Method for detecting oxalic acid content in vitamin C or salt thereof

Abstract

The invention relates to the technical field of analytical chemistry, and provides a method for detecting the content of oxalic acid in vitamin C or a salt thereof, which adopts high performance liquid chromatography, selects octadecylsilane chemically bonded silica to fill a chromatographic column, and takes a mixed solution of 45-55mmol / L monopotassium phosphate solution and methanol in a volume ratio of 99: 1 as a mobile phase. By adopting the content detection method provided by the invention, the oxalic acid in the vitamin C or the salt thereof can be accurately quantified, and after a sample is prepared, rapid testing can be realized, the working intensity of analysts can be reduced, and the sample testing efficiency can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of analytical chemistry, and in particular to a method for detecting the content of oxalic acid in vitamin C or its salt. Background Art

[0002] Oxalic acid is a toxic organic acid. Ingested oxalic acid can combine with calcium ions in the human body to form calcium oxalate, rendering calcium ions unavailable and affecting the body's absorption and utilization of calcium. This may lead to osteoporosis, kidney stones and other calcium metabolism disorders. Therefore, the content of oxalic acid in medicines must be strictly limited.

[0003] Vitamin C and vitamin C sodium are common vitamin drugs or pharmaceutical excipients. The Chinese Pharmacopoeia, the United States Pharmacopoeia (USP) and the European Pharmacopoeia (EP) all have quality standards. The Chinese Pharmacopoeia requires a limit of 0.3% for oxalic acid in vitamin C and vitamin C sodium, and the European Pharmacopoeia requires a limit of 0.2%. Taking the Chinese Pharmacopoeia method as an example, the colorimetric method for determining the limit of oxalic acid is introduced. Weigh 0.25g of sample, add 5.0mL of water, shake to dissolve, add 1.0mL of dilute acetic acid and 0.5mL of calcium chloride test solution, shake well and place for 1 hour as the test solution; accurately weigh 75mg of oxalic acid, place in a 500mL volumetric flask, add water to dissolve and dilute to the scale, shake well and take 5.0mL, add 1.0mL of dilute acetic acid and 0.5mL of calcium chloride test solution, shake well and place for 1 hour as the control solution. The turbidity produced by the test solution shall not be more concentrated than the control solution, that is, 0.3%.

[0004] The current detection method used in the Chinese Pharmacopoeia and the European Pharmacopoeia is the colorimetric method. Its main disadvantage is that there is a large factor of subjective judgment. When the oxalic acid content in the test sample is far below the limit value, it will not affect the judgment of the result. When the oxalic acid content in the test sample is close to the limit, misjudgment is prone to occur. At the same time, the colorimetric method requires the preparation of dilute acetic acid, calcium chloride test solution, test sample solution, and control solution. After preparing the sample, it is necessary to wait for 1 hour for comparison and judgment. The procedure is relatively cumbersome and time-consuming.

[0005] Chinese application CN111157638A discloses a method for detecting the oxalic acid content in vitamin C using a high performance liquid chromatograph, which uses an octadecylsilane bonded silica gel chromatographic column, mobile phase: phosphate buffer solution (containing tetrabutylammonium hydroxide)-acetonitrile, mobile phases A and B according to a gradient program; flow rate 0.9-1.6mL / min; column temperature 28-40°C, detection wavelength 200-210nm, injection volume 20μl-50μl. Although this detection method has high sensitivity, it still has the defect of using toxic tetrabutylammonium hydroxide ion pair reagent, which will cause the modification of the chromatographic column and make the chromatographic column only used for the detection method. Therefore, a new solution needs to be proposed. Summary of the invention

[0006] The present invention provides a method for detecting the content of oxalic acid in vitamin C or its salt, which adopts high performance liquid chromatography and non-toxic components to further improve the detection sensitivity, accuracy and detection efficiency.

[0007] Specifically, the method described in the present invention uses an octadecylsilane bonded silica gel to fill a chromatographic column, and uses a mixed solution of 45-55 mmol / L (preferably 50 mmol / L) potassium dihydrogen phosphate solution and methanol in a volume ratio of 99:1 as the mobile phase for detection.

[0008] When the oxalic acid content in vitamin C or its salt is determined by high performance liquid chromatography, the selection of the mobile phase plays a key role in the sensitivity, accuracy and detection efficiency of the detection result. Specifically, in the mobile phase of the present invention, the concentration of the potassium dihydrogen phosphate solution and the amount of methanol added need to be strictly limited within the above range. When the amount of methanol added is too small, a good detection effect cannot be achieved. If the concentration of the potassium dihydrogen phosphate solution is too high or too low, and the amount of methanol added is too high, the peak separation in the spectrum will be poor, the peak shape will be poor, and the detection efficiency will be affected.

[0009] The method of the present invention uses an octadecylsilane bonded silica gel filled chromatographic column (referred to as C18 column), preferably with a specification of 250 mm×4.6 mm×5 μm or other specifications with equivalent performance, and preferably a Waters Xbride C18 column.

[0010] Preferably, the column temperature is 25-35°C, more preferably 30°C.

[0011] Preferably, the injection volume is 10 μL.

[0012] In the method described in the present invention, the wavelength of the ultraviolet detector is 208-212nm, preferably 210nm. At this wavelength, the component to be detected has strong ultraviolet absorption, which improves the detection sensitivity.

[0013] In some preferred embodiments of the present invention, the following chromatographic conditions are used: Flow rate: 0.6-0.8mL / min; Column temperature: 25-35°C; UV detector wavelength: 208-212nm; Injection volume: 10 μL; Running time: 10 minutes.

[0014] In the method of the present invention, the pH of the potassium dihydrogen phosphate solution of the mobile phase is prepared to be 2.0, so as to better keep the analyte in a non-ionized form and avoid the influence of different compound forms on the detection results.

[0015] The method of the present invention includes the preparation of a test sample, specifically: dissolving vitamin C or a salt thereof in the mobile phase to prepare a test sample solution with a concentration of 1.67-5 mg / mL.

[0016] The method described in the present invention can be applied to the detection of oxalic acid content in vitamin C or its salt, wherein the vitamin C salt includes sodium vitamin C and / or calcium vitamin C.

[0017] In the method described in the present invention, a mobile phase solution is used as a blank solution; an oxalic acid solution (the dissolving medium is the mobile phase) with a concentration of 0.3% of the test solution concentration is used as a reference solution.

[0018] The method of the present invention comprises the following steps: Prepare reference solution; Prepare test sample solution; The HPLC method was used for detection, and the detection conditions included: Chromatographic column: C18, 250×4.6 mm, 5 μm, Waters Xbridge; Mobile phase: a mixed solution of 50mmol / L potassium dihydrogen phosphate solution (pH 2.0) and methanol in a volume ratio of 99:1; Column temperature: 25-35°C; Detection wavelength: 208-212nm; Flow rate: 0.6-0.8mL / min; Injection volume: 10 μL; The solvents of the reference solution and the test solution are both the mobile phase.

[0019] By adopting the detection method provided by the present invention, oxalic acid impurities in vitamin C and its salts whose main components are vitamin C can be rapidly and accurately determined, and significant improvements in detection sensitivity, accuracy and detection time are achieved compared with existing detection methods, and toxic components are avoided in the mobile phase, and there is good separation between the analyte and other components, and the peak shape has good symmetry, which is conducive to accurate quantitative analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is a typical spectrum of the blank solution in Test Example 1 of the present invention.

[0022] Figure 2 This is a typical spectrum of the reference solution in Test Example 1 of the present invention.

[0023] Figure 3 This is a typical spectrum of the mixed solution of the test sample and the reference sample in Test Example 2 of the present invention.

[0024] Figure 4 This is a typical spectrum of the detection limit in Experimental Example 3 of the present invention (S / N 17.12).

[0025] Figure 5 This is a typical spectrum of the quantitative limit in Test Example 3 of the present invention (S / N 61.01).

[0026] Figure 6 It is the linear equation curve of oxalic acid in Test Example 4 of the present invention.

[0027] Figure 7 This is a typical spectrum of the repeatability test product in Test Example 4 of the present invention.

[0028] Figure 8 This is a typical spectrum of the intermediate precision test sample in Test Example 4 of the present invention.

[0029] Fig. 9 This is the background test spectrum of the test sample in Test Example 4 of the present invention.

[0030] Fig.10 This is a typical spectrum of 50% spike recovery in Experimental Example 4 of the present invention.

[0031] Fig.11 This is a typical spectrum of 100% spike recovery in Experimental Example 4 of the present invention.

[0032] Fig.12 This is a typical spectrum of 150% spike recovery in Experimental Example 4 of the present invention.

[0033] Fig.13 This is a typical spectrum of the spiked sample at a flow rate of 0.6 mL / min in Experimental Example 4 of the present invention.

[0034] Fig.14 This is a typical spectrum of the spiked sample at a flow rate of 0.8 mL / min in Experimental Example 4 of the present invention.

[0035] Fig.15 This is a typical spectrum of the spiked sample in Test Example 4 of the present invention at a column temperature of 25°C.

[0036] Fig.16 This is a typical spectrum of the spiked sample in Test Example 4 of the present invention at a column temperature of 35°C.

[0037] Fig.17 This is a typical spectrum of the spiked sample at a detection wavelength of 208 nm in Experimental Example 4 of the present invention.

[0038] Fig.18 This is a typical spectrum of the spiked sample at a detection wavelength of 212 nm in Experimental Example 4 of the present invention.

[0039] Fig.19 This is a typical spectrum of the reference solution in Test Example 4 of the present invention when placed at 4°C for 16 hours.

[0040] Fig. 20 This is a typical spectrum of the spiked test solution in Test Example 4 of the present invention placed at 4°C for 16 hours.

[0041] Fig.21 This is the spectrum tested by the method of patent CN114354812B in comparative example 1 of the present invention.

[0042] Fig. 22 This is a graph obtained by testing the method of the present invention in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Combine the following Figure 1-Figure 22 Describe the technical solution of the present invention and its beneficial effects.

[0045] Example 1 This embodiment 1 provides a method for detecting the oxalic acid content in vitamin C, and the specific steps are as follows: (1) Preparation of reference solution Weigh 10 mg of oxalic acid reference substance, accurately weigh it, place it in a 100 mL volumetric flask, add mobile phase to dissolve and dilute to scale, shake well, and use it as the reference substance stock solution. Transfer 3 mL of the reference substance stock solution to 20 mL, add diluent and dilute to scale, shake well. Prepare 2 copies in parallel.

[0046] (2) Preparation of test solution Take about 50 mg of this product and put it into a 10 mL volumetric flask, add mobile phase to dissolve and dilute to the scale as the test solution; (3) Detection by HPLC The test conditions are as follows: Chromatographic column: C18, 250×4.6 mm, 5 μm, Waters Xbridge; Mobile phase: a mixed solution of 50mmol / L potassium dihydrogen phosphate solution (pH 2.0) and methanol in a volume ratio of 99:1; Column temperature: 30°C; Detection wavelength: 210nm; Flow rate: 0.7 mL / min; Injection volume: 10 μl.

[0047] Test Example 1 System Suitability Verification System suitability requirements: The blank solution (mobile phase) has no interference at the oxalic acid peak; for continuous injection of 6 samples of the reference solution, the relative standard deviation (RSD) of the main peak retention time should be ≤1.0%, and the RSD of the response factor (peak area / reference solution concentration) should be ≤2.0%.

[0048] Calculation of oxalic acid content in the test sample: Where: W STD is the sample weight of oxalic acid reference substance, mg; V STD is the preparation volume of oxalic acid reference solution; P STD is the purity of oxalic acid reference substance; A STD is the average peak area of ​​oxalic acid reference solution; F STD is the ratio of the peak area to the concentration of the reference substance oxalic acid; A S is the peak area of ​​oxalic acid in the test solution; W S is the sample weight of the vitamin C or sodium vitamin C test sample; V S is the preparation volume of the test product.

[0049] Test method: Take one injection of blank solution (mobile phase) and six injections of reference solution. The blank solution spectrum is required to have no interfering peaks at the peak of the analyte; in the six-injection system suitability solution spectrum, the relative standard deviation of the oxalic acid peak retention time should be ≤1.0%, and the relative standard deviation of the peak area response factor should be ≤2.0%.

[0050] The verification results are shown in Table 1, and the blank solution and system suitability spectra are shown in Figure 1-2 shown.

[0051] Table 1 Summary of system suitability verification results

[0052] Summary: The RSD of the main peak retention time of the reference substance was 0.1%, and the RSD of the response factor was 0.2%. The method system had good applicability.

[0053] Test Example 2 Specificity Take blank solution, reference solution, and mixed solution of test sample and reference solution and inject them in sequence. The blank solution is required to have no interference at the oxalic acid peak; in the mixed solution, the separation degree between the vitamin C or sodium vitamin C peak and the oxalic acid peak is ≥1.5. The typical spectra of blank solution and reference solution are shown in Figure 1-Figure 2 The typical spectrum of the mixed solution is shown in Figure 3 shown.

[0054] Summary: There is no interference in the blank solution at the oxalic acid peak. In the mixed solution, the separation degree between the vitamin C or sodium vitamin C peak and the oxalic acid peak is 7.2, which does not interfere with the detection of oxalic acid. The method has good specificity.

[0055] Test Example 3 Verification of detection limit and quantification limit The signal-to-noise ratio (S / N) of the detection limit concentration of oxalic acid should be ≥3, and the signal-to-noise ratio of the quantitative limit concentration should be ≥10. Prepare the detection limit solution and the quantitative limit solution respectively, inject the detection limit solution for 3 consecutive injections, and the signal-to-noise ratio should be ≥3; inject the quantitative limit solution for 6 consecutive injections, and the signal-to-noise ratio should be ≥10, and the relative standard deviation of the peak area should be ≥10%. The verification results are shown in Table 2. The typical spectra of the detection limit and quantitative limit of oxalic acid are shown in Figure 4 , 5 shown.

[0056] Table 2 Summary of verification data of detection limit and quantification limit of oxalic acid

[0057] Test Example 4 Linearity and Range Verification According to 15μg / mL as 100% concentration level (0.3% of the test sample), a series of oxalic acid standard solutions were prepared, and the concentrations were tested from low to high, and the standard curve was drawn with concentration versus peak area. The linear correlation coefficient R is required to be ≥ 0.995, the ratio of the intercept to the 100% concentration response value should be ≤ 5.0%, and the relative standard deviation of the response factor should be ≤ 2.0%. The range value was determined based on the quantitative limit concentration and the linear range concentration. The results are shown in Table 3. Figure 6 shown.

[0058] Table 3 Summary of oxalic acid linearity validation data

[0059] Summary: The linear correlation coefficient R is 0.9999, the ratio of the intercept to the 100% concentration response value is 0.1%, and the RSD of the response factor is 0.6%. According to the linear test results, the detection range of the method for oxalic acid is 5.07~23.03μg / mL.

[0060] Test Example 5: Verification of precision (repeatability and intermediate precision) Repeatability: Prepare a mixed solution of the test sample and oxalic acid with an oxalic acid content of 0.3%, prepare 6 portions in parallel, and inject 1 injection into each. The RSD of the oxalic acid content in the 6 portions of the test sample solution should be ≤5.0%. Intermediate precision: Another experimenter will prepare 6 portions of the mixed solution of the test sample and oxalic acid in parallel according to the repeatability experiment operation, and inject 1 injection into each. The RSD of the oxalic acid content in the 6 portions of the test sample solution should be ≤5.0%, and the RSD of the oxalic acid content in the 12 portions of the test sample for repeatability should be ≤5.0%.

[0061] The precision verification data results are shown in Table 4, and the typical graphs of repeatability and intermediate precision are shown in Figure 7-Figure 8 shown.

[0062] Table 4 Summary of precision verification results

[0063] Summary: Repeatability: The RSD of oxalic acid content in 6 test solutions is 1.7%; Intermediate precision: The RSD of oxalic acid content in 6 test solutions is 3.4%; The RSD of oxalic acid content in 12 test solutions is 2.6%. The method has good precision.

[0064] Test Example 6 Accuracy Reference substance stock solution: Weigh 10 mg of oxalic acid reference substance, accurately weigh, place in a 20 mL volumetric bottle, add diluent and dilute to scale, shake well. Transfer 3 mL to a 50 mL volumetric bottle, add diluent to scale, shake well as reference substance stock solution. Prepare the recovery solution according to Table 5.

[0065] Table 5 Recovery solution preparation

[0066] The recovery rate at each concentration level is required to be between 95.0% and 105.0%; the RSD of the recovery rate of 9 samples should be ≤15.0%. The accuracy verification data is shown in Table 6. The background test spectrum of the test sample is shown in Fig. 9 The typical recovery spectra of 50%, 100%, and 150% spiked recovery are shown in Figure 10-12 shown.

[0067] Table 6 Summary of accuracy (recovery) verification results

[0068] Summary: The accuracy at each concentration level was between 96.90-104.32%, and the RSD of the recoveries of the 9 test samples was 2.2%. The method had good accuracy.

[0069] Test Example 7 Durability The durability of the method was tested using a mixed solution of oxalic acid and the test sample. Based on the standard test conditions, the key test parameters were appropriately adjusted, including flow rate ±0.1mL / min, detection wavelength ±2nm, column temperature ±5℃, and two injections were injected in parallel for each condition. The RSD of the oxalic acid content of each group of 6 test results should be ≤5.0%. The verification results are shown in Table 7. The typical spectrum of the standard test conditions is shown in Figure 7 Typical spectra for other conditions are shown in Figure 13-Figure 18 shown.

[0070] Table 7 Summary of durability verification results

[0071] Summary: Flow rate change ±5°C, flow rate change ±0.1mL / min, wavelength change ±2nm, oxalic acid content RSD ≤ 1.7%. The method has good durability.

[0072] Test Example 8 Solution Stability Place the prepared reference solution and spiked test solution in a 4°C temperature-controlled sample tray, select time points for sample injection testing, and determine the solution stability time period.

[0073] The relative deviation of the oxalic acid peak area of ​​the reference solution compared with the peak area at 0h should be ≤5.0%; the relative deviation of the oxalic acid peak area of ​​the spiked test solution compared with the peak area at 0h should be ≤5.0%. The verification results are shown in Table 8. The typical spectra of the reference solution and the spiked test solution placed at 4℃ for 16 hours are shown in Figure 19-20 shown.

[0074] Table 8 Summary of solution stability validation data

[0075] Summary: The relative deviation of the oxalic acid peak area of ​​the reference solution compared with that at 0h is 3.0% at most; the relative deviation of the oxalic acid peak area of ​​the spiked test solution compared with that at 0h is 2.6% at most, which proves that the test sample can be stable for 16 hours at 4℃.

[0076] Comparative Example 1 Patent CN114354812B uses HILIC (hydrophilic chromatography column) with 10mmol / L sodium dihydrogen phosphate: acetonitrile = 50:50 as the mobile phase to determine the oxalic acid content in vitamin C effervescent tablets. The experimenters conducted a reproducibility experiment on this method, specifically using Cosmosil HILIC 4.6ID×250mm, 5μm hydrophilic chromatography column, preparing samples and setting detection parameters according to its instructions, but failed to achieve the reproducibility of the patented method. Fig.21The results of the Cosmosil HILIC column test show that oxalic acid and tartaric acid can be effectively separated, but the chromatographic peaks have severe tailing, which is not conducive to quantitative analysis. This phenomenon can also be observed in the chromatogram provided in the patent. The retention time of oxalic acid and tartaric acid in this experiment is significantly different from that in the patent, with a difference of about 5 minutes, which prolongs the overall analysis time and causes poor reproducibility of the method.

[0077] The method of the present invention was used to inject oxalic acid, tartaric acid, a mixed solution of oxalic acid and tartaric acid, and a mixed solution of sodium vitamin C and oxalic acid and tartaric acid, and the positioning and system suitability were investigated respectively. The results are shown in Fig. 22 There is good separation between the peaks to be measured, the peak shape is well symmetrical, and there is no tailing phenomenon, which can more accurately perform quantitative detection of impurities.

[0078] In summary, compared with the existing methods, the method of the present invention uses a simple isocratic liquid elution procedure to complete the sample test in a shorter time, and uses a conventional C18 chromatographic column with low cost to achieve better separation between the oxalic acid peak and the main component. At the same time, the mobile phase components do not contain hazardous chemicals such as tetrabutylammonium hydroxide, which is more friendly to personnel and the environment.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the oxalic acid content in vitamin C or its salt, characterized in that: Octadecylsilane bonded silica gel was used to fill the chromatographic column, and a mixed solution of 45-55 mmol / L potassium dihydrogen phosphate solution and methanol in a volume ratio of 99:1 was used as the mobile phase for detection.

2. The method according to claim 1, characterized in that The specifications of the octadecylsilane bonded silica gel filled chromatographic column are 250 mm×4.6 mm×5 μm.

3. The method according to claim 1, characterized in that The column temperature was 25-35°C.

4. The method according to claim 1, characterized in that: The injection volume was 10 μL.

5. The method according to claim 1, characterized in that The wavelength of the UV detector is 208-212nm.

6. The method according to claim 1, characterized in that The pH of the potassium dihydrogen phosphate solution used to prepare the mobile phase is 2.

0.

7. The method according to claim 1, characterized in that Vitamin C or its salt is dissolved in the mobile phase to prepare a test solution with a concentration of 1.67-5 mg / mL.

8. The method according to claim 7, characterized in that The vitamin C salt is sodium vitamin C and / or calcium vitamin C.

9. The method according to claim 1, characterized in that: Use oxalic acid solution with a concentration of 0.3% of the test solution as the reference solution.

10. The method according to any one of claims 1 to 9, characterized in that The steps include: Prepare reference solution; Prepare test sample solution; The HPLC method was used for detection, and the detection conditions included: Chromatographic column: C18, 250×4.6 mm, 5 μm, Waters Xbridge; Mobile phase: a mixed solution of 50mmol / L potassium dihydrogen phosphate solution (pH 2.0) and methanol in a volume ratio of 99:1; Column temperature: 25-35°C; Detection wavelength: 208-212nm; Flow rate: 0.6-0.8mL / min; Injection volume: 10 μL; The solvents of the reference solution and the test solution are both the mobile phase.

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