CN111595969A - Method for detecting compound three-dimensional D-calcium pantothenate syrup B vitamins by HPLC - Google Patents

Method for detecting compound three-dimensional D-calcium pantothenate syrup B vitamins by HPLC Download PDF

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CN111595969A
CN111595969A CN202010430571.4A CN202010430571A CN111595969A CN 111595969 A CN111595969 A CN 111595969A CN 202010430571 A CN202010430571 A CN 202010430571A CN 111595969 A CN111595969 A CN 111595969A
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vitamin
mobile phase
volume fraction
compound
hplc
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CN111595969B (en
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张娟
苏志伟
石贞香
喻喜华
许福春
黄鸣清
许文
钟晓琴
王一
叶水国
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Sinopharm Xingsha Pharmaceuticals Xiamen Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient

Abstract

The invention discloses a method for detecting compound three-dimensional calcium D-pantothenate syrup B vitamins by HPLC, which takes acetonitrile as a mobile phase A and takes a formic acid aqueous solution containing sodium dodecyl sulfate as a mobile phase B; taking the solution to be detected, carrying out HPLC detection, and carrying out vitamin B detection according to the chromatogram1Vitamin B2Vitamin B6And 4 vitamins of nicotinamide are subjected to qualitative and/or quantitative analysis; gradient elution is adopted in the HPLC detection process. Compared with the prior art, the method realizes the rapid qualitative identification and/or quantitative detection of the 4 vitamins in the compound three-dimensional calcium D-pantothenate syrup by an HPLC method, and provides a reliable experimental basis for the development and utilization and quality evaluation of the compound three-dimensional calcium D-pantothenate syrup.

Description

Method for detecting compound three-dimensional D-calcium pantothenate syrup B vitamins by HPLC
The scheme is divided into separate applications by taking an invention patent with the application date of 2017-11-20 and the application number of 201711155001.3, namely a method for detecting 4B vitamins in compound three-dimensional calcium D-pantothenate syrup as a parent case.
Technical Field
The invention relates to the field of quality detection methods of compound three-dimensional calcium D-pantothenate syrups, and particularly relates to a method for detecting B vitamins in compound three-dimensional calcium D-pantothenate syrup by HPLC (high performance liquid chromatography) and application thereof.
Background
The B vitamins are a kind of organic compounds necessary for maintaining human health, promoting growth and development and regulating physiological functions, and cannot be synthesized in human body by itself, and the B vitamins have low content in food and need to be supplemented additionally. Modern pharmacological research shows that vitamin B1Can be used for treating beriberi, peripheral neuritis, dyspepsia, etc.; vitamin B2Can promote intestinal tract tissue development and cell regeneration, and improve intestinal tract absorption capacity. Vitamin B6Can be used for treating malnutrition due to total parenteral nutrition and insufficient intake, and vitamin B in the case of progressive weight loss6Supplementing (1); nicotinamide is closely related to protein metabolism and children intelligence development, and plays an important role in maintaining normal functions of skin and digestive organs. In addition, pharmacological research also shows that the formulaThe vitamin B complex contained in the composition can improve the taste function of children, improve the digestive function of gastric mucosa, stimulate appetite and effectively treat the anorexia of children. The compound three-dimensional calcium D-pantothenate syrup, named vitamin feier paste or vitamin paste, is an important Chinese and western compound preparation for strengthening spleen and stomach clinically, and its main component includes vitamin B1Vitamin B2Vitamin B6The main treatment function of the vitamin B complex such as nicotinamide and the like mixed with the extracts of ten traditional Chinese medicines is 'tonifying middle-jiao and Qi, strengthening spleen and relieving diarrhea, promoting digestion and removing food stagnation'; is especially suitable for infantile malnutrition and vitamin deficiency. The compound three-dimensional calcium D-pantothenate syrup belongs to vitamin medicines in the classification of medicine standards, wherein 4B vitamins are vitamin B respectively1Vitamin B2Vitamin B6And the addition amount of the nicotinamide is higher, and the nicotinamide is also a detection index of the quality standard of the nicotinamide. Although the quality control methods of the B vitamins in the compound three-dimensional calcium D-pantothenate syrup have been reported or have been standardized in the prior art, the methods can only measure the content of 1-2 vitamins, or each vitamin needs to be respectively detected by adopting a plurality of different methods, the B quality of the compound three-dimensional calcium D-pantothenate is difficult to be comprehensively reflected, and the simultaneous measurement of a plurality of vitamins cannot be realized.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the invention provides a High Performance Liquid Chromatography (HPLC) method, which can realize vitamin B with compound three-dimensional calcium D-pantothenate syrup and intermediate thereof as background1Vitamin B2Vitamin B6And a method for simultaneously measuring 4 vitamins of nicotinamide, and provides a new technical means for the quality evaluation of the compound three-dimensional calcium D-pantothenate syrup.
In order to solve the technical problems, the invention adopts the technical scheme that:
a method for detecting compound three-dimensional D-calcium pantothenate syrup B vitamins by HPLC (high performance liquid chromatography) takes acetonitrile as a mobile phase A and takes a formic acid aqueous solution containing sodium dodecyl sulfate as a mobile phase B; taking the solution to be detected, carrying out HPLC detection, and checking the vitamin according to the chromatogramBiotin B1Vitamin B2Vitamin B6And 4 vitamins of nicotinamide are subjected to qualitative and/or quantitative analysis;
gradient elution is adopted in the HPLC detection process, and the elution procedure is as follows:
0-5 min, wherein the volume fraction of the mobile phase A is 10%;
5-20 min, increasing the volume fraction of the mobile phase A from 10% to 20%;
the volume fraction of the mobile phase A is increased from 20% to 35% in 20-30 min;
the volume fraction of the mobile phase A is increased from 35% to 45% within 30-40 min;
the volume fraction of the mobile phase A is increased from 45% to 60% within 40-50 min;
the volume fraction of the mobile phase A is increased from 60% to 75% in 50-60 min;
60-65 min, wherein the volume fraction of the mobile phase A is 75%;
65-70 min, reducing the volume fraction of the mobile phase A phase from 75% to 10%;
70-80 min, and the volume fraction of the mobile phase A is 10%.
The invention has the beneficial effects that: the detection method of the invention can realize vitamin B at the same time1Vitamin B2Vitamin B6And the detection of nicotinamide by the method of the invention has simple operation and accurate and reliable detection result. The detection method provided by the invention not only has higher precision, but also has good reproducibility. The elution gradient of the invention can completely separate four vitamins, and has high separation degree and good separation effect. In the detection process, triethylamine is not needed, so that the method is safer. The method has wide application prospect, and can provide reliable experimental basis for development and quality detection of the compound three-dimensional calcium D-pantothenate syrup.
Drawings
FIG. 1 is an HPLC detection chromatogram of example 1 of the present invention.
FIG. 2 is a chromatogram of HPLC detection using Phenomenex Luna C18 chromatographic column in example 2 of the present invention;
FIG. 3 is a chromatogram of HPLC detection using ZORBAX Bonus-RP column in example 2 of the present invention;
FIG. 4 is a chromatogram of HPLC detection using an Ultimate Polar-RP column in example 2 of the present invention;
FIG. 5 is a chromatogram of HPLC detection using ZORBAX Eclipse XDB-C18 chromatographic column in example 2 of the present invention;
FIG. 6 is a chromatogram of HPLC detection using a Waters X-select CSH C18 column in example 2 of the present invention;
FIG. 7 is a chromatogram of HPLC detection using Thermo Betasil C18 chromatographic column in example 2 of the present invention;
FIG. 8 is a chromatogram of HPLC detection using methanol as mobile phase A in example 2 of the present invention;
FIG. 9 is a chromatogram of HPLC detection using acetonitrile as mobile phase A in example 2 of the present invention;
FIG. 10 is a chromatogram of HPLC detection using 5% acetonitrile as the starting mobile phase in example 2 of the present invention;
FIG. 11 is a chromatogram of HPLC detection using 15% acetonitrile as the starting mobile phase in example 2 of the present invention;
FIG. 12 is a chromatogram of HPLC detection using 10% acetonitrile as the starting mobile phase in example 2 of the present invention;
FIG. 13 is a chromatogram of HPLC detection using acetonitrile-ammonium acetate aqueous solution as mobile phase B in example 2 of the present invention;
FIG. 14 is a chromatogram of HPLC detection using 5mM sodium hexanesulfonate-added formic acid in water as mobile phase B in example 2;
FIG. 15 is a chromatogram of HPLC detection using 5mM sodium heptanesulfonate-added formic acid in water as mobile phase B in example 2;
FIG. 16 is a chromatogram of HPLC detection using 5mM sodium dodecyl sulfate-added formic acid solution as mobile phase B in example 2 of the present invention;
FIG. 17 is a chromatogram of HPLC detection using formic acid aqueous solution with 1mM sodium dodecyl sulfate added as mobile phase B in example 2 of the present invention;
FIG. 18 is a chromatogram of HPLC detection using formic acid aqueous solution with 2.5mM sodium dodecyl sulfate added as mobile phase B in example 2 of the present invention;
FIG. 19 is a chromatogram of HPLC detection using 5mM sodium dodecyl sulfate-added formic acid solution as mobile phase B in example 2 of the present invention;
FIG. 20 is a chromatogram of HPLC detection using 5mM sodium dodecyl sulfate-added aqueous solution as mobile phase B in example 2 of the present invention;
FIG. 21 is a chromatogram of HPLC detection using 5mM sodium dodecyl sulfate-added formic acid (0.01%) in water as mobile phase B in example 2;
FIG. 22 is a chromatogram of HPLC detection using 5mM sodium dodecyl sulfate-added formic acid (0.03%) in water as mobile phase B in example 2;
FIG. 23 is a chromatogram of HPLC detection using 5mM sodium dodecyl sulfate-added formic acid (0.05%) in water as mobile phase B in example 2;
FIG. 24 is a chromatogram of an HPLC assay of a sample prepared in example 2 of the present invention as a solvent with 0.1% aqueous formic acid (pH 2);
FIG. 25 is a chromatogram of an HPLC assay of a sample prepared in example 2 of the present invention as a solvent with 0.1% aqueous triethylamine (pH 6.5);
fig. 26 is a chromatogram of an HPLC assay of a sample prepared according to example 2 of the present invention as a solvent from a 10% aqueous solution of methanol (containing 0.05% formic acid) (pH 4);
FIG. 27 is a chromatogram of HPLC detection at a wavelength of 210nm in example 2 of the present invention;
FIG. 28 is a chromatogram of HPLC detection at a wavelength of 230nm in example 2 of the present invention;
FIG. 29 is a chromatogram of HPLC detection at a wavelength of 290nm in example 2 of the present invention;
FIG. 30 is a chromatogram of HPLC detection at a wavelength of 320nm in example 2 of the present invention;
FIG. 31 is a chromatogram of HPLC detection at a wavelength of 360nm in example 2 of the present invention;
FIG. 32 is a chromatogram of HPLC detection at a wavelength of 260nm in example 2 of the present invention.
Description of reference numerals:
1. vitamin B2(ii) a 2. Nicotinamide; 3. vitamin B6(ii) a 4. Vitamin B1。
Detailed Description
In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.
The most key concept of the invention is as follows: vitamin B by the inventive HPLC gradient elution procedure1Vitamin B2Vitamin B6Completely separated from nicotinamide, thereby realizing the vitamin B in the compound three-dimensional calcium D-pantothenate syrup 14. Vitamin B2Vitamin B63 and nicotinamide.
A method for detecting compound three-dimensional D-calcium pantothenate syrup B vitamins by HPLC (high performance liquid chromatography) takes acetonitrile as a mobile phase A and takes a formic acid aqueous solution containing sodium dodecyl sulfate as a mobile phase B; taking the solution to be detected, carrying out HPLC detection, and carrying out vitamin B detection according to the chromatogram1Vitamin B2Vitamin B6And 4 vitamins of nicotinamide are subjected to qualitative and/or quantitative analysis;
gradient elution is adopted in the HPLC detection process, and the elution procedure is as follows:
0-5 min, wherein the volume fraction of the mobile phase A is 10%;
5-20 min, increasing the volume fraction of the mobile phase A from 10% to 20%;
the volume fraction of the mobile phase A is increased from 20% to 35% in 20-30 min;
the volume fraction of the mobile phase A is increased from 35% to 45% within 30-40 min;
the volume fraction of the mobile phase A is increased from 45% to 60% within 40-50 min;
the volume fraction of the mobile phase A is increased from 60% to 75% in 50-60 min;
60-65 min, wherein the volume fraction of the mobile phase A is 75%;
65-70 min, reducing the volume fraction of the mobile phase A phase from 75% to 10%;
70-80 min, and the volume fraction of the mobile phase A is 10%.
From the above description, the beneficial effects of the present invention are: the detection method of the invention can realize vitamin B at the same time1Vitamin B2Vitamin B6And the detection of nicotinamide by the method of the invention has simple operation and accurate and reliable detection result. The detection method provided by the invention not only has higher precision, but also has good reproducibility. The elution gradient of the invention can completely separate four vitamins, and has high separation degree and good separation effect. In the detection process, triethylamine is not needed, so that the method is safer. The method has wide application prospect, and can provide reliable experimental basis for development and quality detection of the compound three-dimensional calcium D-pantothenate syrup.
The vitamin B1The structural formula of (A) is:
Figure BDA0002500383760000061
the vitamin B2The structural formula of (A) is:
Figure BDA0002500383760000062
the vitamin B6The structural formula of (A) is:
Figure BDA0002500383760000063
the structural formula of the nicotinamide is as follows:
Figure BDA0002500383760000064
further, in the mobile phase B, the volume fraction of formic acid is 0.5%.
Furthermore, in the mobile phase B, the concentration of the sodium dodecyl sulfate is 1-5 mmol/L, preferably 5 mmol/L.
From the above description, the beneficial effects of the present invention are: the four B vitamins can be separated by adding sodium dodecyl sulfate with different concentrations, and the effect is best when 5mM of sodium dodecyl sulfate is added.
Preferably, in the HPLC detection process, the flow rate is 0.8-1.0 mL/min-1。
Further, in the HPLC detection process, the chromatographic column adopted is a C18 chromatographic column with the size of 4.6mm × 250mm and 5 μm, preferably, the C18 chromatographic column is a Betasil C18 chromatographic column, and more preferably, the C18 chromatographic column is a Thermo Betasil C18 chromatographic column.
From the above description, the beneficial effects of the present invention are: the peak capacity of the chromatogram was high and the resolution was the best when measured by Thermo Betasil C18 (250X 4.6mm, 5 μm).
Preferably, in the HPLC detection process, the temperature of the adopted chromatographic column is 25-35 ℃, and preferably 30 ℃.
Further, in the HPLC detection process, the detection wavelength is 230-290 nm, preferably 250-270 nm, and more preferably 260 nm.
Preferably, in the HPLC detection process, the sample injection amount is 1-20 μ l, and preferably 20 μ l.
Further, the detection method also comprises the steps of retention time measurement and standard curve drawing: with vitamin B1Vitamin B2Vitamin B6And a standard solution of four vitamin reference substances of nicotinamide, determining the retention time by using the elution program, and drawing a standard curve corresponding to the vitamin concentration and the peak area.
Further, the solution to be detected is prepared by the following steps: taking a sample to be measured, precisely weighing, adding 10% methanol aqueous solution containing 0.01-0.05% formic acid by volume fraction to dissolve, preparing a solution, filtering with a 0.45 mu m filter membrane, and taking a subsequent filtrate. Preferably, the formic acid volume fraction is 0.05%. Further, the sample to be detected is compound three-dimensional calcium pantothenate syrup.
Preferably, the solution to be tested is prepared by the following steps: taking compound three-dimensional calcium pantothenate syrup, precisely weighing, adding 10% methanol aqueous solution containing 0.01-0.05% formic acid by volume fraction, dissolving, preparing into 0.18-0.22 g/ml solution, filtering with 0.45 μm filter membrane, and taking the subsequent filtrate. Preferably, the formic acid volume fraction is 0.05%.
From the above description, the beneficial effects of the present invention are: the solvent added with formic acid is more favorable for separating the four B vitamins in the sample, and the separation effect of the sample added with the solvent with the volume fraction of 0.5 percent formic acid (pH is 4) is far better than that of the solvent with the volume fraction of 0.1 percent formic acid (pH is 2).
Preferably, the solution to be tested is prepared by the following steps: taking 5g of compound three-dimensional calcium pantothenate syrup, precisely weighing, adding 10ml of 10% methanol aqueous solution containing 0.05% formic acid, shaking for 20min, transferring into a 25ml volumetric flask, rinsing to a constant volume, shaking uniformly, and filtering with a 0.45 μm filter membrane to obtain a subsequent filtrate.
The first embodiment of the invention is as follows: the method for detecting the vitamin B group in the compound three-dimensional D-calcium pantothenate syrup by HPLC comprises the following specific operation processes and experimental conditions:
1. instrument and reagent
Model 1260 high performance liquid chromatograph (agilent, usa); one hundred thousand analytical balances of the CPA225D type (Sartorius, germany); KQ-500E desk ultrasonic cleaner (Kunshan ultrasonic instruments Co., Ltd.); acetonitrile and formic acid are in chromatographic purity (MERCK, Germany); Milli-Q ultra pure water instruments (Millipore, USA).
Control vitamin B1(batch No.: 100390-201505, content: 97.0%), vitamin B2(batch No.: 100369-201504, content: 98.2%), vitamin B6(batch No.: 100116-201404, content 100%) and nicotinamide (batch No.: 100115-201604, content 99.9%) were purchased from the China food and drug testing institute. The 10 batches of compound three-dimensional calcium D-pantothenate syrups are all from the pharmaceutical (Xiamen) company Limited of Chinese medicine Sirkish shark, and one syrup is randomly selected from the syrup for experiments in the determination process.
2. Method and results
2.1 preparation of the solution
2.1.1 control solutions
Precisely weighing vitamin B1Vitamin B2Vitamin B6Mixing with appropriate amount of nicotinamide control solutionDissolving in 10% methanol water (containing 0.05% formic acid) in the same flask, diluting to scale, shaking to obtain mass concentrations of 28.8, 5.8, 8.6, and 28.8 μ g/mL-1Mixed control solution of (4).
2.1.2 preparation of solutions to be tested
Taking 5g of compound three-dimensional calcium D-pantothenate syrup, precisely weighing 5.0011g, placing in a conical flask with a plug, adding 10mL of 10% methanol water (containing 0.05% formic acid), dissolving for 20 minutes by ultrasonic or oscillation, transferring into a 25mL measuring flask, rinsing the conical flask with 10% methanol water (containing 0.05% formic acid) for 3 times, adding the rinsing solution into the measuring flask, adding water to dilute to scale, shaking uniformly, filtering with a 0.45 μm filter membrane, and taking the subsequent filtrate.
2.2 detection of solutions to be tested and of reference solutions
Chromatographic conditions are as follows: a chromatographic column: thermo Betasil C18Analytical (4.6mM × 250mM,5 μm), mobile phase acetonitrile (A) -5mM sodium dodecyl sulfate (containing 0.05% formic acid) water (B) solution, gradient elution (0-5 min, 10% A → 10% A; 5-20 min, 10% A → 20% A; 20-30 min, 20% A → 35% A; 30-40 min, 35% A → 45% A; 40-50 min, 45% A → 60% A; 50-60 min, 60% A → 75% A; 60-65 min, 75% A → 75% A; 65-70 min, 75% A → 10% A; 70-80 min, 10% A → 10% A), flow rate of 1.0mL & min-1(ii) a Detection wavelength: 260 nm; column temperature: 30 ℃; sample introduction amount: 20 μ L, the assay results are shown in FIG. 1, and it can be seen from FIG. 1 that vitamin B can be treated by the method of the present invention1Vitamin B2Vitamin B6The chromatographic peak of each substance is sharp and symmetrical, almost has no tailing, the half peak width is less than 0.2, the baseline is stable and basically has no drift, the noise is low, and the separation degree can reach more than 1.8.
Example 2 of the present invention is an experimental analysis under different detection conditions:
1 chromatographic Condition Experimental analysis
(1) Chromatographic column experiments of different models
The experiment group comprises a control group part and an experiment group part, wherein the control group and the experiment group are subjected to sample injection measurement by using the same sample to be measured. The control group part differs from example 1 in that: the types of the columns were varied and shown in FIGS. 2-6, respectively, Phenomenex Luna C18 (250X 4.6mm, 5 μm), ZORBAX Box-RP (250X 4.6mm, 5 μm), UltimatePolar-RP (250X 4.6mm, 5 μm), ZORBAX Eclipse XDB-C18 (4.6X 250mm,5 μm), Waters XSECTCSH C18 (4.6X 250mm,5 μm), and the results of the measurements are shown in FIGS. Among them, the results of measurement of Phenomenex Luna C18 (250X 4.6mm, 5 μm) are shown in FIG. 2, the results of measurement of ZORBAX Box-RP (250X 4.6mm, 5 μm) are shown in FIG. 3, the results of measurement of Ultimate Polar-RP (250X 4.6mm, 5 μm) are shown in FIG. 4, the results of measurement of ZORBAXeclipse XDB-C18 (4.6X 250mm,5 μm) are shown in FIG. 5, and the results of measurement of Waters XSE CSH C18 (4.6X 250mm,5 μm) are shown in FIG. 6.
In the experimental group, the same as example 1, the measurement results are shown in FIG. 7, and comparison of FIGS. 2 to 7 shows that Thermo Betasil C18 (250X 4.6mm, 5 μm) has a large peak capacity and a high degree of separation as shown in FIG. 1.
(2) Experimental analysis of different organic phase systems
The experiment group comprises a control group part and an experiment group part, wherein the control group and the experiment group are subjected to sample injection measurement by using the same sample to be measured. The control group part differs from example 1 in that: the mobile phase a was methanol, and the measurement results were shown in fig. 8, except that the conditions were the same as in example 1.
The experimental group part was completely the same as example 1, and the measurement results are shown in fig. 9, and it can be seen from a comparison of fig. 8 and 9 that the peak capacity was the best in the acetonitrile system, and both the chromatographic resolution and the peak pattern were far superior to those in the methanol system.
(3) Experimental analysis of acetonitrile concentrations in different initial mobile phases
The experiment group comprises a control group part and an experiment group part, wherein the control group and the experiment group are subjected to sample injection measurement by using the same sample to be measured. The control group part differs from example 1 in that: the initial concentrations of mobile phase a were different, 5% and 15%, respectively, and the results of the measurements are shown in fig. 10 and 11, respectively.
The experimental group was completely the same as example 1, and the measurement results are shown in fig. 12, and it can be seen from a comparison of fig. 10 to 12 that 10% acetonitrile was superior to other concentrations as the initial ratio chromatographic resolution.
(4) Experimental analysis of different types of mobile phase B phase
The experiment group comprises a control group part and an experiment group part, wherein the control group and the experiment group are subjected to sample injection measurement by using the same sample to be measured. The control group part differs from example 1 in that: as mobile phase B, 0.05% acetic acid-5 mM ammonium acetate, 5mM sodium hexane sulfonate (containing 0.05% formic acid), and 5mM sodium heptane sulfonate (containing 0.05% formic acid) were added to water, and the results of the measurements are shown in FIGS. 13, 14, and 15, respectively.
The results of the experimental group were as shown in FIG. 16, which are the same as those in example 1. As can be seen from the comparison of FIGS. 13-16, the sodium dodecyl sulfate is superior to other types of acid aqueous solutions, wherein, as can be seen from FIG. 13, the retention time of nicotinamide in the solution added with sodium hexanesulfonate is greatly advanced to the range of the solvent peak, and the nicotinamide is difficult to separate, and vitamin B is added6The signal of (2) was extremely weak, and therefore, sodium hexanesulfonate was completely unsatisfactory for the analysis of the present sample. In addition, in the experiment with acetic acid, only vitamin B could be detected2And nicotinamide, whereas in the experiment with sodium heptanesulfonate, vitamin B, although four signals were detected2With vitamin B6It cannot be separated.
(5) Experimental analysis of mobile phase B with different concentrations of sodium dodecyl sulfate
The experiment group comprises a control group part and an experiment group part, wherein the control group and the experiment group are subjected to sample injection measurement by using the same sample to be measured. The control group part differs from example 1 in that: the concentration of sodium dodecyl sulfate in phase B of the mobile phase was 1mM or 2.5mM, wherein the experimental determination result of 1mM sodium dodecyl sulfate is shown in FIG. 17, and the experimental determination result of 1mM sodium dodecyl sulfate is shown in FIG. 18.
The results of the experimental group were as shown in FIG. 19, which are the same as those in example 1. As can be seen from a comparison of FIGS. 17-19, the addition of sodium dodecyl sulfate with different concentrations can substantially separate the four B vitamins, but when 1mM sodium dodecyl sulfate is added, vitamin B1Can go outSlight tailing, vitamin B was observed when 2.5mM sodium lauryl sulfate was added1The phenomenon of bifurcation occurs, so that the effect is best when 5mM sodium lauryl sulfate is added.
(6) Experimental analysis of mobile phase B with addition of different concentrations of formic acid
The experiment group comprises a control group part and an experiment group part, wherein the control group and the experiment group are subjected to sample injection measurement by using the same sample to be measured. The control group part differs from example 1 in that: the mobile phase B phase to which 0%, 0.01% and 0.03% by volume of formic acid was added was shown in fig. 20, the mobile phase B phase to which 0% by volume of formic acid was added was shown in fig. 21, and the mobile phase B phase to which 0.03% by volume of formic acid was added was shown in fig. 22.
The results of the experimental group were shown in FIG. 23, which are the same as those in example 1. As shown in comparison of FIGS. 20 to 23, vitamin B was found in the measurement of the mobile phase B without addition of formic acid2The retention time of the vitamin B also advances to the peak-out time of the solvent peak, and the vitamin B6It cannot be detected at all; when the mobile phase B added with formic acid is detected, 4 vitamins can be simultaneously detected and basically separated, wherein the separation effect of the mobile phase B added with formic acid with the volume fraction of 0.05% is the best.
2. And (3) experimental analysis of a test sample solution by a solvent:
the experiment group comprises a control group part and an experiment group part, wherein the control group and the experiment group are subjected to sample injection measurement by using the same sample to be measured. The control group part differs from example 1 in that: fig. 24 and 25 show the results of measurement of 0.1% by volume formic acid (pH 2) in 10% aqueous methanol or 0.1% by volume triethylamine (pH 6.5) in 10% aqueous methanol.
The results of the experimental group were as shown in FIG. 26, which is the same as example 1. Comparison of FIGS. 24-26 shows that although 4 vitamins can signal with triethylamine, vitamin B6Can hardly be separated out. The four B vitamins can be separated by adding formic acid, and the separation effect of the solvent with the addition of 0.5% formic acid (pH 4) by volume fraction is far better than that of the solvent with the addition of 0.1% formic acid (pH 2).
3. Experimental analysis of different detection wavelengths
The experiment group comprises a control group part and an experiment group part, wherein the control group and the experiment group are subjected to sample injection measurement by using the same sample to be measured. The control group part differs from example 1 in that: the detection wavelengths are 210nm, 230nm, 290nm, 320nm or 360nm respectively, and the measurement results are shown in the sequence of fig. 27-31.
The results of the measurement in the experimental group were shown in FIG. 32, which are the same as those in example 1. Comparing fig. 27-32, it was found that when 210nm or 230nm was used as the detection wavelength, the baseline drift was severe, wherein vitamin B was found at 210nm2The response of (2) is extremely low. At 290nm, the signal is weaker, while at 320nm or 360nm, niacinamide and vitamin B appear1The signal is very weak and the baseline is stable when detected at a wavelength of 260 nm.
The embodiment 3 of the invention is the experimental analysis of the method precision and accuracy:
1. and (3) precision test: the operation steps are completely the same as the first embodiment, 20 μ L of the reference solution is precisely absorbed, sample introduction is performed for 6 times, the precision of the detection method is determined, and the detection results are shown in Table 1.
TABLE 1 precision test results table
Figure BDA0002500383760000121
As can be seen from Table 1, the RSD of the 4 vitamins measured by the method is less than 0.5%, which indicates that the method of the invention has good precision.
2. And (3) accuracy test: the operation steps are completely the same as the first embodiment, sample injection analysis is carried out, the content of the reference substance is calculated according to an external standard one-point method of the concentration and the peak area of the reference substance, the addition of the reference substance at three levels of low, medium and high is carried out according to the measurement result of the sample, the calculation recovery rate represents the accuracy, the result is shown in table 2, and the table 2 is a result table of the sample addition recovery rate at three levels of low, medium and high.
TABLE 2 sample recovery results at three levels, low, medium and high
Figure BDA0002500383760000122
Figure BDA0002500383760000131
As can be seen from Table 2, the standard addition rate of the vitamin B group standard substance is 96-101% by adopting the detection method, which shows that the detection method has high accuracy.
In conclusion, the invention provides a method for detecting B vitamins in compound three-dimensional calcium D-pantothenate syrup by HPLC (high performance liquid chromatography), and the 4 vitamins (vitamin B) in the compound three-dimensional calcium D-pantothenate syrup can be rapidly and qualitatively identified and/or quantitatively detected by the HPLC method 21. Nicotinamide 2 and vitamin B6And vitamin B1) And a reliable experimental basis is provided for development and utilization and quality evaluation of the compound three-dimensional calcium D-pantothenate syrup.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.

Claims (6)

1. A method for detecting B vitamins in compound three-dimensional D-calcium pantothenate syrup by HPLC is characterized in that: acetonitrile is taken as a mobile phase A phase, and an acidic aqueous solution containing sodium dodecyl sulfate is taken as a mobile phase B phase; taking the solution to be detected, carrying out HPLC detection, and carrying out vitamin B detection according to the chromatogram1Vitamin B2Vitamin B6And 4 vitamins of nicotinamide are subjected to qualitative and/or quantitative analysis;
gradient elution is adopted in the HPLC detection process, and the elution procedure is as follows:
0-5 min, wherein the volume fraction of the mobile phase A is 10%;
5-20 min, increasing the volume fraction of the mobile phase A from 10% to 20%;
the volume fraction of the mobile phase A is increased from 20% to 35% in 20-30 min;
the volume fraction of the mobile phase A is increased from 35% to 45% within 30-40 min;
the volume fraction of the mobile phase A is increased from 45% to 60% within 40-50 min;
the volume fraction of the mobile phase A is increased from 60% to 75% in 50-60 min;
60-65 min, wherein the volume fraction of the mobile phase A is 75%;
65-70 min, reducing the volume fraction of the mobile phase A phase from 75% to 10%;
70-80 min, wherein the volume fraction of the mobile phase A is 10%;
the pH value of the mobile phase B is 4;
in the HPLC detection process, the temperature of the adopted chromatographic column is 30 ℃.
2. The method for detecting vitamin B in compound three-dimensional calcium D-pantothenate syrup according to claim 1, comprising the following steps: and in the mobile phase B phase, the concentration of the sodium dodecyl sulfate is 1-5 mmol/L.
3. The method for detecting vitamin B in compound three-dimensional calcium D-pantothenate syrup according to claim 1, comprising the following steps: in the HPLC detection process, the adopted chromatographic column is a C18 chromatographic column, the size is 4.6mm multiplied by 250mm, and the size is 5 μm.
4. The method for detecting vitamin B in compound three-dimensional calcium D-pantothenate syrup according to claim 1, comprising the following steps: in the HPLC detection process, the adopted detection wavelength is 250-270 nm.
5. The method for detecting vitamin B in compound three-dimensional calcium D-pantothenate syrup according to claim 1, comprising the following steps: the detection method further comprises the step of protectingAnd (3) determining the retention time and drawing a standard curve: with vitamin B1Vitamin B2Vitamin B6And a standard solution of four vitamin reference substances of nicotinamide, determining the retention time by using the elution program, and drawing a standard curve corresponding to the vitamin concentration and the peak area.
6. The method for detecting vitamin B in compound three-dimensional calcium D-pantothenate syrup according to claim 1, comprising the following steps: the solution to be detected is prepared by the following steps: taking a sample to be measured, precisely weighing, adding 10% methanol aqueous solution containing 0.01-0.05% formic acid by volume fraction to dissolve, preparing into 0.18-0.22 g/ml solution, filtering with a 0.45 mu m filter membrane, and taking the subsequent filtrate.
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