Quantitative analysis of four anions in cream preparations

Abstract

The present invention provides a method for simultaneously determining the content of four or more anions in a cream preparation by using a liquid chromatography coupled to a charged aerosol detector (CAD), wherein the four or more anions include chloride ions, nitrate ions, phosphate ions and sulfate ions, and the liquid chromatography conditions include: a chromatographic column: a C18 column, a mobile phase A: a 10 mM n-octylamine aqueous solution, the pH value is adjusted to 4.5 ± 0.5, a mobile phase B: acetonitrile, gradient elution is adopted, and the detector is a charged aerosol detector (CAD). The determination method of the present invention can accurately quantify the four anions in the cream preparation, has low detection cost, simple sample preparation, short total detection time, high specificity and accuracy.

Description

Technical Field

[0001] The invention relates to a method for simultaneously quantitatively analyzing more than four anions in a cream preparation by using HPLC-CAD, in particular to a method for simultaneously quantitatively analyzing chloride ions, nitrate radicals, phosphate radicals and sulfate radicals. Background Art

[0002] Creams are uniform semisolid preparations formed by dissolving or dispersing raw materials in an emulsion-type matrix. The acid radical ions in creams mainly come from impurities in raw materials or excipients with acid radicals (such as anhydrous calcium hydrogen phosphate, sodium dihydrogen phosphate, sodium dodecyl sulfate, etc.). The types of acid radical ions include Cl-, NO3-, SO4 2- PO4 3- Etc. Quantitative analysis of acid radical ions in creams can effectively control product quality.

[0003] Cl-, NO3-, SO4 2- PO4 3- At the same time, ion chromatography is generally used for detection. In addition, the above anions can also be determined by spectrophotometry, chemical titration (GB / T 50123-2019 "Standard for Geotechnical Test Methods"), gas phase-mass spectrometry (a method for determining the phosphate content in the culture medium CN202111518722.2), fluorescence detection (a cyclodextrin-molybdate europium / lanthanum hybrid microsphere and its preparation method and application in fluorescence detection of phosphorus-containing compounds CN202110853706.2, a method for detecting phosphate ions CN202110677304.1), etc. Ion chromatography can basically meet the requirements for the detection of acid ions in terms of detection accuracy, but this method is mainly used in water, atmosphere, soil and other fields. There is no report on the detection of acid ions in creams, and the above methods involve precise and expensive detection equipment or complex pretreatment processes, which leads to certain deficiencies in detection cost and detection efficiency.

[0004] Due to Cl-, NO3-, SO4 2- PO4 3- There is no group (chromophore) that can cause characteristic absorption of electronic spectrum, so it is impossible to detect by ultraviolet-visible light detection method. In addition, due to its large polarity, it is difficult to retain in ordinary chromatographic columns. At present, the mainstream basically adopts ion chromatography detection. For example, Chinese patent application CN113109480A adopts ion chromatography-conductivity detection method, which requires ion chromatograph and related consumables (such as ion exchange chromatographic column, suppressor, etc.), which is expensive and has certain shortcomings in detection cost.

[0005] In summary, it is necessary to develop a method for detecting the acid radical ion content as a new detection method to accurately quantify the acid radical ions in the cream to ensure the quality of the product.

[0006] Charged aerosol detector (CAD) is a universal detector, which is used in conjunction with HPLC. The eluent is atomized by the nitrogen in the atomizer, and the larger droplets flow out through the waste pipe under the action of the collision device, and the smaller solute droplets dry at room temperature to form solute particles. At the same time, the second nitrogen flow formed by the nitrogen diversion used for carrier gas passes through the corona device (including high-voltage platinum wire electrodes) to form positively charged nitrogen particles, which collide with the solute particles in the opposite direction to make the solute particles positively charged. It does not depend on the structure of the analyte, nor does it need to ionize the analyte. As long as the substance is a semi-volatile or non-volatile compound, it can be detected, and the sensitivity can reach the pg level, with good reproducibility. It is a powerful supplement to commonly used detectors such as ultraviolet (UV), mass spectrometry (MS), differential refractive index (RI) and evaporative light scattering (ELSD). This project uses HPLC-CAD detection technology, which can not only meet the simultaneous determination of multiple acid radical ions, but also has simple operation and low maintenance and use costs compared to ion chromatography, GC, MS and other analytical techniques. Summary of the invention

[0007] Technical Problems to be Solved by the Invention

[0008] As mentioned above, Cl-, NO3-, SO4 2- PO4 3- The content of acid radical ions may affect the properties of the cream, for example, the safety of the cream, therefore, it is necessary to analyze and determine the acid radical ions in the cream in order to control the product quality of the cream preparation. However, the currently commonly used ion chromatography method requires an ion chromatograph and related consumables (such as ion exchange chromatography columns, suppressors, etc.), which are expensive and have certain shortcomings in terms of detection costs.

[0009] In view of the above technical problems, the object of the present invention is to provide a method which is low in cost, simple, and highly accessible and can simultaneously detect the contents of phosphate ions, chloride ions, nitrate ions, and sulfate ions in a cream.

[0010] Technical solutions to technical issues

[0011] The present invention includes the following technical solutions.

[0012] A method for simultaneously determining the content of four or more ions in a cream preparation by liquid chromatography, wherein the four or more ions include chloride ions, nitrate ions, phosphate ions and sulfate ions, characterized in that the liquid chromatography conditions are as follows:

[0013] Chromatographic column: C18 column,

[0014] Mobile phase A: 10 mM n-octylamine aqueous solution,

[0015] Adjust the pH to 4.5±0.5, preferably 4.5±0.3, more preferably 4.5±0.2,

[0016] Mobile phase B: acetonitrile,

[0017] Gradient elution program:

[0018] Time (min) Mobile phase A (volume %) Mobile phase B (volume %) 0.0 80±2 20±2 5.0±1.0 80±2 20±2 12.0±1.0 70±2 30±2 15.0±1.0 70±2 30±2 15.1±0.2 80±2 20±2 20.0±1.0 80±2 20±2

[0019] Detector: CAD detector.

[0020] In the determination method of the present invention, the chromatographic conditions also include detection wavelength: 210±5nm, preferably 210±2nm, flow rate: 1.0±0.2mL / min, column temperature: 30±5°C, preferably column temperature is 30±3°C, more preferably 30±2°C.

[0021] The determination method of the present invention further comprises the steps of preparing a sample of the cream preparation, weighing the cream preparation, adding a diluent, uniformly dispersing, and filtering, preferably filtering through a microporous filter membrane.

[0022] The determination method of the present invention calculates the contents of four ions in the cream preparation by an external standard method.

[0023] In the determination method of the present invention, the chromatographic conditions also include: an evaporator temperature of 35° C., a power law function value (PowerFunction) of 1.00, and a collection frequency of 5 Hz.

[0024] In the measurement method of the present invention, the pH of the mobile phase A is preferably adjusted with glacial acetic acid.

[0025] In the measuring method of the present invention, the cream preparation is lidocaine cream.

[0026] Beneficial technical effects

[0027] Beneficial effects of the present invention:

[0028] 1. The electrospray detector (CAD) can accurately quantify the contents of phosphate, chloride, nitrate and sulfate ions in cream preparations, providing a new method for the detection of acid ions.

[0029] 2. Using HPLC and conventional C18 reversed phase chromatography column, the method is highly popularizable and has low detection cost.

[0030] 3. This method has simple sample preparation, short total detection time, and is convenient for quickly issuing test results.

[0031] 4. This method has been verified to meet the requirements of specificity and accuracy, and provides a new detection method for the determination of acid radical ion content. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 .Chromatogram using Aglilent Poroshell 120HILIC-Z (150×3.0mm, 2.7μm) column;

[0033] Figure 2 .Chromatograms of the diluent, four ion references, and cream formulation samples. DETAILED DESCRIPTION

[0034] The present invention provides a method for simultaneously determining the contents of four kinds of acid radical ions in a cream preparation based on a high performance liquid chromatography-electrospray detector (HPLC-CAD) coupling technology. The four kinds of acid radical ions are Cl - 、NO3-、SO4 2- PO4 3- .

[0035] The specific steps are as follows:

[0036] 1. Screening of chromatographic conditions: Using mixed ion reference solution, the separation of target ions on different chromatographic columns (Aglilent Poroshell 120 HILIC-Z (150×3.0 mm, 2.7 μm), Welch Ultimate AQ-C18 (250×4.6 mm, 5 μm), and Waters Xselect HSS-T3 (150×4.6 mm, 3.5 μm)) was investigated, and the appropriate chromatographic conditions were determined based on peak symmetry and peak separation.

[0037] Aglilent Poroshell 120HILIC-Z (150×3.0 mm, 2.7 μm) column was used. Figure 1 As shown, the peak positions of chloride, sulfate, phosphate and nitrate ions can be determined, but the peak shapes are poor.

[0038] Using the Welch Ultimate AQ-C18 (250×4.6mm, 5μm) chromatographic column, the peak positions of chloride, sulfate, phosphate and nitrate ions can be determined, and the peak shape is also acceptable, but the peak position and the chromatographic gradient fluctuation overlap slightly. Although it can also be used, the overall chromatogram effect is not as good as Waters Xselect HSS-T3 (150×4.6mm, 3.5μm). The separation of target ions by different mobile phase systems (100mM ammonium formate aqueous solution (adjusted to pH=3.0 with formic acid)-acetonitrile, 10mM n-octylamine aqueous solution (adjusted to pH=4.5 with glacial acetic acid)-acetonitrile), different diluents (50% acetonitrile, water), and different gradient elution sequences were investigated. According to the peak symmetry and peak separation, the appropriate mobile phase was determined as follows: A: 10mM n-octylamine aqueous solution, adjusted to pH 4.5±0.5, mobile phase B: acetonitrile, gradient elution program:

[0039] Time (min) Mobile phase A (volume %) Mobile phase B (volume %) 0.0 80±2 20±2 5.0±1.0 80±2 20±2 12.0±1.0 70±2 30±2 15.0±1.0 70±2 30±2 15.1±0.2 80±2 20±2 20.0±1.0 80±2 20±2

[0040] 2. Determination of sample preparation method: According to the content range of target ions in the cream preparation, accurately weigh an appropriate amount of the cream preparation, place it in a centrifuge tube, add an appropriate amount of diluent, vortex to make it evenly dispersed, dilute it with the diluent to an appropriate concentration, shake well, filter (preferably using a 0.22 μm nylon 66 filter membrane), and take the filtrate as the test solution.

[0041] 3. Sample determination

[0042] (1) Sample preparation

[0043] 1) Accurately measure appropriate amounts of chloride ion standard solution, nitrate ion standard solution, sulfate ion standard solution, and phosphate ion standard solution, and use water as solvent to prepare reference substance stock solution;

[0044] 2) Accurately measure the reference substance stock solution and use water as solvent to prepare the reference substance solution;

[0045] 3) Accurately weigh an appropriate amount of cream preparation, add an appropriate amount of water, vortex to disperse, transfer to a volumetric flask, dilute with water to the scale, shake well, filter with a 0.22μm filter membrane, and take the filtrate, which is the test solution.

[0046] (2) Testing conditions

[0047] 1) Chromatographic conditions:

[0048]

[0049] 4. Calculation of results

[0050] According to the external standard method, the contents of the four acid radical ions were calculated based on the peak areas.

[0051] Example

[0052] The present invention is described in detail below with reference to the examples. The examples illustrate detailed implementation methods and processes to fully disclose and demonstrate that the invention can be implemented. The examples do not represent limitations on the invention, and the protection scope of the present invention is not limited to the following examples. Based on the specific embodiments of the present invention, all other implementation methods obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0053] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art. The progress of the reaction of the present invention can be monitored by conventional monitoring methods in the art (such as TLC, LCMS or NMR), and the reaction end point is generally when the reaction substrate disappears. The experimental methods for which specific conditions are not indicated in the embodiments of the specification are generally in accordance with conventional conditions in the art or the conditions recommended by the manufacturer.

[0054] In the present invention, unless otherwise specified, "above", "below" or "within" means that the number itself is included.

[0055] Example 1: Quantitative analysis of four acid radical ions in cream preparations using HPLC-CAD method;

[0056] Solution preparation

[0057] Accurately measure appropriate amounts of chloride ion standard solution, nitrate standard solution, sulfate standard solution, and phosphate standard solution, and use water as solvent to prepare reference substance stock solution (stock solution concentration is 200 μg / mL);

[0058] name source batch number Validity Content / concentration Chloride ion standard solution Aladdin J2010109 2021.11.30 1000.00μg / ml Phosphate standard solution Aladdin K2011080 2021.12.14 1000.00μg / ml Sulfate standard solution Aladdin H2006174 2021.12.14 1000.00μg / ml Nitrate standard solution Aladdin H2017213 2021.12.14 1000.00μg / ml

[0059] Accurately measure the reference substance stock solution, use water as solvent, and prepare the reference substance solution (the concentration of the reference substance solution is 50 μg / mL);

[0060] Accurately weigh about 100 mg of lidocaine cream preparation, add appropriate amount of water (about 5 mL), vortex to disperse, transfer to a 10 mL volumetric flask, dilute to scale with water, shake well, filter with a 0.22 μm nylon filter membrane, and take the filtrate, which is the test solution;

[0061] The diluent is water.

[0062] Test conditions

[0063] Chromatographic parameters:

[0064]

[0065]

[0066] Injection order

[0067] Inject the diluent (water), reference solution and test solution into the liquid chromatograph respectively and record the chromatogram.

[0068] Test Results

[0069] The diluent, reference solution and test solution were analyzed separately, and the obtained chromatograms were as follows: Figure 2 As shown, Cl-, PO4 3- 、NO3-、SO4 2- The retention times of the four acid radicals were 3.4min, 3.9min, 4.9min and 12.3min respectively. The contents of the four acid radicals were calculated by the external standard method.

[0070] The calculation formula is as follows:

[0071]

[0072] Table 1 Detection results of 4 kinds of acid radical ions in the preparation

[0073]

[0074] Embodiment 2:

[0075] Specificity investigation of the test method provided in Example 1 of the present invention

[0076] 1) Solution preparation

[0077] Diluent: Water.

[0078] Blank solution: diluent.

[0079] Mixed solution (200 μg / ml): Accurately measure 5 ml each of chloride ion standard solution (1000 μg / ml), nitrate standard solution (1000 μg / ml), sulfate standard solution (1000 μg / ml), and phosphate standard solution (1000 μg / ml), place them in a 25 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0080] Mixed solution of reference substance (50 μg / ml): Accurately measure 2.5 ml of the mixed solution (200 μg / ml), place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0081] Chloride ion positioning solution (200μg / ml): Accurately measure 1ml of chloride ion standard solution (1000μg / ml), place it in a 5ml volumetric flask, dilute to the scale with diluent, and shake well.

[0082] Nitrate ion positioning solution (200μg / ml): Accurately measure 1ml of nitrate standard solution (1000μg / ml), place it in a 5ml volumetric flask, dilute to the scale with diluent, and shake well.

[0083] Sulfate ion positioning solution (200μg / ml): Accurately measure 1ml of sulfate standard solution (1000μg / ml), place it in a 5ml volumetric flask, dilute to the scale with diluent, and shake well.

[0084] Phosphate ion positioning solution (200μg / ml): Accurately measure 1ml of phosphate standard solution (1000μg / ml), place it in a 5ml volumetric flask, dilute to the scale with diluent, and shake well.

[0085] Test solution (10 mg / ml): Accurately weigh about 100 mg of the cream preparation, place it in a 5 ml centrifuge tube, add 2.5 ml of diluent, vortex to make it evenly dispersed, transfer to a 10 ml volumetric flask, dilute to the scale with diluent, shake well, filter with a 0.22 μm filter membrane (nylon 66), and take the filtrate.

[0086] 2) Take samples of each ion localization solution, mixed reference solution and test solution, and the results are as follows Figure 2 As shown. Figure 2 It can be seen that the blank solution has no interference at each target peak, and the method of the present invention has good specificity.

[0087] Embodiment 3:

[0088] Study on the injection precision of the test method provided in Example 1

[0089] 1) Take 6 injections of the reference solution and get the following results:

[0090] Table 2 Phosphate ion injection precision results

[0091]

[0092] Table 3 Chloride ion injection precision results

[0093]

[0094]

[0095] Table 4 Nitrate ion injection precision results

[0096]

[0097] Table 5 Sulfate ion injection precision results

[0098]

[0099] 2) Results of injection precision investigation: The reference solution was injected 6 times in a row, and the retention time RSDs of phosphate ion, chloride ion, nitrate ion, and sulfate ion were all less than 0.10%, and the peak area RSDs were all less than 2.0%, indicating good injection precision.

[0100] Embodiment 4:

[0101] Solution stability study of the test method provided in Example 1

[0102] 1) Take the reference solution and the spiked test solution and inject them at different time points to examine the stability of the solution. The results are as follows:

[0103] Table 6 Phosphate ion solution stability results

[0104]

[0105]

[0106] N / A: Not applicable

[0107] Table 7 Chloride ion solution stability results

[0108]

[0109] Table 8 Nitrate ion solution stability results

[0110]

[0111] Table 9 Sulfate ion solution stability results

[0112]

[0113] Conclusion of stability investigation: the ratio of the peak area of ​​phosphate ion, chloride ion, nitrate ion and sulfate ion reference solution measured after 2, 9, 12 and 14 hours to the peak area at 0h was all between 90% and 110%, indicating that the reference solution was stable within 14h; the ratio of the peak area of ​​the spiked test solution measured after 1, 3, 7 and 11.5 hours to the peak area at 0h was all between 90% and 110%, indicating that the spiked test solution was stable within 11.5h.

[0114] Embodiment 5:

[0115] Investigation of the detection limit and quantification limit of the test method provided in Example 5

[0116] 1) Solution preparation

[0117] Quantitative limit solution: Accurately measure 1 ml of the reference substance mixed solution (50 μg / ml), place it in a 10 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0118] Detection limit solution: Accurately measure 5 ml of LOQ solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0119] 2) Take the detection limit and quantification limit solutions and inject them, and get the following results:

[0120] Table 10 Results of detection limit and quantification limit of phosphate ion

[0121]

[0122] Table 11 Results of detection limit and quantification limit of chloride ion

[0123]

[0124]

[0125] Table 12 Results of detection limit and quantification limit of nitrate ion

[0126]

[0127] Table 13 Results of detection limit and quantification limit of sulfate ion

[0128]

[0129] 3) Results of detection limit and quantification limit precision: The S / N of phosphate ion, chloride ion, nitrate ion and sulfate ion in the detection limit solution was greater than 3; the quantification limit solution was sampled 6 times continuously, and the S / N of phosphate ion, chloride ion, nitrate ion and sulfate ion was greater than 10, and the peak area RSD was no more than 10%. This shows that the detection limit of this method is low and the precision is good. The detection limit of this method for phosphate ion, chloride ion, nitrate ion and sulfate ion is 2.50μg / ml, and the quantification limit is 5μg / ml.

[0130] Embodiment 6:

[0131] Investigation of the linear range of the test method provided in Example 1

[0132] 1) Linear solution preparation

[0133] A mixed reference stock solution (Stock STD) was prepared to prepare a linear solution containing chloride ion, nitrate ion, phosphate ion, and sulfate ion with a LOQ of 150 μg / ml.

[0134] 2) Linear solution sample detection

[0135] Take a series of linear standard solutions (LOQ, L-50%, L-100%, L-150%, L-200%, L-300%), and inject each solution once. Take the ion concentration as the abscissa and the peak area as the ordinate to perform linear fitting. The results are shown in the table below.

[0136] Table 14 Linear results of each ion

[0137]

[0138]

[0139] 3) Linearity test results

[0140] The test results show that the peak area and concentration of phosphate ion, chloride ion, nitrate ion and sulfate ion are linear in the concentration range of 5-150μg / ml, and the correlation coefficient is greater than 0.990, which meets the acceptable standard.

[0141] Embodiment 7:

[0142] Accuracy of the test method provided in Example 1

[0143] 1) Preparation of 0.5% spiked test solution

[0144] Accurately weigh about 100 mg of the cream preparation, place it in a 5 ml centrifuge tube, add 2.5 ml of the mixed reference substance stock solution (200 μg / ml), vortex to disperse it evenly, transfer it to a 10 ml volumetric flask, dilute it to the scale with a diluent, shake it well, filter it with a 0.22 μm filter membrane (nylon 66), and take the filtrate. Prepare 6 portions in parallel.

[0145] 2) Take the spiked sample and inject it. The results are as follows:

[0146] Table 15 Phosphate ion accuracy results

[0147]

[0148] Table 16 Chloride ion accuracy results

[0149]

[0150] Table 17 Nitrate ion accuracy results

[0151]

[0152] Table 18 Sulfate ion accuracy results

[0153]

[0154] 3) Conclusion of accuracy investigation: When the spiked test solution was injected, the recoveries of phosphate ion, chloride ion, nitrate ion and sulfate ion were all in the range of 90% to 110%, indicating that the method had good accuracy.

[0155] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and substitutions that can be thought of by those skilled in the art are included in the present invention, and the protection scope is defined by the attached claims.

Claims

1. A method for simultaneously determining the contents of chloride ions, nitrate ions, phosphate ions and sulfate ions in an ointment preparation by liquid chromatography, characterized in that: The liquid chromatography conditions are as follows: Chromatographic column: C18 column, Mobile phase A: 10±1mM n-octylamine aqueous solution, pH adjusted to 4.5±0.5, Mobile phase B: acetonitrile, Gradient elution program: Detector: CAD detector; The cream preparation sample was prepared according to the following steps: weighing the cream preparation, adding 50% acetonitrile or water as a diluent, uniformly dispersing, and filtering.

2. The method of claim 1, wherein the liquid chromatography conditions further comprise: Flow rate: 1.0±0.2mL / min, and / or, Column temperature: 30±5℃.

3. The method according to claim 1 or 2, wherein the content of chloride ion, nitrate ion, phosphate ion and sulfate ion in the cream preparation is calculated by external standard method.

4. The method according to claim 1 or 2, wherein the liquid chromatography conditions further comprise: The evaporator temperature is 35°C, and / or The power law function value is 1.00, and / or, The acquisition frequency is 5 Hz.

5. The method according to claim 1 or 2, wherein: The pH value of mobile phase A was adjusted with glacial acetic acid.

6. The method according to claim 1 or 2, wherein the pH value of mobile phase A is adjusted to 4.5±0.2.

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