Method for detecting aluminum content in calcium gluconate sodium chloride injection

Abstract

The application discloses a kind of detection methods of the aluminium content in calcium gluconate sodium chloride injection, the detection method is to take 200ml calcium gluconate sodium chloride injection and heat concentration, then take the concentrate and carry out incineration ashing or wet digestion or microwave digestion pretreatment;Again, the sample after pretreatment is with 1% 3-amino-5-hydroxy pyrazole derivative solution, in the aqueous solution system containing acetic acid-ammonium acetate buffer (pH4.5) Derivative reaction occurs, reaction time is about 15 minutes;Finally, with ZORBAXEclipse Plus (C18) (250*4.6mm, 5 μm) chromatographic column, methanol-10mM ammonium acetate containing 0.1% formic acid aqueous solution (40:60) mixed solution as mobile phase, 254nm detection wavelength under high performance liquid chromatography-ultraviolet detection method is detected.The detection method has the advantages of high sensitivity, almost no detection interference, good accuracy, stable aluminum derivative formed, no high toxicity reagent in test process, shorter analysis time, etc., can effectively solve the deficiencies existing in the prior art.Can effectively control the aluminum in calcium gluconate sodium chloride injection, and very good ensure the quality of calcium gluconate sodium chloride injection.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analytical chemistry and relates to a method for detecting aluminum content in calcium gluconate sodium chloride injection. Specifically, it relates to a method for separating and determining aluminum content in calcium gluconate sodium chloride injection using sample pretreatment-derivation-high performance liquid chromatography-ultraviolet detection. Background Technology

[0002] The Center for Drug Evaluation of the National Medical Products Administration (NMPA) clearly states in its list of common issues concerning generic chemical drugs that aluminum has certain nephrotoxicity, necessitating research and control of aluminum content in large-volume parenteral nutrition (TPN) injections, with a control limit of no more than 25 μg / L. Therefore, research and control of aluminum content in calcium gluconate sodium chloride injection are required; however, there are currently no literature reports on methods for determining aluminum content in calcium gluconate sodium chloride injection.

[0003] Existing methods for detecting trace / ultra-trace aluminum content in various industries (such as metallurgy, food, water, and pharmaceuticals) mainly include direct detection methods such as ICP-AES (inductively coupled plasma atomic emission spectrometry), ICP-OES (inductively coupled plasma atomic emission spectrometry), ICP-MS (inductively coupled plasma mass spectrometry), X-ray fluorescence spectrometry, and atomic absorption spectrophotometry.

[0004] ICP-AES, ICP-OES, ICP-MS, and X-ray fluorescence spectrometry instruments are uncommon, and most pharmaceutical companies do not possess them, requiring outsourcing for testing. This leads to delays in drug release, causing significant inconvenience. Furthermore, the equipment and testing costs are high, making them unsuitable for pharmaceutical QC testing. Industry feedback generally indicates that atomic absorption spectrophotometry suffers from instability due to the low atomization rate of aluminum, resulting in numerous deviations during pharmaceutical QC testing and hindering accurate and timely feedback on drug quality, thus affecting drug release.

[0005] Few studies report the detection of trace / ultra-aluminum in samples by high-performance liquid chromatography after reacting with derivatizing reagents. Detectors include fluorescence detectors, mass spectrometry detectors, evaporative light scattering detectors, differential refractive index detectors, and ultraviolet-visible spectrophotometers (including diode array detectors).

[0006] Evaporative light scattering (ELS) detectors, differential refractive index (DRI) detectors, mass spectrometry (MS) detectors, and fluorescence detectors are all uncommon. ELS and DRI detectors are general-purpose detectors with low selectivity, primarily suitable for compounds lacking UV absorption and fluorescence. Their sensitivity is low, often requiring increased sample concentration to achieve detection. Furthermore, for complex matrix solutions formed by trace / ultra-level aluminum reactions with derivatizing reagents, every component can be detected indiscriminately, leading to significant interference, especially at higher sample concentrations. This results in inaccurate quantitative detection and poor reproducibility. Mass spectrometry detectors are only used by a few companies and are expensive. The high-salt, non-volatile matrix in derivatized solutions easily contaminates the ion source, increasing instrument maintenance costs and making them unsuitable for the high-frequency release testing required by manufacturers. Additionally, their narrow linear range makes it difficult to control trace / constant amounts of aluminum effectively. Fluorescence detectors have limited light source lifespan, requiring periodic replacement, increasing maintenance costs. The instruments themselves are also relatively expensive, increasing initial costs. The derivatization reaction requires a fluorescent reagent to react with trace amounts of aluminum. However, the fluorescence intensity of the fluorescent derivatized product is easily affected by environmental factors such as solvent polarity, pH value, and temperature, requiring strict control of experimental conditions. Under light irradiation, photolysis or photobleaching can easily occur, leading to signal attenuation and a narrow linear range, making it difficult to control trace amounts of aluminum effectively.

[0007] Literature reports the use of stilbene (also known as stilbene-4,4-bis(1-azo)-3,4-dihydroxybenzene-2,2'-disulfonate ammonium or stilbene-4,4-bis(1-azo)-3,4-dihydroxybenzene-2,2'-disulfonate ammonium) as a derivatizing reagent to react with aluminum in water samples collected from ores or oil fields. High-performance liquid chromatography-ultraviolet (HPLC-UV) detection was then used to determine the aluminum content in these water samples (Reference 1: Separation and determination of scandium, tin, and aluminum by reversed-phase ion-pair HPLC, colorimetric analysis). References 1-474 (Reference 1: Separation and Determination of Aluminum, Gallium and Indium by Reversed-Phase Ion-Pair High Performance Liquid Chromatography, Analytical Laboratory, Vol. 18, No. 2, March 1999; Reference 2: Determination of Aluminum, Gallium and Indium Mixtures by High Performance Liquid Chromatography, Liaoning Chemical Industry, Vol. 36, No. 9, September 2007: 645-647) clearly indicate that the complexes in this method have poor stability, requiring 15 minutes of color development before injection, and separation and detection must be completed within 135 minutes. For multi-batch release testing, this requires constant monitoring of the samples, greatly increasing testing costs and causing significant inconvenience.

[0008] Literature reports the use of chlorosulfonylphenol S [also known as chlorosulfonylphenol S or chlorosulfonic acid C, chemical name 2,7-bis(5-chloro-2-hydroxy-3-sulfophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid or 2,7-bis(5-chloro-2-hydroxy-3-sulfophenylazo)chromotropic acid] as a derivatizing reagent to react with aluminum in water / flour / rice / human hair in a boiling water bath. The aluminum content in water / flour / rice / human hair was then detected by high performance liquid chromatography-ultraviolet detection [Literature: Separation and determination of aluminum(III), chromium(III), niobium(V) and copper(II) by reversed-phase high performance liquid chromatography with chlorosulfonylphenol S, Journal of Southwest Normal University (Natural Science Edition), Vol.19, No.3, June 1994: 260-264]. In this method, the detection wavelength for aluminum derivatives is 570 nm, which falls within the visible light region. In routine high-performance liquid chromatography-ultraviolet (HPLC-UV) detection methods, the detection wavelength is typically in the UV range of 200-400 nm. The visible light region has lower sensitivity, and for the detection of trace / ultra-level aluminum, it is difficult to avoid interference from the complex matrix of the derivatized solution at low concentrations, leading to inaccurate determination.

[0009] Literature reports the use of 8-hydroxyquinine as a derivatizing reagent to react with aluminum in water, followed by extraction with chloroform, and then detection of aluminum salt content in water using high-performance liquid chromatography-ultraviolet detection [Reference 1: Extraction of aluminum in drinking water - high-performance liquid chromatography analysis, Yunnan Environmental Science, Vol. 19 Supplement, August 2000: 259-261; Reference 2: Extraction and high-performance liquid chromatography analysis of tris(8-hydroxyquinine)aluminum(III) complexes, Journal of Inorganic Chemistry, Vol. 16, No. 4, July 2000: 637-640]. This literature reports that the method underwent an interference test with calcium (Ca), with calcium interfering with aluminum detection by a factor of 50, and that the method used highly toxic chloroform as the extractant.

[0010] In general, there are currently no literature reports on methods for determining the aluminum content in calcium gluconate sodium chloride injection. There are also almost no reports on the determination of aluminum using high-performance liquid chromatography-ultraviolet (HPLC-UV) after a derivatization reaction between a derivatizing agent and aluminum in the pharmaceutical field. The few existing reports on the determination of aluminum using HPLC-UV after a derivatization reaction between a derivatizing agent and aluminum in other fields (such as water and ores) are problematic. These methods suffer from various issues: either the aluminum derivatives are unstable; the detection wavelength is in the visible light region, leading to low sensitivity and significant interference; or even if the aluminum derivative formed by 8-hydroxyquinine as a derivatizing agent is stable and its absorption wavelength is in the ultraviolet region, it is easily interfered with by calcium (experimental results show that calcium interference can be up to 50 times greater), and various other problems arise from the use of highly toxic extractant chloroform. Summary of the Invention

[0011] The purpose of this invention is to address the aforementioned problems by providing a method for detecting aluminum content in calcium gluconate sodium chloride injection. This method employs sample pretreatment-derivation-high performance liquid chromatography-ultraviolet (UV) chromatography. This method offers advantages such as high sensitivity, virtually no detection interference, good accuracy, stable aluminum derivatives formed, no highly toxic reagents involved in the experiment, and short analysis time, effectively overcoming the shortcomings of existing technologies. It can effectively control aluminum content in calcium gluconate sodium chloride injection, ensuring the quality of the injection.

[0012] The inventors screened many derivatizing reagents and found from a large amount of experimental data that the aluminum derivative formed by the derivatization reaction of 3-amino-5-hydroxypyrazole with aluminum has good stability, is not affected by the matrix such as calcium ions and sodium ions in calcium gluconate sodium chloride injection, has good sensitivity, and the optimal absorption wavelength is in the ultraviolet absorption region.

[0013] The inventors conducted numerous experiments, and the experimental data showed that pretreatment of calcium gluconate sodium chloride injection solution, similar to ignition ashing, wet digestion, and microwave digestion, yields better accuracy, less interference, and better specificity.

[0014] The inventors screened the derivatization reaction conditions. Experimental data showed that adding 10 ml of acetate-ammonium acetate buffer (pH 4.5), 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) and the sample to be derivatized, then diluting with water to 50 ml, shaking well, and letting stand for 15 minutes, the derivatization reaction could be completed. The resulting derivatized product had good stability, less interference, and better specificity.

[0015] The inventors screened multiple brands and models of chromatographic columns and under different chromatographic conditions. Experimental data showed that when the chromatographic column was a ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm) and the mobile phase was a mixture of methanol and buffer solution, the detection method had good sensitivity and specificity. The buffer solution was a 0.1% formic acid aqueous solution, a 0.1% formic acid aqueous solution containing 10mM ammonium acetate, or a 0.1% formic acid aqueous solution containing 10mM potassium dihydrogen phosphate.

[0016] In an embodiment of the present invention, the present invention provides a method for detecting aluminum content in calcium gluconate sodium chloride injection, wherein the detection method employs sample pretreatment-derivation-high performance liquid chromatography-ultraviolet light method.

[0017] In an embodiment of the present invention, the detection method of the present invention includes sample pretreatment by heating and concentrating 200 ml of calcium gluconate sodium chloride injection; further processing the concentrate, wherein the further processing method is wet digestion, ignition ashing, and microwave digestion, preferably ignition ashing and microwave digestion, and more preferably microwave digestion.

[0018] In an embodiment of the present invention, the detection method of the present invention includes the following detailed steps for wet digestion: transfer the concentrate to a digestion tube, add 5 ml of nitric acid, gently shake, let stand for 30 minutes, then add 5 ml of hydrogen peroxide, gently shake, let stand for 30 minutes, place in a heating plate, and heat at 150°C for 2 hours.

[0019] In an embodiment of the present invention, the detection method of the present invention includes the following detailed steps for ignition ashing: the concentrate is transferred to a crucible, slowly ignited until completely carbonized, cooled, moistened with sulfuric acid, heated at a low temperature until the sulfuric acid vapor is removed, and then ignited at 500-550°C to achieve complete ashing.

[0020] In an embodiment of the present invention, the detection method of the present invention includes the following detailed steps for microwave digestion: The concentrate is transferred to a digestion tube, 5 ml of nitric acid is added, and microwave digestion is performed for 1 hour at a temperature of 200°C, a power of 1200 W, and a pressure of 10 MPa. Then, the tube is placed on a hot plate and subjected to acid removal at 150°C for 1 hour, followed by cooling for later use.

[0021] In an embodiment of the present invention, the detection method of the present invention, wherein the derivatization is to react a pretreated sample with a 3-amino-5-hydroxypyrazole derivatizing reagent to form an aluminum derivative with the 3-amino-5-hydroxypyrazole derivatizing reagent.

[0022] In an embodiment of the present invention, the detection method of the present invention, wherein the specific conditions for the derivatization reaction are as follows: 10 ml of acetate-ammonium acetate buffer (pH 4.5), 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) and the sample to be derivatized are placed in a volumetric flask, diluted with water to 50 ml, shaken well, and left to stand for 15 minutes to complete the derivatization reaction.

[0023] In an embodiment of the present invention, the detection method of the present invention, wherein the high performance liquid chromatography-ultraviolet method is performed using a high performance liquid chromatograph, an ultraviolet-visible spectrophotometer (including a diode array detector) as the detector, a chromatographic column packed with octadecylsilane-bonded silica gel, and a mobile phase of a mixed solution of methanol and buffer solution.

[0024] In an embodiment of the present invention, the detection method of the present invention uses a ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm) chromatographic column.

[0025] In an embodiment of the present invention, the detection method of the present invention includes a buffer solution of 0.1% formic acid aqueous solution, 0.1% formic acid aqueous solution containing 10mM ammonium acetate, or 0.1% formic acid aqueous solution containing 10mM potassium dihydrogen phosphate, preferably 0.1% formic acid aqueous solution containing 10mM ammonium acetate.

[0026] In an embodiment of the present invention, the detection method of the present invention wherein the mobile phase is a mixed solution of methanol-buffered buffer (35-45:65-55), preferably a mixed solution of methanol-buffered buffer (40:60).

[0027] In an embodiment of the present invention, the detailed steps of the detection method of the present invention, namely, sample pretreatment by wet digestion-derivation-high performance liquid chromatography-ultraviolet light method, are as follows:

[0028] (1) Solution preparation

[0029] Acetic acid-ammonium acetate buffer (pH 4.5): Dissolve 77g of ammonium acetate in 500ml of water, then add 60ml of glacial acetic acid and an appropriate amount of water to make 1000ml. Shake well to obtain the buffer.

[0030] Diluent (required for preparing derivatizing agent solution): Take 500ml of methanol, add 100ml of acetate-ammonium acetate buffer (pH 4.5), then dilute with water to 1000ml, shake well, and the solution is ready.

[0031] Derivatizing agent solution (1% 3-amino-5-hydroxypyrazole): Weigh 5g of 3-amino-5-hydroxypyrazole accurately, place it in a 500ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute to the mark with diluent. Shake well to obtain the solution.

[0032] Mobile phase: Take 1600 ml of acetonitrile and 2400 ml of 0.1% formic acid aqueous solution containing 10 mM ammonium acetate, place them together in a mobile phase bottle, shake well, and sonicate to obtain the mobile phase.

[0033] (2) Preparation of blank solution

[0034] Blank solution: Accurately transfer 10 ml of acetate-ammonium acetate buffer (pH 4.5) and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the blank solution.

[0035] (3) Preparation of reference solution

[0036] Reference solution: Accurately transfer 1 ml of 5 μg / ml aluminum single element standard solution, 10 ml of acetate-ammonium acetate buffer (pH 4.5), and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the solution.

[0037] (4) Sample pretreatment

[0038] Concentration: Take 200ml of calcium gluconate sodium chloride injection solution and place it in a beaker. Heat it on an electric furnace to concentrate it to about 5ml.

[0039] Wet digestion: Transfer the above concentrate to a digestion tube, add 5 ml of nitric acid, shake gently, let stand for 30 minutes, then add 5 ml of hydrogen peroxide, shake gently, let stand for 30 minutes, place in a heating plate, and heat at 150°C for 2 hours.

[0040] (5) Preparation of test solution

[0041] Test solution: Transfer the pretreated sample to a 50ml volumetric flask, then accurately transfer 10ml of acetate-ammonium acetate buffer (pH 4.5) and 10ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the test solution.

[0042] (6) Measurement

[0043] Column type: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); Detection wavelength: 254nm; Injection volume: 20μl; Column temperature: 35℃; Flow rate: 0.8ml / min; Run time: isocratic run for 30 minutes. Calculated by peak area using the external standard method.

[0044] In an embodiment of the present invention, the detailed steps of the detection method of the present invention, namely the sample pretreatment by incineration ashing-derivation-high performance liquid chromatography-ultraviolet light method, are as follows:

[0045] (1) Solution preparation

[0046] Acetic acid-ammonium acetate buffer (pH 4.5): Dissolve 77g of ammonium acetate in 500ml of water, then add 60ml of glacial acetic acid and an appropriate amount of water to make 1000ml. Shake well to obtain the buffer.

[0047] Diluent (required for preparing derivatizing agent solution): Take 500ml of methanol, add 100ml of acetate-ammonium acetate buffer (pH 4.5), then dilute with water to 1000ml, shake well, and the solution is ready.

[0048] Derivatizing agent solution (1% 3-amino-5-hydroxypyrazole): Weigh 5g of 3-amino-5-hydroxypyrazole accurately, place it in a 500ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute to the mark with diluent. Shake well to obtain the solution.

[0049] Mobile phase: Take 1600 ml of acetonitrile and 2400 ml of 0.1% formic acid aqueous solution containing 10 mM ammonium acetate, place them together in a mobile phase bottle, shake well, and sonicate to obtain the mobile phase.

[0050] (2) Preparation of blank solution

[0051] Blank solution: Accurately transfer 10 ml of acetate-ammonium acetate buffer (pH 4.5) and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the blank solution.

[0052] (3) Preparation of reference solution

[0053] Reference solution: Accurately transfer 1 ml of 5 μg / ml aluminum single element standard solution, 10 ml of acetate-ammonium acetate buffer (pH 4.5), and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the solution.

[0054] (4) Sample pretreatment

[0055] Concentration: Take 200ml of calcium gluconate sodium chloride injection solution and place it in a beaker. Heat it on an electric furnace to concentrate it to about 5ml.

[0056] Incineration and ashing: Transfer the above concentrate to a crucible, slowly incinerate until completely carbonized, cool, add sulfuric acid to moisten, heat at low temperature until sulfuric acid vapor is removed, and then incinerate at 500-550℃ to completely ashing.

[0057] (5) Preparation of test solution

[0058] Test solution: Transfer the pretreated sample to a 50ml volumetric flask, then accurately transfer 10ml of acetate-ammonium acetate buffer (pH 4.5) and 10ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the test solution.

[0059] (6) Measurement

[0060] Column type: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); Detection wavelength: 254nm; Injection volume: 20μl; Column temperature: 35℃; Flow rate: 0.8ml / min; Run time: isocratic run for 30 minutes. Calculated by peak area using the external standard method.

[0061] In an embodiment of the present invention, the detailed steps of the detection method of the present invention, namely, sample pretreatment using microwave digestion-derivation-high performance liquid chromatography-ultraviolet light method, are as follows:

[0062] (1) Solution preparation

[0063] Acetic acid-ammonium acetate buffer (pH 4.5): Dissolve 77g of ammonium acetate in 500ml of water, then add 60ml of glacial acetic acid and an appropriate amount of water to make 1000ml. Shake well to obtain the buffer.

[0064] Diluent (required for preparing derivatizing agent solution): Take 500ml of methanol, add 100ml of acetate-ammonium acetate buffer (pH 4.5), then dilute with water to 1000ml, shake well, and the solution is ready.

[0065] Derivatizing agent solution (1% 3-amino-5-hydroxypyrazole): Weigh 5g of 3-amino-5-hydroxypyrazole accurately, place it in a 500ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute to the mark with diluent. Shake well to obtain the solution.

[0066] Mobile phase: Take 1600 ml of acetonitrile and 2400 ml of 0.1% formic acid aqueous solution containing 10 mM ammonium acetate, place them together in a mobile phase bottle, shake well, and sonicate to obtain the mobile phase.

[0067] (2) Preparation of blank solution

[0068] Blank solution: Accurately transfer 10 ml of acetate-ammonium acetate buffer (pH 4.5) and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the blank solution.

[0069] (3) Preparation of reference solution

[0070] Reference solution: Accurately transfer 1 ml of 5 μg / ml aluminum single element standard solution, 10 ml of acetate-ammonium acetate buffer (pH 4.5), and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the solution.

[0071] (4) Sample pretreatment

[0072] Concentration: Take 200ml of calcium gluconate sodium chloride injection solution and place it in a beaker. Heat it on an electric furnace to concentrate it to about 5ml.

[0073] Microwave digestion: Transfer the above concentrate to a digestion tube, add 5 ml of nitric acid, and microwave digest for 1 hour at 200℃, 1200W, and 10MPa. Then place it on a hot plate and remove the acid at 150℃ for 1 hour. Cool and set aside for later use.

[0074] (5) Preparation of test solution

[0075] Test solution: Transfer the pretreated sample to a 50ml volumetric flask, then accurately transfer 10ml of acetate-ammonium acetate buffer (pH 4.5) and 10ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the test solution.

[0076] (6) Measurement

[0077] Column type: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); Detection wavelength: 254nm; Injection volume: 20μl; Column temperature: 35℃; Flow rate: 0.8ml / min; Run time: isocratic run for 30 minutes. Calculated by peak area using the external standard method. Attached Figure Description

[0078] Figure 1 This represents the spectrum of the blank excipient solution under the "Specificity" item in the method verification described in Example 1.

[0079] Figure 2 This represents the chromatogram of the reference solution under the "Specificity" item in the method verification described in Example 1.

[0080] Figure 3 The image shows the chromatogram of the 100% spiked test solution under the "Specificity" item in the method verification described in Example 1.

[0081] Figure 4 This represents the spectrum of the limit-of-quantity solution under the "Linearity and Range" section in the method verification described in Example 1. Detailed Implementation

[0082] 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.

[0083] The reagents used in the following examples are commercially available.

[0084] Example 1

[0085] 1.1 Detection Method

[0086] 1.1.1 Solution Preparation

[0087] Acetic acid-ammonium acetate buffer (pH 4.5): Dissolve 77g of ammonium acetate in 500ml of water, then add 60ml of glacial acetic acid and an appropriate amount of water to make 1000ml. Shake well to obtain the buffer.

[0088] Diluent (required for preparing derivatizing agent solution): Take 500ml of methanol, add 100ml of acetate-ammonium acetate buffer (pH 4.5), then dilute with water to 1000ml, shake well, and the solution is ready.

[0089] Derivatizing agent solution (1% 3-amino-5-hydroxypyrazole): Weigh 5g of 3-amino-5-hydroxypyrazole accurately, place it in a 500ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute to the mark with diluent. Shake well to obtain the solution.

[0090] 1.1.2 Blank solution

[0091] Blank solution: Accurately transfer 10 ml of acetate-ammonium acetate buffer (pH 4.5) and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the blank solution.

[0092] 1.1.3 Reference Solution

[0093] Reference solution: Accurately transfer 1 ml of 5 μg / ml aluminum single element standard solution, 10 ml of acetate-ammonium acetate buffer (pH 4.5), and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the solution.

[0094] 1.1.4 Sample Pretreatment

[0095] Concentration: Take 200ml of calcium gluconate sodium chloride injection solution and place it in a beaker. Heat it on an electric furnace to concentrate it to about 5ml.

[0096] Microwave digestion: Transfer the above concentrate to a digestion tube, add 5 ml of nitric acid, and microwave digest for 1 hour at 200℃, 1200W, and 10MPa. Then place it on a hot plate and remove the acid at 150℃ for 1 hour. Cool and set aside for later use.

[0097] 1.1.5 Test solution

[0098] Test solution: Transfer the pretreated sample to a 50ml volumetric flask, then accurately transfer 10ml of acetate-ammonium acetate buffer (pH 4.5) and 10ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the test solution.

[0099] 1.1.6 Chromatographic conditions

[0100] Mobile phase: Take 1600 ml of acetonitrile and 2400 ml of 0.1% formic acid aqueous solution containing 10 mM ammonium acetate, put them together in a mobile phase bottle, shake well, and sonicate to obtain the mobile phase.

[0101] Column type: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); Detection wavelength: 254nm; Injection volume: 20μl; Column temperature: 35℃; Flow rate: 0.8ml / min; Run time: 30 minutes of isocratic running.

[0102] 1.1.7 Measurement

[0103] Determination: Inject 20 μL of the blank solution, reference solution, and test solution (one injection each) into the high-performance liquid chromatograph (HPLC) under the chromatographic conditions described above, and record the chromatogram. Calculate the aluminum content using the external standard method based on peak area. The aluminum content must not exceed 25 μg / L.

[0104] 1.2 Method Validation

[0105] 1.2.1 Specificity

[0106] 1.2.1.1 Blank solution 1

[0107] Blank solution 1: Accurately transfer 10 ml of acetate-ammonium acetate buffer (pH 4.5) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the blank solution.

[0108] 1.2.1.2 Blank solution 2

[0109] Blank solution 2: Accurately transfer 10 ml of acetate-ammonium acetate buffer (pH 4.5) and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the blank solution.

[0110] 1.2.1.3 Blank excipient solution

[0111] Preparation of blank excipients: Weigh 450 mg calcium gluconate and 337.5 mg sodium chloride accurately, place them in a 500 ml volumetric flask, add an appropriate amount of water to dissolve, and dilute with water to the mark. Shake well to obtain the blank excipient.

[0112] Blank excipient pretreatment: Place 200 ml of blank excipient in a beaker and heat on an electric furnace to concentrate to approximately 5 ml. Transfer the concentrate to a digestion tube, add 5 ml of nitric acid, and microwave digest for 1 hour at 200°C, 1200 W, and 10 MPa. Then place on a hot plate and remove acid at 150°C for 1 hour. Cool and set aside for later use.

[0113] Blank excipient solution: Transfer the pretreated sample to a 50ml volumetric flask, then accurately transfer 10ml of acetate-ammonium acetate buffer (pH 4.5) and 10ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the blank excipient solution.

[0114] 1.2.1.4 Reference Solution

[0115] Reference solution: Prepare the same reference solution as described in section "1.1.3" of Example 1.

[0116] 1.2.1.5 Test solution

[0117] Test solution: Prepare the test solution as described in section "1.1.5" of Example 1.

[0118] 1.2.1.6 100% spiked test solution

[0119] 100% spiked test solution: Take the sample obtained by the same operation as the pretreated sample under "1.4" in Example 1, place it in a 50ml volumetric flask, then accurately transfer 1ml of 5μg / ml aluminum single element standard solution, 10ml of acetate-ammonium acetate buffer (pH 4.5) and 10ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the solution.

[0120] 1.2.1.7 Measurement

[0121] Determination: Inject 20 μl of each of the following solutions into the high-performance liquid chromatograph (HPLC): blank solution 1, blank solution 2, blank excipient solution, reference solution, test solution, and 100% spiked test solution (1 injection each). Inject under the chromatographic conditions described above and record the chromatogram. Note that blank solution 1, blank solution 2, blank excipient solution, and test solution must not interfere with the determination of aluminum derivatives in this product.

[0122] 1.2.2 System Applicability

[0123] 1.2.2.1 Reference solution

[0124] Reference solution: Prepare the same reference solution as described in section "1.1.3" of Example 1.

[0125] 1.2.2.2 100% spiked test solution

[0126] 100% spiked test solution: Prepare the 100% spiked test solution as described in section “1.2.1.6” of Example 1.

[0127] 1.2.2.3 Measurement

[0128] Determination: Inject 20 μl of the above reference solution (5 injections) and 100% spiked test solution (1 injection) into the high-performance liquid chromatograph (HPLC) under the above chromatographic conditions and record the chromatogram. The RSD of the peak area of ​​the aluminum derivative in the 5 consecutive injections of the reference solution should not exceed 2%, and the resolution between the aluminum derivative peak and adjacent peaks in the 100% spiked test solution should be greater than 1.5.

[0129] 1.2.3 Limit of Quantitation and Limit of Detection

[0130] 1.2.3.1 Quantitative limit solution

[0131] Limit of Quantification Solution: Take an appropriate amount of the reference solution, add an appropriate amount of acetate-ammonium acetate buffer (pH 4.5), and dilute with water until the signal-to-noise ratio of the aluminum salt derivative peak is about 10.

[0132] 1.2.3.2 Detection Limit Solution

[0133] Detection limit solution: Take an appropriate amount of the reference solution, add an appropriate amount of acetate-ammonium acetate buffer (pH 4.5), and dilute with water until the signal-to-noise ratio of the aluminum salt derivative peak is about 3.

[0134] 1.2.3.4 Measurement

[0135] Determination: Inject 20 μl of the above-mentioned limit of quantitation (LOQ) solution (6 injections) and limit of detection (LOD) solution (3 injections) into the high-performance liquid chromatograph (HPLC) under the chromatographic conditions described above, and record the chromatogram. For the LQ solution, inject 6 injections consecutively, ensuring the peak area RSD of the aluminum derivative peak is not greater than 20%, the signal-to-noise ratio is not less than 10, and the concentration of the LQ solution is not greater than 10% of the limit concentration. For the LOD solution, inject 3 injections consecutively, ensuring the signal-to-noise ratio is not less than 3.

[0136] 1.2.4 Linearity and Range

[0137] 1.2.4.1 Linear Solutions

[0138] 30% linear solution: Accurately transfer 0.3 ml of 5 μg / ml aluminum single element standard solution, 10 ml of acetate-ammonium acetate buffer (pH 4.5), and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the solution.

[0139] 50% linear solution: Accurately transfer 0.5 ml of 5 μg / ml aluminum single element standard solution, 10 ml of acetate-ammonium acetate buffer (pH 4.5), and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the solution.

[0140] 100% linear solution: Accurately transfer 1 ml of 5 μg / ml aluminum single-element standard solution, 10 ml of acetate-ammonium acetate buffer (pH 4.5), and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the solution.

[0141] 150% linear solution: Accurately transfer 1.5 ml of 5 μg / ml aluminum single-element standard solution, 10 ml of acetate-ammonium acetate buffer (pH 4.5), and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the solution.

[0142] 200% linear solution: Accurately transfer 2 ml of 5 μg / ml aluminum single-element standard solution, 10 ml of acetate-ammonium acetate buffer (pH 4.5), and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the solution.

[0143] 1.2.4.2 Measurement

[0144] Determination: Inject 20 μl of each of the above linear solutions (one injection each) into the high-performance liquid chromatograph (HPLC) under the chromatographic conditions described above, and record the chromatogram. The solution should be linear within the range of the limit of quantitation (LOQ) to 200% of the limit concentration, with a regression correlation coefficient (r) ≥ 0.999, and the ratio of the absolute value of the Y-axis intercept to the response value at 100% of the limit concentration should be within 25%.

[0145] 1.2.5 Accuracy

[0146] 1.2.5.1 Reference Solution

[0147] Reference solution: Prepare the same reference solution as described in section "1.1.3" of Example 1.

[0148] 1.2.5.2 Background Solution

[0149] Background solution: Prepare the same test solution as in item "1.1.5" of Example 1.

[0150] 1.2.5.3 Recovery rate solution

[0151] 50% Spiked Test Solution: Take the sample obtained by the same procedure as the pretreated sample under section "1.4" in Example 1, place it in a 50 ml volumetric flask, and then accurately transfer 0.5 ml of 5 μg / ml aluminum single-element standard solution, 10 ml of acetate-ammonium acetate buffer (pH 4.5), and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into the flask. Dilute to the mark with water, shake well, and let stand for 15 minutes. (Prepare 3 parallel portions)

[0152] 100% spiked test solution: Prepare the 100% spiked test solution as described in section “1.2.1.6” of Example 1. (Prepare 3 portions in parallel)

[0153] 150% Spiked Test Solution: Take the sample obtained by the same procedure as the pretreated sample under section "1.4" in Example 1, place it in a 50 ml volumetric flask, and then accurately transfer 1.5 ml of 5 μg / ml aluminum single-element standard solution, 10 ml of acetate-ammonium acetate buffer (pH 4.5), and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into the flask. Dilute to the mark with water, shake well, and let stand for 15 minutes. (Prepare 3 parallel portions)

[0154] 1.2.5.4 Measurement

[0155] Determination: Inject 20 μl of the above-mentioned reference solution, background solution, and spiked solutions (recovery solutions) (one injection each) into the high-performance liquid chromatograph (HPLC) under the chromatographic conditions described above, and record the chromatograms. The recoveries of the 150%, 100%, and 50% recovery solutions at the limit concentrations were all between 80% and 120%, and the RSDs of the recovered solutions were no greater than 20%.

[0156] 1.2.6 Precision - Repeatability (Operator A)

[0157] 1.2.6.1 Blank solution

[0158] Blank solution: Prepare the blank solution as described in section "1.1.2" of Example 1.

[0159] 1.2.6.2 Reference Solution

[0160] Reference solution: Prepare the same reference solution as described in section "1.1.3" of Example 1.

[0161] 1.2.6.3 100% spiked test solution

[0162] 100% spiked test solution: Prepare the 100% spiked test solution as described in section “1.2.1.6” of Example 1. (Prepare 6 portions in parallel)

[0163] 1.2.6.4 Measurement

[0164] Determination: Inject 20 μl of the above blank solution, reference solution, and 100% spiked test solution (one injection of each) into the high-performance liquid chromatograph (HPLC) under the above chromatographic conditions and record the chromatogram. The RSD of aluminum salt content in the six parallel 100% spiked test solutions prepared by operator A should not exceed 20%.

[0165] 1.2.7 Precision - Intermediate Precision (Operator B)

[0166] 1.2.7.1 Blank solution

[0167] Blank solution: Prepare the blank solution as described in section "1.1.2" of Example 1.

[0168] 1.2.7.2 Reference Solution

[0169] Reference solution: Prepare the same reference solution as described in section "1.1.3" of Example 1.

[0170] 1.2.7.3 100% spiked test solution

[0171] 100% spiked test solution: Prepare the 100% spiked test solution as described in section “1.2.1.6” of Example 1. (Prepare 6 portions in parallel)

[0172] 1.2.7.4 Measurement

[0173] Determination: Inject 20 μl of the above blank solution, reference solution, and each 100% spiked test solution (one injection each) into the high-performance liquid chromatograph (HPLC) under the chromatographic conditions described above, and record the chromatograms. For operator B, the RSD of aluminum salt content in 6 parallel 100% spiked test solutions should not exceed 20%; the RSD of aluminum salt content in 12 parallel solutions should not exceed 20%.

[0174] 1.2.8 Solution stability

[0175] 1.2.8.1 Reference solution

[0176] Reference solution: Prepare the same reference solution as described in section "1.1.3" of Example 1.

[0177] 1.2.8.2 100% spiked test solution

[0178] 100% spiked test solution: Prepare the 100% spiked test solution as described in section “1.2.1.6” of Example 1.

[0179] 1.2.8.3 Measurement

[0180] Determination: Under room temperature conditions, at approximately 0h, 2h, 4h, 8h, 16h, 24h, 48h, and 72h, inject 20μl of the reference solution and each 100% spiked test solution (one injection each) into the high-performance liquid chromatograph (HPLC), and record the chromatograms according to the chromatographic conditions described above. The recoveries of the reference solution at each time point should be within the range of 90–110%; the relative deviation of the aluminum salt content in the 100% spiked test solution measured at each time point from that at 0h should not exceed 20%.

[0181] 1.2.9 Durability

[0182] 1.2.9.1 Blank solution

[0183] Blank solution: Prepare the blank solution as described in section "1.1.2" of Example 1.

[0184] 1.2.9.2 Reference Solution

[0185] Reference solution: Prepare the same reference solution as described in section "1.1.3" of Example 1.

[0186] 1.2.9.3 100% spiked test solution

[0187] 100% spiked test solution: Prepare the 100% spiked test solution as described in section “1.2.1.6” of Example 1.

[0188] 1.2.9.4 Measurement

[0189] Determination: Inject 20 μl of the above blank solution, reference solution, and 100% spiked test solution (one injection of each) into the high-performance liquid chromatograph (HPLC). Inject under each robustness chromatographic condition (robustness condition 1: column temperature 40℃; robustness condition 2: column temperature 30℃; robustness condition 3: flow rate 0.7 ml / min; robustness condition 4: flow rate 0.9 ml / min) and record the chromatogram. The RSD of the aluminum salt content in the 100% spiked test solution under each robustness condition compared to the normal condition should not exceed 20%.

[0190] 1.3 Statistical Analysis of Method Validation Results

[0191] Example 2

[0192] 2.1 Detection Method

[0193] 2.1.1 Solution Preparation

[0194] Acetic acid-ammonium acetate buffer (pH 4.5): Dissolve 77g of ammonium acetate in 500ml of water, then add 60ml of glacial acetic acid and an appropriate amount of water to make 1000ml. Shake well to obtain the buffer.

[0195] Diluent (required for preparing derivatizing agent solution): Take 500ml of methanol, add 100ml of acetate-ammonium acetate buffer (pH 4.5), then dilute with water to 1000ml, shake well, and the solution is ready.

[0196] Derivatizing agent solution (1% 3-amino-5-hydroxypyrazole): Weigh 5g of 3-amino-5-hydroxypyrazole accurately, place it in a 500ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute to the mark with diluent. Shake well to obtain the solution.

[0197] 2.1.2 Blank solution

[0198] Blank solution: Accurately transfer 10 ml of acetate-ammonium acetate buffer (pH 4.5) and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the blank solution.

[0199] 2.1.3 Reference Solution

[0200] Reference solution: Accurately transfer 1 ml of 5 μg / ml aluminum single element standard solution, 10 ml of acetate-ammonium acetate buffer (pH 4.5), and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the solution.

[0201] 2.1.4 Sample Pretreatment

[0202] Concentration: Take 200ml of calcium gluconate sodium chloride injection solution and place it in a beaker. Heat it on an electric furnace to concentrate it to about 5ml.

[0203] Incineration and ashing: Transfer the above concentrate to a crucible, slowly incinerate until completely carbonized, cool, add sulfuric acid to moisten, heat at low temperature until sulfuric acid vapor is removed, and then incinerate at 500-550℃ to completely ashing.

[0204] 2.1.5 Test solution

[0205] Test solution: Transfer the pretreated sample to a 50ml volumetric flask, then accurately transfer 10ml of acetate-ammonium acetate buffer (pH 4.5) and 10ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the test solution.

[0206] 2.1.6 Chromatographic conditions

[0207] Mobile phase: Take 1600 ml of acetonitrile and 2400 ml of 0.1% formic acid aqueous solution containing 10 mM ammonium acetate, put them together in a mobile phase bottle, shake well, and sonicate to obtain the mobile phase.

[0208] Column type: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); Detection wavelength: 254nm; Injection volume: 20μl; Column temperature: 35℃; Flow rate: 0.8ml / min; Run time: 30 minutes of isocratic running.

[0209] 2.1.7 Measurement

[0210] Determination: Inject 20 μl of each of the above blank solution, reference solution, and test solution (one injection each) into the high-performance liquid chromatograph (HPLC) under the chromatographic conditions described above, and record the chromatogram. Calculate the concentration by peak area using the external standard method. The aluminum salt content must not exceed 25 μg / L.

[0211] 2.2 Method Validation

[0212] The method verification was performed according to the method verification method under "1.2" in Example 1, wherein only the indicators involving the test sample were tested, and the verification results under "1.2" were used for items not involving the test sample.

[0213] 2.3 Statistical Analysis of Method Validation Results

[0214] Example 3

[0215] 3.1 Detection Method

[0216] 3.1.1 Solution Preparation

[0217] Acetic acid-ammonium acetate buffer (pH 4.5): Dissolve 77g of ammonium acetate in 500ml of water, then add 60ml of glacial acetic acid and an appropriate amount of water to make 1000ml. Shake well to obtain the buffer.

[0218] Diluent (required for preparing derivatizing agent solution): Take 500ml of methanol, add 100ml of acetate-ammonium acetate buffer (pH 4.5), then dilute with water to 1000ml, shake well, and the solution is ready.

[0219] Derivatizing agent solution (1% 3-amino-5-hydroxypyrazole): Weigh 5g of 3-amino-5-hydroxypyrazole accurately, place it in a 500ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute to the mark with diluent. Shake well to obtain the solution.

[0220] 3.1.2 Blank solution

[0221] Blank solution: Accurately transfer 10 ml of acetate-ammonium acetate buffer (pH 4.5) and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the blank solution.

[0222] 3.1.3 Reference Solution

[0223] Reference solution: Accurately transfer 1 ml of 5 μg / ml aluminum single element standard solution, 10 ml of acetate-ammonium acetate buffer (pH 4.5), and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the solution.

[0224] 3.1.4 Sample Pretreatment

[0225] Concentration: Take 200ml of calcium gluconate sodium chloride injection solution and place it in a beaker. Heat it on an electric furnace to concentrate it to about 5ml.

[0226] Wet digestion: Transfer the above concentrate to a digestion tube, add 5 ml of nitric acid, shake gently, let stand for 30 minutes, then add 5 ml of hydrogen peroxide, shake gently, let stand for 30 minutes, place in a heating plate, and heat at 150°C for 2 hours.

[0227] 3.1.5 Test solution

[0228] Test solution: Transfer the pretreated sample to a 50ml volumetric flask, then accurately transfer 10ml of acetate-ammonium acetate buffer (pH 4.5) and 10ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the test solution.

[0229] 3.1.6 Chromatographic conditions

[0230] Mobile phase: Take 1600 ml of acetonitrile and 2400 ml of 0.1% formic acid aqueous solution containing 10 mM ammonium acetate, put them together in a mobile phase bottle, shake well, and sonicate to obtain the mobile phase.

[0231] Column type: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); Detection wavelength: 254nm; Injection volume: 20μl; Column temperature: 35℃; Flow rate: 0.8ml / min; Run time: 30 minutes of isocratic running.

[0232] 3.1.7 Measurement

[0233] Determination: Inject 20 μl of each of the above blank solution, reference solution, and test solution (one injection each) into the high-performance liquid chromatograph (HPLC) under the chromatographic conditions described above, and record the chromatogram. Calculate the concentration by peak area using the external standard method. The aluminum salt content must not exceed 25 μg / L.

[0234] 3.2 Method Validation

[0235] The method verification was performed according to the method verification method under "1.2" in Example 1, wherein only the indicators involving the test sample were tested, and the verification results under "1.2" were used for items not involving the test sample.

[0236] 3.3 Statistical Analysis of Method Validation Results

[0237] Example 4

[0238] 4.1 Detection Method

[0239] 4.1.1 Solution Preparation

[0240] Acetic acid-ammonium acetate buffer (pH 4.5): Dissolve 77g of ammonium acetate in 500ml of water, then add 60ml of glacial acetic acid and an appropriate amount of water to make 1000ml. Shake well to obtain the buffer.

[0241] Diluent (required for preparing derivatizing agent solution): Take 500ml of methanol, add 100ml of acetate-ammonium acetate buffer (pH 4.5), then dilute with water to 1000ml, shake well, and the solution is ready.

[0242] Derivatizing agent solution (1% 3-amino-5-hydroxypyrazole): Weigh 5g of 3-amino-5-hydroxypyrazole accurately, place it in a 500ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute to the mark with diluent. Shake well to obtain the solution.

[0243] 4.1.2 Blank solution

[0244] Blank solution: Accurately transfer 10 ml of acetate-ammonium acetate buffer (pH 4.5) and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the blank solution.

[0245] 4.1.3 Reference Solution

[0246] Reference solution: Accurately transfer 1 ml of 5 μg / ml aluminum single element standard solution, 10 ml of acetate-ammonium acetate buffer (pH 4.5), and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the solution.

[0247] 4.1.4 Sample Pretreatment

[0248] Concentration: Take 200ml of calcium gluconate sodium chloride injection solution and place it in a beaker. Heat it on an electric furnace to concentrate it to about 5ml.

[0249] Microwave digestion: Transfer the above concentrate to a digestion tube, add 5 ml of nitric acid, and microwave digest for 1 hour at 200℃, 1200W, and 10MPa. Then place it on a hot plate and remove the acid at 150℃ for 1 hour. Cool and set aside for later use.

[0250] 4.1.5 Test solution

[0251] Test solution: Transfer the pretreated sample to a 50ml volumetric flask, then accurately transfer 10ml of acetate-ammonium acetate buffer (pH 4.5) and 10ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the test solution.

[0252] 4.1.6 Chromatographic conditions

[0253] Mobile phase: Take 1600 ml of acetonitrile and 2400 ml of 0.1% formic acid solution, place them together in a mobile phase bottle, shake well, and sonicate to obtain the mobile phase.

[0254] Column type: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); Detection wavelength: 254nm; Injection volume: 20μl; Column temperature: 35℃; Flow rate: 0.8ml / min; Run time: 30 minutes of isocratic running.

[0255] 4.1.7 Measurement

[0256] Determination: Inject 20 μl of each of the above blank solution, reference solution, and test solution (one injection each) into the high-performance liquid chromatograph (HPLC) under the chromatographic conditions described above, and record the chromatogram. Calculate the concentration by peak area using the external standard method. The aluminum salt content must not exceed 25 μg / L.

[0257] 4.2 Method Validation

[0258] The method verification was performed according to the method verification method under item "1.2" in Example 1.

[0259] 4.3 Statistical Analysis of Method Validation Results

[0260] Example 5

[0261] 5.1 Detection Method

[0262] 5.1.1 Solution Preparation

[0263] Acetic acid-ammonium acetate buffer (pH 4.5): Dissolve 77g of ammonium acetate in 500ml of water, then add 60ml of glacial acetic acid and an appropriate amount of water to make 1000ml. Shake well to obtain the buffer.

[0264] Diluent (required for preparing derivatizing agent solution): Take 500ml of methanol, add 100ml of acetate-ammonium acetate buffer (pH 4.5), then dilute with water to 1000ml, shake well, and the solution is ready.

[0265] Derivatizing agent solution (1% 3-amino-5-hydroxypyrazole): Weigh 5g of 3-amino-5-hydroxypyrazole accurately, place it in a 500ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute to the mark with diluent. Shake well to obtain the solution.

[0266] 5.1.2 Blank solution

[0267] Blank solution: Accurately transfer 10 ml of acetate-ammonium acetate buffer (pH 4.5) and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the blank solution.

[0268] 5.1.3 Reference Solution

[0269] Reference solution: Accurately transfer 1 ml of 5 μg / ml aluminum single element standard solution, 10 ml of acetate-ammonium acetate buffer (pH 4.5), and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the solution.

[0270] 5.1.4 Sample Pretreatment

[0271] Concentration: Take 200ml of calcium gluconate sodium chloride injection solution and place it in a beaker. Heat it on an electric furnace to concentrate it to about 5ml.

[0272] Microwave digestion: Transfer the above concentrate to a digestion tube, add 5 ml of nitric acid, and microwave digest for 1 hour at 200℃, 1200W, and 10MPa. Then place it on a hot plate and remove the acid at 150℃ for 1 hour. Cool and set aside for later use.

[0273] 5.1.5 Test solution

[0274] Test solution: Transfer the pretreated sample to a 50ml volumetric flask, then accurately transfer 10ml of acetate-ammonium acetate buffer (pH 4.5) and 10ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the test solution.

[0275] 5.1.6 Chromatographic conditions

[0276] Mobile phase: Take 1600 ml of acetonitrile and 2400 ml of 0.1% formic acid aqueous solution containing 10 mM potassium dihydrogen phosphate, put them together in a mobile phase bottle, shake well, and sonicate to obtain the mobile phase.

[0277] Column type: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); Detection wavelength: 254nm; Injection volume: 20μl; Column temperature: 35℃; Flow rate: 0.8ml / min; Run time: 30 minutes of isocratic running.

[0278] 5.1.7 Measurement

[0279] Determination: Inject 20 μl of each of the above blank solution, reference solution, and test solution (one injection each) into the high-performance liquid chromatograph (HPLC) under the chromatographic conditions described above, and record the chromatogram. Calculate the concentration by peak area using the external standard method. The aluminum salt content must not exceed 25 μg / L.

[0280] 5.2 Method Validation

[0281] The method verification was performed according to the method verification method under item "1.2" in Example 1.

[0282] 5.3 Statistical Analysis of Method Validation Results

[0283] Example 6

[0284] 6.1 Detection Method

[0285] 6.1.1 Solution Preparation

[0286] Acetic acid-ammonium acetate buffer (pH 4.5): Dissolve 77g of ammonium acetate in 500ml of water, then add 60ml of glacial acetic acid and an appropriate amount of water to make 1000ml. Shake well to obtain the buffer.

[0287] Diluent (required for preparing derivatizing agent solution): Take 500ml of methanol, add 100ml of acetate-ammonium acetate buffer (pH 4.5), then dilute with water to 1000ml, shake well, and the solution is ready.

[0288] Derivatizing agent solution (1% 3-amino-5-hydroxypyrazole): Weigh 5g of 3-amino-5-hydroxypyrazole accurately, place it in a 500ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute to the mark with diluent. Shake well to obtain the solution.

[0289] 6.1.2 Blank solution

[0290] Blank solution: Accurately transfer 10 ml of acetate-ammonium acetate buffer (pH 4.5) and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the blank solution.

[0291] 6.1.3 Reference Solution

[0292] Reference solution: Accurately transfer 1 ml of 5 μg / ml aluminum single element standard solution, 10 ml of acetate-ammonium acetate buffer (pH 4.5), and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the solution.

[0293] 6.1.4 Sample Pretreatment

[0294] Concentration: Take 200ml of calcium gluconate sodium chloride injection solution and place it in a beaker. Heat it on an electric furnace to concentrate it to about 5ml.

[0295] Microwave digestion: Transfer the above concentrate to a digestion tube, add 5 ml of nitric acid, and microwave digest for 1 hour at 200℃, 1200W, and 10MPa. Then place it on a hot plate and remove the acid at 150℃ for 1 hour. Cool and set aside for later use.

[0296] 6.1.5 Test solution

[0297] Test solution: Transfer the pretreated sample to a 50ml volumetric flask, then accurately transfer 10ml of acetate-ammonium acetate buffer (pH 4.5) and 10ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the test solution.

[0298] 6.1.6 Chromatographic conditions

[0299] Mobile phase: Take 1400 ml of acetonitrile and 2600 ml of 0.1% formic acid aqueous solution containing 10 mM ammonium acetate, put them together in a mobile phase bottle, shake well, and sonicate to obtain the mobile phase.

[0300] Column type: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); Detection wavelength: 254nm; Injection volume: 20μl; Column temperature: 35℃; Flow rate: 0.8ml / min; Run time: 40 minutes of isocratic running.

[0301] 6.1.7 Measurement

[0302] Determination: Inject 20 μl of each of the above blank solution, reference solution, and test solution (one injection each) into the high-performance liquid chromatograph (HPLC) under the chromatographic conditions described above, and record the chromatogram. Calculate the concentration by peak area using the external standard method. The aluminum salt content must not exceed 25 μg / L.

[0303] 6.2 Method Validation

[0304] The method verification was performed according to the method verification method under item "1.2" in Example 1.

[0305] 6.3 Statistical Analysis of Method Validation Results

[0306] Example 7

[0307] 7.1 Detection Method

[0308] 7.1.1 Solution Preparation

[0309] Acetic acid-ammonium acetate buffer (pH 4.5): Dissolve 77g of ammonium acetate in 500ml of water, then add 60ml of glacial acetic acid and an appropriate amount of water to make 1000ml. Shake well to obtain the buffer.

[0310] Diluent (required for preparing derivatizing agent solution): Take 500ml of methanol, add 100ml of acetate-ammonium acetate buffer (pH 4.5), then dilute with water to 1000ml, shake well, and the solution is ready.

[0311] Derivatizing agent solution (1% 3-amino-5-hydroxypyrazole): Weigh 5g of 3-amino-5-hydroxypyrazole accurately, place it in a 500ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute to the mark with diluent. Shake well to obtain the solution.

[0312] 7.1.2 Blank solution

[0313] Blank solution: Accurately transfer 10 ml of acetate-ammonium acetate buffer (pH 4.5) and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the blank solution.

[0314] 7.1.3 Reference Solution

[0315] Reference solution: Accurately transfer 1 ml of 5 μg / ml aluminum single element standard solution, 10 ml of acetate-ammonium acetate buffer (pH 4.5), and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the solution.

[0316] 7.1.4 Sample Pretreatment

[0317] Concentration: Take 200ml of calcium gluconate sodium chloride injection solution and place it in a beaker. Heat it on an electric furnace to concentrate it to about 5ml.

[0318] Microwave digestion: Transfer the above concentrate to a digestion tube, add 5 ml of nitric acid, and microwave digest for 1 hour at 200℃, 1200W, and 10MPa. Then place it on a hot plate and remove the acid at 150℃ for 1 hour. Cool and set aside for later use.

[0319] 7.1.5 Test solution

[0320] Test solution: Transfer the pretreated sample to a 50ml volumetric flask, then accurately transfer 10ml of acetate-ammonium acetate buffer (pH 4.5) and 10ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the test solution.

[0321] 7.1.6 Chromatographic conditions

[0322] Mobile phase: Take 1800 ml of acetonitrile and 2200 ml of 0.1% formic acid aqueous solution containing 10 mM ammonium acetate, put them together in a mobile phase bottle, shake well, and sonicate to obtain the mobile phase.

[0323] Column type: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); Detection wavelength: 254nm; Injection volume: 20μl; Column temperature: 35℃; Flow rate: 0.8ml / min; Run time: 40 minutes of isocratic running.

[0324] 7.1.7 Measurement

[0325] Determination: Inject 20 μl of each of the above blank solution, reference solution, and test solution (one injection each) into the high-performance liquid chromatograph (HPLC) under the chromatographic conditions described above, and record the chromatogram. Calculate the concentration by peak area using the external standard method. The aluminum salt content must not exceed 25 μg / L.

[0326] 7.2 Method Validation

[0327] The method verification was performed according to the method verification method under item "1.2" in Example 1.

[0328] 7.3 Statistical Analysis of Method Validation Results

[0329] Comparative Example 1

[0330] The aluminum salts in this product were investigated according to the method described in the reference "Extraction of Aluminum in Drinking Water - High Performance Liquid Chromatography Analysis, Yunnan Environmental Science, Vol. 19 Supplement, August 2000: 259-261".

[0331] Blank solution: Take 160 ml and place it in a separatory funnel. Add 1.0 ml of acetate-sodium acetate buffer solution (1 mol / L, pH 5.8), add 1.5 ml of acetic acid solution of 8-hydroxyquinoline (0.145%), then adjust the pH to 8.5 with 1 mol / L tris(hydroxymethyl)aminomethane (Tris) solution. Extract with 5 ml of chloroform in three portions. Filter the extract through filter paper into a 25 ml volumetric flask and dilute to the mark with chloroform.

[0332] Reference solution: Accurately transfer 1 ml of 5 μg / ml aluminum single element standard solution and dilute with water to 160 ml. Transfer to a separatory funnel, add 1.0 ml of acetate-sodium acetate buffer solution (1 mol / L, pH 5.8), add 1.5 ml of acetic acid solution of 8-hydroxyquinoline (0.145%), then adjust the pH to 8.5 with 1 mol / L tris(hydroxymethyl)aminomethane (Tris) solution. Extract three times with 5 ml of chloroform. Filter the extract through filter paper into a 25 ml volumetric flask and dilute to the mark with chloroform.

[0333] Test solution: Accurately transfer 160 ml of calcium gluconate sodium chloride injection into a separatory funnel, add 1.0 ml of acetate-sodium acetate buffer solution (1 mol / L, pH 5.8), add 1.5 ml of acetic acid solution of 8-hydroxyquinoline (0.145%), then adjust the pH to 8.5 with 1 mol / L tris(hydroxymethyl)aminomethane (Tris) solution, extract three times with 5 ml of chloroform, filter the extract through filter paper into a 25 ml volumetric flask, and dilute to the mark with chloroform.

[0334] 100% spiked test solution: Accurately transfer 1 ml of 5 μg / ml aluminum single-element standard solution and 160 ml of calcium gluconate sodium chloride injection into a separatory funnel, add 1.0 ml of acetate-sodium acetate buffer solution (1 mol / L, pH 5.8), add 1.5 ml of 8-hydroxyquinoline acetic acid solution (0.145%), then adjust the pH to 8.5 with 1 mol / L tris(hydroxymethyl)aminomethane (Tris) solution, extract three times with 5 ml of chloroform, filter the extract through filter paper into a 25 ml volumetric flask, and dilute to the mark with chloroform.

[0335] Mobile phase: methanol-ethyl acetate-water (40:20:40), containing 2.5 mol / L lithium lactate and 0.05 mmol / L EDTA.

[0336] Chromatographic conditions: Column type: ZORBAX Eclipse Plus (C8) (250*4.6mm, 5μm); Detection wavelength: 390nm; Injection volume: 10μl; Column temperature: 30℃; Flow rate: 1ml / min; Run time: isocratic run for 30 minutes.

[0337] Determination: Inject 10 μl of the above blank solution, reference solution, test solution, and 100% spiked test solution (1 injection each) into the high performance liquid chromatograph, and inject under the above chromatographic conditions, and record the chromatogram.

[0338] Results: The results showed that the blank solution had no interference, but the 100% recovery rate was as high as 6700%, indicating that the matrix severely interfered with the detection.

[0339] Comparative Example 2

[0340] The aluminum salts in this product were investigated according to the method described in the reference "Separation and determination of aluminum(III), chromium(III), niobium(V) and copper(II) by chlorosulfonyl S reversed-phase high performance liquid chromatography, Journal of Southwest Normal University (Natural Science Edition), Vol.19, No.3, June 1994: 260-264".

[0341] Blank solution: Take 2.5 ml of acetate-sodium acetate buffer solution (pH 3.5) and 4 ml of 0.05% chlorosulfonylphenol S solution and place them in a 25 ml volumetric flask. Dilute with water to 20 ml, shake well, heat in a boiling water bath for 15 minutes, cool to room temperature, dilute with water to the mark, and shake well.

[0342] Reference solution: Accurately transfer 1 ml of 5 μg / ml aluminum single element standard solution into a 25 ml volumetric flask, then add 2.5 ml of acetate-sodium acetate buffer solution (pH 3.5) and 4 ml of 0.05% chlorosulfonylphenol S solution, dilute with water to 20 ml, shake well, heat in a boiling water bath for 15 minutes, cool to room temperature, dilute with water to the mark, and shake well.

[0343] Test solution: Take the sample obtained by the same operation as the pretreated sample under "1.4" in Example 1 and place it in a 25 ml volumetric flask. Add 2.5 ml of acetate-sodium acetate buffer solution (pH 3.5) and 4 ml of 0.05% chlorosulfonylphenol S solution. Dilute with water to 20 ml, shake well, heat in a boiling water bath for 15 minutes, cool to room temperature, dilute with water to the mark, and shake well.

[0344] 100% spiked test solution: Take the sample obtained by the same operation as the pretreated sample under "1.4" in Example 1 and place it in a 25 ml volumetric flask. Then accurately transfer 1 ml of 5 μg / ml aluminum single element standard solution, 2.5 ml of acetate-sodium acetate buffer solution (pH 3.5) and 4 ml of 0.05% chlorosulfonylphenol S solution into it, dilute with water to 20 ml, shake well, heat in a boiling water bath for 15 minutes, cool to room temperature, dilute with water to the mark, and shake well.

[0345] Mobile phase: Acetonitrile (38%)-tetrahydrofuran (2%)-water (60%) solution containing 0.06 mol / L tetraethylammonium bromide and 0.01 mol / L acetate buffer (pH 3.5).

[0346] Chromatographic conditions: Column type: Shim-pack ODS(C18) (250*4.6mm, 5μm); Detection wavelength: 570nm; Injection volume: 20μl; Column temperature: 30℃; Flow rate: 1ml / min; Run time: isocratic run for 30 minutes.

[0347] Determination: Inject 20 μl of the above blank solution, reference solution, test solution, and 100% spiked test solution (1 injection each) into the high performance liquid chromatograph, and inject under the above chromatographic conditions, and record the chromatogram.

[0348] Results: The results showed that the blank solution had no interference, but the response of the reference solution was very low, with a signal-to-noise ratio of only 5.6. In contrast, there were strong interference peaks near the aluminum derivative peaks in the test solution and the 100% spiked test solution.

Claims

1. A method for detecting aluminum content in calcium gluconate sodium chloride injection, characterized in that... This detection method employs a sample pretreatment-derivation-high performance liquid chromatography-ultraviolet detection approach, including the following steps: Sample pretreatment: Take 200 ml of calcium gluconate sodium chloride injection and heat to concentrate; further process the concentrate, wherein the further processing method is microwave digestion; Derivatization: The pretreated sample is derivatized with a 3-amino-5-hydroxypyrazole derivatizing agent, wherein aluminum in the sample forms an aluminum derivative with the 3-amino-5-hydroxypyrazole derivatizing agent; High performance liquid chromatography-ultraviolet detection: A high performance liquid chromatograph with an ultraviolet-visible spectrophotometer was used, with an octadecylsilane-bonded silica gel column as the packing material and a mixed solution of methanol and buffer solution as the mobile phase for separation and detection. The buffer solution is a 0.1% formic acid aqueous solution, a 0.1% formic acid aqueous solution containing 10mM ammonium acetate, or a 0.1% formic acid aqueous solution containing 10mM potassium dihydrogen phosphate. The buffer solution is a 0.1% formic acid aqueous solution containing 10 mM ammonium acetate; The chromatographic column packed with octadecylsilane-bonded silica gel is a ZORBAX Eclipse Plus C18. The mobile phase is a mixed solution of methanol and buffer solution containing 40% methanol; The steps are as follows: (1) Solution preparation Acetic acid-ammonium acetate buffer solution with pH 4.5: Dissolve 77g of ammonium acetate in 500ml of water, add 60ml of glacial acetic acid and an appropriate amount of water to make 1000ml, shake well, and you have the solution. Diluent required for preparing derivatizing agent solution: Take 500ml of methanol, add 100ml of acetate-ammonium acetate buffer solution with pH 4.5, then dilute with water to 1000ml, shake well, and the solution is ready; 1% 3-amino-5-hydroxypyrazole derivatizing agent solution: Weigh 5g of 3-amino-5-hydroxypyrazole accurately, place it in a 500ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute to the mark with diluent. Shake well to obtain the solution. Mobile phase: Take 1600 ml of methanol and 2400 ml of 0.1% formic acid aqueous solution containing 10 mM ammonium acetate, put them together in a mobile phase bottle, shake well, and sonicate to obtain the mobile phase. (2) Preparation of blank solution Blank solution: Accurately transfer 10 ml of pH 4.5 acetate-ammonium acetate buffer and 10 ml of 1% 3-amino-5-hydroxypyrazole derivatizing agent solution into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the blank solution. (3) Preparation of reference solution Reference solution: Accurately transfer 1 ml of 5 µg / ml aluminum single element standard solution, 10 ml of pH 4.5 acetate-ammonium acetate buffer solution, and 10 ml of 1% 3-amino-5-hydroxypyrazole derivatizing agent solution into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the solution. (4) Sample pretreatment Concentration: Place 200ml of calcium gluconate sodium chloride injection in a beaker and heat it on an electric furnace to concentrate it to about 5ml; Microwave digestion: Transfer the above concentrate to a digestion tube, add 5 ml of nitric acid, and microwave digest for 1 hour at 200℃, 1200W, and 10MPa; then place it on a hot plate and remove the acid at 150℃ for 1 hour, cool, and set aside for use. (5) Preparation of test solution Test solution: Transfer the pretreated sample to a 50ml volumetric flask, then accurately transfer 10ml of a pH 4.5 acetate-ammonium acetate buffer solution and 10ml of a 1% 3-amino-5-hydroxypyrazole derivatizing agent solution into it, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the test solution. (6) Measurement Column type: ZORBAX Eclipse Plus C18, 250*4.6mm, 5μm; Detection wavelength: 254nm; Injection volume: 20μl; Column temperature: 35℃; Flow rate: 0.8ml / min; Run time: 30 minutes of isocratic running; Calculated by peak area using external standard method.

2. The detection method according to claim 1, comprising the following steps: (1) Solution preparation Acetic acid-ammonium acetate buffer solution with pH 4.5: Dissolve 77g of ammonium acetate in 500ml of water, add 60ml of glacial acetic acid and an appropriate amount of water to make 1000ml, shake well, and you have the solution. Diluent required for preparing the derivatizing agent solution: Take 500 ml of methanol, add 100 ml of acetate-ammonium acetate buffer solution with pH 4.5, then dilute with water to 1000 ml, shake well, and the solution is ready. 1% 3-amino-5-hydroxypyrazole derivatizing agent solution: Weigh 5g of 3-amino-5-hydroxypyrazole accurately, place it in a 500ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute to the mark with diluent. Shake well to obtain the solution. Mobile phase: Take 1600 ml of methanol and 2400 ml of 0.1% formic acid aqueous solution containing 10 mM ammonium acetate, put them together in a mobile phase bottle, shake well, and sonicate to obtain the mobile phase. (2) Preparation of blank solution Blank solution: Accurately transfer 10 ml of pH 4.5 acetate-ammonium acetate buffer and 10 ml of 1% 3-amino-5-hydroxypyrazole derivatizing agent solution into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the blank solution. (3) Preparation of reference solution Reference solution: Accurately transfer 1 ml of 5 µg / ml aluminum single element standard solution, 10 ml of acetate-ammonium acetate buffer solution with pH 4.5, and 10 ml of 1% 3-amino-5-hydroxypyrazole derivatizing agent solution into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the solution. (4) Sample pretreatment Concentration: Place 200ml of calcium gluconate sodium chloride injection in a beaker and heat it on an electric furnace to concentrate it to about 5ml; Wet digestion: Transfer the above concentrate to a digestion tube, add 5 ml of nitric acid, shake gently, let stand for 30 minutes, then add 5 ml of hydrogen peroxide, shake gently, let stand for 30 minutes, place on a heating plate, and heat at 150°C for 2 hours. (5) Preparation of test solution Test solution: Transfer the pretreated sample to a 50 ml volumetric flask, then accurately transfer 10 ml of pH 4.5 acetate-ammonium acetate buffer and 10 ml of 1% 3-amino-5-hydroxypyrazole derivatizing agent solution into the flask. Dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the product; (6) Measurement Column type: ZORBAX Eclipse Plus, C18, 250*4.6mm, 5μm; Detection wavelength: 254nm; Injection volume: 20μl; Column temperature: 35℃; Flow rate: 0.8ml / min; Run time: 30 minutes of isocratic running; Calculated by peak area using the external standard method.

3. The detection method according to claim 1, comprising the following steps: (1) Solution preparation Acetic acid-ammonium acetate buffer solution with pH 4.5: Dissolve 77g of ammonium acetate in 500ml of water, add 60ml of glacial acetic acid and an appropriate amount of water to make 1000ml, shake well, and you have the solution. Diluent required for preparing the derivatizing agent solution: Take 500 ml of methanol, add 100 ml of acetate-ammonium acetate buffer solution with pH 4.5, then dilute with water to 1000 ml, shake well, and the solution is ready. 1% 3-amino-5-hydroxypyrazole derivatizing agent solution: Weigh 5g of 3-amino-5-hydroxypyrazole accurately, place it in a 500ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute to the mark with diluent. Shake well to obtain the solution. Mobile phase: Take 1600 ml of methanol and 2400 ml of 0.1% formic acid aqueous solution containing 10 mM ammonium acetate, put them together in a mobile phase bottle, shake well, and sonicate to obtain the mobile phase. (2) Preparation of blank solution Blank solution: Accurately transfer 10 ml of pH 4.5 acetate-ammonium acetate buffer and 10 ml of 1% 3-amino-5-hydroxypyrazole derivatizing agent solution into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the blank solution. (3) Preparation of reference solution Reference solution: Accurately transfer 1 ml of 5 µg / ml aluminum single element standard solution, 10 ml of acetate-ammonium acetate buffer solution with pH 4.5, and 10 ml of 1% 3-amino-5-hydroxypyrazole derivatizing agent solution into a 50 ml volumetric flask, dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the solution. (4) Sample pretreatment Concentration: Place 200ml of calcium gluconate sodium chloride injection in a beaker and heat it on an electric furnace to concentrate it to about 5ml; Incineration and ashing: Transfer the above concentrate to a crucible, slowly incinerate until completely carbonized, cool, add sulfuric acid to moisten, heat at low temperature until sulfuric acid vapor is removed, and then incinerate at 500-550℃ to completely ashing. (5) Preparation of test solution Test solution: Transfer the pretreated sample to a 50 ml volumetric flask, then accurately transfer 10 ml of pH 4.5 acetate-ammonium acetate buffer and 10 ml of 1% 3-amino-5-hydroxypyrazole derivatizing agent solution into the flask. Dilute with water to the mark, shake well, and let stand for 15 minutes to obtain the product; (6) Measurement Column type: ZORBAX Eclipse Plus, C18, 250*4.6mm, 5μm; Detection wavelength: 254nm; Injection volume: 20μl; Column temperature: 35℃; Flow rate: 0.8ml / min; Run time: 30 minutes of isocratic running; Calculated by peak area using the external standard method.

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