CN115856123A - Method for simultaneously detecting citric acid, L-malic acid and D-malic acid in calcium citrate malate - Google Patents
Method for simultaneously detecting citric acid, L-malic acid and D-malic acid in calcium citrate malate Download PDFInfo
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Abstract
The invention belongs to the technical field of analysis and detection, and particularly relates to a method for simultaneously detecting citric acid, L-malic acid and D-malic acid in calcium citrate malate. The determination method comprises the following steps: preparing a mixed reference solution for detecting citric acid, L-malic acid and D-malic acid, and detecting by using an HPLC method, wherein a chromatographic column is a Phenomenex3126 (D) chiral chromatographic column; the mobile phase is a mixed solution of copper sulfate solution containing ammonium acetate and isopropanol. According to the invention, by optimizing HPLC chromatographic conditions and adopting a chiral chromatographic column, the separation of L-malic acid and D-malic acid is realized, the contents of citric acid, L-malic acid and D-malic acid in the calcium citrate malate can be detected simultaneously, the blank of the prior art is filled, and meanwhile, the quality of the calcium citrate malate can be judged by detecting the content of D-malic acid in the calcium citrate malate, so that a basis is provided for quality control of the calcium citrate malate.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and particularly relates to a method for simultaneously detecting citric acid, L-malic acid and D-malic acid in calcium citrate malate.
Background
Calcium Citrate Malate, also known as Calcium Malate, having the english name Calcium Citrate Maleate (CCM). CCM is a chelate formed by mixing calcium, citric acid and malic acid according to a certain proportion as a high-quality calcium source in developed countries such as European and American pushi, and is a preferred high-absorptivity calcium raw material in the United states, western Europe, japan and the like.
CCM has moderate calcium content in similar products and obviously better solubility than other products, the solubility of CCM increases along with the reduction of pH, and the CCM still has better solubility in slightly alkaline and near-neutral environments. The high solubility of calcium levulinate over a wide pH range is an important feature associated with high absorption. According to the research progress of Wuzhengqi, lingxu chrysanthemum, calcium fortifier and calcium fortified food [ J ]. Food industry science and technology, 2001 (04): 81-82, it is pointed out that the components citric acid and malic acid of calcium malate have optical left-handed structures (the left-handed structures are optically active compound structures that can be recognized by biological membranes) and are intermediate metabolites of the in vivo tricarboxylic acid cycle (TAC cycle), and can ensure that calcium ions are slowly released along with the oxidation of citric acid and malic acid in the body. This property results in CCM with high bioavailability.
Since L-malic acid having a physiological function of levogyration is more expensive than racemic DL-malic acid, D-malic acid is not physiologically functional. The calcium citrate malate tablet on the market at home does not indicate which configuration the chemical configuration of the contained malic acid belongs to, and the situation that D-malic acid is used as L-malic acid is easy to exist, which directly influences the efficacy of the L-malic acid. In addition, although related documents report DL-malic acid and citric acid determination, the content of total malic acid can only be determined, and the structural proportion and the content of L-malic acid and D-malic acid cannot be obtained. If the structural proportion and the content of the L-malic acid and the D-malic acid need to be obtained, a malic acid enantiomer separation method is additionally used, but the related malic acid enantiomer separation method only can separate the malic acid, and no report of citric acid determination exists. Therefore, it is necessary to develop a method for separating the malic acid enantiomer in the calcium citrate malate tablet and simultaneously detecting the contents of citric acid, L-malic acid and D-malic acid.
Disclosure of Invention
The invention aims to provide a method for simultaneously detecting citric acid, L-malic acid and D-malic acid in calcium citrate malate, the method has the advantages of simple solution preparation, convenient operation, capability of effectively separating the L-malic acid and the D-malic acid, high speed and high precision, and can be used for measuring the content of the citric acid, the L-malic acid and the D-malic acid in the calcium citrate malate.
The invention provides a method for simultaneously detecting citric acid, L-malic acid and D-malic acid in calcium citrate malate, which comprises the steps of preparing a mixed reference solution for detecting the citric acid, the L-malic acid and the D-malic acid, and detecting by adopting an HPLC method, wherein a chromatographic column is a Phenomenex3126 (D) chiral chromatographic column; the mobile phase is a mixed solution of copper sulfate solution containing ammonium acetate and isopropanol.
The inventor originally used Phenomenex3126 (D) chiral stationary phase chromatographic column to realize resolution of antofloxacin and dextroantofloxacin. However, the research process finds that the analysis time for resolving the malic acid enantiomer is particularly long only by using the copper sulfate-containing solution isopropanol as a mobile phase, and the D-malic acid does not peak at 60min even when the concentration of the isopropanol is increased to the maximum tolerated ratio of the chromatographic column. And the addition of ammonium acetate in the copper sulfate solution can lead the D-malic acid to generate a peak in advance. Therefore, the inventor further optimizes the chromatographic conditions, finds that the method not only can split and detect the L-malic acid and the D-malic acid in the calcium citrate malate, but also can simultaneously detect the citric acid in the calcium citrate malate, just fills the blank of simultaneously detecting the contents of the citric acid, the L-malic acid and the D-malic acid in the calcium citrate malate, and can judge the quality of the calcium citrate malate by detecting the content of the D-malic acid in the calcium citrate malate. Specifically, the Phenomenex3126 (D) chiral chromatography column is 250X 4.6mm in size.
Preferably, in the above technical solution, the copper sulfate concentration in the copper sulfate solution containing ammonium acetate is 2.8mmol/L.
Preferably, in the above technical scheme, the copper sulfate solution containing ammonium acetate contains 30-40mmol/L ammonium acetate, and glacial acetic acid is used for adjusting the pH to 4.6-5.0, preferably the pH is 5.
Preferably, in the above technical scheme, the volume ratio of the copper sulfate solution containing ammonium acetate to the isopropanol in the mobile phase is 85-87: 13-15.
The Phenomenex3126 (D) chiral chromatographic column requires copper sulfate solution as mobile phase, the concentration of copper sulfate is controlled at 1-3mmol/L, and Cu content is increased 2+ The sample retention time can be shortened by concentration or addition of an organic modifier such as isopropanol, but the proportion of isopropanol needs to be controlled within 15 percent. In the technical scheme, ammonium acetate is further added into the copper sulfate solution, so that the retention time of malic acid can be effectively shortened, the separation speed is high, and the analysis time is shortened.
Preferably, in the above technical solution, the volume ratio of the copper sulfate solution containing ammonium acetate to the isopropanol in the mobile phase is 87: 13.
Preferably, in the above technical solution, the chromatographic conditions further include a column temperature of 40 ℃, a flow rate of 1.0mL/min, and a sample volume of 20 μ L.
Preferably, in the above technical solution, the detection wavelength is 340nm.
The L-malic acid, citric acid and D-malic acid have maximum absorption near 270-280nm, but the baseline noise of the waveband is large, and the solutions of the test substance and the reference substance have large peak inversions in 10-11min, and the peak inversions are larger along with the larger concentration of the substance to be detected. And the baseline noise is smaller after the wavelength is larger than 320nm, and the peak inversion of the solutions of the test sample and the reference sample is smaller in 10-11 min. Therefore, the detection wavelength is selected to be 340nm in comprehensive consideration.
The invention also provides an application of the detection method in quality control of the calcium citrate malate tablets. The method can determine the content of citric acid, L-malic acid and D-malic acid in the calcium citrate malate tablet, and control the product quality by reducing the amount of the D-malic acid.
Compared with the prior art, the method has the beneficial effects that:
1. according to the invention, by optimizing HPLC chromatographic conditions, the chiral chromatographic column is adopted, so that the separation of L-malic acid and D-malic acid is realized, the contents of citric acid, L-malic acid and D-malic acid in the calcium citrate malate can be detected simultaneously, the blank of the prior art is filled, and meanwhile, the quality of the calcium citrate malate can be judged by detecting the content of D-malic acid in the calcium citrate malate, so that a basis is provided for the quality control of the calcium citrate malate;
2. the method has simple and convenient pretreatment, and has good accuracy, precision and repeatability; the chromatographic condition adopted has good separation degree and high speed, and can be used for simultaneously determining the contents of citric acid, L-malic acid and D-malic acid in the calcium citrate malate.
Drawings
FIG. 1 is a chromatogram of example 1 of the present invention, in which peak No. 1 is L-malic acid, peak No. 2 is citric acid, and peak No. 3 is D-malic acid;
FIG. 2 is a chromatogram of example 2 of the present invention, in which peak No. 1 is L-malic acid, peak No. 2 is citric acid, and peak No. 3 is D-malic acid;
FIG. 3 is a chromatogram of example 3 of the present invention, in which peak No. 1 is L-malic acid, peak No. 2 is citric acid, and peak No. 3 is D-malic acid;
FIG. 4 is a chromatogram of comparative example 1 of the present invention, in which peak No. 1 is L-malic acid;
FIG. 5 is a chromatogram of comparative example 2 of the present invention, in which peak No. 1 is L-malic acid, peak No. 2 is citric acid, and peak No. 3 is D-malic acid;
FIG. 6 is a typical chromatogram of the sample solution of the present invention, in which peak 1 is L-malic acid, peak 2 is citric acid, and peak 3 is D-malic acid.
Detailed Description
The technical features of the present invention described above and those described in detail below (as an embodiment) can be combined with each other to form a new or preferred technical solution, but the present invention is not limited to these embodiments, and the embodiments also do not limit the present invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. The formulations according to the following examples are all commercially available products and are commercially available, unless otherwise specified.
The invention is described in further detail below with reference to the figures and examples:
1. instruments and reagents
1.1 Instrument: LC-20AD XR hplc, with diode array detector (shimadzu corporation, japan), METTLER MS205DU electronic balance;
1.2 reagent and reagent: citric acid control (source: stanfordAnalytical Chemicals Inc., batch No.: SM181125-06, content: 99.9%), L-malic acid control (source: macklin, batch No.: C13236954, content: 98.0%), D-malic acid control (source: macklin, batch No.: C13626178, content: 99.0%), isopropanol were chromatographically pure, and the remaining reagents were analytically pure. Calcium citrate malate tablets (production company: hainan general union pharmaceutical Co., ltd., specification: 0.5g, batch No. 20220306).
2. Solution preparation
Precisely weighing 50.02mg of citric acid reference substance, 51.00mg of L-malic acid reference substance and 50.68mg of D-malic acid reference substance, placing in a 50mL measuring flask, adding mobile phase for dissolving, and diluting to scale to obtain mixed reference substance stock solution (containing three reference substances with concentration of about 1000 μ g/mL). Taking appropriate amount of the above solution, adding mobile phase to dilute into mixed control solution containing three control substances with concentration of about 500, 400, 300, 200, 150, 100, 50, 20 μ g/mL respectively.
Grinding calcium citrate malate tablets, precisely weighing about 50mg of the fine powder, placing the fine powder in a 50mL measuring flask, adding a mobile phase for dissolving, and diluting to a scale to obtain a test solution.
Example 1
A method for simultaneously detecting citric acid, L-malic acid and D-malic acid in calcium citrate malate comprises the following steps: taking a mixed reference solution with the concentrations of citric acid, L-malic acid and D-malic acid all being 200 mug/mL, and detecting by an HPLC method, wherein the chromatographic conditions are as follows:
a chromatographic column: phenomenex3126 (D) chiral chromatography column (250X 4.6 mm);
mobile phase: 2.8mmol/L ammonium acetate-containing copper sulfate solution (containing 40mmol/L ammonium acetate, adjusted to pH 5.0 with glacial acetic acid) -isopropanol (87;
flow rate: 1.0mL/min;
detection wavelength: 340nm;
sample introduction amount: 20 mu L of the solution;
column temperature: at 40 ℃.
The chromatogram was recorded and is shown in FIG. 1.
Example 2
A method for simultaneously detecting citric acid, L-malic acid and D-malic acid in calcium citrate malate comprises the following steps: taking a mixed reference solution with the concentrations of citric acid, L-malic acid and D-malic acid of 200 mug/mL, and detecting by using an HPLC method, wherein the chromatographic conditions are as follows:
and (3) chromatographic column: phenomenex3126 (D) chiral chromatography column (250X 4.6 mm);
mobile phase: 2.8mmol/L ammonium acetate-containing copper sulfate solution (containing 40mmol/L ammonium acetate, adjusted to pH 4.6 with glacial acetic acid) -isopropanol (87;
flow rate: 1.0mL/min;
detection wavelength: 340nm;
sample injection amount: 20 mu L of the solution;
column temperature: at 40 ℃.
The chromatogram was recorded and is shown in FIG. 2.
Example 3
A method for simultaneously detecting citric acid, L-malic acid and D-malic acid in calcium citrate malate comprises the following steps: taking a mixed reference substance solution of which the concentrations of three reference substances, namely citric acid, L-malic acid and D-malic acid are all 200 mug/mL, and detecting by adopting an HPLC method, wherein the chromatographic conditions are as follows:
a chromatographic column: phenomenex3126 (D) chiral chromatography column (250X 4.6 mm);
mobile phase: 2.8mmol/L ammonium acetate-containing copper sulfate solution (containing 30mmol/L ammonium acetate, adjusted to pH 5.0 with glacial acetic acid) -isopropanol (87;
flow rate: 1.0mL/min;
detection wavelength: 340nm;
sample injection amount: 20 mu L of the solution;
column temperature: at 40 ℃.
The chromatogram was recorded and is shown in FIG. 3.
Comparative example 1
A method for simultaneously detecting citric acid, L-malic acid and D-malic acid in calcium citrate malate comprises the following steps: taking a mixed reference solution with the concentrations of citric acid, L-malic acid and D-malic acid all being 200 mug/mL, and detecting by an HPLC method, wherein the chromatographic conditions are as follows:
a chromatographic column: phenomenex3126 (D) chiral chromatography column (250X 4.6 mm);
mobile phase: 2.8mmol/L copper sulfate solution-isopropanol (85;
flow rate: 1.0mL/min;
detection wavelength: 340nm;
sample introduction amount: 20 mu L of the solution;
column temperature: at 40 ℃.
The chromatogram was recorded and is shown in FIG. 4.
Comparative example 2
A method for simultaneously detecting citric acid, L-malic acid and D-malic acid in calcium citrate malate comprises the following steps: taking a mixed reference solution with the concentrations of citric acid, L-malic acid and D-malic acid of 200 mug/mL, and detecting by using an HPLC method, wherein the chromatographic conditions are as follows:
a chromatographic column: phenomenex3126 (D) chiral chromatography column (250X 4.6 mm);
mobile phase: 2.8mmol/L ammonium acetate-containing copper sulfate solution (containing 20mmol/L ammonium acetate, adjusted to pH 5.0 with glacial acetic acid) -isopropanol (87;
flow rate: 1.0mL/min;
detection wavelength: 340nm;
sample introduction amount: 20 mu L of the solution;
column temperature: at 40 ℃.
The chromatogram was recorded and is shown in FIG. 5.
From the results of examples 1-3, it can be seen that the chromatographic conditions of the present invention can be used to separate L-malic acid and D-malic acid, and can simultaneously separate citric acid, and the three substances have good separation degree, short time, high speed and high sensitivity.
In comparative example 1, in which a copper sulfate solution-isopropyl alcohol mobile phase containing no ammonium acetate and having no pH adjustment was used, the L-malic acid retention time was about 22min, no peak was observed at 60min of the D-malic acid, and the entire analysis time was too long, so that the chromatographic condition of comparative example 1 was not suitable for analytical determination of citric acid, L-malic acid, and D-malic acid even when the amount of isopropyl alcohol was increased.
In comparative example 2, the copper sulfate solution-isopropanol mobile phase containing ammonium acetate at a concentration of 20mmol/L is used, although the separation degree of the three substances is good, the citric acid peak and the 10min peak inversion are overlapped to a certain extent, the determination of the citric acid content is interfered, the retention time of the D-malic acid peak is 21min, the peak is latest, and the analysis time is prolonged.
Methodology investigation
1. Linear range
Taking mixed reference substance solutions with the concentrations of the three reference substances respectively being about 500, 400, 300, 200, 150, 100, 50 and 20 mug/mL,the measurement was carried out under the chromatographic conditions in example 1, respectively, and the chromatogram was recorded. And (3) performing linear regression by taking the concentration (x) of the object to be measured as an abscissa and the peak area of the object to be measured as an ordinate (y). The concentrations of the L-malic acid, the citric acid and the D-malic acid are in good linear relation with the peak areas of corresponding substances to be measured between 20 and 500 mu g/mL, wherein the linear equation is as follows: l-malic acid: y =2037.9x-6922.4 2 =0.9999; citric acid: y =1628.7x-19305,r 2 =0.9994; d-malic acid: y =2034.4x-6142.9,r 2 ＝0.9998。
2. Precision degree
A mixed control solution containing three controls each at a concentration of about 200. Mu.g/mL was taken, measured under the chromatographic conditions of example 1, and the chromatogram was recorded. The mixed reference substance solution is continuously injected for 6 needles, and the RSD of the peak areas of the L-malic acid, the citric acid and the D-malic acid is 0.5 percent, 0.8 percent and 0.5 percent, which indicates that the precision of the method is good.
3. Quantitative limit and detection limit
The mixed control solutions containing the three controls at concentrations of about 20. Mu.g/mL, respectively, were diluted with mobile phase to about 1.2, 4, and 6. Mu.g/mL for each of the three controls, and the chromatograms were recorded as determined under the chromatographic conditions described in example 1, respectively. Results show that the signal-to-noise ratio of L-malic acid peaks is larger than 3: 1 and 10: 1 when the concentration of L-malic acid is 1.2 mu g/mL and 4 mu g/mL, and the detection limit and the quantification limit of L-malic acid can be positioned at 1.2 mu g/mL and 4 mu g/mL; at 6 mug/mL and 20 mug/mL, the signal to noise ratio of the citric acid and D-malic acid peaks is larger than 3: 1 and 10: 1, so that the detection limit and the quantification limit of the citric acid and D-malic acid can be positioned at 6 mug/mL and 20 mug/mL.
4. Content reproducibility
Grinding calcium citrate malate tablets, precisely weighing about 50mg of the fine powder, placing the fine powder into a 50mL measuring flask, adding mobile phase ultrasound for dissolving, filtering to obtain a sample solution, and parallelly preparing 6 parts. The mixed control solution containing three controls with the concentration of about 200 mug/mL respectively is taken as the control solution. The test solution and the control solution were taken, measured under the chromatographic conditions in example 1, and chromatograms were recorded, and typical chromatograms thereof are shown in fig. 6. The contents of L-malic acid, citric acid and D-malic acid in the calcium citrate malate tablets are calculated according to an external standard one-point method, the RSD of the content results of the 3 substances to be detected in a repeatability experiment is less than 1.5%, and the results are shown in Table 1.
TABLE 1 content repeatability measurements
5. Accuracy of
Precisely weighing calcium citrate malate tablets, grinding the calcium citrate malate tablets to about 10mg, putting the calcium citrate malate tablets into a 20mL measuring flask, adding 2mL of mixed reference stock solution (the concentration of each of the three reference substances is about 1000 mu g/mL), adding mobile phase ultrasound to dissolve the calcium citrate malate solution, filtering the solution to serve as an accuracy determination solution, and parallelly preparing 6 parts. The control solutions were mixed to a concentration of about 200. Mu.g/mL for each of the three controls. The accuracy measurement solution and the control solution were taken, measured under the chromatographic conditions in example 1, respectively, and the chromatogram was recorded. The amounts of L-malic acid, citric acid and D-malic acid in the solution were measured with accuracy by the one-point method of external standard, and the recovery rates were calculated, with the results shown in table 2.
TABLE 2 results of accuracy measurements
In conclusion, the method adopts a Phenomenex3126 (D) chiral stationary phase chromatographic column, realizes the simultaneous detection of citric acid, L-malic acid and D-malic acid by optimizing chromatographic conditions, and can determine corresponding substances in the calcium citrate malate citrate tablets; the determination method can be suitable for detecting citric acid, L-malic acid and D-malic acid in the calcium citrate malate tablet, is simple and convenient in pretreatment, and has good accuracy, precision and repeatability.
Finally, it should be emphasized that the above-described preferred embodiments of the present invention are merely examples of implementations, rather than limitations, and that many variations and modifications of the invention are possible to those skilled in the art, without departing from the spirit and scope of the invention.
Claims (10)
1. A method for simultaneously detecting citric acid, L-malic acid and D-malic acid in calcium citrate malate is characterized in that a mixed reference solution for detecting the citric acid, the L-malic acid and the D-malic acid is prepared, and HPLC (high performance liquid chromatography) is adopted for detection, wherein a chromatographic column is a Phenomenex3126 (D) chiral chromatographic column; the mobile phase is a mixed solution of copper sulfate solution containing ammonium acetate and isopropanol.
2. The method for simultaneously detecting citric acid, L-malic acid and D-malic acid in calcium citrate malate as claimed in claim 1, wherein the copper sulfate concentration in the copper sulfate solution containing ammonium acetate is 2.8mmol/L.
3. The method for simultaneously detecting citric acid, L-malic acid and D-malic acid in calcium citrate malate according to claim 1, wherein the copper sulfate solution containing ammonium acetate contains 30-40mmol/L ammonium acetate, and the pH is adjusted to 4.6-5.0, preferably to 5 with glacial acetic acid.
4. The method for simultaneously detecting citric acid, L-malic acid and D-malic acid in calcium citrate malate as claimed in claim 1, wherein the volume ratio of ammonium acetate-containing copper sulfate solution to isopropanol in the mobile phase is 85-87: 13-15.
5. The method for simultaneously detecting citric acid, L-malic acid and D-malic acid in calcium citrate malate as claimed in claim 4, wherein the volume ratio of the copper sulfate solution containing ammonium acetate to isopropanol in the mobile phase is 87: 13.
6. The method for simultaneous detection of citric acid, L-malic acid and D-malic acid in calcium citrate malate according to claim 1, wherein the column temperature is 40 ℃.
7. The method for simultaneously detecting citric acid, L-malic acid and D-malic acid in calcium citrate malate according to claim 1, wherein the flow rate is 1.0mL/min.
8. The method for simultaneously detecting citric acid, L-malic acid and D-malic acid in calcium citrate malate according to claim 1, wherein the sample size is 20 μ L.
9. The method of claim 1, wherein the detection wavelength is 340nm.
10. Use of the method of any one of claims 1-9 for quality control of calcium citrate malate tablets.
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