A method for simultaneously detecting multiple water-soluble vitamins and its application

Abstract

The invention discloses a method for simultaneously detecting multiple water-soluble vitamins, comprising: preparing a mixed standard working solution, wherein the mixed standard working solution comprises L(+)-ascorbic acid, D-isoascorbic acid, vitamin B1, nicotinic acid, nicotinamide, pyridoxal, pyridoxamine, pyridoxine, vitamin B2, pantothenic acid, biotin, vitamin B 12 and folic acid; the mixed standard working solution is detected by high performance liquid chromatography-diode array detector, octadecylsilane bonded silica gel is used as filler, acetonitrile is used as mobile phase A, and potassium dihydrogen phosphate solution is used as mobile phase B for gradient elution, and the corresponding standard curve between the concentration and peak area of ​​each water-soluble vitamin is obtained; the test solution is taken, and the concentration of each water-soluble vitamin is detected under the same conditions by high performance liquid chromatography-diode array detector. The present invention has good reproducibility, is accurate and reliable, and can simultaneously detect the content of 13 water-soluble vitamins in a sample.

Description

Technical Field

[0001] The present invention relates to the technical field of water-soluble vitamin detection, and in particular to a method for simultaneously detecting multiple water-soluble vitamins and its application. Background Art

[0002] Water-soluble vitamins mainly include vitamin B1, vitamin B2, vitamin B3 (niacin, niacinamide), pantothenic acid, vitamin B6 (pyridoxine, pyridoxal, pyridoxamine), folic acid, vitamin B 12 , biotin, vitamin C (L(+)-ascorbic acid, D-isoascorbic acid). Traditional analytical methods for water-soluble vitamins include ultraviolet spectrophotometry, fluorescence photometry, and microbiological methods, and their determination is mostly based on a single vitamin. The 2020 edition of the Chinese Pharmacopoeia and national standard methods are mostly based on the determination of a single vitamin, with cumbersome steps, long detection cycles, and time-consuming operations. In addition, some of these methods are greatly affected by interference factors and have low sensitivity, and cannot meet the actual needs of rapid and accurate detection of vitamins. For methods for simultaneously determining the content of multiple vitamins, high performance liquid chromatography or high performance liquid chromatography-tandem mass spectrometry is generally used. Among them, although high performance liquid chromatography-tandem mass spectrometry has higher detection sensitivity, it is prone to matrix effects, thereby affecting the accuracy of the results, and the mass spectrometry detector is expensive and not suitable for the promotion of the method. Therefore, high performance liquid chromatography is mostly used to determine water-soluble vitamins. However, the types of water-soluble vitamins that can be simultaneously determined by HPLC are relatively small, and generally cannot cover the simultaneous determination of all water-soluble vitamins in multivitamin drugs and foods. Moreover, since most water-soluble vitamins exist in aqueous solution in the form of ions, their retention capacity in reversed-phase chromatography is poor, so most of them adopt the method of adding ion pair reagents with greater column efficiency and pump damage to the mobile phase to improve peak shape and separation. The addition of ion pair reagents not only damages the instrument and chromatographic column, but also makes it difficult to balance the chromatographic column, which can easily cause changes in the retention time and elution order of vitamins with similar properties, affecting the accuracy and reproducibility of the measurement results. In addition, since the amount of water-soluble vitamins added varies greatly, from μg / 100g to g / 100g, it is inevitable that various components to be measured must have high sensitivity. However, due to the different chemical formula structures and functional groups of different water-soluble vitamins, the maximum absorption wavelengths of different vitamins are inconsistent. The use of ordinary HPLC-UV detectors cannot meet the random switching of multiple maximum wavelengths to ensure high sensitivity. Therefore, there are still few studies that can simultaneously determine 10 or more water-soluble vitamins.

[0003] With the development of modern analytical technology, high performance liquid chromatography and liquid chromatography-mass spectrometry can be used to simultaneously determine a variety of water-soluble vitamins, but the varieties are generally incomplete, and many require the use of ion pair reagents such as sodium octane sulfonate and hexadecyltrimethylammonium bromide. Ion pair reagents not only damage the instrument and chromatographic column, but also make the chromatographic column difficult to balance, which can easily cause retention time drift and changes in elution order, affecting the accuracy and reproducibility of the measurement results. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for simultaneously detecting multiple water-soluble vitamins. The method has good reproducibility, is accurate and reliable, and can simultaneously detect at least 13 water-soluble vitamins.

[0005] In order to solve the above technical problems, the present invention provides a method for simultaneously detecting multiple water-soluble vitamins, comprising the following steps:

[0006] Prepare a mixed standard working solution, the mixed standard working solution includes L (+) -ascorbic acid, D-isoascorbic acid, vitamin B1, niacin, niacinamide, pyridoxal, pyridoxamine, pyridoxine, vitamin B2, pantothenic acid, biotin, vitamin B 12 and folic acid;

[0007] The mixed standard working solution is detected by a high performance liquid chromatograph-diode array detector, wherein the liquid chromatograph uses octadecylsilane bonded silica gel as a filler, acetonitrile as a mobile phase A, and potassium dihydrogen phosphate solution as a mobile phase B for gradient elution to obtain a standard curve corresponding to the concentration and peak area of ​​each water-soluble vitamin;

[0008] Take the test solution and use a high performance liquid chromatography-diode array detector to detect it under the same conditions to obtain the detection peak area of ​​each water-soluble vitamin in the test solution, and obtain the concentration of each water-soluble vitamin in the test solution according to the standard curve.

[0009] In one embodiment, the gradient elution is performed according to the following procedure:

[0010] 0-5min, mobile phase A is 0%, mobile phase B is 100%;

[0011] 5min-15min, mobile phase A from 0→15%, mobile phase B from 100%→85%;

[0012] 15min~25min, mobile phase A from 15→33%, mobile phase B from 85%→67%;

[0013] 25min~35min, mobile phase A from 33%→10%, mobile phase B from 67%→90%;

[0014] 35min~40min, mobile phase A changes from 10%→0%, and mobile phase B changes from 90%→100%.

[0015] In one embodiment, the concentration of the potassium dihydrogen phosphate solution is 0.05 mol / L to 0.15 mol / L.

[0016] In one embodiment, the pH value of the potassium dihydrogen phosphate solution is 2.7-2.8.

[0017] In one embodiment, when gradient elution is performed, the flow rate of the mobile phase is 0.5 mL / min to 0.6 mL / min.

[0018] In one embodiment, when gradient elution is performed, the column temperature is 28°C to 30°C.

[0019] In one embodiment, the detection wavelength for each water-soluble vitamin is as follows:

[0020] The detection wavelength for pantothenic acid and biotin is 205 nm;

[0021] The detection wavelength for L(+)-ascorbic acid and D-isoascorbic acid was 260 nm;

[0022] The detection wavelength of vitamin B1, vitamin B2, niacin and niacinamide is 260nm;

[0023] The detection wavelength for pyridoxine, pyridoxal, pyridoxamine, and folic acid is 290 nm;

[0024] Vitamin B 12 The detection wavelength is 362nm.

[0025] In one embodiment, the mixed standard working solution is prepared by the following method:

[0026] L(+)-ascorbic acid, D-isoascorbic acid, vitamin B1, niacin, niacinamide, pyridoxal, pyridoxamine and pyridoxine are mixed with hydrochloric acid solution to prepare a mixed solution A;

[0027] Mix vitamin B2 with hydrochloric acid solution to prepare mixed solution B;

[0028] Take pantothenic acid, biotin, vitamin B 12 Mix with water to obtain mixed solution C;

[0029] Mix folic acid and ammonia water to prepare mixed solution D;

[0030] The mixed solution A, mixed solution B, mixed solution C and mixed solution D are mixed to obtain the mixed standard working solution.

[0031] In one embodiment, the test solution is prepared by the following method:

[0032] Take vitamin B1, vitamin B2, niacin, niacinamide, pyridoxal, pyridoxamine and pyridoxine samples, mix them with hydrochloric acid solution, and filter them after ultrasonic treatment;

[0033] Take L(+)-ascorbic acid and D-isoascorbic acid samples, mix them with metaphosphoric acid solution, and filter after ultrasonic treatment;

[0034] Take pantothenic acid, biotin and B vitamins 12 The sample was mixed with water, treated with ultrasound and then filtered;

[0035] Take a folic acid sample and mix it with ammonia water, perform ultrasonic treatment and then filter it.

[0036] In one embodiment, during the ultrasonic treatment, the power is 400W to 600W, and the treatment time is 20min to 30min.

[0037] The present invention also provides the application of the above method for simultaneously detecting multiple water-soluble vitamins in (1):

[0038] (1) Simultaneously detect L(+)-ascorbic acid, D-isoascorbic acid, vitamin B1, niacin, niacinamide, pyridoxal, pyridoxamine, pyridoxine, vitamin B2, pantothenic acid, biotin, and vitamin B 12 And folic acid, a total of 13 water-soluble vitamins.

[0039] The implementation of the present invention has the following beneficial effects:

[0040] The invention establishes a method for simultaneously detecting multiple water-soluble vitamins, adopts high performance liquid chromatography-diode array detector (HPLC-DAD), and realizes simultaneous separation and determination of multiple water-soluble vitamins under acetonitrile-potassium dihydrogen phosphate gradient elution conditions, so as to solve the problems that the types of water-soluble vitamins to be simultaneously determined are too few and incomplete, the sensitivity of common ultraviolet detectors is low, and the chromatographic column is difficult to balance due to the use of ionic reagents, the retention time of index components is easy to drift, the chromatographic column is easy to wear and has a short life, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the chromatogram of gradient elution condition 1;

[0042] Figure 2 is the chromatogram of gradient elution condition 2;

[0043] Figure 3 is the chromatogram of gradient elution condition 3;

[0044] Figure 4is a partial view of the chromatogram of gradient elution condition 3;

[0045] Figure 5 is the chromatogram of gradient elution condition 4;

[0046] Figure 6 is the chromatogram of gradient elution condition 5;

[0047] Figure 7 is the chromatogram of gradient elution condition 6;

[0048] Figure 8 This is the chromatogram at a flow rate of 0.4 ml / min;

[0049] Fig. 9 This is the chromatogram at a flow rate of 0.5 ml / min;

[0050] Fig.10 This is the chromatogram at a flow rate of 0.6 ml / min;

[0051] Fig.11 This is the chromatogram with a column temperature of 25°C;

[0052] Fig.12 This is the chromatogram with a column temperature of 28°C;

[0053] Fig.13 This is the chromatogram with a column temperature of 30°C;

[0054] Fig.14 It is the specificity investigation chromatogram of the blank solution;

[0055] Fig.15 It is the specificity investigation chromatogram of the mixed reference solution;

[0056] Fig.16 It is a specificity investigation chromatogram of the test solution. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solutions and advantages clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0058] 1. Instruments, reagents and test drugs

[0059] Instruments: High performance liquid chromatography-diode array detector (1260, Agilent), with octadecylsilane bonded silica gel as filler [Shimadzu Shim-pack Scepter C18-120 (4.6 mm × 250 mm, 5 μm) chromatographic column]; 1 / 10,000 electronic analytical balance (ME204E, Mettler-Toledo), 1 / 100,000 electronic analytical balance (XS105DU, Mettler-Toledo), CNC ultrasonic cleaner (KQ500DE, Kunshan Ultrasonic Instrument Co., Ltd.).

[0060] Reagents: hydrochloric acid (Guangzhou Chemical Reagent Factory) and ammonia (Xilong Scientific Co., Ltd.) were of analytical grade; phosphoric acid (Komi Chemical Reagent Co., Ltd.) and acetonitrile (Merck Co., Ltd.) were of chromatographic grade; and water was purified water (China Resources C'estbon Beverages (Holdings) Co., Ltd.).

[0061] Reagents: potassium dihydrogen phosphate (Shanghai MacLean Biochemical Technology Co., Ltd.) analytical grade; vitamin B1 (thiamine hydrochloride, (batch number: 22080797, purity 99.6%); vitamin B2 (batch number: 23050842, purity 97.8%); pyridoxine hydrochloride (batch number: 22070406, purity 99.7%); pyridoxal hydrochloride (batch number: 22050759, purity 99.9%); pyridoxamine hydrochloride (batch number: 22060330, purity 99.8%); L(+)-ascorbic acid ( Batch number: 21110703, purity 99.9%); D-isoascorbic acid (Batch number: 22120414, purity 87.7%); niacinamide (Batch number: 23030718, purity 99.8%); pantothenic acid (D-calcium pantothenate) (Batch number: 22090823, purity 98.8%); folic acid (Batch number: 22110991, purity 94.8%) are all from Tanmo Quality Inspection Technology Co., Ltd. Niacin (Batch number: 100434-201603, purity 99.8%); vitamin B 12 (Batch number: 100248-202006, purity 88.6%); biotin (Batch number: 100291-201804, purity 99.5%) are all from the China Food and Drug Inspection Institute. Compound vitamins (GlanbiaRE35490 / A) (Batch number: FS2304004, from Shanghai Lanjian Biotechnology Co., Ltd.); multivitamins (Ruipu 221025B-02-20) (Batch number: FS2302027, from Zhengzhou Ruipu Bioengineering Co., Ltd.); multivitamin mineral tablets (Batch number: YF23032405, from Guangdong Qifeng Health Industry Co., Ltd.); vitamin B tablets (Batch number: YF23032404, from Guangdong Qifeng Health Industry Co., Ltd.)

[0062] 2 Chromatographic conditions

[0063] Gradient elution, flow rate, column temperature and wavelength were investigated to determine the optimal chromatographic conditions.

[0064] (1) Investigation of gradient conditions

[0065] Acetonitrile was used as mobile phase A, 0.1 mol / L potassium dihydrogen phosphate solution (pH adjusted to 2.7-2.8 with phosphoric acid) was used as mobile phase B, flow rate was 0.5 ml / min, column temperature was 30°C, and 6 different gradient conditions in Tables 1-6 were investigated. The test results are shown in Figure 1-Figure 7 As can be seen from the figure, the components under gradient elution condition 1 were not separated; the components under gradient elution condition 2 were not separated; in the chromatogram under gradient elution condition 3, except for pyridoxine and nicotinamide, the separation degree between pyridoxine and nicotinamide was only 0.33, which did not meet the requirements, and the other components basically achieved good separation; the separation degree between pyridoxine and nicotinamide in the chromatogram under gradient elution condition 4 was 1.02, which did not meet the requirements; the separation degree between pyridoxine and nicotinamide in the chromatogram under gradient elution condition 5 was 0.51, which did not meet the requirements; the separation degree between pyridoxine and nicotinamide under gradient elution condition 6 was significantly improved to 1.34; according to the separation effect of the characteristic peaks, gradient elution condition 6 was selected.

[0066] Table 1 Gradient elution conditions 1

[0067]

[0068] Table 2 Gradient elution conditions 2

[0069]

[0070] Table 3 Gradient elution conditions 3

[0071]

[0072] Table 4 Gradient elution conditions 4

[0073]

[0074]

[0075] Table 5 Gradient elution conditions 5

[0076]

[0077] Table 6 Gradient elution conditions 6

[0078]

[0079] (2) Investigation of flow rate

[0080] Acetonitrile was used as mobile phase A, 0.1 mol / L potassium dihydrogen phosphate solution (pH adjusted to 2.7-2.8 with phosphoric acid) was used as mobile phase B, gradient elution condition 6, column temperature 30°C, flow rates 0.4 ml / min, 0.5 ml / min, 0.6 ml / min respectively. Test results are shown in Figure 8-Figure 10 As can be seen from the figure, when the flow rate is 0.4ml / min, the separation degree of pyridoxine and nicotinamide is 0.37, which does not meet the requirements; when the flow rate is 0.5ml / min, the separation degree of pyridoxine and nicotinamide is 1.44; when the flow rate is 0.6ml / min, the separation degree of pyridoxine and nicotinamide is 1.44; according to the separation effect of the characteristic peaks, the flow rate is selected to be 0.5ml / min.

[0081] (3) Investigation of column temperature

[0082] Acetonitrile was used as mobile phase A, 0.1 mol / L potassium dihydrogen phosphate solution (pH adjusted to 2.7-2.8 with phosphoric acid) was used as mobile phase B, gradient elution condition 6, flow rate 0.6 ml / min, column temperature 25°C, 28°C, 30°C respectively. Test results are shown in Figure 11-13 As can be seen from the figure, when the column temperature is 25°C, the separation degree of pyridoxine and nicotinamide is 1.34; when the column temperature is 28°C, the separation degree of pyridoxine and nicotinamide is 1.56, which meets the requirements; when the column temperature is 30°C, the separation degree of pyridoxine and nicotinamide is 1.44; according to the separation effect of the characteristic peaks, the column temperature is selected to be 28°C.

[0083] (4) Investigation of wavelength

[0084] Each component is scanned by a DAD detector in the wavelength range of 190 to 500 nm to obtain an absorption spectrum of each component. The spectrum of pantothenic acid has a strong absorption peak at 202 nm, the spectrum of biotin has a strong absorption peak at 200 nm, the spectrum of L(+)-ascorbic acid has a strong absorption peak at 252 nm, the spectrum of D-isoascorbic acid has a strong absorption peak at 252 nm, the spectrum of vitamin B1 has a strong absorption peak at 246 nm, the spectrum of vitamin B2 has a strong absorption peak at 268 nm, the spectrum of nicotinic acid has a strong absorption peak at 261 nm, the spectrum of nicotinamide has a strong absorption peak at 258 nm, the spectrum of pyridoxine has a strong absorption peak at 291 nm, the spectrum of pyridoxal has a strong absorption peak at 295 nm, the spectrum of pyridoxamine has a strong absorption peak at 289 nm, the spectrum of folic acid has a strong absorption peak at 284 nm, and the spectrum of vitamin B 12The spectrum of the product has a strong absorption peak at 362nm. Taking into account the simultaneous detection and sensitivity of each component, the detection wavelength of pantothenic acid and biotin is determined to be 205nm; the detection wavelength of L(+)-ascorbic acid and D-isoascorbic acid is determined to be 260nm; the detection wavelength of vitamin B1, vitamin B2, niacin, and niacinamide is determined to be 260nm; the detection wavelength of pyridoxine, pyridoxal, pyridoxamine, and folic acid is determined to be 290nm; and the detection wavelength of vitamin B 12 The detection wavelength is 362nm.

[0085] (5) Determination of chromatographic conditions

[0086] Based on the results of the gradient elution conditions, flow rate, column temperature and wavelength, the optimal chromatographic conditions were determined to be gradient elution condition 6, column temperature of 28°C, and flow rate of 0.5 ml / min, as follows:

[0087] Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, 0.1 mol / L potassium dihydrogen phosphate solution (pH adjusted to 2.7-2.8 with phosphoric acid) was used as mobile phase B, and gradient elution was performed according to the provisions in Table 7; the flow rate was 0.5 ml / min; the column temperature was 28°C for gradient elution; the injection volume was 10 μl; the detection wavelength for pantothenic acid and biotin was 205 nm; the detection wavelength for L(+)-ascorbic acid and D-isoascorbic acid was 260 nm; the detection wavelength for vitamin B1, vitamin B2, niacin, and niacinamide was 260 nm; the detection wavelength for pyridoxine, pyridoxal, pyridoxamine, and folic acid was 290 nm; the detection wavelength for vitamin B 12 The detection wavelength is 362nm.

[0088] Table 7 Gradient elution conditions determined

[0089]

[0090] 3. Preparation of standard stock solution

[0091] Stock solution A: Accurately weigh 33.125 mg of L(+)-ascorbic acid, 28.05 mg of D-isoascorbic acid, 4.98 mg of vitamin B1, 3.76 mg of nicotinic acid, 5.36 mg of nicotinamide, 7.57 mg of pyridoxal, 8.14 mg of pyridoxamine and 5.67 mg of pyridoxine, place in a 25 ml volumetric flask, add 0.1% hydrochloric acid solution to prepare a mixed stock solution containing 132.3675 μg of L(+)-ascorbic acid, 98.3994 μg of D-isoascorbic acid, 229.39392 μg of nicotinic acid, 5347.928 μg of nicotinamide, 98.4032 μg of vitamin B1, 302.4972 μg of pyridoxal, 324.9488 μg of pyridoxamine and 226.1196 μg of pyridoxine per ml.

[0092] Stock solution B: Accurately weigh 5.27 mg of vitamin B2 and place it in a 25 ml volumetric flask. Add 2 ml of hydrochloric acid solution (1+1) to dissolve it, then add 0.1% hydrochloric acid solution to make a stock solution containing 226.1196 μg per ml.

[0093] Stock solution C: accurately weigh 29.3 mg pantothenic acid, 4.34 mg biotin and vitamin B 12 1.38 mg, placed in a 50 ml volumetric flask, dissolved in 50-60 ° C water to make each ml contain 578.968 μg of pantothenic acid, 86.366 μg of biotin, and vitamin B 12 24.5422 μg of stock solution is obtained.

[0094] Stock solution D: Accurately weigh 2.78 mg of folic acid and place it in a 25 ml volumetric flask. Add 0.5% ammonia solution to make a solution containing 105.4176 μg of folic acid per 1 ml.

[0095] 4. Preparation of standard series working solutions

[0096] Accurately pipette 0.1ml, 0.2ml, 0.4ml, 0.6ml, 0.8ml and 1ml of stock solutions A, B, C and D respectively into 10ml volumetric flasks, dilute to the scale with water to obtain a series of mixed standard working solutions, and filter through a 0.45μm aqueous microporous filter membrane for use.

[0097] 5. Preparation of test solution

[0098] Vitamin B1, vitamin B2, niacin, niacinamide, pyridoxal, pyridoxamine and pyridoxine: Take 0.05-0.2g of uniform sample, weigh accurately, put into a 50ml brown volumetric flask, add about 30ml of 0.1% hydrochloric acid solution, ultrasonic (power 500W) for 20min, cool, add 0.1% hydrochloric acid solution to the scale, shake well and centrifuge, take the supernatant and filter it through a 0.45μm microporous filter membrane.

[0099] L(+)-ascorbic acid, D-isoascorbic acid: Take 0.05-0.2g of uniform sample, accurately weigh, place in a 50ml brown volumetric flask, add about 30ml of 20g / L metaphosphoric acid solution, ultrasonic (power 500W) for 20min, cool, add 20g / L metaphosphoric acid solution to the scale, shake well and centrifuge, take the supernatant and filter through a 0.45μm microporous filter membrane to obtain.

[0100] Biotin and B Vitamins 12 : Take 1-5g of uniform sample, weigh accurately, place in a 50ml brown volumetric flask, add about 30ml of 60℃ water, ultrasonic (power 500W) for 20min, cool, add water to the scale, shake and centrifuge, take the supernatant and filter through a 0.45μm microporous membrane.

[0101] Pantothenic acid: Precision measurement of biotin and vitamin B 12 "Put 1 ml of the supernatant in a 10 ml volumetric flask, dilute to the mark with water, and filter the supernatant through a 0.45 μm microporous membrane to obtain the product.

[0102] Folic acid: Take 0.5-1g of uniform sample, weigh accurately, place in a 50ml brown volumetric flask, add about 30ml of 0.5% ammonia solution, ultrasonic (power 500W) for 20min, let cool, add water to the scale, shake well and centrifuge, take the supernatant and filter it through a 0.45μm microporous membrane.

[0103] 6 Methodological Validation

[0104] (1) Specificity inspection

[0105] Take the reagent blank, mixed standard solution and test solution, and perform the analysis according to the chromatographic conditions under "2(5)". The results are shown in Figure 14 to Figure 16 The results showed that the test sample chromatogram had the same chromatographic peak at the corresponding retention time as the reference sample chromatogram, and there was no interference from the negative blank, indicating that the method had good specificity.

[0106] (2) Linear investigation

[0107] Accurately pipette the series of mixed standard working solutions under item "4" and inject them into the liquid chromatograph. Draw a series of standard curves with the concentration of 13 water-soluble vitamins as the horizontal axis and the peak area response value as the vertical axis. The results are shown in Table 8. The results show that the concentration of each component has a good linear relationship with the peak area within the linear range of the above table.

[0108] Table 8 Linear regression equation, linear relationship and linear range of 13 vitamins

[0109] Serial number project Linear regression equation <![CDATA[Coefficient of correlation R 2 > Linear range (μg / ml) 1 Pantothenic acid y=10713.574x+2273.6 1.000 11.6～69.5 2 Biotin y=11457.584x+2977.8 0.999 1.7～10.4 3 <![CDATA[Vitamin B1]]> y=36334.235x-8456.333 0.999 2.0～11.9 4 <![CDATA[Vitamin B2]]> y=64265.342x+766.267 1.000 2.1～12.4 5 L(+)-Ascorbic Acid y=29345.071x-3375.933 0.998 26.5～159.2 6 D-isoascorbic acid y=35495.759x+19719 1.000 19.7～118.1 7 niacin y=89294.518x-4901.533 1.000 4.6～27.6 8 Niacinamide y=32991.246x+1714.267 1.000 10.7～64.2 9 Pyridoxine y=39260.247x-123.400 1.000 2.3～13.6 10 Pyridoxamine y=23983.418x-2378.733 1.000 3.2～19.5 11 Pyridoxal y=38497.546x-5442.000 1.000 3.0～18.1 12 Folic acid y=51846.507x-1472.533 1.000 1.1～6.3 13 <![CDATA[Vitamin B 12 > y=1816.498x-188.267 1.000 0.5～2.9

[0110] (3) Precision inspection

[0111] Accurately pipette the mixed standard working solution under item "4" and repeat the injection 6 times according to the chromatographic conditions under item "2(5)". The injection volume is 10 μl. The peak area of ​​each component is measured and the peak area RSD is calculated. The results are shown in Table 9. The results show that the RSD of the precision of each component is less than 3%, indicating that the precision of the instrument is good.

[0112] Table 9 Summary of precision investigation results of 13 vitamins

[0113] Serial number project Precision RSD (%) 1 Pantothenic acid 0.21 2 Biotin 2.28 3 <![CDATA[Vitamin B1]]> 1.38 4 <![CDATA[Vitamin B2]]> 0.41 5 L(+)-Ascorbic Acid 1.26 6 D-isoascorbic acid 0.36 7 niacin 0.19 8 Niacinamide 1.89 9 Pyridoxine 1.86 10 Pyridoxamine 1.22 11 Pyridoxal 1.58 12 Folic acid 0.18 13 <![CDATA[Vitamin B 12 > 0.71

[0114] (4) Repeatability study

[0115] Take compound vitamins (GlanbiaRE35490 / A) (batch number: FS2304004) and prepare "L(+)-ascorbic acid, vitamin B1, vitamin B2, nicotinamide, pyridoxine", "biotin and vitamin B 12 ”, “pantothenic acid” and “folic acid” test solutions were prepared in parallel for 6 times respectively, and the samples were injected according to the chromatographic conditions under “2(5)” with an injection volume of 10 μl. The peak area of ​​each component was determined, and the content of each component and RSD were calculated. The results are shown in Table 10. The results showed that the same batch of samples was measured 6 times, and the repeatability RSD of each component met the RSD limit requirements specified in the “Guidelines for Validation of Analytical Methods for Drug Quality Standards” of the 2020 edition of the “Chinese Pharmacopoeia”, and the repeatability of this analytical method was good.

[0116] Table 10 Summary of the repeatability test results of various vitamins

[0117]

[0118]

[0119] 7. Sample determination

[0120] Take compound vitamins (Glanbia RE35490 / A) (batch number: FS2304004), multivitamins (Ruipu 221025B-02-20) (batch number: FS2302027); multivitamin mineral tablets (batch number: YF23032405); vitamin B tablets (batch number: YF23032404) samples according to the preparation method of the test solution under "5", prepare the corresponding test solution, and inject the above test solution into the chromatographic conditions under "2(5)" to measure, record the peak area of ​​each component of the chromatogram of the test solution, and obtain the concentration of each water-soluble vitamin in the test solution according to the standard curve drawn under "6(2)". The results are shown in 11.

[0121] Table 11 Summary of test results of each component

[0122]

[0123]

[0124] In summary, the present invention can realize the synthesis of vitamin B1, vitamin B2, niacin, niacinamide, pyridoxine, pyridoxal, pyridoxamine, ... 12, pantothenic acid, biotin, folic acid, L (+) -ascorbic acid, D-isoascorbic acid, a total of 13 kinds of water-soluble vitamins are determined simultaneously, the operation is simple, and the inspection cycle is short. The diode array detector (DAD) can be switched to the maximum absorption wavelength of each component at any time, and the detection method has high sensitivity and is suitable for the detection of vitamins with different addition amounts. At the same time, the present invention uses methanol-potassium dihydrogen phosphate buffer gradient elution to avoid the use of traditional ionic reagents, the chromatographic column is easy to reach equilibrium, the peak shape of each component is good, the separation is good, the instrument and chromatographic column used in the detection method are low in cost, and the popularity is good, and the application is promoted.

[0125] The above is a preferred embodiment of the invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the invention, and these improvements and modifications are also considered to be within the scope of protection.

Claims

1. A method for simultaneously detecting multiple compounds, characterized in that: The following steps are involved: Prepare a mixed standard working solution, the mixed standard working solution includes L (+) -ascorbic acid, D-isoascorbic acid, vitamin B1, niacin, niacinamide, pyridoxal, pyridoxamine, pyridoxine, vitamin B2, pantothenic acid, biotin, vitamin B 12 and folic acid; The mixed standard working solution was detected by a high performance liquid chromatograph-diode array detector, wherein the chromatographic column of the liquid chromatograph was Shimadzu Shim-pack Scepter C18-120, the inner diameter of the chromatographic column was 4.6 mm, the column length was 250 mm, the particle size was 5 μm, acetonitrile was used as mobile phase A, and potassium dihydrogen phosphate solution was used as mobile phase B for gradient elution to obtain a standard curve corresponding to the concentration and peak area of ​​each compound; Taking the test solution, using a high performance liquid chromatograph-diode array detector to detect under the same conditions, obtaining the detection peak area of ​​each compound in the test solution, and obtaining the concentration of each compound in the test solution according to the standard curve; The gradient elution was carried out according to the following procedure: 0~5min, mobile phase A is 0%, mobile phase B is 100%; 5min~15min, mobile phase A from 0→15%, mobile phase B from 100%→85%; 15min~25min, mobile phase A from 15→33%, mobile phase B from 85%→67%; 25min~35min, mobile phase A from 33% to 10%, mobile phase B from 67% to 90%; 35min~40min, mobile phase A from 10%→0%, mobile phase B from 90%→100%; The concentration of the potassium dihydrogen phosphate solution is 0.05 mol / L to 0.15 mol / L; The pH value of the potassium dihydrogen phosphate solution is 2.7-2.8; When performing gradient elution, the flow rate of the mobile phase is 0.5 mL / min~0.6 mL / min; When gradient elution is performed, the column temperature is 28°C~30°C; The detection wavelengths for each compound are as follows: The detection wavelength for pantothenic acid and biotin is 205 nm; The detection wavelength for L(+)-ascorbic acid and D-isoascorbic acid was 260 nm; The detection wavelength of vitamin B1, vitamin B2, niacin and niacinamide is 260nm; The detection wavelength for pyridoxine, pyridoxal, pyridoxamine, and folic acid is 290 nm; Vitamin B 12 The detection wavelength is 362nm; The test solution was prepared by the following method: When testing vitamin B1, vitamin B2, niacin, niacinamide, pyridoxal, pyridoxamine and pyridoxine, take a sample and mix it with a hydrochloric acid solution, and then filter it after ultrasonic treatment; When detecting L(+)-ascorbic acid and D-isoascorbic acid, take a sample and mix it with a metaphosphoric acid solution, and then filter it after ultrasonic treatment; In the detection of pantothenic acid, biotin and vitamin B 12 When the sample is mixed with water, it is filtered after ultrasonic treatment. When testing folic acid, take the sample and mix it with ammonia water, treat it with ultrasound and then filter it; The sample is a vitamin B tablet.

2. The method for simultaneously detecting multiple compounds according to claim 1, characterized in that: The mixed standard working solution was prepared using the following method: L(+)-ascorbic acid, D-isoascorbic acid, vitamin B1, niacin, niacinamide, pyridoxal, pyridoxamine and pyridoxine are mixed with hydrochloric acid solution to prepare a mixed solution A; Mix vitamin B2 with hydrochloric acid solution to prepare mixed solution B; Take pantothenic acid, biotin, vitamin B 12 Mix with water to obtain mixed solution C; Mix folic acid and ammonia water to prepare mixed solution D; The mixed solution A, mixed solution B, mixed solution C and mixed solution D are mixed to obtain the mixed standard working solution.

3. The method for simultaneously detecting multiple compounds according to claim 1, characterized in that: In the ultrasonic treatment, the power is 400W~600W, and the treatment time is 20min~30min.

4. Use of the method for simultaneously detecting multiple compounds as claimed in any one of claims 1 to 3 in (1): (1) Simultaneously detect L(+)-ascorbic acid, D-isoascorbic acid, vitamin B1, niacin, niacinamide, pyridoxal, pyridoxamine, pyridoxine, vitamin B2, pantothenic acid, biotin, and vitamin B 12 The content of 13 compounds in total including vitamin C and folic acid.

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