Method for detecting related substances of calcium gluconate
By using high-performance liquid chromatography-electrospray detection with a cation exchange resin column and an acidic mobile phase, the problem of separating and detecting 13 impurities in calcium gluconate was solved, achieving high-sensitivity and low-cost quality control.
Patent Information
- Application Number
- CN202511948941.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-23
- Publication Date
- 2026-02-10
AI Technical Summary
Existing technologies are insufficient to effectively separate and detect 13 known impurities in calcium gluconate simultaneously, resulting in low detection sensitivity, significant baseline interference, and high costs, which fail to meet the requirements of drug quality control.
A high-performance liquid chromatography-electro-spray detection method was adopted, using a chromatographic column with cation exchange resin as the packing material and an acidic solution as the mobile phase, combined with an electro-spray detector, to optimize the detection conditions and achieve effective separation and high-sensitivity detection of various impurities.
This method enables efficient separation and accurate detection of 13 known impurities in calcium gluconate, reducing detection costs, improving detection sensitivity and specificity, and ensuring drug quality.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of pharmaceutical analysis chemistry, and relates to a detection method of calcium gluconate related substances, in particular to a detection method of separating and detecting calcium gluconate related substances by using high performance liquid chromatography-electrospray detection method. BACKGROUND
[0002] Calcium is an inorganic substance necessary for maintaining the normal functions of human nerve, muscle, skeletal system, cell membrane and capillary permeability. Calcium gluconate is the raw material of calcium gluconate sodium chloride injection, calcium gluconate injection, calcium gluconate granules, calcium gluconate tablets, calcium gluconate oral solution, calcium gluconate tablets, and compound calcium gluconate oral solution.
[0003] The production methods of calcium gluconate include electrolysis method, fermentation method, homogeneous oxidation method, electro-oxidation method, and heterogeneous oxidation method. At present, calcium gluconate is mainly prepared by fermenting glucose. The impurities possibly contained in the starting material glucose include fructose, maltose, isomaltose, and maltotriose. Glucose is prone to produce the genotoxic impurity 5-hydroxymethyl-2-furfural under high temperature. After fermentation, the aldehyde group of glucose is oxidized to carboxyl group, thereby forming gluconic acid, and the gluconic acid and calcium salt form calcium gluconate. During the fermentation process, the alcohol group in glucose may be oxidized to aldehyde group, thereby producing gluconic dialdehyde impurity. The aldehyde group in gluconic dialdehyde impurity may be oxidized to carboxyl group, thereby producing D-glucuronic acid impurity. Meanwhile, the impurities maltose and isomaltose in glucose will undergo similar reactions to glucose, and the aldehyde groups thereof are oxidized to carboxyl groups, thereby producing maltobionic acid impurity and isomaltobionic acid impurity (isomaltose acid). Among them, fructose and maltotriose in glucose cannot produce similar acid-forming impurities due to the absence of aldehyde groups in their structures. If the impurity removal process is not good, fructose and maltotriose will remain in calcium gluconate. Gluconic acid will undergo similar reactions to glucose under high temperature, thereby producing the genotoxic impurity 5-hydroxymethyl-2-furancarboxylic acid. Gluconic acid may condense to produce D-gluconic acid-δ-lactone impurity and D-gluconic acid-γ-lactone impurity. D-glucuronic acid may condense to produce D-glucuronolactone impurity.
[0004] At present, the quality standards of calcium gluconate are collected in the United States Pharmacopoeia, the European Pharmacopoeia / British Pharmacopoeia, the Japanese Pharmacopoeia, and the Chinese Pharmacopoeia. Among them, the quality standards of various countries specify that “sucrose or reducing sugars” are investigated by physical and chemical tests, without specifying the specific impurity names; “organic impurities and boric acid” are investigated by physical and chemical tests, without specifying the specific impurity names; “oxalate” is investigated by ion chromatography.
[0005] The study and control of impurities are related to the clinical safety of drugs and are the key quality attributes of drug quality control. The adverse reactions in the clinical use of drugs depend not only on the pharmacological activity of the main components, but also on the impurities in the drugs. Therefore, the impurities in the drugs need to be studied and controlled. High-purity raw materials are the key factor for the control of drug impurities. However, the impurity spectrum of calcium gluconate is not clearly defined in the pharmacopoeias of various countries, and only a general control of one type of impurity is performed. If the impurities exceed the limit, the synthesis process of calcium gluconate cannot be optimized for targeted impurity removal, and because the sensitivity of the physicochemical test is not high enough, the control limit is high, which cannot well control the quality of calcium gluconate, and the potential genotoxic impurities are not controlled.
[0006] In summary, through the analysis of the impurity spectrum of calcium gluconate and the relevant inspection items in the pharmacopoeias of various countries, it is necessary to study and control the impurities such as D-glucuronic acid, isomaltose acid, maltose acid, fructose, D-glucuronic acid lactone, glucose, D-glucuronic acid-γ-lactone, glucose dialdehyde, D-glucuronic acid-δ-lactone, oxalic acid, maltotriose, 5-hydroxymethyl-2-furancarboxylic acid, and 5-hydroxymethyl-2-furancarboxaldehyde in calcium gluconate. All impurities are investigated in their molecular state, such as the impurities of oxalate are named as oxalic acid molecules.
[0007] CN112611821 reports the determination of the contents of maltotriose acid calcium, isomaltose acid calcium, maltose acid calcium, and glucose diacid calcium in calcium gluconate by high performance liquid chromatography-differential refractive detection method; the chromatographic column is a hydrogen ion chromatographic column, the mobile phase is formic acid solution, and the elution is isocratic. However, the sensitivity of the differential refractive detector is low, and the limit of oxalate in calcium gluconate is as low as 0.01%. Under the requirement of sensitivity, the concentration of the test solution of calcium gluconate needs to be as high as 100 mg / ml, while the saturation concentration of calcium gluconate is about 20 mg / ml.
[0008] CN113156002 reported the determination of D-glucuronic acid, D-glucuronolactone, D-gluconic acid-γ-lactone, D-gluconic acid-δ-lactone and 5-hydroxymethylfurfural (5-hydroxymethyl-2-furfuraldehyde) in calcium gluconate by high performance liquid chromatography-ultraviolet detection method, the chromatographic column was C18 column, the mobile phase was a solution containing ion pair reagent, and isocratic elution. CN117741001 reported the determination of D-gluconic acid-γ-lactone, 5-hydroxymethylfurfural, 5-hydroxymethylfuroic acid (5-hydroxymethyl-2-furoic acid), calcium lactate and calcium gluconate in compound calcium gluconate oral solution by high performance liquid chromatography-ultraviolet detection method, the chromatographic column was C18 column, the mobile phase A was buffer salt solution and the mobile phase B was methanol, and gradient elution. However, the known impurities in calcium gluconate are polar impurities, the retention of C18 column is weak, even if the mobile phase containing ion pair reagent is used, the classification effect of such impurities is still poor; the retention of impurities is even worse and the separation effect is also worse with the mobile phase of ordinary buffer salt; and because the known impurities in calcium gluconate are almost weak in ultraviolet absorption, the detection wavelength is short, and the detection is easily interfered by the mobile phase containing ion pair reagent or buffer salt, thereby causing a large baseline fluctuation and failing to well control the quality; at the same time, because of the weak ultraviolet absorption, the sensitivity is low, a high concentration of calcium gluconate test solution is needed, and the chromatographic column is greatly damaged.
[0009] CN117147736 reported the determination of the content of related substances in D-gluconic acid-δ-lactone by high performance liquid chromatography-electrospray detection method, the chromatographic column was HILIC column, the mobile phase A was trifluoroacetic acid solution and the mobile phase B was acetonitrile, and gradient elution. In the above analysis, there are as many as 13 known impurities in calcium gluconate, and the HILIC column in this document cannot effectively separate and determine the 13 known impurities at the same time; if the analysis is separated, the economic and time cost will be increased, and the efficiency will be greatly reduced.
[0010] In general, there is no relevant literature report on the simultaneous determination of the above-mentioned 13 known impurities in calcium gluconate. Some of the above-mentioned 13 known impurities in calcium gluconate have large polarity, weak ultraviolet absorption, poor solubility, low response / sensitivity in most detectors, poor retention in most chromatographic columns, large baseline interference in most detection methods, and cannot be effectively separated from each other and the main component in most chromatographic columns. At present, the detection of sugar compounds mostly uses an amino column, which has the disadvantages of serious column loss and poor durability. Based on the above reasons, the current control method is to split the 13 impurities into multiple methods for detection control, but this will increase the economic and time cost and greatly reduce the efficiency. Therefore, there is an urgent need for a detection method for simultaneously determining the 13 known impurities in calcium gluconate, so as to obtain high-quality and high-safety calcium gluconate raw material with higher efficiency, thereby reducing the cost of raw material, and further reducing the cost of each preparation, so as to contribute to the patients to obtain economic, affordable, safe and effective preparations. SUMMARY
[0011] The purpose of the present application is to solve the above-mentioned problems, and provide a detection method for the related substances of calcium gluconate, which adopts high performance liquid chromatography-electrospray detection method for detection. Each impurity has good retention in the chromatographic column, high sensitivity, small baseline interference, good accuracy, good specificity (each impurity can be effectively separated from each other and the main component), low concentration of test solution, almost no column loss, good durability, and can simultaneously analyze and detect 13 known impurities, etc. The above-mentioned problems existing in the prior art can be effectively solved. The related substances in calcium gluconate (especially the above-mentioned 13 known impurities) can be effectively controlled, and the quality and low cost of calcium gluconate can be ensured.
[0012] The present inventors have conducted a large number of experiments, and screened a lot of detectors, chromatographic columns and mobile phases. The experimental data show that the detection method of high performance liquid chromatography-electrospray detection, using a chromatographic column with cation exchange resin as the filler and an acidic solution as the mobile phase, can effectively solve the above-mentioned problems. In the obtained method, each impurity has good retention in the chromatographic column, good sensitivity, small baseline interference, good specificity (each impurity can be effectively separated from each other and the main component), good accuracy, etc.
[0013] In an embodiment of the present application, the present application provides a detection method for the related substances of calcium gluconate, which adopts high performance liquid chromatography-electrospray detection method for detection; comprising the following steps:
[0014] (1) The chromatographic parameters are set as follows:
[0015] The chromatographic column is a chromatographic column with cation exchange resin as the filler.
[0016] The detector is an electrical fog detector.
[0017] The evaporation temperature is 50-60 DEG C.
[0018] The collection frequency is 8-12 Hz.
[0019] The filter constant is 4.5-5.5 s.
[0020] The power function value is 1.8-2.2.
[0021] The flow rate is 0.4-1.5 ml / min.
[0022] The column temperature is 45-55 DEG C.
[0023] The sample size is 10-50 mu l.
[0024] The elution procedure is isocratic elution.
[0025] The mobile phase is selected from 0.09-0.11% formic acid solution, 0.09-0.11% acetic acid solution, 0.09-0.11% trifluoroacetic acid solution.
[0026] (2) The solution is prepared as follows:
[0027] The diluent is the mobile phase.
[0028] The system suitability solution: the same amount of test sample calcium gluconate, D-glucuronate, isomaltose acid, maltobionic acid, fructose, D-glucuronolactone, glucose, D-gluconic acid-gamma-lactone, glucaric acid, D-gluconic acid-delta-lactone, oxalic acid, maltotriose, 5-hydroxymethyl-2-furancarboxylic acid, 5-hydroxymethyl-2-furancarboxaldehyde are placed in the same amount bottle, and the diluent is used to prepare the system suitability solution.
[0029] The test sample solution: the test sample calcium gluconate is placed in the amount bottle, and the diluent is used to prepare the test sample solution.
[0030] The self-control solution: the test sample solution is placed in the amount bottle, and the diluent is used to dilute and prepare the self-control solution.
[0031] (3) The determination method is as follows:
[0032] The system suitability solution, the self-control solution and the test sample solution are precisely measured respectively, and are injected into the liquid chromatograph respectively, and the chromatogram is recorded, and the content of each impurity is calculated according to the main component self-control method with correction factor.
[0033] The calculation formula of the related substance detection method is as follows:
[0034] Content of each known impurity (%) = peak area of each known impurity in the test solution / peak area of the self-control solution / dilution multiple of the test solution when the self-control solution is prepared x correction factor of each impurity;
[0035] Content of the largest unknown impurity (%) = peak area of the largest unknown impurity in the test solution / peak area of the self-control solution / dilution multiple of the test solution when the self-control solution is prepared;
[0036] Total unknown impurity content (%) = sum of peak areas of unknown impurities in the test solution / peak area of the self-control solution / dilution multiple of the test solution when the self-control solution is prepared;
[0037] Total impurity (%) is the sum of the content of each known impurity and the total unknown impurity content.
[0038] In an embodiment of the present application, the correction factor and structural formula information of each known impurity of the related substances are shown in the following table:
[0039] In an embodiment of the present application, the chromatographic column is selected from JADE-PAK Carbohydrate Ca 2+ 8% (7.8mm*300mm, 8μm), Hi-Plex Ca 8% (7.7mm*300mm, 8μm), ItoISep COA Monosaccharide H + 8% (300*7.8mm, 8μm), Hi-Plex H 8% (300*7.7mm, 8μm), Dionex TM IonPac TM CS12A (250*4mm, 5μm); preferably Hi-Plex Ca 8% (7.7mm*300mm, 8μm), ItoISep COA Monosaccharide H + 8% (300*7.8mm, 8μm), Dionex TM IonPac TM CS12A (250*4mm, 5μm); further preferably Dionex TM IonPac TM CS12A (250*4mm, 5μm);
[0040] In an embodiment of the present application, the chromatographic parameters are preferably as follows:
[0041] The evaporation temperature is preferably 55°C;
[0042] The acquisition frequency is preferably 10 Hz;
[0043] The filter constant is preferably 5.0 s;
[0044] The power function value is preferably 2.0;
[0045] The flow rate is preferably 0.7-1.2 ml / min, further preferably 1.0 ml / min;
[0046] The column temperature is preferably 50°C;
[0047] The injection volume is preferably 20 μl;
[0048] The mobile phase is preferably 0.09-0.11% trifluoroacetic acid solution, further preferably 0.1% trifluoroacetic acid solution.
[0049] In an embodiment of the present application, the specific detailed steps of the high performance liquid chromatography-electrospray detection method are as follows:
[0050] (1) The chromatographic parameters are set as follows:
[0051] Chromatographic column: chromatographic column with cation exchange resin as filler;
[0052] The chromatographic column is Dionex TM IonPac TM CS12A (250*4 mm, 5 μm);
[0053] Detector: electrospray detector;
[0054] The electrospray detector is Corona Veo RS electrospray detector;
[0055] Evaporation temperature: 55°C;
[0056] Acquisition frequency: 10 Hz;
[0057] Filter constant: 5.0 s;
[0058] Power function value: 2.0;
[0059] Flow rate: 1.0 ml / min;
[0060] Column temperature: 50°C;
[0061] Injection volume: 20 μl;
[0062] Isocratic elution, running for 70 minutes;
[0063] Mobile phase: 0.1% trifluoroacetic acid solution;
[0064] (2) The solution is prepared as follows:
[0065] Diluent: 0.1% trifluoroacetic acid solution;
[0066] D-glucuronic acid reference standard and glucuronide reference standard mixed stock solution (stock solution 1): Weigh 10 mg of D-glucuronic acid reference standard and 10 mg of glucuronide reference standard accurately, place them in a 200 ml volumetric flask, add diluent to dissolve and dilute to the mark with diluent, shake well to obtain the solution;
[0067] Stock solution of isomaltocolic acid reference standard / maltodextrin reference standard / D-glucono-γ-lactone reference standard / D-glucono-δ-lactone reference standard (Stock Solution 2): Weigh 10 mg each of isomaltocolic acid reference standard, 10 mg of maltodextrin reference standard, 10 mg of D-glucono-γ-lactone reference standard and 10 mg of D-glucono-δ-lactone reference standard accurately, place them in a 100 ml volumetric flask, dissolve them with diluent and dilute to the mark with diluent, shake well, and the solution is ready.
[0068] Stock solution of fructose reference standard / glucose reference standard / maltotriose reference standard / D-glucuronide reference standard (Stock Solution 3): Weigh 10 mg of fructose reference standard, 10 mg of glucose reference standard, 10 mg of maltotriose reference standard and 10 mg of D-glucuronide reference standard respectively, place them in a 100 ml volumetric flask, dissolve them with diluent and dilute to the mark with diluent, shake well, and the solution is ready.
[0069] Oxalic acid reference standard stock solution (stock solution 4): Weigh 10 mg of oxalic acid reference standard accurately, place it in a 100 ml volumetric flask, dissolve it with diluent and dilute to the mark with diluent, and shake well; then accurately transfer 1 ml of this solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution;
[0070] 5-Hydroxymethyl-2-furanic acid reference standard / 5-hydroxymethyl-2-furanaldehyde reference standard mixed stock solution (Stock Solution 5): Accurately weigh 20 mg of 5-hydroxymethyl-2-furanic acid reference standard and 20 mg of 5-hydroxymethyl-2-furanaldehyde reference standard, place them in a 100 ml volumetric flask, dissolve them with diluent and dilute to the mark with diluent, and shake well; then accurately transfer 1 ml of this solution to a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution;
[0071] Stock solution 6: Accurately transfer 1 ml of stock solution 1, 1 ml of stock solution 2, 1 ml of stock solution 3, 1 ml of stock solution 4 and 1 ml of stock solution 5 into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution;
[0072] System suitability solution: Weigh 10 mg of calcium gluconate for testing accurately, place it in a 10 ml volumetric flask, add diluent to dissolve, then accurately transfer 1 ml of stock solution 6 into it, dilute to the mark with diluent, and shake well to obtain the solution;
[0073] Test solution: Weigh 10 mg of calcium gluconate accurately, place it in a 10 ml volumetric flask, add diluent to dissolve it, and dilute to the mark with diluent. Shake well to obtain the solution.
[0074] Self-control solution: Accurately transfer 1 ml of the test solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well; then accurately transfer 1 ml of this solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution;
[0075] (3) The determination method is as follows:
[0076] Accurately measure the system suitability solution, self-control solution, and test solution respectively, inject them into the liquid chromatograph, record the chromatograms, and calculate the content of each impurity according to the principal component self-control method with correction factor added;
[0077] The calculation formula for the related substance detection method is as follows:
[0078] The content (%) of each known impurity = peak area of each known impurity in the test solution / peak area of the self-control solution / dilution factor of the test solution when preparing the self-control solution × correction factor of each impurity;
[0079] The content of the largest unknown impurity (%) = the peak area of the largest unknown impurity in the test solution / the peak area of the self-control solution / the dilution factor of the test solution when preparing the self-control solution;
[0080] Unknown total impurity content (%) = Sum of peak areas of unknown impurities in the test solution / Peak area of the self-control solution / Dilution factor of the test solution when preparing the self-control solution;
[0081] Total impurities (%) are the sum of the contents of all known impurities and the contents of unknown total impurities;
[0082] The correction factors and structural information of the known impurities of the relevant substances are shown in the table below: Attached Figure Description
[0083] Figure 1 The graph represents the system suitability solution (100% spiked test solution) of the method described in Example 1.
[0084] Figure 2The graph represents the sensitivity solution obtained by the method described in Example 1. Detailed Implementation
[0085] The present invention will be further illustrated by the following embodiments, but it should be understood that the following embodiments are not limited to the scope of the present invention.
[0086] The reagents used in the following examples are commercially available.
[0087] Example 1
[0088] 1.1 Detection Method
[0089] 1.1.1 Solution Preparation
[0090] Mobile phase (0.1% trifluoroacetic acid solution): Take 5 ml of trifluoroacetic acid and place it in a 5 L mobile phase bottle. Add 5000 ml of filtered purified water, sonicate, and shake well to obtain the mobile phase.
[0091] Diluent: Mobile phase.
[0092] D-glucuronic acid reference standard and glucuronide reference standard mixed stock solution (stock solution 1): Weigh 10 mg of D-glucuronic acid reference standard and 10 mg of glucuronide reference standard accurately, place them in a 200 ml volumetric flask, add diluent to dissolve and dilute to the mark with diluent, shake well to obtain the solution.
[0093] Stock solution of isomaltocolic acid reference standard / maltodextrin reference standard / D-glucono-γ-lactone reference standard / D-glucono-δ-lactone reference standard (Stock Solution 2): Weigh 10 mg of isomaltocolic acid reference standard, 10 mg of maltodextrin reference standard, 10 mg of D-glucono-γ-lactone reference standard and 10 mg of D-glucono-δ-lactone reference standard respectively, place them in a 100 ml volumetric flask, dissolve them with diluent and dilute to the mark with diluent, shake well, and the solution is ready.
[0094] Stock solution of fructose reference standard / glucose reference standard / maltotriose reference standard / D-glucuronide reference standard (Stock Solution 3): Weigh 10 mg of fructose reference standard, 10 mg of glucose reference standard, 10 mg of maltotriose reference standard and 10 mg of D-glucuronide reference standard accurately, place them in a 100 ml volumetric flask, dissolve them with diluent and dilute to the mark with diluent, shake well, and the solution is ready.
[0095] Oxalic acid reference standard stock solution (stock solution 4): Weigh 10 mg of oxalic acid reference standard accurately, place it in a 100 ml volumetric flask, add diluent to dissolve it and dilute to the mark with diluent, and shake well; then accurately transfer 1 ml of the solution to a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0096] 5-Hydroxymethyl-2-furanic acid reference standard / 5-hydroxymethyl-2-furanaldehyde reference standard mixed stock solution (Stock Solution 5): Accurately weigh 20 mg of 5-hydroxymethyl-2-furanic acid reference standard and 20 mg of 5-hydroxymethyl-2-furanaldehyde reference standard, place them in a 100 ml volumetric flask, add diluent to dissolve and dilute to the mark with diluent, and shake well; then accurately transfer 1 ml of this solution to a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the final solution.
[0097] Stock solution 6: Accurately transfer 1 ml of stock solution 1, 1 ml of stock solution 2, 1 ml of stock solution 3, 1 ml of stock solution 4 and 1 ml of stock solution 5 into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the stock solution.
[0098] System suitability solution: Weigh 10 mg of calcium gluconate for testing accurately, place it in a 10 ml volumetric flask, dissolve it with diluent, then accurately transfer 1 ml of stock solution 6 into it, dilute to the mark with diluent, and shake well to obtain the solution.
[0099] Test solution: Weigh 10 mg of calcium gluconate accurately, place it in a 10 ml volumetric flask, add diluent to dissolve it, and dilute to the mark with diluent. Shake well to obtain the test solution.
[0100] Self-control solution: Accurately transfer 1 ml of the test solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well; then accurately transfer another 1 ml of the solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0101] 1.1.2 Chromatographic parameters
[0102] Chromatographic column: A chromatographic column packed with cation exchange resin, such as Dionex. TM IonPac TM CS12A (250*4mm, 5μm); Detector: Electro-fogging detector, CoronaVeo RS electro-fogging detector; Evaporation temperature: 55℃; Acquisition frequency: 10Hz; Filtration constant: 5.0s; Power function value: 2.0; Flow rate: 1.0ml / min; Column temperature: 50℃; Injection volume: 20μl; Isocratic elution, run for 70 minutes.
[0103] 1.1.3 Measurement
[0104] Accurately measure the system suitability solution, self-control solution, and test solution, respectively, and inject them into the liquid chromatograph. Record the chromatograms and calculate the content of each impurity according to the principal component self-control method with correction factors.
[0105] 1.1.4 Calculation Formula
[0106] The content (%) of each known impurity = peak area of each known impurity in the test solution / peak area of the self-control solution / dilution factor of the test solution when preparing the self-control solution × correction factor of each impurity.
[0107] The content of the largest unknown impurity (%) = the peak area of the largest unknown impurity in the test solution / the peak area of the self-control solution / the dilution factor of the test solution when preparing the self-control solution.
[0108] Unknown total impurity content (%) = Sum of peak areas of unknown impurities in the test solution / Peak area of the self-control solution / Dilution factor of the test solution when preparing the self-control solution.
[0109] Total impurities (%) is the sum of the contents of all known impurities and the contents of unknown total impurities.
[0110] The correction factor for D-glucuronic acid is 1.04, for isomaltocolic acid it is 0.53, for maltobionic acid it is 0.55, for fructose it is 0.53, for D-glucuronide it is 0.82, for glucose it is 0.60, for D-gluconic acid-γ-lactone it is 0.82, for glucuronide dialdehyde it is 0.50, for D-gluconic acid-δ-lactone it is 0.83, for oxalic acid it is 0.30, for maltotriose it is 0.66, for 5-hydroxymethyl-2-furanic acid it is 0.47, and for 5-hydroxymethyl-2-furanaldehyde it is 0.47.
[0111] 1.2 Method Validation
[0112] 1.2.1 Solution Preparation
[0113] 1.2.1.1 Specificity
[0114] 3% hydrogen peroxide solution: Take 1 ml of 30% hydrogen peroxide solution, place it in a 10 ml volumetric flask, dilute with water to the mark, and shake well.
[0115] 1 mol / L sodium hydroxide solution: Weigh 400 mg of sodium hydroxide into a 50 ml beaker, add 10 ml of water, dissolve, and shake well.
[0116] 1 mol / L hydrochloric acid solution: Take about 1 ml of hydrochloric acid solution into a 10 ml volumetric flask, dilute with water to the mark, and shake well.
[0117] Hydrogen peroxide blank solution: Take 1 ml of 3% hydrogen peroxide solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the blank solution.
[0118] Sodium hydroxide-hydrochloric acid blank solution: Take 1 ml of 1 mol / L sodium hydroxide solution into a 10 ml volumetric flask, add 1 ml of 1 mol / L hydrochloric acid solution to neutralize, dilute to the mark with diluent, and shake well to obtain the solution.
[0119] Blank solution: diluent.
[0120] D-glucuronic acid stock solution: Accurately weigh 10 mg of D-glucuronic acid reference standard, place it in a 200 ml volumetric flask, dissolve it with diluent and dilute to the mark with diluent, and shake well; then accurately transfer 10 ml of this solution to a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0121] D-glucuronic acid positioning solution: Accurately transfer 1 ml of D-glucuronic acid stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0122] Glucodialdehyde stock solution: Weigh 10 mg of glucosaldehyde reference standard accurately, place it in a 200 ml volumetric flask, dissolve it with diluent and dilute to the mark with diluent, and shake well; then accurately transfer 10 ml of this solution to a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0123] Glucodialdehyde positioning solution: Accurately transfer 1 ml of glucosaldehyde stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0124] Isomaltocolic acid stock solution: Weigh 10 mg of isomaltocolic acid reference standard accurately, place it in a 100 ml volumetric flask, dissolve it with diluent and dilute to the mark with diluent, and shake well; then accurately transfer 10 ml of this solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0125] Isomaltocolic acid positioning solution: Accurately transfer 1 ml of isomaltocolic acid stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0126] Maltodextrin stock solution: Weigh 10 mg of maltodextrin reference standard accurately, place it in a 100 ml volumetric flask, dissolve it with diluent and dilute to the mark with diluent, and shake well; then accurately transfer 10 ml of this solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0127] Maltodextrin positioning solution: Accurately transfer 1 ml of maltodextrin stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0128] D-gluconate-γ-lactone stock solution: Accurately weigh 10 mg of D-gluconate-γ-lactone reference standard, place it in a 100 ml volumetric flask, dissolve it with diluent and dilute to the mark with diluent, and shake well; then accurately transfer 10 ml of this solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0129] D-gluconate-γ-lactone positioning solution: Accurately transfer 1 ml of D-gluconate-γ-lactone stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0130] D-glucono-δ-lactone stock solution: Accurately weigh 10 mg of D-glucono-δ-lactone reference standard, place it in a 100 ml volumetric flask, dissolve it with diluent and dilute to the mark with diluent, and shake well; then accurately transfer 10 ml of this solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0131] D-gluconate-δ-lactone positioning solution: Accurately transfer 1 ml of D-gluconate-δ-lactone stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0132] Fructose stock solution: Accurately weigh 10 mg of fructose reference standard, place it in a 100 ml volumetric flask, dissolve it with diluent and dilute to the mark with diluent, and shake well; then accurately transfer 10 ml of this solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0133] Fructose positioning solution: Accurately transfer 1 ml of fructose stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0134] Glucose stock solution: Weigh 10 mg of glucose reference standard accurately, place it in a 100 ml volumetric flask, dissolve it with diluent and dilute to the mark with diluent, and shake well; then accurately transfer 10 ml of this solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0135] Glucose positioning solution: Accurately transfer 1 ml of glucose stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0136] Maltotriose stock solution: Accurately weigh 10 mg of maltotriose reference standard, place it in a 100 ml volumetric flask, dissolve it with diluent and dilute to the mark with diluent, and shake well; then accurately transfer 10 ml of this solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0137] Maltotriose positioning solution: Accurately transfer 1 ml of maltotriose stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0138] D-Glucuronide Stock Solution: Accurately weigh 10 mg of D-glucuronide reference standard, place it in a 100 ml volumetric flask, dissolve it with diluent and dilute to the mark with diluent, and shake well; then accurately transfer 10 ml of this solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0139] D-Glucuronide Positioning Solution: Accurately transfer 1 ml of D-glucuronide stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0140] Oxalic acid stock solution: Weigh 10 mg of oxalic acid reference standard accurately, place it in a 100 ml volumetric flask, dissolve it with diluent and dilute to the mark with diluent, and shake well; then accurately transfer 1 ml of the solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0141] Oxalic acid positioning solution: Accurately transfer 1 ml of oxalic acid stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0142] 5-Hydroxymethyl-2-furanic acid stock solution: Accurately weigh 20 mg of 5-hydroxymethyl-2-furanic acid reference standard, place it in a 100 ml volumetric flask, dissolve it with diluent and dilute to the mark with diluent, and shake well; then accurately transfer 1 ml of this solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0143] 5-Hydroxymethyl-2-furanic acid positioning solution: Accurately transfer 1 ml of 5-hydroxymethyl-2-furanic acid stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0144] 5-Hydroxymethyl-2-furancarbaldehyde stock solution: Accurately weigh 20 mg of 5-hydroxymethyl-2-furancarbaldehyde reference standard, place it in a 100 ml volumetric flask, dissolve it with diluent and dilute to the mark with diluent, and shake well; then accurately transfer 1 ml of this solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0145] 5-Hydroxymethyl-2-furanaldehyde positioning solution: Accurately transfer 1 ml of 5-hydroxymethyl-2-furanaldehyde stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0146] D-glucuronic acid reference standard and glucuronide reference standard mixed stock solution (stock solution 1): Weigh 10 mg of D-glucuronic acid reference standard and 10 mg of glucuronide reference standard accurately, place them in a 200 ml volumetric flask, add diluent to dissolve and dilute to the mark with diluent, shake well to obtain the solution.
[0147] Stock solution of isomaltocolic acid reference standard / maltodextrin reference standard / D-glucono-γ-lactone reference standard / D-glucono-δ-lactone reference standard (Stock Solution 2): Weigh 10 mg of isomaltocolic acid reference standard, 10 mg of maltodextrin reference standard, 10 mg of D-glucono-γ-lactone reference standard and 10 mg of D-glucono-δ-lactone reference standard respectively, place them in a 100 ml volumetric flask, dissolve them with diluent and dilute to the mark with diluent, shake well, and the solution is ready.
[0148] Stock solution of fructose reference standard / glucose reference standard / maltotriose reference standard / D-glucuronide reference standard (Stock Solution 3): Weigh 10 mg of fructose reference standard, 10 mg of glucose reference standard, 10 mg of maltotriose reference standard and 10 mg of D-glucuronide reference standard accurately, place them in a 100 ml volumetric flask, dissolve them with diluent and dilute to the mark with diluent, shake well, and the solution is ready.
[0149] Oxalic acid reference standard stock solution (stock solution 4): Weigh 10 mg of oxalic acid reference standard accurately, place it in a 100 ml volumetric flask, add diluent to dissolve it and dilute to the mark with diluent, and shake well; then accurately transfer 1 ml of the solution to a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0150] 5-Hydroxymethyl-2-furanic acid reference standard / 5-hydroxymethyl-2-furanaldehyde reference standard mixed stock solution (Stock Solution 5): Accurately weigh 20 mg of 5-hydroxymethyl-2-furanic acid reference standard and 20 mg of 5-hydroxymethyl-2-furanaldehyde reference standard, place them in a 100 ml volumetric flask, add diluent to dissolve and dilute to the mark with diluent, and shake well; then accurately transfer 1 ml of this solution to a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the final solution.
[0151] Stock solution 6: Accurately transfer 1 ml of stock solution 1, 1 ml of stock solution 2, 1 ml of stock solution 3, 1 ml of stock solution 4 and 1 ml of stock solution 5 into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the stock solution.
[0152] Impurity mixture solution: Accurately transfer 1 ml of stock solution 6 into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0153] Calcium gluconate reference solution: Weigh 10 mg of calcium gluconate reference standard accurately, place it in a 10 ml volumetric flask, dissolve it with diluent, and dilute to the mark with diluent. Shake well to obtain the solution.
[0154] Calcium gluconate reference standard stock solution: Accurately transfer 1 ml of calcium gluconate reference standard solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0155] Calcium gluconate self-reference solution: Accurately transfer 1 ml of calcium gluconate reference stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0156] Background solution: Weigh 10 mg of calcium gluconate, place it in a 10 ml volumetric flask, dissolve it with diluent, dilute to the mark with diluent, and shake well.
[0157] Self-control solution: Accurately transfer 1 ml of the background solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well; then accurately transfer 1 ml of this solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0158] 100% spiked test solution: Weigh 10 mg of calcium gluconate accurately, place it in a 10 ml volumetric flask, dissolve it with diluent, then accurately transfer 1 ml of stock solution 6 into it, dilute to the mark with diluent, and shake well to obtain the solution.
[0159] Oxidative degradation: Weigh 10 mg of calcium gluconate accurately, place it in a 10 ml volumetric flask, add 1 ml of 3% hydrogen peroxide solution, heat in a water bath for 4 hours, cool, dilute to the mark with diluent, and shake well to obtain the final product.
[0160] Alkali degradation: Weigh 10 mg of calcium gluconate accurately, place it in a 10 ml volumetric flask, add 1 ml of 1 mol / L sodium hydroxide solution, heat in a water bath for 4 hours, cool, add 1 ml of 1 mol / L hydrochloric acid solution to neutralize, dilute to the mark with diluent, and shake well to obtain the final product.
[0161] Acid degradation: Weigh 10 mg of calcium gluconate accurately, place it in a 10 ml volumetric flask, add 1 ml of 1 mol / L hydrochloric acid solution, heat in a water bath for 4 hours, cool, add 1 ml of 1 mol / L sodium hydroxide solution to neutralize, dilute to the mark with diluent, and shake well to obtain the final product.
[0162] High-temperature degradation: Accurately weigh 10 mg of calcium gluconate sample that has been placed at 105℃ for 7 days, place it in a 10 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the product is obtained.
[0163] Photodegradation: Accurately weigh 10 mg of calcium gluconate sample that has been exposed to light for 30 days, place it in a 10 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the product is obtained.
[0164] High temperature and high humidity degradation: Weigh 10 mg of calcium gluconate accurately, place it in a 10 ml volumetric flask, add 2 ml of diluent, heat in a water bath for 8 hours, cool, dilute to the mark with diluent, and shake well to obtain the final product.
[0165] 1.2.1.2 System Applicability
[0166] System suitability solution: Prepare the same 100% spiked test solution as described in "1.2.1 Specificity" in Example 1.
[0167] Self-control solution: Prepare the same self-control solution as described in "1.2.1 Specificity" in Example 1.
[0168] Sensitivity solution: Accurately transfer 5 ml of the self-control solution into a 200 ml volumetric flask, dilute to the mark with diluent, and shake well.
[0169] 1.2.1.3 Limit of Quantitation and Limit of Detection
[0170] 100% limit solutions for each impurity: Prepare the same location solutions for each impurity as described in "1.2.1 Specificity" in Example 1.
[0171] Quantitative limit solutions for each impurity: Accurately transfer 5 ml of 100% limit solutions for each impurity into a 20 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0172] Detection limit solution for each impurity: Accurately transfer 3 ml of the quantitation limit solution for each impurity into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0173] Calcium gluconate self-reference solution: Prepare the same calcium gluconate self-reference solution as described in "1.2.1 Specificity" in Example 1.
[0174] Limit of Quantification (LOQ) Solution of Main Component: Accurately transfer 5 ml of calcium gluconate self-reference solution into a 200 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0175] Limit of detection solution for main component: Accurately transfer 3 ml of the limit of detection solution for main component into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0176] 1.2.1.4 Linearity and Range
[0177] 30% linear solution: Accurately transfer 0.3 ml of each reference standard stock solution under "1.2.1 Specificity" in Example 1 into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0178] 50% linear solution: Accurately transfer 0.5 ml of each reference standard stock solution under "1.2.1 Specificity" in Example 1 into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0179] 100% linear solution: Accurately transfer 1 ml of each reference standard stock solution under "1.2.1 Specificity" in Example 1 into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0180] 150% linear solution: Accurately transfer 1.5 ml of each reference standard stock solution under "1.2.1 Specificity" in Example 1 into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0181] 200% linear solution: Accurately transfer 2 ml of each reference standard stock solution under "1.2.1 Specificity" in Example 1 into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0182] 1.2.1.5 Accuracy
[0183] Reference solutions for each impurity: Prepare the same positioning solutions for each impurity reference standard as described in "1.2.1 Specificity" in Example 1.
[0184] Calcium gluconate self-reference solution: Prepare the same calcium gluconate self-reference solution as described in "1.2.1 Specificity" in Example 1.
[0185] Background solution: Prepare the same background solution as described in "1.2.1 Specificity" in Example 1.
[0186] 50% Spiked Test Solution: Accurately weigh 10 mg of calcium gluconate test sample and place it in a 10 ml volumetric flask. Dissolve the solution in diluent, then accurately transfer 0.5 ml of each impurity stock solution from section "1.2.1 Specificity" in Example 1 to the flask. Dilute to the mark with diluent and mix well. (Prepare 3 parallel portions)
[0187] 100% Spiked Test Solution: Accurately weigh 10 mg of calcium gluconate test solution and place it in a 10 ml volumetric flask. Dissolve the solution in diluent, then accurately transfer 1 ml of each impurity stock solution from "1.2.1 Specificity" in Example 1 into the solution. Dilute to the mark with diluent and mix well. (Prepare 3 parallel portions)
[0188] 150% Spiked Test Solution: Accurately weigh 10 mg of calcium gluconate test sample and place it in a 10 ml volumetric flask. Dissolve the sample in diluent, then accurately transfer 1.5 ml of each impurity stock solution from section "1.2.1 Specificity" in Example 1 into the flask. Dilute to the mark with diluent and mix well. (Prepare 3 parallel portions)
[0189] 1.2.1.6 Precision - Repeatability (Operator A)
[0190] Calcium gluconate self-reference solution: Prepare the same calcium gluconate self-reference solution as described in "1.2.1 Specificity" in Example 1.
[0191] 100% Spiked Test Solution: Accurately weigh 10 mg of calcium gluconate test solution and place it in a 10 ml volumetric flask. Dissolve the solution in diluent, then accurately transfer 1 ml of each impurity stock solution from "1.2.1 Specificity" in Example 1 into the solution. Dilute to the mark with diluent and mix well. (Prepare 6 parallel portions)
[0192] 1.2.1.7 Precision - Intermediate Precision (Operator B)
[0193] Calcium gluconate self-reference solution: Prepare the same calcium gluconate self-reference solution as described in "1.2.1 Specificity" in Example 1.
[0194] 100% Spiked Test Solution: Accurately weigh 10 mg of calcium gluconate test solution and place it in a 10 ml volumetric flask. Dissolve the solution in diluent, then accurately transfer 1 ml of each impurity stock solution from "1.2.1 Specificity" in Example 1 into the solution. Dilute to the mark with diluent and mix well. (Prepare 6 parallel portions)
[0195] 1.2.1.8 Solution stability
[0196] Calcium gluconate self-reference solution: Prepare the same calcium gluconate self-reference solution as described in "1.2.1 Specificity" in Example 1.
[0197] 100% spiked test solution: Weigh 10 mg of calcium gluconate test sample accurately, place it in a 10 ml volumetric flask, add diluent to dissolve it, then accurately transfer 1 ml of each impurity stock solution under "1.2.1 Specificity" in Example 1 into it, dilute to the mark with diluent, shake well, and the solution is ready.
[0198] 1.2.1.9 Durability
[0199] Calcium gluconate self-reference solution: Prepare the same calcium gluconate self-reference solution as described in "1.2.1 Specificity" in Example 1.
[0200] 100% spiked test solution: Weigh 10 mg of calcium gluconate test sample accurately, place it in a 10 ml volumetric flask, add diluent to dissolve it, then accurately transfer 1 ml of each impurity stock solution under "1.2.1 Specificity" in Example 1 into it, dilute to the mark with diluent, shake well, and the solution is ready.
[0201] 1.2.2 Measurement
[0202] In accordance with the relevant guidelines of the Chinese Pharmacopoeia and ICH on method validation, the chromatographic conditions of this method were followed for injection, and the chromatograms were recorded to evaluate the method.
[0203] 1.2.3 Statistical Analysis of Method Validation Results
[0204] Example 2
[0205] 2.1 Solution Preparation
[0206] Mobile phase (0.1% formic acid solution): Take 5 ml of formic acid and place it in a 5 L mobile phase bottle. Add 5000 ml of filtered purified water, sonicate, and shake well to obtain the mobile phase.
[0207] Diluent: Mobile phase.
[0208] Blank solution: diluent.
[0209] System suitability solution: Prepare the same system suitability solution as described in "1.1.1 Solution Preparation" in Example 1.
[0210] Test solution: Prepare the test solution as described in "1.1.1 Solution Preparation" in Example 1.
[0211] Self-control solution: Accurately transfer 1 ml of the test solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well; then accurately transfer another 1 ml of the solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0212] Sensitivity solution: Accurately transfer 5 ml of the self-control solution into a 200 ml volumetric flask, dilute to the mark with diluent, and shake well.
[0213] 100% spiked test solution: Prepare the system suitability solution as described in "1.1.1 Solution Preparation" in Example 1.
[0214] 2.2 Chromatographic parameters
[0215] Chromatographic column: A chromatographic column packed with cation exchange resin, such as Dionex. TM IonPac TMCS12A (250*4mm, 5μm); Detector: Electro-fogging detector, CoronaVeo RS electro-fogging detector; Evaporation temperature: 55℃; Acquisition frequency: 10Hz; Filtration constant: 5.0s; Power function value: 2.0; Flow rate: 1.0ml / min; Column temperature: 50℃; Injection volume: 20μl; Isocratic elution, run for 70 minutes.
[0216] 2.3 Measurement
[0217] Accurately measure blank solution, sensitivity solution, system suitability solution, self-control solution, test solution, and 100% spiked test solution, respectively, and inject them into the liquid chromatograph. Record the chromatograms and calculate the content of each impurity according to the principal component self-control method with correction factor.
[0218] 2.4 Results
[0219] In this method, the blank solution does not interfere with the detection of known impurities. The signal-to-noise ratio of the sensitivity solution is 50.4. The minimum resolution between adjacent impurities and between the main component and adjacent impurities in the hot solution is 1.7. In the self-control solution, the RSD of the retention time of the main component in 5 consecutive injections is 0.01%, and the RSD of the peak area is 0.9%. The recoveries of impurities in the 100% spiked test solution are 91.3%–108.6%.
[0220] Example 3
[0221] 3.1 Solution Preparation
[0222] Mobile phase (0.1% acetic acid solution): Take 5 ml of acetic acid and place it in a 5 L mobile phase bottle. Add 5000 ml of filtered purified water, sonicate, and shake well to obtain the mobile phase.
[0223] Diluent: Mobile phase.
[0224] Blank solution: diluent.
[0225] System suitability solution: Prepare the same system suitability solution as described in "1.1.1 Solution Preparation" in Example 1.
[0226] Test solution: Prepare the test solution as described in "1.1.1 Solution Preparation" in Example 1.
[0227] Self-control solution: Accurately transfer 1 ml of the test solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well; then accurately transfer another 1 ml of the solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.
[0228] Sensitivity solution: Accurately transfer 5 ml of the self-control solution into a 200 ml volumetric flask, dilute to the mark with diluent, and shake well.
[0229] 100% spiked test solution: Prepare the system suitability solution as described in "1.1.1 Solution Preparation" in Example 1.
[0230] 3.2 Chromatographic parameters
[0231] Chromatographic column: A chromatographic column packed with cation exchange resin, such as Dionex. TM IonPac TM CS12A (250*4mm, 5μm); Detector: Electro-fogging detector, CoronaVeo RS electro-fogging detector; Evaporation temperature: 55℃; Acquisition frequency: 10Hz; Filtration constant: 5.0s; Power function value: 2.0; Flow rate: 1.0ml / min; Column temperature: 50℃; Injection volume: 20μl; Isocratic elution, run for 70 minutes.
[0232] 3.3 Measurement
[0233] Accurately measure blank solution, sensitivity solution, system suitability solution, self-control solution, test solution, and 100% spiked test solution, respectively, and inject them into the liquid chromatograph. Record the chromatograms and calculate the content of each impurity according to the principal component self-control method with correction factor.
[0234] 3.4 Results
[0235] In this method, the blank solution does not interfere with the detection of known impurities. The signal-to-noise ratio of the sensitivity solution is 46.8. The minimum resolution between adjacent impurities and between the main component and adjacent impurities in the hot solution is 1.6. In the self-control solution, the RSD of the retention time of the main component in 5 consecutive injections is 0.01%, and the RSD of the peak area is 0.7%. The recoveries of impurities in the 100% spiked test solution are 92.1%–106.7%.
[0236] Example 4
[0237] 4.1 Solution Preparation
[0238] Mobile phase (0.1% trifluoroacetic acid solution): Prepare the mobile phase as described in "3.1 Solution Preparation" in Example 3.
[0239] Diluent: Mobile phase.
[0240] Blank solution: diluent.
[0241] System suitability solution: Prepare the same system suitability solution as described in "3.1 Solution Preparation" in Example 3.
[0242] Test solution: Prepare the test solution as described in "3.1 Solution Preparation" in Example 3.
[0243] Self-control solution: Prepare the same self-control solution as in "3.1 Solution Preparation" in Example 3.
[0244] Sensitivity solution: Prepare the same sensitivity solution as described in "3.1 Solution Preparation" in Example 3.
[0245] 100% spiked test solution: Prepare the 100% spiked test solution as described in "3.1 Solution Preparation" in Example 3.
[0246] 4.2 Chromatographic parameters
[0247] Column: Hi-Plex Ca 8% (7.7mm*300mm, 8μm) column packed with cation exchange resin; Detector: CoronaVeo RS electro-fogging detector; Evaporation temperature: 55℃; Acquisition frequency: 10Hz; Filtration constant: 5.0s; Power function value: 2.0; Flow rate: 0.7ml / min; Column temperature: 50℃; Injection volume: 20μl; Isocratic elution, run for 70 minutes.
[0248] 4.3 Measurement
[0249] Accurately measure blank solution, sensitivity solution, system suitability solution, self-control solution, test solution, and 100% spiked test solution, respectively, and inject them into the liquid chromatograph. Record the chromatograms and calculate the content of each impurity according to the principal component self-control method with correction factor.
[0250] 4.4 Results
[0251] In this method, the blank solution does not interfere with the detection of known impurities. The signal-to-noise ratio of the sensitivity solution is 59.8. The minimum resolution between adjacent impurities and between the main component and adjacent impurities in the hot solution is 1.9. In the self-control solution, the RSD of the retention time of the main component in 5 consecutive injections is 0.05%, and the RSD of the peak area is 1.2%. The recoveries of impurities in the 100% spiked test solution are 89.4%–109.7%.
[0252] Example 5
[0253] 5.1 Solution Preparation
[0254] Mobile phase (0.1% trifluoroacetic acid solution): Prepare the mobile phase as described in "3.1 Solution Preparation" in Example 3.
[0255] Diluent: Mobile phase.
[0256] Blank solution: diluent.
[0257] System suitability solution: Prepare the same system suitability solution as described in "3.1 Solution Preparation" in Example 3.
[0258] Test solution: Prepare the test solution as described in "3.1 Solution Preparation" in Example 3.
[0259] Self-control solution: Prepare the same self-control solution as in "3.1 Solution Preparation" in Example 3.
[0260] Sensitivity solution: Prepare the same sensitivity solution as described in "3.1 Solution Preparation" in Example 3.
[0261] 100% spiked test solution: Prepare the 100% spiked test solution as described in "3.1 Solution Preparation" in Example 3.
[0262] 5.2 Chromatographic parameters
[0263] Chromatographic column: A chromatographic column packed with cation exchange resin, ItoISep COA Monosaccharide H + 8% (300*7.8mm, 8μm); Detector: Electro-mist detector, CoronaVeo RS electro-mist detector; Evaporation temperature: 55℃; Acquisition frequency: 10Hz; Filtration constant: 5.0s; Power function value: 2.0; Flow rate: 0.7ml / min; Column temperature: 50℃; Injection volume: 20μl; Isocratic elution, run for 70 minutes.
[0264] 5.3 Measurement
[0265] Accurately measure blank solution, sensitivity solution, system suitability solution, self-control solution, test solution, and 100% spiked test solution, respectively, and inject them into the liquid chromatograph. Record the chromatograms and calculate the content of each impurity according to the principal component self-control method with correction factor.
[0266] 5.4 Results
[0267] In this method, the blank solution does not interfere with the detection of known impurities. The signal-to-noise ratio of the sensitivity solution is 49.6. The minimum resolution between adjacent impurities and between the main component and adjacent impurities in the hot solution is 1.8. In the self-control solution, the RSD of the retention time of the main component in 5 consecutive injections is 0.01%, and the RSD of the peak area is 0.6%. The recoveries of impurities in the 100% spiked test solution are 91.4%–106.2%.
[0268] Example 6
[0269] 6.1 Solution Preparation
[0270] Mobile phase (0.1% trifluoroacetic acid solution): Prepare the mobile phase as described in "3.1 Solution Preparation" in Example 3.
[0271] Diluent: Mobile phase.
[0272] Blank solution: diluent.
[0273] System suitability solution: Prepare the same system suitability solution as described in "3.1 Solution Preparation" in Example 3.
[0274] Test solution: Prepare the test solution as described in "3.1 Solution Preparation" in Example 3.
[0275] Self-control solution: Prepare the same self-control solution as in "3.1 Solution Preparation" in Example 3.
[0276] Sensitivity solution: Prepare the same sensitivity solution as described in "3.1 Solution Preparation" in Example 3.
[0277] 100% spiked test solution: Prepare the 100% spiked test solution as described in "3.1 Solution Preparation" in Example 3.
[0278] 6.2 Chromatographic parameters
[0279] Column: Hi-Plex H 8% (300*7.7mm, 8μm) packed with cation exchange resin; Detector: CoronaVeo RS electro-vaporization detector; Evaporation temperature: 55℃; Acquisition frequency: 10Hz; Filtration constant: 5.0s; Power function value: 2.0; Flow rate: 0.7ml / min; Column temperature: 50℃; Injection volume: 20μl; Isocratic elution, run for 70 minutes.
[0280] 6.3 Measurement
[0281] Accurately measure blank solution, sensitivity solution, system suitability solution, self-control solution, test solution, and 100% spiked test solution, respectively, and inject them into the liquid chromatograph. Record the chromatograms and calculate the content of each impurity according to the principal component self-control method with correction factor.
[0282] 6.4 Results
[0283] In this method, the blank solution does not interfere with the detection of known impurities. The signal-to-noise ratio of the sensitivity solution is 50.3. The minimum resolution between adjacent impurities and between the main component and adjacent impurities in the hot solution is 1.4. In the self-control solution, the RSD of the retention time of the main component in 5 consecutive injections is 0.02%, and the RSD of the peak area is 0.7%. The recoveries of impurities in the 100% spiked test solution are 85.7%–115.2%.
[0284] Example 7
[0285] 7.1 Solution Preparation
[0286] Mobile phase (0.1% trifluoroacetic acid solution): Prepare the mobile phase as described in "3.1 Solution Preparation" in Example 3.
[0287] Diluent: Mobile phase.
[0288] Blank solution: diluent.
[0289] System suitability solution: Prepare the same system suitability solution as described in "3.1 Solution Preparation" in Example 3.
[0290] Test solution: Prepare the test solution as described in "3.1 Solution Preparation" in Example 3.
[0291] Self-control solution: Prepare the same self-control solution as in "3.1 Solution Preparation" in Example 3.
[0292] Sensitivity solution: Prepare the same sensitivity solution as described in "3.1 Solution Preparation" in Example 3.
[0293] 100% spiked test solution: Prepare the 100% spiked test solution as described in "3.1 Solution Preparation" in Example 3.
[0294] 7.2 Chromatographic parameters
[0295] Chromatographic column: A chromatographic column packed with cation exchange resin, JADE-PAK Carbohydrate Ca 2+ 8% (7.8mm*300mm, 8μm); Detector: Electro-mist detector, CoronaVeo RS electro-mist detector; Evaporation temperature: 55℃; Acquisition frequency: 10Hz; Filtration constant: 5.0s; Power function value: 2.0; Flow rate: 0.7ml / min; Column temperature: 50℃; Injection volume: 20μl; Isocratic elution, run for 70 minutes.
[0296] 7.3 Measurement
[0297] Accurately measure blank solution, sensitivity solution, system suitability solution, self-control solution, test solution, and 100% spiked test solution, respectively, and inject them into the liquid chromatograph. Record the chromatograms and calculate the content of each impurity according to the principal component self-control method with correction factor.
[0298] 7.4 Results
[0299] In this method, the blank solution does not interfere with the detection of known impurities. The signal-to-noise ratio of the sensitivity solution is 48.6. The minimum resolution between adjacent impurities and between the main component and adjacent impurities in the hot solution is 1.3. In the self-control solution, the RSD of the retention time of the main component in 5 consecutive injections is 0.01%, and the RSD of the peak area is 0.5%. The recoveries of impurities in the 100% spiked test solution are 88.7%–117.2%.
[0300] Comparative Example 1
[0301] The method described in reference CN117147736 was used to investigate related substances in this product, calcium gluconate. The optimal diluent in the method described in CN117147736 is a 70% acetonitrile aqueous solution. However, during the experiment, it was found that calcium gluconate and most known impurities in this product did not dissolve well in the 70% acetonitrile aqueous solution, nor did they dissolve well in the initial mobile phase (a mixture of 0.05% (ml / ml) trifluoroacetic acid solution and acetonitrile (90:10) (v / v)) described in CN117147736. Therefore, the method disclosed in CN117147736 cannot be used for the detection of related substances in this product, calcium gluconate.
[0302] Comparative Example 2
[0303] The method described in reference CN112611821 was used to investigate related substances in this product's calcium gluconate. The test results showed that, using the method described in CN112611821, the oxalate concentration at a signal-to-noise ratio of 10 was 10 μg / ml, and the limit for oxalate was 0.01%, therefore the concentration of the test sample should be 100 mg / ml. However, the saturation concentration of calcium gluconate is approximately 20-30 mg / ml; therefore, the method disclosed in CN112611821 cannot be used for the detection of related substances in this product's calcium gluconate.
[0304] Comparative Example 3
[0305] The method described in reference "CN113156002" was used to investigate related substances in this product's calcium gluconate. The test results showed that, using the method described in CN113156002, the detection of known impurities in the blank solution was interfered with, and the oxalate concentration at a signal-to-noise ratio of 10 was 50 μg / ml. Since the limit for oxalate is 0.01%, the concentration of the test sample should be 500 mg / ml. However, the saturation concentration of calcium gluconate is approximately 20-30 mg / ml. Therefore, the method disclosed in CN113156002 cannot be used for the detection of related substances in this product's calcium gluconate.
Claims
1. A method for detecting calcium gluconate-related substances, characterized in that... This detection method employs high-performance liquid chromatography-electrofoaming detection and includes the following steps: (1) The chromatographic parameters are set as follows: The chromatographic column is a chromatographic column packed with cation exchange resin; The detector is an electro-fog detector; The evaporation temperature is 50–60℃; The sampling frequency is 8–12 Hz; The filtration constant is 4.5–5.5 s; The power function value is 1.8 to 2.2; The flow rate is 0.4–1.5 ml / min; The column temperature is 45–55℃; The injection volume is 10–50 μl; The elution procedure is isocratic elution; The mobile phase is selected from 0.09–0.11% formic acid solution, 0.09–0.11% acetic acid solution, and 0.09–0.11% trifluoroacetic acid solution; (2) The solution is prepared as follows: Diluent: Mobile phase; System suitability solution: Take the test samples calcium gluconate, D-glucuronic acid, isomaltoconic acid, maltonic acid, fructose, D-glucuronide, glucose, D-gluconic acid-γ-lactone, glucidated dialdehyde, D-gluconic acid-δ-lactone, oxalic acid, maltotriose, 5-hydroxymethyl-2-furanic acid, and 5-hydroxymethyl-2-furanaldehyde into the same volumetric flask and dilute with diluent to prepare a system suitability solution; Test solution: Place the calcium gluconate sample in a volumetric flask and dilute it with a diluent to prepare the test solution; Self-control solution: Take the test solution and place it in a volumetric flask, then dilute it with diluent to prepare a self-control solution; (3) The determination method is as follows: Accurately measure the system suitability solution, self-control solution, and test solution respectively, inject them into the liquid chromatograph, record the chromatograms, and calculate the content of each impurity according to the principal component self-control method with correction factor added; The calculation formula for the relevant substance detection method is as follows: The content (%) of each known impurity = peak area of each known impurity in the test solution / peak area of the self-control solution / dilution factor of the test solution when preparing the self-control solution × correction factor of each impurity; The content of the largest unknown impurity (%) = the peak area of the largest unknown impurity in the test solution / the peak area of the self-control solution / the dilution factor of the test solution when preparing the self-control solution; Unknown total impurity content (%) = Sum of peak areas of unknown impurities in the test solution / Peak area of the self-control solution / Dilution factor of the test solution when preparing the self-control solution; Total impurities (%) is the sum of the contents of all known impurities and the contents of unknown total impurities.
2. The detection method according to claim 1, wherein, The correction factors and structural information of the known impurities of the substances are shown in the table below:
3. The detection method according to claim 1, wherein, The chromatographic column is selected from JADE-PAK CarbohydrateCa 2+ 8% (7.8mm*300mm, 8μm), Hi-Plex Ca 8% (7.7mm*300mm, 8μm), ItoISep COAMonosaccharide H + 8% (300*7.8mm, 8μm), Hi-Plex H 8% (300*7.7mm, 8μm), Dionex TM IonPac TM CS12A (250*4mm, 5μm); preferably Hi-Plex Ca 8% (7.7mm*300mm, 8μm), ItoISep COA Monosaccharide H + 8% (300*7.8mm, 8μm), Dionex TM IonPac TM CS12A (250*4mm, 5μm); Dionex is further preferred. TM IonPac TM CS12A (250*4mm, 5μm).
4. The detection method according to claim 1, wherein, The preferred chromatographic parameters are as follows: The preferred evaporation temperature is 55℃; The optimal sampling frequency is 10Hz; The preferred filtration constant is 5.0 s; The preferred value for the power function is 2.0; The flow rate is preferably 0.7–1.2 ml / min, and more preferably 1.0 ml / min; The preferred column temperature is 50℃; The preferred injection volume is 20 μl; The preferred mobile phase is a 0.09–0.11% trifluoroacetic acid solution.
5. The detection method according to any one of claims 1 to 4, characterized in that... The detection method employs high-performance liquid chromatography-electrofoaming detection and includes the following steps: (1) The chromatographic parameters are set as follows: Chromatographic column: A chromatographic column packed with cation exchange resin; The chromatographic column mentioned is a Dionex. TM IonPac TM CS12A (250*4mm, 5μm); Detector: Electro-fog detector; The electro-fogging detector is the Corona Veo RS electro-fogging detector. Evaporation temperature: 55℃; Sampling frequency: 10Hz; Filtration constant: 5.0s; Power function value: 2.0; Flow rate: 1.0 ml / min; Column temperature: 50℃; Injection volume: 20 μl; Isocratic elution, run for 70 minutes; Mobile phase: 0.1% trifluoroacetic acid solution; (2) The solution is prepared as follows: Diluent: 0.1% trifluoroacetic acid solution; D-glucuronic acid reference standard and glucuronide reference standard mixed stock solution (stock solution 1): Weigh 10 mg of D-glucuronic acid reference standard and 10 mg of glucuronide reference standard accurately, place them in a 200 ml volumetric flask, add diluent to dissolve and dilute to the mark with diluent, shake well to obtain the solution; Stock solution of isomaltocolic acid reference standard / maltodextrin reference standard / D-glucono-γ-lactone reference standard / D-glucono-δ-lactone reference standard (Stock Solution 2): Weigh 10 mg each of isomaltocolic acid reference standard, 10 mg of maltodextrin reference standard, 10 mg of D-glucono-γ-lactone reference standard and 10 mg of D-glucono-δ-lactone reference standard accurately, place them in a 100 ml volumetric flask, dissolve them with diluent and dilute to the mark with diluent, shake well, and the solution is ready. Stock solution of fructose reference standard / glucose reference standard / maltotriose reference standard / D-glucuronide reference standard (Stock Solution 3): Weigh 10 mg of fructose reference standard, 10 mg of glucose reference standard, 10 mg of maltotriose reference standard and 10 mg of D-glucuronide reference standard respectively, place them in a 100 ml volumetric flask, dissolve them with diluent and dilute to the mark with diluent, shake well, and the solution is ready. Oxalic acid reference standard stock solution (stock solution 4): Weigh 10 mg of oxalic acid reference standard accurately, place it in a 100 ml volumetric flask, dissolve it with diluent and dilute to the mark with diluent, and shake well; then accurately transfer 1 ml of this solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution; 5-Hydroxymethyl-2-furanic acid reference standard / 5-hydroxymethyl-2-furanaldehyde reference standard mixed stock solution (Stock Solution 5): Accurately weigh 20 mg of 5-hydroxymethyl-2-furanic acid reference standard and 20 mg of 5-hydroxymethyl-2-furanaldehyde reference standard, place them in a 100 ml volumetric flask, dissolve them with diluent and dilute to the mark with diluent, and shake well; then accurately transfer 1 ml of this solution to a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution; Stock solution 6: Accurately transfer 1 ml of stock solution 1, 1 ml of stock solution 2, 1 ml of stock solution 3, 1 ml of stock solution 4 and 1 ml of stock solution 5 into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution; System suitability solution: Weigh 10 mg of calcium gluconate for testing accurately, place it in a 10 ml volumetric flask, add diluent to dissolve, then accurately transfer 1 ml of stock solution 6 into it, dilute to the mark with diluent, and shake well to obtain the solution; Test solution: Weigh 10 mg of calcium gluconate accurately, place it in a 10 ml volumetric flask, add diluent to dissolve it, and dilute to the mark with diluent. Shake well to obtain the solution. Self-control solution: Accurately transfer 1 ml of the test solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well; then accurately transfer 1 ml of this solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution; (3) The determination method is as follows: Accurately measure the system suitability solution, self-control solution, and test solution respectively, inject them into the liquid chromatograph, record the chromatograms, and calculate the content of each impurity according to the principal component self-control method with correction factor added; The calculation formula for the relevant substance detection method is as follows: The content (%) of each known impurity = peak area of each known impurity in the test solution / peak area of the self-control solution / dilution factor of the test solution when preparing the self-control solution × correction factor of each impurity; The content of the largest unknown impurity (%) = the peak area of the largest unknown impurity in the test solution / the peak area of the self-control solution / the dilution factor of the test solution when preparing the self-control solution; Unknown total impurity content (%) = Sum of peak areas of unknown impurities in the test solution / Peak area of the self-control solution / Dilution factor of the test solution when preparing the self-control solution; Total impurities (%) are the sum of the contents of all known impurities and the contents of unknown total impurities; The correction factors and structural information of the known impurities of the relevant substances are shown in the table below: