A method for simultaneously determining the contents of multiple components in oral five-dimensional lysine hydrochloride glucose

Abstract

The invention discloses a method for simultaneously determining the contents of multiple components in oral five-dimensional lysine hydrochloride glucose, and the determination method adopts HPLC-multi-wavelength method to simultaneously determine the contents of 8 components in oral five-dimensional lysine hydrochloride glucose, including chromatographic conditions, preparation of reference solution, preparation and determination of test solution and other steps. The method of the invention can accurately and effectively determine the contents of 8 components of lysine hydrochloride, calcium pantothenate, vitamin B1, vitamin B2, nicotinamide, vanillin, vitamin B6 and 10-hydroxy-2-decene acid in oral five-dimensional lysine glucose at the same time, the method is simple, accurate, the result is reliable, and the practicability is high, which can provide a basis for comprehensive and effective evaluation and control of the quality of oral five-dimensional lysine hydrochloride glucose, make up for the defects of the current quality standard of oral five-dimensional lysine glucose, and is conducive to quality control of the product to ensure clinical efficacy.

Description

Technical Field

[0001] The invention relates to the technical field of medical detection, and in particular to a method for simultaneously determining the contents of multiple components in oral five-dimensional lysine hydrochloride glucose by using an HPLC-multi-wavelength method. Background Art

[0002] Oral five-dimensional lysine hydrochloride glucose is included in the fifth volume of "Chemical Drug Local Standards Upgraded to National Standards". The main ingredients are: glucose, lysine hydrochloride, vitamin B1, vitamin B2, vitamin B6, niacinamide, calcium pantothenate, sucrose; according to the collected information, the excipients added by various companies are: full-fat milk powder, vanillin, sodium carboxymethylcellulose, lemon yellow, royal jelly, wetting agent (75% ethanol). Oral five-dimensional lysine hydrochloride glucose is suitable for malnutrition caused by chronic diseases such as anemia and hepatitis: glucose is one of the main sources of calories for the human body, used to supplement calories and treat hypoglycemia; lysine hydrochloride maintains body nitrogen balance, promotes protein synthesis and metabolism, and promotes human growth and intellectual development; vitamin B1 is a coenzyme necessary for carbohydrate metabolism, which can maintain the normal function of the heart, nervous and digestive systems; vitamin B2 is an important coenzyme for tissue respiration, which is related to maintaining the integrity of red blood cells; vitamin B6 acts as a coenzyme on various metabolic functions of proteins, carbohydrates and lipids.

[0003] In addition, calcium pantothenate is a component of coenzyme A in the human body and organisms, and participates in the metabolism of carbohydrates, fats, and proteins. If it is insufficient, it will cause metabolic disorders. Nicotinamide is closely related to protein metabolism and children's intellectual development, and plays an important role in maintaining the normal functions of the skin and digestive organs. 10-Hydroxy-2-decene acid (10-HDA) is an important main component of royal jelly and is often used as a quality indicator of royal jelly. Vanillin is an important synthetic fragrance, often used as a fixative and flavor enhancer. Since the aldehyde group of vanillin is easily oxidized and has poor stability, the content analysis of vanillin is of practical significance. For the above reasons, it is of great significance to control the content of each component. However, the current quality standard of oral five-dimensional lysine hydrochloride glucose does not control the above components, lacks key detection items, cannot effectively control the content of each active ingredient of this product, and cannot fully reflect the quality of oral five-dimensional lysine hydrochloride glucose, resulting in serious defects in product quality control. At present, there are no relevant research reports on the method for determining the content of multiple components in five-dimensional lysine hydrochloride glucose. Therefore, if a method is developed that can simultaneously determine the content of multiple components in oral five-dimensional lysine hydrochloride glucose, the quality of oral five-dimensional lysine hydrochloride glucose can be evaluated more comprehensively and accurately, thereby effectively controlling its quality and ensuring clinical efficacy. Summary of the invention

[0004] The purpose of the present invention is to provide a method for simultaneously determining the contents of multiple components in oral five-dimensional lysine hydrochloride glucose in view of the deficiencies of the prior art. The determination method uses HPLC-multi-wavelength method to simultaneously determine the contents of 8 components in oral five-dimensional lysine hydrochloride glucose. The method is simple, accurate, and the results are reliable, which can provide a basis for comprehensive and effective evaluation and control of the quality of oral five-dimensional lysine hydrochloride glucose.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] A method for simultaneously determining the contents of multiple components in oral five-dimensional lysine hydrochloride glucose, using HPLC-multi-wavelength method to simultaneously determine the contents of 8 components including lysine hydrochloride, calcium pantothenate, vitamin B1, vitamin B2, nicotinamide, vanillin, vitamin B6 and 10-hydroxy-2-decenoic acid in oral five-dimensional lysine hydrochloride glucose, specifically comprising the following steps:

[0007] (1) Chromatographic conditions: Alltima-C18, 250 mm × 4.6 mm, 5 μm column, 5 mmol / L sodium heptane sulfonate solution adjusted to pH 2.5 with 20% phosphoric acid solution as mobile phase A, acetonitrile as mobile phase B, gradient elution, flow rate of 1 ml / min, column temperature of 40 °C, detection wavelengths of 200 nm (lysine hydrochloride, calcium pantothenate), 211 nm (10-HDA), 261 nm (vitamin B1, vitamin B2, nicotinamide, vanillin), 290 nm (vitamin B6), injection volume of 20 μL;

[0008] (2) Preparation of reference solution:

[0009] ① Stock solutions of each reference substance (1): Accurately weigh 52.26 mg of lysine hydrochloride reference substance, 10.56 mg of nicotinamide reference substance, 18.93 mg of vanillin reference substance, and 17.82 mg of 10-HDA reference substance into a 50 ml volumetric flask, add about 30 ml of 0.1% phosphoric acid solution, sonicate and shake to dissolve, and set aside;

[0010] ②Reference substance stock solution (2): Accurately weigh 10.70 mg of vitamin B1 reference substance, 13.03 mg of vitamin B6 reference substance, and 10.11 mg of calcium pantothenate reference substance, respectively, and place them in 10 ml volumetric flasks, add 0.1% phosphoric acid solution to dissolve and dilute to scale, shake well, and obtain; place 10.89 mg of vitamin B2 reference substance in a 20 ml volumetric flask, add 5 ml of water and 1 ml of phosphoric acid, heat in a water bath and shake to dissolve, take out, cool to room temperature, dilute to scale with water, shake well, and obtain;

[0011] ③ Mixed reference substance stock solution: Accurately measure 4, 1, 3, and 2 ml of vitamin B1, vitamin B6, calcium pantothenate, and vitamin B2 reference substance stock solutions respectively and place them in a 50 ml volumetric bottle of reference substance stock solution (1) under item ①. Dilute to the mark with 0.1% phosphoric acid solution and shake well to obtain;

[0012] ④ Preparation of mixed reference solution: Accurately measure 2 ml of the mixed reference stock solution under item ③ and place it in a 10 ml volumetric flask, dilute to the mark with 0.1% phosphoric acid solution, shake well, and the solution is ready;

[0013] (3) Preparation of test solution: Take the contents of 10 packets of this product, mix and grind, weigh 2 g, accurately weigh, place in a 10 ml volumetric flask, add 6 ml of 0.1% phosphoric acid solution, sonicate and shake to dissolve, cool to room temperature, dilute to scale with 0.1% phosphoric acid solution, shake well, filter, and take the filtrate to obtain; if the test solution contains whole milk powder, it is necessary to first centrifuge at 9000 rpm and 0°C for 10 min, take the supernatant and filter it with a filter membrane while it is cold, and take the filtrate to obtain;

[0014] (4) Determination: Accurately pipette 20 μL of the mixed reference solution and test solution respectively, inject into the HPLC, and perform determination under the above-mentioned chromatographic conditions. Record the chromatogram and peak area value. Calculate the contents of 8 components in the sample, including lysine hydrochloride, calcium pantothenate, vitamin B1, vitamin B2, niacinamide, vanillin, vitamin B6 and 10-hydroxy-2-decenoic acid, based on the peak area.

[0015] The above-mentioned method for simultaneously determining the contents of multiple components in oral five-dimensional lysine hydrochloride glucose, in the chromatographic conditions of step (1), the gradient elution program for gradient elution is: 0-45min, the volume percentage of mobile phase A is 95%-77%, and the volume percentage of mobile phase B is 5%-23%; 45-57min, the volume percentage of mobile phase A is 77%, and the volume percentage of mobile phase B is 23%; 57-58min, the volume percentage of mobile phase A is 77%-95%, and the volume percentage of mobile phase B is 23%-5%; 58-65min, the volume percentage of mobile phase A is 695%, and the volume percentage of mobile phase B is 5%.

[0016] The beneficial effects of the present invention are:

[0017] The present invention establishes a method for simultaneously determining the contents of 8 components in oral five-dimensional lysine hydrochloride glucose by HPLC-multi-wavelength method. When the method is used for detection, each auxiliary material has no interference with the 8 components to be tested, the separation between each substance can reach baseline separation, and each substance is linear in its own linear range (r is 0.9999), with good precision (RSD is 0.04% to 0.30%), good reproducibility (RSD is 0.24% to 1.89%), high sample recovery rate, and the test solution is stable within 24 hours. After inspection, the established method is accurate and has good specificity.

[0018] The method for simultaneously determining the content of multiple components in oral five-dimensional lysine hydrochloride glucose provided by the present invention can accurately and effectively simultaneously determine the contents of 8 components of lysine hydrochloride, calcium pantothenate, vitamin B1, vitamin B2, nicotinamide, vanillin, vitamin B6 and 10-hydroxy-2-decene acid in oral five-dimensional lysine glucose, the method is simple, easy to operate, accurate, reliable, practical, and stable, and can provide a basis for comprehensive and effective evaluation and control of the quality of oral five-dimensional lysine hydrochloride glucose, effectively make up for the defects of the current quality standard of oral five-dimensional lysine glucose, and can be effectively used for the quality control of the product. The method of the present invention can be used to more comprehensively and accurately evaluate the quality of oral five-dimensional lysine hydrochloride glucose, so as to facilitate quality control thereof and ensure clinical efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Blank excipient solution spectrum (200nm), in the figure, 1, calcium pantothenate, 2, niacinamide, 3, vitamin B6, 4, lysine hydrochloride, 5, vitamin B2, 6, vanillin, 7, vitamin B1, 8, 10-HDA; A, mixed reference solution, B, whole milk powder, C, glucose, D, sucrose, E, royal jelly, F, sodium carboxymethyl cellulose, G, ethanol, H, tartrazine;

[0020] Figure 2 Spectra of reference solution at different wavelengths, in the figure, 1, calcium pantothenate, 2, nicotinamide, 3, vitamin B6, 4, lysine hydrochloride, 5, vitamin B2, 6, vanillin, 7, vitamin B1, 8, 10-HDA; A, 200 nm, B.211 nm, C, 261 nm, D, 290 nm;

[0021] Figure 3Spectra of mixed reference solution and test solution (200nm), in which: 1. Calcium pantothenate, 2. Nicotinamide, 3. Vitamin B6, 4. Lysine hydrochloride, 5. Vitamin B2, 6. Vanillin, 7. Vitamin B1, 8. 10-HDA; A. mixed reference solution, B. Test solution from manufacturer 1, C. Test solution from manufacturer 2, D. Test solution from manufacturer 3, E. Test solution from manufacturer 4. DETAILED DESCRIPTION

[0022] Example 1

[0023] 1 Materials

[0024] 1.1 Main instruments

[0025] LC-20AT HPLC (Shimadzu, Japan), XS205 electronic analytical balance (Mettler Toledo, Switzerland); 9983 ultrasonic instrument (Ningbo Haishu Kesheng Ultrasonic Equipment Co., Ltd.), UNIVERSAL320 R centrifuge (Hettich Technology Co., Ltd., Germany).

[0026] 1.2 Main drugs and reagents

[0027] Nicotinamide reference substance (batch number: 100115-202005, calculated as C6H6N2O, content is 99.9%), calcium pantothenate reference substance (batch number: 100369-201905, calculated as C 17 H 20 N4O6, the content is 96.8%), vitamin B6 reference substance (batch number: 100116-201906, according to C8H 11 NO3·HCl, content is 99.9%), vitamin B1 reference substance (batch number: 100390-201806, according to C 12 H 17 ClN4OS·HCl, content is 97.9%), vitamin B2 reference substance (batch number: 100369-201905, according to C 17 H 20 N4O6, the content is 98.2%), lysine hydrochloride reference substance (batch number: 140673-202010, according to C6H 14 N2O2·HCl, the content is 99.9%), vanillin reference substance (Batch No.: 100491-202203, C8H8O3, the content is 99.3%) were purchased from China Food and Drug Inspection Institute; 10-hydroxy-2-decenoic acid reference substance (Shanghai Anpu Experiment Technology Co., Ltd., Lot: 2105646, 1 content is 98.7%); acetonitrile (chromatographic grade, Merck), MILLIOPE water, and other reagents were of analytical grade.

[0028] The samples come from the national random evaluation work in 2022, 6 batches from each of the 4 companies, totaling 24 batches. The prescription table of each company is shown in Table 1. According to the document "National Standard of the People's Republic of China Royal Jelly GB9697-2008", the qualified royal jelly contains 10-HDA ≥ 1.4%, and the superior royal jelly ≥ 1.8%, so the prescription amount of 10-HDA is calculated as 0.28g / 1000g.

[0029]

[0030] 2 Methods and Results

[0031] 2.1 Chromatographic conditions

[0032] Alltima-C18 (250 mm ×4.6 mm, 5 μm) was used as the chromatographic column, 5 mmol / L sodium heptane sulfonate solution (adjusted to pH 2.5 with 20% phosphoric acid solution) was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed according to the table below (elution program is shown in Table 2). The flow rate was 1 ml / min, the column temperature was 40 °C, the detection wavelengths were 200 nm (lysine hydrochloride, calcium pantothenate), 211 nm (10-HDA), 261 nm (vitamin B1, vitamin B2, niacinamide, vanillin), and 290 nm (vitamin B6), and the injection volume was 20 μL.

[0033]

[0034] 2.2 Solution preparation

[0035] 2.2.1 Stock solutions of various reference substances (1)

[0036] Accurately weigh 52.26 mg of lysine hydrochloride reference substance, 10.56 mg of nicotinamide reference substance, 18.93 mg of vanillin reference substance, and 17.82 mg of 10-HDA reference substance and place them in the same 50 ml volumetric flask. Add about 30 ml of 0.1% phosphoric acid solution, sonicate and shake at the same time to dissolve, and set aside.

[0037] 2.2.2 Stock solutions of each reference substance (2)

[0038] Accurately weigh 10.70 mg of vitamin B1 reference substance, 13.03 mg of vitamin B6 reference substance, and 10.11 mg of calcium pantothenate reference substance, and place them in 10 ml volumetric flasks respectively. Add 0.1% phosphoric acid solution to dissolve and dilute to the scale, shake well, and serve. Place 10.89 mg of vitamin B2 reference substance in a 20 ml volumetric flask, add 5 ml of water and 1 ml of phosphoric acid, heat and shake in a water bath to dissolve, take out, cool to room temperature, dilute to the scale with water, shake well, and serve.

[0039] 2.2.3 Mixed reference stock solution

[0040] Accurately measure 4, 1, 3, and 2 ml of the reference stock solutions of vitamin B1, vitamin B6, calcium pantothenate, and vitamin B2, respectively, and place them in a 50 ml volumetric flask of “reference stock solution (1) under 2.2.1”. Dilute to the mark with 0.1% phosphoric acid solution and shake well.

[0041] 2.2.4 Preparation of mixed reference solution

[0042] Accurately measure 2 ml of the mixed reference stock solution under "2.2.3" and place it in a 10 ml volumetric flask. Dilute to the mark with 0.1% phosphoric acid solution and shake well.

[0043] 2.2.5 Test solution

[0044] Take the contents of 10 packets of this product, mix them, grind them into powder, weigh about 2g, weigh them accurately, put them in a 10ml volumetric bottle, add about 6ml of 0.1% phosphoric acid solution, ultrasonicate and shake to dissolve, cool to room temperature, dilute to scale with 0.1% phosphoric acid solution, shake well, filter, and take the filtrate. (If the test solution contains whole milk powder, it is necessary to first centrifuge at 9000rpm and 0℃ for 10 minutes, take the supernatant and filter it with a filter membrane while it is cold, and take the filtrate).

[0045] 2.3 Specificity experiment

[0046] The solutions of each component were prepared according to the prescriptions provided by each company. The results showed that the excipients had no interference with the 8 components to be tested, and the separation between the substances could reach baseline separation. The chromatograms are shown in Figures 1 to 3 (Since all substances have absorption at 200nm, it is relatively clear. Figure 1 , 3 Using 200 nm as a representative).

[0047] 2.4 Linear relationship experiment

[0048] Accurately measure 0.2, 0.5, 0.7, 1.0, 1.6, 2.0, 3.0, and 4.0 ml of the mixed reference stock solution under item "2.2.3" and place them in 10 ml volumetric flasks respectively. Dilute to the scale with 0.1% phosphoric acid solution, shake well, and use them as standard curve solutions 1 to 8 for determination. The results show that the linear relationship between each component is established. The results are shown in Table 3.

[0049]

[0050] 2.5 Precision experiment

[0051] 2.5.1 Instrument precision: Take the standard curve solution 6 under "2.4 Linear relationship experiment" and continuously inject it according to the chromatographic conditions under "2.1" and record the peak area. The results are shown in Table 4. The results show that the instrument precision is good.

[0052]

[0053] 2.5.2 Repeatability: Take one batch of samples from each of the enterprises 2 and 3 and prepare the test solution for content determination according to the method in “2.2.5”. Prepare 6 samples in parallel and then inject and measure according to the chromatographic conditions in “2.1”. Record the peak area. The results have good repeatability. The results are shown in Table 5.

[0054]

[0055] 2.6 Recovery rate experiment

[0056] Prepare mixed blank excipients without the ingredients to be tested according to the prescriptions of each company, weigh 2g, and place them in 10ml volumetric flasks, accurately add 2ml of the mixed reference stock solution under "2.2.3", prepare the recovery test solution according to the test solution under "2.2.5" and measure the results. The results are shown in Table 6, which show that the recovery rate is good.

[0057]

[0058] 2.7 Solution stability test

[0059] The recovery rate of the test solution was measured at 0, 4, 8, 16 and 14 hours respectively. The results showed that each substance was stable within 24 hours. See Table 7.

[0060]

[0061] 2.8 Detection limit and quantification limit experiments

[0062] The standard curve solution 1 under “2.4 Linear relationship experiment” was diluted and injected according to the chromatographic conditions under “2.1”. The detection limit and quantification limit were calculated according to the signal-to-noise ratio of 1:3 and 1:10 respectively. The results are shown in Table 8.

[0063]

[0064] 2.9 Sample determination

[0065] A total of 12 batches of samples from 4 manufacturers were taken and measured according to the method of the present invention, and the labeled amount percentage of each component was calculated. The results are shown in Table 9.

[0066]

[0067] 3. Discussion

[0068] 3.1 Selection and optimization of chromatographic conditions

[0069] At the beginning of the experiment, the pH value of the mobile phase without adjustment (pH value 7.2) and the pH value of the mobile phase adjusted to 4.2, 3.1, and 2.5 were compared, and it was found that the peak shape and separation effect of each substance were affected by the pH value: at pH 7.2, each peak was sail-shaped, and the retention time of calcium pantothenate was only 2.8 minutes; in the process of pH value from 4.2→3.1→2.5, the symmetry of each peak became better, the retention time of calcium pantothenate was prolonged, and the order of vitamin B6 and vitamin B2 peaks changed (vitamin B6 was in front at pH 4.2 and 2.5, and vitamin B2 was in front at pH 3.1); when the pH was 2.5, the peak shape and separation degree of each peak were optimal, so the mobile phase pH value of 2.5 was finally selected. After comparing a variety of different gradient elution methods, the gradient used in the present invention is the best, and the chromatographic conditions used in the present invention are established.

[0070] 3.2 Wavelength selection

[0071] After using a diode array detector to collect the chromatogram of the mixed reference solution, the UV spectrum of each substance was extracted. The results showed that lysine hydrochloride and calcium pantothenate had no maximum absorption, and the maximum absorption wavelengths of niacinamide, vitamin B6, vitamin B2, vanillin, vitamin B1, and 10-hydroxy-2-decenoic acid were 261, 290, 267, 279, 246, and 212 nm, respectively. Since the concentration and response of each substance varied greatly, in order to better determine the eight substances at the same time, the measurement wavelength under "2.1 Chromatographic Conditions" was used after comprehensive consideration.

[0072] 3.3 The content of multiple ingredients in this product is low

[0073] Among the measurement results of 24 batches of samples, niacinamide and vitamin B2 contents were relatively high, followed by vitamin B1; the contents of lysine hydrochloride, calcium pantothenate, vitamin B6, vanillin, and 10-hydroxy-2-decenoic acid were relatively poor, all of which were lower than 70% of the labeled amount. Among them, the content of lysine hydrochloride was seriously low, and there was a certain correlation between the batch number and the content: the content of products with old batches was generally lower than that of products with new batches, indicating that there are serious problems with the product quality of this product and it needs urgent attention.

[0074] 3.4 Analysis of the causes of low content

[0075] The prescription of this product is complex, containing a large amount of reducing sugar glucose and amino compounds such as lysine hydrochloride, calcium pantothenate, and nicotinamide. According to existing literature, glucose can undergo Maillard reaction with amino compounds under hot and humid conditions, resulting in a decrease in the content of such compounds and the generation of a large amount of brown substances; calcium pantothenate has high hygroscopicity and poor chemical stability, and is sensitive to water, heat, acid, and alkali. When mixed with other acidic vitamins such as ascorbic acid, thiamine, and pyridoxine hydrochloride, it will cause calcium pantothenate to degrade and lose its activity; vanillin has poor stability due to the easy oxidation of the aldehyde group of vanillin. The production process of this product is wet granulation and hot air drying, which provide conditions for the Maillard reaction. Combined with the results of accelerated stability and raw material compatibility research, it can be basically determined that the reason for the decrease in the content of this product is that the ingredients undergo Maillard reaction with each other during production and storage, resulting in a decrease in content.

[0076] In addition, the current quality standard of this product lacks key testing items and cannot effectively control the content of each active ingredient of this product. The manufacturer also did not pay attention to the content of other main ingredients in the product except glucose, and has been using this standard for inspection, resulting in serious defects in product quality control.

[0077] 3.4 Summary

[0078] In summary, the HPLC-DAD method established in the present invention can effectively and simultaneously determine the eight components in the oral five-dimensional lysine glucose granules. After inspection, the contents of multiple components are low. The established method is accurate and has good specificity, which effectively makes up for the defects of the current quality standards and can be effectively used for the quality control of the product.

[0079] Example 2

[0080] A method for simultaneously determining the contents of multiple components in oral five-dimensional lysine hydrochloride glucose, using HPLC-multi-wavelength method to simultaneously determine the contents of 8 components including lysine hydrochloride, calcium pantothenate, vitamin B1, vitamin B2, nicotinamide, vanillin, vitamin B6 and 10-hydroxy-2-decenoic acid in oral five-dimensional lysine hydrochloride glucose, specifically comprising the following steps:

[0081] (1) Chromatographic conditions: Alltima-C18 (250 mm × 4.6 mm, 5 μm) was used as the chromatographic column, 5 mmol / L sodium heptane sulfonate solution (adjusted to pH 2.5 with 20% phosphoric acid solution) was used as the mobile phase A, and acetonitrile was used as the mobile phase B. Gradient elution was performed. The gradient elution program was as follows: 0-45 min, the volume percentage of mobile phase A was 95%-77%, and the volume percentage of mobile phase B was 5%-23%; 45-57 min, the volume percentage of mobile phase A was 77%, and the volume percentage of mobile phase B was 23%; 57-58 min, the volume percentage of mobile phase A was 100%, and the volume percentage of mobile phase B was 15%. The volume percentage is 77% to 95%, and the volume percentage of mobile phase B is 23% to 5%; 58 to 65 min, the volume percentage of mobile phase A is 695%, and the volume percentage of mobile phase B is 5%; the flow rate is 1 ml / min, the column temperature is 40 ° C, the detection wavelength is 200 nm (lysine hydrochloride, calcium pantothenate), 211 nm (10-HDA), 261 nm (vitamin B1, vitamin B2, nicotinamide, vanillin), 290 nm (vitamin B6), and the injection volume is 20 μL;

[0082] (2) Preparation of reference solution:

[0083] ① Stock solutions of each reference substance (1): Accurately weigh 52.26 mg of lysine hydrochloride reference substance, 10.56 mg of nicotinamide reference substance, 18.93 mg of vanillin reference substance, and 17.82 mg of 10-HDA reference substance into a 50 ml volumetric flask, add about 30 ml of 0.1% phosphoric acid solution, sonicate and shake to dissolve, and set aside;

[0084] ②Reference substance stock solution (2): Accurately weigh 10.70 mg of vitamin B1 reference substance, 13.03 mg of vitamin B6 reference substance, and 10.11 mg of calcium pantothenate reference substance, respectively, and place them in 10 ml volumetric flasks, add 0.1% phosphoric acid solution to dissolve and dilute to scale, shake well, and obtain; place 10.89 mg of vitamin B2 reference substance in a 20 ml volumetric flask, add 5 ml of water and 1 ml of phosphoric acid, heat in a water bath and shake to dissolve, take out, cool to room temperature, dilute to scale with water, shake well, and obtain;

[0085] ③ Mixed reference substance stock solution: Accurately measure 4, 1, 3, and 2 ml of vitamin B1, vitamin B6, calcium pantothenate, and vitamin B2 reference substance stock solutions respectively and place them in a 50 ml volumetric bottle of reference substance stock solution (1) under item ①. Dilute to the mark with 0.1% phosphoric acid solution and shake well to obtain;

[0086] ④ Preparation of mixed reference solution: Accurately measure 2 ml of the mixed reference stock solution under item ③ and place it in a 10 ml volumetric flask, dilute to the mark with 0.1% phosphoric acid solution, shake well, and the solution is ready;

[0087] (3) Preparation of test solution:

[0088] Take the contents of 10 packets of this product, mix them, grind them into powder, weigh 2g, weigh accurately, put them in a 10ml volumetric flask, add 6ml of 0.1% phosphoric acid solution, ultrasonicate and shake to dissolve, cool to room temperature, dilute to scale with 0.1% phosphoric acid solution, shake well, filter, and take the filtrate to obtain; if the test solution contains whole milk powder, it is necessary to first centrifuge at 9000rpm and 0℃ for 10 min, take the supernatant and filter it with a filter membrane while it is cold, and take the filtrate to obtain;

[0089] (4) Determination: Accurately pipette 20 μL of the mixed reference solution and test solution respectively, inject into the HPLC, and perform determination under the above-mentioned chromatographic conditions. Record the chromatogram and peak area value. Calculate the contents of 8 components in the sample, including lysine hydrochloride, calcium pantothenate, vitamin B1, vitamin B2, niacinamide, vanillin, vitamin B6 and 10-hydroxy-2-decenoic acid, based on the peak area.

Claims

1. A method for simultaneously determining the contents of multiple components in oral five-dimensional lysine hydrochloride glucose, characterized in that: The HPLC-multi-wavelength method is used to simultaneously determine the contents of 8 ingredients, including lysine hydrochloride, calcium pantothenate, vitamin B1, vitamin B2, nicotinamide, vanillin, vitamin B6 and 10-hydroxy-2-decenoic acid, in the oral five-dimensional lysine hydrochloride glucose, which specifically includes the following steps: (1) Chromatographic conditions: Alltima-C18, 250 mm × 4.6 mm, 5 μm column was used as the chromatographic column, 5 mmol / L sodium heptane sulfonate solution adjusted to pH 2.5 with 20% phosphoric acid solution was used as the mobile phase A, and acetonitrile was used as the mobile phase B. Gradient elution was performed. The gradient elution program was as follows: 0-45 min, the volume percentage of mobile phase A was 95%-77%, and the volume percentage of mobile phase B was 5%-23%; 45-57 min, the volume percentage of mobile phase A was 77%, and the volume percentage of mobile phase B was 23%; 57-58 min, The volume percentage of mobile phase A is 77% to 95%, and the volume percentage of mobile phase B is 23% to 5%; 58 to 65 min, the volume percentage of mobile phase A is 695%, and the volume percentage of mobile phase B is 5%; the flow rate is 1 ml / min, the column temperature is 40°C, the detection wavelengths are: lysine hydrochloride and calcium pantothenate 200nm, 10-HDA 211nm, vitamin B1, vitamin B2, nicotinamide, vanillin 261nm, vitamin B6 290nm, and the injection volume is 20 μL; (2) Preparation of reference solution: ① Stock solutions of each reference substance (1): Accurately weigh 52.26 mg of lysine hydrochloride reference substance, 10.56 mg of nicotinamide reference substance, 18.93 mg of vanillin reference substance, and 17.82 mg of 10-HDA reference substance into a 50 ml volumetric flask, add about 30 ml of 0.1% phosphoric acid solution, sonicate and shake to dissolve, and set aside; ②Reference substance stock solution (2): Accurately weigh 10.70 mg of vitamin B1 reference substance, 13.03 mg of vitamin B6 reference substance, and 10.11 mg of calcium pantothenate reference substance, respectively, and place them in 10 ml volumetric flasks, add 0.1% phosphoric acid solution to dissolve and dilute to scale, shake well, and obtain; place 10.89 mg of vitamin B2 reference substance in a 20 ml volumetric flask, add 5 ml of water and 1 ml of phosphoric acid, heat in a water bath and shake to dissolve, take out, cool to room temperature, dilute to scale with water, shake well, and obtain; ③ Mixed reference substance stock solution: Accurately measure 4, 1, 3, and 2 ml of vitamin B1, vitamin B6, calcium pantothenate, and vitamin B2 reference substance stock solutions respectively and place them in a 50 ml volumetric bottle of reference substance stock solution (1) under item ①. Dilute to the mark with 0.1% phosphoric acid solution and shake well to obtain; ④ Preparation of mixed reference solution: Accurately measure 2 ml of the mixed reference stock solution under item ③ and place it in a 10 ml volumetric flask, dilute to the mark with 0.1% phosphoric acid solution, shake well, and the solution is ready; (3) Preparation of test solution: Take the contents of 10 packets of this product, mix and grind, weigh 2 g, accurately weigh, place in a 10 ml volumetric flask, add 6 ml of 0.1% phosphoric acid solution, sonicate and shake to dissolve, cool to room temperature, dilute to scale with 0.1% phosphoric acid solution, shake well, filter, and take the filtrate to obtain; if the test solution contains whole milk powder, it is necessary to first centrifuge at 9000 rpm and 0°C for 10 min, take the supernatant and filter it with a filter membrane while it is cold, and take the filtrate to obtain; (4) Determination: Accurately pipette 20 μL of the mixed reference solution and test solution respectively, inject into the HPLC, and perform determination under the above-mentioned chromatographic conditions. Record the chromatogram and peak area value. Calculate the contents of 8 components in the sample, including lysine hydrochloride, calcium pantothenate, vitamin B1, vitamin B2, niacinamide, vanillin, vitamin B6 and 10-hydroxy-2-decenoic acid, based on the peak area.

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