientific and effective and can achieve the purpose of quality control of biotin content in Pankangjian Gu® B Vitamin Tablets.

[0071] Example 2:

[0072] A method for determining the biotin content in a multivitamin supplement, comprising the following steps: 1. Instruments Electronic balance, ultrasonic cleaner, high performance liquid chromatograph, centrifuge.

[0073] 2. Reagents Potassium dihydrogen phosphate (AR), phosphoric acid (AR), diethylenetriaminepentaacetic acid, DTPA (AR), acetonitrile (chromatographic grade); Biotin standard (Source: China Institute for Food and Drug Control, content 99.5%, CAS: 58-85-5).

[0074] Compound nutrient supplement samples: Pangkangjian®B vitamin tablets, containing vitamin B1 13.76-20 mg, vitamin B2 2.8-20 mg, vitamin B6 6.88-10 mg, vitamin B12 2.5-10 mg, folic acid 20-100 mg, biotin 60-100 mg, pantothenic acid 13.76-20 mg, calcium 110.5-120 mg, magnesium 30-40.75 mg, manganese 0.1-1.5 mg, iron 3-12 mg, zinc 4-10 mg, selenium 5-15 mg, copper 0.1-0.5 mg per 0.5 g. 12 6.4-10 mg, nicotinamide 34-50 mg, folic acid 320-500 mg, biotin 60-100 mg, pantothenic acid 13.76-20 mg, calcium 110.5-120 mg, magnesium 30-40.75 mg, manganese 0.1-1.5 mg, iron 3-12 mg, zinc 4-10 mg, selenium 5-15 mg, copper 0.1-0.5 mg.

[0075] 3. Preparation of solution: 0.02 mol / L potassium dihydrogen phosphate solution (mobile phase A): accurately weigh 2.72 g of potassium dihydrogen phosphate, dissolve in 900 mL of water, adjust the pH value to 2.5 with phosphoric acid, and dilute to 1000 mL with water, filter, and degas to obtain.

[0076] Acetonitrile (mobile phase B): take 500 mL of acetonitrile, and ultrasonic for 20 min to obtain.

[0077] 4. Analysis method 4.1. Instrument conditions and parameters Chromatographic conditions: Chromatographic column: XB-C8, 150 mm*4.6 mm, 5 µm; Detection wavelength: 210 nm; Column temperature: 35 °C; Injection volume: 50 µL; Flow rate of mobile phase: 1.0 ml / min; Detector: diode array detector; Mobile phase A: 0.02 mol / L potassium dihydrogen phosphate solution, pH value 2.5; Mobile phase B: acetonitrile; Use isocratic elution, the volume ratio of mobile phase A and mobile phase B is 92:8.

[0078] 4.2. Preparation of sample solution Mix 1 g of mineral-containing compound vitamin tablets, 0.1 g of diethylene triamine pentaacetic acid, and 3 g of water to obtain pretreated mineral-containing compound vitamin tablets.

[0079] The pre-processed mineral-containing multivitamin tablet sample was pre-frozen at -40℃ for 4 hours, and then vacuum freeze-dried at 18℃ and 13 Pa for 2.5 hours; 0.25 g of the vacuum freeze-dried sample was placed in a 10 mL brown volumetric flask, 6 mL of water at 55℃ was added, and the mixture was oscillated in an ultrasonic water bath for 20 minutes at an ultrasonic power of 60 W. After cooling to 25℃, the mixture was diluted to 10 mL with water and shaken well. The solution was diluted with water to a concentration of 1.783 μg / mL to 35.66 μg / mL, shaken well, filtered with a 0.45 µm filter membrane, and used as needed to obtain a sample solution.

[0080] 4.3 Preparation of standard solution 8.96 mg of biotin standard was precisely weighed into a 25 ml brown volumetric flask, dissolved with 60℃ hot water by ultrasonic, and cooled to constant volume. The solution was shaken well to ensure complete transparency. The solution was stored at 2-8℃ for 7 days. The concentration of the biotin standard stock solution was 356.61 μg / ml.

[0081] An appropriate amount of biotin standard stock solution was taken and diluted with water to prepare standard working curve solutions with concentrations of 1.783 μg / mL, 3.566 μg / mL, 8.195 μg / mL, 17.830 μg / mL, 26.745 μg / mL, and 35.66 μg / mL.

[0082] 4.4 Sample determination 20 μL of the sample solution and the standard working curve solution were precisely taken and injected into the liquid chromatograph, and the determination was performed, i.e. Figure 5 as shown in the table.

[0083] 4.5 Calculation The regression equation of the standard curve was obtained, and the peak area corresponding to the sample was brought into the regression equation to calculate the sample concentration. The content was calculated according to the following formula: ; In the formula: X—biotin content in the sample, μg / tablet; C—biotin concentration in the sample according to the regression equation, μg / mL; V—dilution volume of the product, mL; m—product specification, g / tablet; W—sample mass, g.

[0084] Methodology verification: The determination method of Example 2 was subjected to methodology verification: 1. Method blank test 1.1 Test method Preparation of blank solution: Without taking sample, the blank solution was prepared according to the method of sample treatment in Example 2 4.2; Preparation of standard solution: Precisely weigh 8.96 mg of biotin standard into a 25 ml brown volumetric flask, dissolve with 60°C hot water by ultrasonic, cool and dilute to the mark after cooling, shake well to ensure the solution is completely transparent; store at 2-8°C for 7 days. As biotin standard stock solution, the concentration is 356.61 μg / ml.

[0085] Take an appropriate amount of biotin standard stock solution, use water as solvent, prepare standard working curve solutions with concentrations of 1.783 μg / mL, 3.566 μg / mL, 8.195 μg / mL, 17.830 μg / mL, 26.745 μg / mL, 35.66 μg / mL.

[0086] Precisely take 20 μL of blank solution and standard working curve solution respectively, inject into liquid chromatograph for determination, compare the peak retention time of blank solution with that of biotin standard solution.

[0087] 1.2, Test results The HPLC chromatogram of blank solution is shown in Figure 6 The HPLC chromatogram of biotin standard solution is shown in Figure 7 .

[0088] 1.3, Test conclusion According to Figure 6 , Figure 7 , the blank solution has no absorption peak at the biotin peak retention time of standard solution, indicating that the blank solution has no interference with the determination results.

[0089] 2, Linear range confirmation 2.1, Test data Precisely take 20 μL of each of the 6 different concentrations of standard working curve solution, inject into liquid chromatograph, and the test results are shown in Table 4.

[0090] Table 4 Standard curve data table

[0091] 2.3, Linear test conclusion Linear evaluation: the linear equation is Y=20.79X+7.1584, the correlation coefficient (R 2) is 0.9999; therefore, the method shows good linearity at a concentration of 1.783 μg / mL to 35.66 μg / mL, meeting the requirements of GB / T 27417-2017 "Guidelines for Chemical Analysis Method Confirmation and Verification": the correlation is required to be ≥0.99.

[0092] 3. Detection limit and quantification limit The detection limit DL and quantification limit QL of the analysis method are calculated by the signal-to-noise ratio (S / N). DL is defined as the concentration corresponding to S / N = 3, and QL is defined as the concentration corresponding to S / N = 10. 3.1. Detection limit When the signal-to-noise ratio (S / N) is 3, the detection limit of biotin is 0.043 μg / mL.

[0093] 3.2. Quantification limit When the signal-to-noise ratio (S / N) is 10, the quantification limit of biotin is 0.115 μg / mL.

[0094] 4. Precision test 4.1. Test method Six samples were weighed, the sample solution was prepared according to the sample solution preparation method of Example 2, the sample content was detected, and the RSD (%) was calculated.

[0095] 4.2. Test data Table 5 Test data table

[0096] 4.3. Test conclusion The RSD of the biotin content of the six samples is 1.1%, indicating that the method has good precision, meeting the requirements of GB / T 27417-2017 "Guidelines for Chemical Analysis Method Confirmation and Verification": the RSD (%) is required to be ≤2.0%.

[0097] 5. Recovery rate test 5.1. Test method 8.96 mg of biotin standard was precisely weighed in a 25 ml brown volumetric flask, dissolved with 60°C hot water under ultrasonic, and after cooling, the volume was adjusted to the mark, shaken well to ensure the solution was completely transparent; stored at 2-8°C for 7 days. As a biotin standard stock solution, the concentration is 356.61 μg / ml.

[0098] Spike addition: Accurately weigh 9 portions of sample, divide them into three groups of 3 portions each, and accurately add 0.6 mL, 1.0 mL, and 1.6 mL of biotin standard stock solution with a concentration of 35.66 μg / mL to each group respectively to prepare sample solutions according to the method in Example 2.

[0099] 5.2 The test data are as follows: Table 6 Recovery rate test data

[0100] The amount added = the amount measured in the spiked sample - the amount measured in the whole sample; Recovery rate (%) = Measured amount added / Theoretical amount added × 100%.

[0101] 5.3 Experimental Conclusions The average recovery rate was 97.1%, and the relative standard deviation (RSD) was 1.0%, which meets the requirements of GB / T27417-2017 "Guideline for Conformity Assessment and Validation of Chemical Analysis Methods": the analyte > 100 mg / kg, the recovery rate is 95~105%.

[0102] 6. Conclusion The biotin content determination method was tested for linearity, precision, blank, limit of detection, limit of quantitation, and recovery rate. All results met the requirements of GB / T27417-2017 "Guideline for Conformity Assessment and Validation of Chemical Analysis Methods", proving that the content determination method is scientific and effective and can achieve the purpose of quality control of biotin content in Pankangjian Gu® B Vitamin Tablets.

Claims

1. A method for determining the biotin content of a multiple nutrient supplement, characterized by: It comprises the following steps: Prepare sample solution and standard solution; Inject the sample solution and the standard solution into a high performance liquid chromatograph for liquid chromatography analysis to determine the content of biotin; The conditions of the high performance liquid chromatography analysis include: The chromatographic column is a C8 column; The mobile phase A is a 0.01-0.02 mol / L potassium dihydrogen phosphate solution with a pH value of 2.5; The mobile phase B is acetonitrile; The elution is performed in an isocratic elution mode, and the volume ratio of the mobile phase A to the mobile phase B is (90-95):(10-5).

2. A method of determining the amount of biotin in a multi-nutritional supplement according to claim 1, characterized in that: The conditions of the high performance liquid chromatography analysis further include: The flow rate of the mobile phase is 0.5-1.0 mL / min; The injection volume is 20-50 μL; The detection wavelength is 205-210 nm; The column temperature is 30-35℃; The detector is a diode array detector.

3. The method of determining the amount of biotin in a multinutrient supplement according to claim 1, wherein: The length of the chromatographic column is 150 mm, the diameter is 4.6 mm, and the particle size of the filler is 5 μm.

4. The method of determining the amount of biotin in a nutritional supplement according to claim 1, wherein: The preparation method of the sample solution comprises the following steps: Pre-freeze the composite nutrient supplement sample at -40℃ to -80℃ for 3-5 hours, and then perform vacuum freeze drying; Take 0.1-0.5 g of the sample after vacuum freeze drying, add 5-6 mL of water with a temperature of 50-60℃, perform ultrasonic hydrolysis for 15-25 minutes, cool to 20-30℃, and then dilute to 10 mL with water, and shake well; Dilute with water to obtain a sample solution with a concentration of 1.783-35.66 μg / mL.

5. A method of determining the amount of biotin in a multi-nutritional supplement according to claim 4, characterized in that: During the vacuum freeze drying process, the temperature is 15-20℃, the vacuum pressure is 10-15 Pa, and the vacuum drying time is 2-3 hours; During the ultrasonic hydrolysis process, the ultrasonic power is 55-65 W.

6. The method of determining the amount of biotin in a nutritional supplement according to claim 1, wherein: The composite nutrient supplement sample is any one of a composite vitamin tablet without minerals and a composite vitamin tablet with minerals.

7. A method of determining the amount of biotin in a multinutrient supplement according to claim 6, characterized in that: The mineral-free multivitamin tablet includes vitamin Bl, vitamin B2, vitamin B6, vitamin B 12 , biotin, folic acid, nicotinamide, and pantothenic acid.

8. The method of determining the amount of biotin in a nutritional supplement according to claim 6, wherein: When the composite nutrient supplement sample is the composite vitamin tablet with minerals, the composite vitamin tablet with minerals is pretreated, and the steps are as follows: mix 0.5-1.5 parts by mass of the composite vitamin tablet with minerals with 0.01-0.5 parts by mass of diethylenetriamine pentaacetic acid to obtain the pretreated composite vitamin tablet with minerals.

9. A method of determining the amount of biotin in a multinutrient supplement according to claim 6 or 8, characterised in that: The mineral-containing multivitamin tablet includes vitamin Bl, vitamin B2, vitamin B6, vitamin B 12 , biotin, folic acid, nicotinamide, pantothenic acid, calcium, magnesium, manganese, iron, zinc, selenium, and copper.

10. The method of determining the amount of biotin in a nutritional supplement according to claim 1, wherein: The preparation method of the standard solution comprises the following steps: Precisely weigh 8.96 mg of biotin standard, ultrasonically dissolve it with 60℃ hot water, dilute to 25 mL after cooling, and obtain a biotin standard stock solution; Take an appropriate amount of the standard stock solution, use water as the solvent, and prepare standard working curve solutions with concentrations of 1.783 μg / mL, 3.566 μg / mL, 8.195 μg / mL, 17.830 μg / mL, 26.745 μg / mL, and 35.66 μg / mL.

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