Vitamin B 12 Determination method

Abstract

This application relates to the technical field of analytical chemistry, specifically to a vitamin B 12 The determination method comprises the following steps: taking a sample containing vitamin B 12 The test sample is added with potassium ferricyanide solution, hydrochloric acid solution and the first solvent to obtain a mixed solution, which is then dispersed at 38°C to 42°C and then kept warm at 100±2°C to obtain a test solution. The test solution is subjected to liquid chromatography analysis to determine the content of vitamin B 12 The determination method has high sensitivity, good selectivity, high safety, easy preparation, simple operation, cost saving and reliable detection results.

Description

Technical Field

[0001] This application relates to the field of analytical chemistry, in particular to a vitamin B 12 determination method. Background Art

[0002] Vitamin B 12 It is an indispensable trace element for the human body. Its main functions include: 1) promoting the development and maturation of red blood cells, keeping the body's hematopoietic function in a normal state, and preventing pernicious anemia; maintaining the health of the nervous system; 2) existing in the form of a coenzyme, which can increase the utilization rate of folic acid and promote the metabolism of carbohydrates, fats and proteins; 3) it has the function of activating amino acids and promoting the biosynthesis of nucleic acids, which can promote protein synthesis. It plays an important role in the growth and development of infants and young children; 4) it metabolizes fatty acids, so that fats, carbohydrates and proteins can be properly used by the body; 5) it eliminates irritability, concentrates attention, enhances memory and sense of balance; 6) it is an indispensable vitamin for the healthy function of the nervous system, and participates in the formation of a lipoprotein in nerve tissue.

[0003] Vitamin B 12 It can be obtained from food on a daily basis, such as fish, eggs, meat and animal offal, but it is not found in plants. Therefore, some special groups such as vegetarians and people with poor absorption and metabolism need to take vitamin B regularly. 12 Supplements; in some areas, vitamin B 12 Food sources are scarce, so people in these areas need to pay attention to vitamin B 12 Therefore, the development of vitamin B 12 The content determination method is of great significance.

[0004] Current determination of vitamin B in food 12 Methods for determining the content include microbiological assay and chromatography. Microbiological assay relies on vitamin B 12 The growth principle of microorganisms is gradually being replaced by chromatography due to its long detection time, lack of selectivity and low accuracy.

[0005] The State Food and Drug Administration's Import Drug Registration Standard Method (Standard No. JX20170332) records a vitamin B 12The HPLC determination method is as follows: the chromatographic conditions and system suitability test conditions are as follows: octadecylsilane bonded silica gel is used as the filler (ACE 3AQ, 150×4.6mm, 3μm), phosphate buffer (13.6g of anhydrous dipotassium hydrogen phosphate is dissolved in 1000ml of water)-acetonitrile (88:12) is used as the mobile phase, the flow rate is 1.0ml / min, the column temperature is 25℃, the injector temperature is 8℃, and the detection wavelength is 550nm; the determination method includes: taking 20 tablets of this product, accurately weighing, grinding, accurately weighing an appropriate amount (equivalent to vitamin B 12 20μg), placed in a 100ml volumetric flask, accurately added 50ml of mobile phase, vigorously shaken for 30 seconds, immediately ultrasonicated for 2min, and shaken from time to time, immediately filtered, accurately measured 100μl of the filtrate, injected into the liquid chromatograph, and recorded the chromatogram; separately took vitamin B 12 An appropriate amount of reference substance was accurately weighed, dissolved in the mobile phase and quantitatively diluted to make a solution containing about 0.5 μg per 1 ml as the reference substance solution. The solution was determined in the same way and calculated by the peak area according to the external standard method.

[0006] Vitamin B 12 It exists in various forms and contains the mineral cobalt. It has vitamin B 12 The active compounds are collectively called "cobalamin". 12 They are methylcobalamin (MeCbl), adenosylcobalamin (AdCb1) and hydroxocobalamin (OHCbl). These forms have been shown in clinical studies to improve the body's vitamin B 12 Cyanocobalamin (CNCbl) can also be converted like OHCbl, but is rarely found in nature. However, for vitamin B 12 For quantitative analysis, CNCbl is very important because it is the most stable B 12 Vitamins to aid in sample preparation.

[0007] There is a literature report that a vitamin B 12 Ultra-high performance liquid chromatography (UPLC) is used to detect the remaining forms of vitamin B in the test sample. 12 The method uses a highly toxic reagent, potassium cyanide, in the preparation of the test sample, and its safety needs to be improved. The national standard law stipulates a similar detection method, which also requires the use of a toxic cyanide reagent as a vitamin B 12 Moreover, the above methods all use solid phase extraction columns or immunoaffinity extraction columns in the detection process, resulting in higher detection costs and possible errors due to the instability of the extraction column.

[0008] In view of this, the technical content of this application is specially proposed. Summary of the Invention

[0009] Based on this, the purpose of this application includes providing a vitamin B 12 The method has high sensitivity, good selectivity, high safety, simple operation, cost saving and reliable detection results.

[0010] Specifically, the vitamin B 12 The determination method includes the following steps:

[0011] Take vitamin B 12 The test sample is mixed with potassium ferrocyanide, hydrochloric acid and the first solvent to prepare a mixed solution.

[0012] The mixed solution is dispersed at 38°C to 42°C to obtain a dispersion.

[0013] The dispersion was kept warm at 100±2°C to prepare a test solution;

[0014] Take the test solution and perform liquid chromatography analysis to determine the vitamin B 12 content;

[0015] The conditions of the liquid chromatography analysis include:

[0016] (1) Mobile phase A is acetonitrile, and mobile phase B comprises: 1) dipotassium hydrogen phosphate, 2) sodium heptanesulfonate, and 3) water, wherein the pH value of mobile phase B is 6.5 to 6.9;

[0017] (2) Gradient elution is used, and the procedure of the gradient elution includes:

[0018] 0-5min, maintain the volume percentage of the mobile phase B at 100%,

[0019] 5min~15min, the volume percentage of the mobile phase B decreases from 100% to 85%,

[0020] 15min~20min, the volume percentage of the mobile phase B decreases from 85% to 80%,

[0021] 20min~35min, the volume percentage of the mobile phase B decreases from 80% to 60%,

[0022] 35min~39min, maintain the volume percentage of the mobile phase B at 60%,

[0023] 39min~40min, the volume percentage of the mobile phase B increases from 60% to 100%,

[0024] 40 min to 50 min, the volume percentage of the mobile phase B is maintained at 100%; and

[0025] (3) The detection wavelengths include 355nm~365nm and 545nm~555nm.

[0026] The above-mentioned determination method has at least the following advantages:

[0027] (1) Traditional vitamin B 12 In the determination method, the quantitative limit concentration is ≥20ng / ml. When the sample concentration is too low, vitamin B cannot be accurately quantified. 12 The concentration of vitamin B 12 The content in nature is generally low, so when using these methods to determine, the sample needs to be enriched and pre-treated, which is complicated and may affect the accuracy of the determination. This application improves the sensitivity of the detection method by establishing appropriate chromatographic conditions without affecting the specificity of the method and the interference of complex matrices. 12 The concentration can be as low as 10 ng / ml, at which point the signal-to-noise ratio (S / N) can reach 10 and the RSD% of the peak area can be less than 10%.

[0028] (2) Traditional vitamin B 12 The assay method cannot effectively distinguish B vitamins in multivitamins 12 This application uses dual detection wavelengths and suitable chromatographic conditions to separate vitamin B from complex multivitamin preparations. 12 The chromatographic peaks are well separated from other vitamin components.

[0029] (3) The test sample of the present application has good stability, which can ensure the sensitivity and accuracy of the detection. At the same time, the preparation of the test sample does not require the use of highly toxic cyaniding reagents such as potassium cyanide and sodium cyanide, and is safe, reliable and easy to implement.

[0030] (4) This application does not require the use of solid phase extraction columns or immunoaffinity extraction columns to pretreat the test sample, which greatly saves costs and reduces errors. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific implementation methods or the description of the prior art. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0032] Figure 1 This is a comparison chart of the chromatographic detection results of Example 1 of the present application, wherein A is the chromatographic detection chart of the blank solvent, B is the chromatographic detection chart of vitamin B12 A is the chromatogram of the reference substance, and C is the chromatogram of the multivitamin tablets used for testing. DETAILED DESCRIPTION

[0033] The present application will be further described below in conjunction with the embodiments, examples and accompanying drawings. It should be understood that these examples are intended only to illustrate the present application and are not intended to limit the scope of the present application. In addition, it should be understood that after reading the content taught in this application, those skilled in the art may make various changes or modifications to the present application, and these equivalent forms also fall within the scope of protection of the claims appended hereto.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0035] the term

[0036] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0037] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the any and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND" and technical solutions connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0038] Herein, "preferred", "better", "better", etc. are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0039] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0040] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0041] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution within the above numerical interval is considered continuous and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0042] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within the ranges of ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, and ±0.1°C are permitted.

[0043] In this application, weight can be mass units known in the chemical industry, such as μg, mg, g, and kg.

[0044] The stable placement mentioned in this application refers to the stable existence of the sample within the detection time limit. For example, the test solution will not affect the test results due to deterioration within 0 to 24 hours after preparation. More specifically, it means that sampling and testing at any time point within 0 to 24 hours after preparation is completed will not affect the accuracy of the test. In some embodiments, the test solutions with different placement times were tested, and the results showed that the test was completed within 0 to 8 hours, the test result was 111%, the test was completed within 12 to 16 hours, the test result was 110%, and the test was completed within 18 to 24 hours, and the test result was 108%, indicating that the test results of the test sample were basically stable after 24 hours. This application has no special requirements for the placement temperature, and it can be placed at room temperature. The room temperature described in this application refers to 0°C to 35°C, preferably 20°C to 35°C. Example temperatures include but are not limited to 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, and 30°C.

[0045] This application relates to a vitamin B 12 The method has high sensitivity, good selectivity, high safety, easy preparation, simple operation, cost saving and reliable detection results.

[0046] In some embodiments, vitamin B 12 The determination method includes the following steps:

[0047] Take vitamin B 12 The test sample is mixed with potassium ferrocyanide, hydrochloric acid and the first solvent to prepare a mixed solution.

[0048] The mixed solution was dispersed at 38°C to 42°C to obtain a dispersion.

[0049] Take the dispersion and keep it at 100±2℃ to prepare the test solution;

[0050] Take the test solution for liquid chromatography analysis to determine the vitamin B 12 content;

[0051] Preferably, the conditions for liquid chromatography analysis include:

[0052] (1) Mobile phase A is acetonitrile, and mobile phase B comprises: 1) dipotassium hydrogen phosphate, 2) sodium heptanesulfonate, and 3) water, and the pH value of mobile phase B is 6.5-6.9;

[0053] (2) Gradient elution is used. The procedure of gradient elution includes:

[0054] 0~5min, maintain the volume percentage of mobile phase B at 100%,

[0055] 5min~15min, the volume percentage of mobile phase B decreased from 100% to 85%,

[0056] 15min~20min, the volume percentage of mobile phase B decreased from 85% to 80%,

[0057] From 20 to 35 minutes, the volume percentage of mobile phase B decreased from 80% to 60%.

[0058] 35min~39min, maintain the volume percentage of mobile phase B at 60%,

[0059] From 39 to 40 minutes, the volume percentage of mobile phase B increased from 60% to 100%.

[0060] 40-50 min, the volume percentage of mobile phase B is maintained at 100%; and

[0061] (3) The detection wavelengths include 355nm~365nm and 545nm~555nm.

[0062] In some embodiments, the injection volume for liquid chromatography analysis is 180 μL to 220 μL, further selected from 190 μL to 210 μL, and preferably 200 μL.

[0063] In some embodiments, the injection volume for liquid chromatography analysis is 200 μL; and / or

[0064] The test product contains vitamin B 12 multivitamin tablets; and / or

[0065] The dispersing step includes: taking the mixed liquid and dispersing it at 80 r / min to 100 r / min.

[0066] In some embodiments, vitamin B 12 The content is 0.4μg / g~2.8μg / g, for example, 0.4μg / g, 0.6μg / g, 0.8μg / g, 1.2μg / g, 1.5μg / g, 1.8μg / g, 2μg / g, 2.2μg / g, 2.4μg / g, 2.6μg / g, etc.

[0067] In some embodiments, the conditions for liquid chromatography analysis further include at least one of the following:

[0068] (1) Liquid chromatography analysis is ultra-high performance liquid chromatography analysis;

[0069] (2) The flow rate of gradient elution is 0.8 mL / min to 1.2 mL / min;

[0070] (3) The column temperature of the liquid chromatography analysis is 25℃~35℃;

[0071] (4) The chromatographic column for liquid chromatography analysis is a C18 column;

[0072] (5) In mobile phase B: the molar concentration of dipotassium hydrogen phosphate in mobile phase B is 40 mmol / L to 60 mmol / L.

[0073] The mass concentration of sodium heptanesulfonate in mobile phase B is 0.08% to 0.12%.

[0074] In some embodiments, the chromatographic column is of Agilent Zorbax SB-C18 model, with specifications of 4.6×250 mm and 3.5 μm.

[0075] In some embodiments, the detection wavelengths are 361 nm and 550 nm.

[0076] In some embodiments, the molar concentration of dipotassium hydrogen phosphate in mobile phase B is 40 mmol / L to 60 mmol / L, and the molar concentration can further be 50 mmol / L to 60 mmol / L;

[0077] The mass concentration of sodium heptane sulfonate is 0.08% to 0.12%;

[0078] Furthermore, the pH value of mobile phase B is 6.5-6.9.

[0079] In some embodiments, mobile phase B contains an appropriate amount of potassium hydroxide for adjusting the pH value.

[0080] In some specific embodiments, the molar concentration of dipotassium hydrogen phosphate in mobile phase B is 50 mmol / L, and the mass concentration of sodium heptanesulfonate is 0.1%.

[0081] In some embodiments, the pH value of mobile phase B is 6.5 to 6.9, further 6.8 to 6.9.

[0082] In some embodiments, the step of preparing the mixed solution comprises:

[0083] Provide an aqueous solution of potassium ferrocyanide, wherein the mass concentration of potassium ferrocyanide is 2.8% to 3.2%.

[0084] Providing an aqueous solution of hydrochloric acid, wherein the concentration of hydrochloric acid is 0.8 mol / L to 1.2 mol / L,

[0085] Providing an aqueous solution of acetonitrile, wherein the volume concentration of acetonitrile is 4% to 6%,

[0086] Mix the test sample, an aqueous solution of potassium cyanide, an aqueous solution of hydrochloric acid and an aqueous solution of acetonitrile to prepare a mixed solution;

[0087] wherein the volume ratio of the potassium ferricyanide solution, the hydrochloric acid solution, and the first solvent is 1:(0.8-1.2):(10-12);

[0088] The mass volume ratio of the test sample and the mixed solution is 0.01g / mL~0.02g / mL.

[0089] In some embodiments, in the step of dispersing the mixed solution, the dispersion time is 20 min to 40 min; and / or

[0090] In the step of keeping the dispersion liquid warm, the keeping time is 0.5h to 2h.

[0091] In some embodiments, the test solution can be stably placed for 0 to 24 hours; further, the temperature at which it can be stably placed is 0°C to 35°C.

[0092] In some embodiments, vitamin B 12 The assay steps include:

[0093] Vitamin B6 was used as a control 12 , add the second solvent to dissolve and prepare a reference solution;

[0094] Take reference solution of different concentrations, measure them according to the conditions of liquid chromatography analysis and record the peak area, and establish a linear equation based on the peak area of ​​different concentrations;

[0095] Substitute the test results of the test sample into the linear equation for calculation.

[0096] In some embodiments, in the step of preparing the reference solution:

[0097] The second solvent comprises acetonitrile and water, the volume concentration of acetonitrile in the second solvent is 4% to 6%, and / or

[0098] The reference solution contains vitamin B 12 The mass volume concentration is 0.03μg / mL~0.05μg / mL.

[0099] In some embodiments, the linear equation is y = 183.93x - 0.4348.

[0100] In some embodiments, the limit of quantification of the assay is 10 ng / mL to 10.2 ng / mL, and the limit of detection is 3.3 ng / mL to 3.5 ng / mL.

[0101] The following are some specific examples.

[0102] For experimental parameters not specified in the following specific examples, reference is made to the guidance given in this application document, and reference may also be made to experimental manuals in the art or other experimental methods known in the art, or to the experimental conditions recommended by the manufacturer.

[0103] The raw materials and reagents involved in the following specific examples can be obtained commercially, or can be prepared by those skilled in the art according to known methods.

[0104] In the following examples, unless otherwise specified, the samples were tested using the following assay method C1:

[0105] 1) Chromatographic conditions

[0106] Chromatographic column: Agilent Zorbax SB-C18, 4.6×250 mm, 3.5 μm,

[0107] Flow rate: 1.0ml / min,

[0108] Column temperature: 25°C,

[0109] Detection wavelength: 361nm and 550nm,

[0110] Injection volume: 200 μl,

[0111] Mobile phase A: pure acetonitrile,

[0112] Mobile phase B: 50 mM potassium hydrogen phosphate solution, pH = 6.8, containing 0.1% sodium heptane sulfonate,

[0113] Gradient elution program:

[0114] 0~5min, maintain the volume percentage of mobile phase B at 100%,

[0115] 5min~15min, the volume percentage of mobile phase B decreased from 100% to 85%,

[0116] 15min~20min, the volume percentage of mobile phase B decreased from 85% to 80%,

[0117] From 20 to 35 minutes, the volume percentage of mobile phase B decreased from 80% to 60%.

[0118] 35min~39min, maintain the volume percentage of mobile phase B at 60%,

[0119] From 39 to 40 minutes, the volume percentage of mobile phase B increased from 60% to 100%.

[0120] From 40 to 50 minutes, the volume percentage of mobile phase B was maintained at 100%;

[0121] 2) Preparation of test samples

[0122] Take an appropriate amount of the test sample and place it in a 50ml volumetric flask. Add 4ml of 3% potassium ferrocyanide and 4ml of 1mol / L hydrochloric acid to soak the sample. Then add 42ml of 5% acetonitrile and weigh the sample. Place the sample on a shaker at 40°C and shake at 90rpm / min for 30min. Then place the sample in a 100°C water bath for 1 hour. Remove the sample and bring it to room temperature. Add 5% acetonitrile to make up for the loss in weight. Filter the solution through a 0.22μm filter membrane and use the filtrate as the test sample solution.

[0123] 3) Preparation of reference substances

[0124] Take vitamin B 12 An appropriate amount of reference substance was dissolved and diluted with 5% acetonitrile to make a 0.04 μg / ml solution as the reference substance solution;

[0125] 4) Determination

[0126] Take the test solution prepared in 2) and the reference solution prepared in 3) respectively, and measure according to the chromatographic conditions in 1), record the peak areas of the reference and test samples in the chromatogram, and calculate the content of vitamin B in the test sample based on the peak area. 12 The advantages of this method are good chromatographic separation performance, no need for other auxiliary extraction and purification steps, stable sample preparation, and no impact on stability after injection after 24 hours at room temperature.

[0127] 1. Validation of the assay method

[0128] Example 1

[0129] Determination of vitamin B1 in the test sample using assay C1 12 The specific preparation method of the test sample is as follows:

[0130] Bayer Pharmaceuticals Multivitamin tablets (tablet weight 1.5g, batch number JS15578) were used as test sample (equivalent to vitamin B 12Containing 2μg, about 0.75g), placed in a 50ml volumetric flask, add 4ml of 3% potassium ferrocyanide, then add 4ml of 1mol / L hydrochloric acid, soak the test sample, add 42ml of 5% acetonitrile, and weigh the weight. Place it on a 40℃ shaker at a frequency of 90rpm / min for 30min, then place it in a 100℃ water bath for 1 hour, take it out and let it cool to room temperature, add 5% acetonitrile to make up for the loss in weight. Take the solution and filter it with a 0.22μm filter membrane, and the filtrate is used as the test sample solution. Perform double comparison of repeatability operations, and the experimental results are as shown in the following table. The RSD% specified value of the biological method test results is less than or equal to 15%, and the maximum and minimum values ​​in the detection process are discarded to artificially reduce the error. This method meets RSD% < 10%.

[0131] Table 1

[0132]

[0133] Example 2

[0134] Determination of vitamin B1 in the test sample using assay C1 12 The specific preparation method of the test sample is as follows:

[0135] Take Beijing Silian Pharmaceutical Multivitamin tablets (tablet weight 0.93g, containing vitamin B 12 Specification 1.2μg, batch number 5170801) test sample (equivalent to vitamin B 12 Containing 2μg, about 0.75g), placed in a 50ml volumetric flask, add 4ml of 3% potassium ferrocyanide, then add 4ml of 1mol / L hydrochloric acid, soak the test sample, add 42ml of 5% acetonitrile, and weigh the weight. Place it on a shaker at 40℃ and shake at a frequency of 90rpm / min for 30min, then place it in a 100℃ water bath for 1 hour, take it out and let it cool to room temperature, add 5% acetonitrile to make up for the lost weight. Take the solution and filter it with a 0.22μm filter membrane, and the filtrate is used as the test solution. It has been tested that this product contains vitamin B 12 The amount is 98% of the labeled amount. After checking with the preparation process data, the test results and the error of the preparation process addition amount are within 5%.

[0136] Example 3

[0137] Determination of vitamin B1 in the test sample using assay C1 12 The specific preparation method of the test sample is as follows:

[0138] Take Hangzhou Sanofi Minsheng Health Pharmaceutical Co., Ltd. Multivitamin tablets (tablet weight 1.15g, containing vitamin B12 specification 2.3μg, batch number H22E009) are used as the test sample (equivalent to vitamin B 12Contains 2μg, about 1.0g), placed in a 50ml volumetric flask, add 4ml of 3% potassium ferrocyanide, then add 4ml of 1mol / L hydrochloric acid, soak the test sample, add 42ml of 5% acetonitrile, and weigh the weight. Place it on a shaker at 40℃ and shake at a frequency of 90rpm / min for 30min, then place it in a 100℃ water bath for 1 hour, take it out and let it cool to room temperature, add 5% acetonitrile to make up for the lost weight. Take the solution and filter it with a 0.22μm filter membrane, and the filtrate is used as the test solution. It has been tested that this product contains vitamin B 12 138% of the labeled amount.

[0139] Example 4

[0140] Determination of vitamin B1 in the test sample using assay C1 12 The specific preparation method of the test sample is as follows:

[0141] Take Beijing Silian Health Technology Co., Ltd. (Tablet weight 0.258g, containing vitamin B 12 Specification 1.3μg, batch number 05200806) test sample (equivalent to vitamin B 12 Contains 2μg, about 0.3969g), placed in a 50ml volumetric flask, add 4ml of 3% potassium ferrocyanide, then add 4ml of 1mol / L hydrochloric acid, soak the test sample, add 42ml of 5% acetonitrile, and weigh the weight. Place it on a shaker at 40℃ and shake at a frequency of 90rpm / min for 30min, then place it in a 100℃ water bath for 1 hour, take it out and let it cool to room temperature, add 5% acetonitrile to make up for the lost weight. Take the solution and filter it with a 0.22μm filter membrane, and the filtrate is used as the test solution. It has been tested that this product contains vitamin B 12 The amount is 120% of the labeled amount. After checking with the preparation process data, the test results show that the error between the preparation process addition amount and the preparation process addition amount is within 5%.

[0142] Example 5

[0143] Determination of vitamin B1 in the test sample using assay C1 12 The specific preparation method of the test sample is as follows:

[0144] Take Beijing Silian Health Technology Co., Ltd. (Tablet weight 1.2g, containing vitamin B 12 Specification 2.4μg, batch number 05220504) test sample (equivalent to vitamin B 12Contains 2μg, about 1g), placed in a 50ml volumetric flask, add 4ml of 3% potassium ferrocyanide, then add 4ml of 1mol / L hydrochloric acid, soak the test sample, add 42ml of 5% acetonitrile, and weigh the weight. Place on a 40℃ shaker at a frequency of 90rpm / min for 30min, then place in a 100℃ water bath for 1 hour, take out and let it cool to room temperature, add 5% acetonitrile to make up for the lost weight. Take the solution and filter it with a 0.22μm filter membrane, and the filtrate is used as the test solution. It has been tested that this product contains vitamin B 12 The amount is 129% of the labeled amount. After checking with the preparation process data, the test results show that the error between the preparation process addition amount and the preparation process addition amount is within 5%.

[0145] Example 6

[0146] Determination of vitamin B1 in the test sample using assay C1 12 The specific preparation method of the test sample is as follows:

[0147] Take Beijing Silian Health Technology Co., Ltd. (Tablet weight 1.03g, containing vitamin B 12 Specification 0.9μg, batch number 05210102) test sample (equivalent to vitamin B 12 Contains 2μg, about 2.2g), placed in a 50ml volumetric flask, add 4ml of 3% potassium ferrocyanide, then add 4ml of 1mol / L hydrochloric acid, soak the test sample, add 42ml of 5% acetonitrile, and weigh the weight. Place it on a shaker at 40℃ and shake at a frequency of 90rpm / min for 30min, then place it in a 100℃ water bath for 1 hour, take it out and let it cool to room temperature, add 5% acetonitrile to make up for the lost weight. Take the solution and filter it with a 0.22μm filter membrane, and the filtrate is used as the test solution. It has been tested that this product contains vitamin B 12 The amount is 152% of the labeled amount. After checking with the preparation process data, the test results show that the error between the preparation process addition amount and the preparation process addition amount is within 5%.

[0148] 2. Chromatographic method validation

[0149] The test samples in Examples 7 to 14 were selected from the Elevit multivitamin and mineral tablets produced by Bayer, with batch number JS15578 and a specification of 1.5 g.

[0150] Example 7

[0151] The determination was carried out using a method substantially the same as that of method C1, except that the chromatographic analysis conditions were as follows: mobile phase B was a 40 mM solution of dipotassium hydrogen phosphate at pH 6.8 containing 0.09% sodium heptanesulfonate; the test results were as follows Figure 1 , Figure 1 A is the blank group, B is the reference substance, and C is the test substance. Figure 1 It can be seen that the test sample contains vitamin B 12 The amount is equivalent to 112% of the labeled amount.

[0152] Example 8

[0153] The determination was performed using a method essentially the same as that of method C1, except that the chromatographic analysis conditions were as follows: mobile phase B was a 60 mM solution of dipotassium hydrogen phosphate at pH 6.8 containing 0.12% sodium heptanesulfonate. 12 The amount is equivalent to 108% of the labeled amount.

[0154] Example 9

[0155] The determination was carried out using a method that was essentially the same as that of method C1, with the following differences: the column temperature was 30°C; the content of vitamin B in the test sample was detected. 12 The amount is equivalent to 109% of the labeled amount.

[0156] Example 10

[0157] The determination was carried out using a method that was essentially the same as that of method C1, with the following differences: the column temperature was 35°C; the content of vitamin B in the test sample was detected. 12 The amount is equivalent to 105% of the labeled amount.

[0158] Example 11

[0159] The determination was carried out using a method that was essentially the same as that of method C1, except that the chromatographic analysis conditions were as follows: mobile phase B was a 50 mM solution of dipotassium hydrogen phosphate at pH 6.6 containing 0.1% sodium heptane sulfonate; the sample was tested for vitamin B 12 The amount is equivalent to 108% of the labeled amount.

[0160] Example 12

[0161] The determination was carried out using a method essentially the same as that of method C1, except that the chromatographic analysis conditions were as follows: mobile phase B was a 50 mM solution of dipotassium hydrogen phosphate at pH 7.0 containing 0.1% sodium heptane sulfonate; the content of vitamin B in the test sample was determined. 12 The amount is equivalent to 113% of the labeled amount.

[0162] Example 13

[0163] The determination was carried out using a method that was essentially the same as that of method C1, with the difference that the flow rate in the chromatographic analysis was 1.05 ml / min and the content of vitamin B in the test sample was detected. 12 The amount is equivalent to 108% of the labeled amount.

[0164] Example 14

[0165] The determination was carried out using a method that was essentially the same as that of method C1, with the difference that the flow rate in the chromatographic analysis was 0.95 ml / min and the content of vitamin B in the test sample was detected. 12 The amount is equivalent to 107% of the labeled amount.

[0166] Table 2 shows the vitamin B under different chromatographic conditions of Examples 1 to 8. 12 Content test results.

[0167] Table 2

[0168]

[0169] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0170] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the description in this specification.

[0171] The embodiments described above only express several implementation methods of the present application, but they should not be understood as limiting the scope of the patent application. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent in this application shall be based on the attached claims, and the description and drawings may be used to explain the contents of the claims.

Claims

1. A method for determining vitamin B in multivitamins 12 The method for determining the content of is characterized in that, The steps include: A test sample, an aqueous solution of potassium ferricyanide, an aqueous solution of hydrochloric acid, and an aqueous solution of acetonitrile are mixed to prepare a mixed solution; the test sample is selected from Bayer Pharmaceuticals' Aleve Multivitamin Tablets, Sanofi Minsheng Health Pharmaceuticals' 21 Gold Vita Multivitamin Tablets, Silian Pharmaceuticals' Kingsleyan Multivitamin Tablets, or a multivitamin mineral tablet; The potassium ferrocyanide aqueous solution contains potassium ferrocyanide at a mass concentration of 2.8% to 3.2%, the hydrochloric acid concentration in the hydrochloric acid aqueous solution is 0.8 mol / L to 1.2 mol / L, the acetonitrile concentration in the acetonitrile aqueous solution is 4% to 6%, and the volume ratio of the potassium ferrocyanide aqueous solution, the hydrochloric acid aqueous solution, and the acetonitrile aqueous solution is 1:0.8-1.2:10-12; the mass-to-volume ratio of the test sample to the mixed solution is 0.01 g / mL to 0.02 g / mL; The mixed solution was dispersed at 38°C to 42°C and 80r / min to 100r / min for 20min to 40min to prepare a dispersion solution, and the dispersion solution was kept at 100±2°C for 0.5h to 2h to prepare a test solution; Take the test solution and perform ultra-high performance liquid chromatography to determine the content of vitamin B 12 content; The conditions for the ultra-high performance liquid chromatography analysis include: mobile phase A is acetonitrile, mobile phase B is composed of dipotassium hydrogen phosphate, sodium heptane sulfonate and water, the molar concentration of dipotassium hydrogen phosphate in the mobile phase B is 40 mmol / L to 60 mmol / L, the mass concentration of sodium heptane sulfonate in the mobile phase B is 0.08% to 0.12%, and the pH value of the mobile phase B is 6.5 to 6.9; gradient elution is used, and the gradient elution procedure includes: 0 to 5 minutes, maintaining the volume percentage of the mobile phase B at 100%, 5 minutes to 15 minutes, the volume percentage of the mobile phase B decreases from 100% to 85%, and 15 minutes to 20 minutes. n, the volume percentage of the mobile phase B decreases from 85% to 80% in 20 min to 35 min, the volume percentage of the mobile phase B decreases from 80% to 60% in 35 min to 39 min, and the volume percentage of the mobile phase B is maintained at 60% in 39 min to 40 min, the volume percentage of the mobile phase B increases from 60% to 100% in 40 min to 50 min, and the volume percentage of the mobile phase B is maintained at 100%; the flow rate of the gradient elution is 0.8 mL / min to 1.2 mL / min; the detection wavelengths include 355 nm to 365 nm and 545 nm to 555 nm; the chromatographic column is an Agilent Zorbax SB-C18 chromatographic column, and the column temperature is 25°C to 35°C; the injection volume is 200 μL.

2. The method for determining vitamin B1 in vitamin B complex according to claim 1 12 The method for determining the content of is characterized in that, The determination of vitamin B 12 The assay steps include: Vitamin B6 was used as a control 12 , add the second solvent to dissolve and prepare a reference solution; Taking the reference solution of different concentrations, measuring them according to the conditions of the liquid chromatography analysis and recording the peak areas, and establishing a linear equation based on the peak areas of different concentrations; Substitute the test results of the test sample into the linear equation for calculation.

3. The method for determining vitamin B in vitamin complex according to claim 1. 12 The method for determining the content of is characterized in that, The stable placement time of the test solution is 0 to 24 hours; the stable placement temperature is 0° C. to 35° C.

4. The method for determining vitamin B1 in vitamin B complex according to claim 2 12 The method for determining the content of is characterized in that, In the preparation steps of the reference solution: The second solvent comprises acetonitrile and water, the volume concentration of the acetonitrile in the second solvent is 4% to 6%, and / or The reference solution contains the vitamin B 12 The mass volume concentration is 0.03μg / mL~0.05μg / mL.

5. The method for determining vitamin B in multivitamins according to any one of claims 1 to 4. 12 The method for determining the content of is characterized in that, The limit of quantification of the assay is 10 ng / mL to 10.2 ng / mL, and the limit of detection is 3.3 ng / mL to 3.5 ng / mL.

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