Method for detecting aluminum content in calcium gluconate and sodium chloride injection

Abstract

The invention discloses a method for detecting the content of aluminum in a calcium gluconate and sodium chloride injection, which comprises the following steps of: heating and concentrating 200ml of calcium gluconate and sodium chloride injection, and carrying out pre-treatment of ignition ashing or wet digestion or microwave digestion on concentrate; then taking the pretreated sample and a 1% 3-amino-5-hydroxypyrazole derivative agent solution, and carrying out a derivative reaction in an aqueous solution system containing an acetic acid-ammonium acetate buffer solution (pH 4.5) for about 15 minutes; finally, a ZORBAXEclipse Plus (C18) (250 * 4.6 mm, 5 [mu] m) chromatographic column is used, a mixed solution of methanol and a 0.1% formic acid aqueous solution containing 10 mM ammonium acetate (40: 60) is used as a mobile phase, and a high performance chromatography-ultraviolet detection method is carried out under the detection wavelength of 254 nm. The detection method has the advantages of high sensitivity, almost no detection interference, good accuracy, stable formed aluminum derivative, no high-toxicity reagent in the test process, short analysis time and the like, and can effectively overcome the defects in the prior art. The aluminum in the calcium gluconate and sodium chloride injection can be effectively controlled, and the quality of the calcium gluconate and sodium chloride injection is well ensured.

Description

Technical Field

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

[0002] The National Drug Administration Drug Evaluation Center's common problems with generic chemical drugs clearly pointed out that aluminum has certain nephrotoxicity, and it is necessary to study and control the aluminum element in large-volume total parenteral nutrition (TPN) injections, with a control limit of no more than 25 μg / L. Therefore, it is necessary to study and control the aluminum content in calcium gluconate and sodium chloride injections, but there is currently no literature report on the determination method of aluminum content in calcium gluconate and sodium chloride injections.

[0003] The existing detection methods for trace aluminum content in various industries (such as metallurgy, food, water, pharmaceuticals, etc.) are mainly direct detection methods such as ICP-AES (inductively coupled plasma atomic emission spectrometry), ICP-OES (inductively coupled plasma 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 are all uncommon instruments. Pharmaceutical companies generally do not have such instruments and need to outsource testing, which results in the inability to release drugs in a timely manner, which is extremely inconvenient. In addition, the equipment costs are high, and the testing costs are high, so they are not suitable for pharmaceutical QC testing. The industry generally feedbacks that the atomic absorption spectrophotometry method is unstable due to the low atomization rate of aluminum, and there are many deviations in the QC testing of pharmaceutical companies, which cannot accurately and timely feedback the quality of drugs, thus affecting the release of drugs.

[0005] There are few literature reports on the detection of trace aluminum in samples by high performance liquid chromatography after reacting with a derivatization reagent. The detectors include fluorescence detector, mass spectrometer detector, evaporative light scattering detector, differential refractometer detector, and ultraviolet-visible spectrophotometer (including diode array detector).

[0006] Evaporative light scattering detector, differential refractive index detector, mass spectrometer detector, fluorescence detector are all uncommon detectors. Among them, evaporative light scattering detector and differential refractive index detector are universal detectors with low selectivity. They are mainly suitable for compounds without ultraviolet absorption and fluorescence characteristics. They have low sensitivity and often need to increase the concentration of the test solution to achieve the detection effect. For the complex matrix solution formed by the reaction of trace / trace aluminum with the derivatization reagent, each component can be detected indiscriminately, resulting in greater interference. Especially when the concentration of the test solution is higher, the interference is stronger, resulting in the inability to accurately perform quantitative detection and poor method reproducibility. Mass spectrometers are only equipped in a few companies, and the equipment is expensive. The high-salt non-volatile matrix in the derivatization solution can easily cause the ion source to be quickly contaminated, increasing the maintenance cost of the instrument, and is not suitable for daily high-frequency release testing by production companies. In addition, the linear range is narrow and cannot control trace / constant aluminum well. The light source of the fluorescence detector has a limited life span and needs to be replaced regularly, which increases the maintenance cost. The instrument itself is also relatively expensive, increasing the original cost. A derivatization reagent with a fluorescent group is required to react with trace / trace aluminum. The fluorescence intensity of the fluorescent derivative product is easily affected by environmental factors such as solvent polarity, pH value, and temperature, and the experimental conditions need to be strictly controlled. Under light, photolysis or photobleaching is prone to occur, resulting in signal attenuation and a narrow linear range, which makes it difficult to control trace / constant aluminum.

[0007] There are literature reports that stilbeneazole [also known as stilbene azo, chemical name is 1,2-diphenylethylene-4,4-bis(1-azo)-3,4-dihydroxybenzene-2,2'-disulfonic acid ammonium or stilbene-4,4-bis(1-azo)-3,4-dihydroxybenzene-2,2'-disulfonic acid ammonium] is used as a derivatization reagent to react with aluminum in water samples collected from ores or oil fields, and the aluminum content in water samples collected from ores or oil fields is detected by high performance liquid chromatography-ultraviolet detection (Reference 1: Separation and determination of scandium, tin and aluminum by reversed phase ion pair high performance liquid chromatography, color Spectrum, Vol. 19, No. 5, September 2001: 472-474; Document 2: 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: 31-34; Document 2: Determination of aluminum, gallium and indium mixture by high performance liquid chromatography, Liaoning Chemical Industry, Vol. 36, No. 9, September 2007: 645-647), the document clearly points out that the stability of the complex in this method is poor, the sample needs to be injected after 15 minutes of color development, and the separation and detection need to be completed within 135 minutes. For multiple batches of release inspection, someone needs to be on duty all the time, which greatly increases the inspection cost and is extremely inconvenient.

[0008] There are literature reports on using chlorosulfonphenol S [also known as chlorosulfonphenol S or chlorosulfonic acid C, chemical name 2,7-bis(5-chloro-2-hydroxy-3-sulfonylphenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid or 2,7-bis(5-chloro-2-hydroxy-3-sulfonylphenylazo) chromotropic acid] as a derivatization reagent, which undergoes a derivatization reaction with aluminum in water / flour / rice / human hair in a boiling water bath, and the aluminum content in water / flour / rice / human hair is 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 using chlorosulfonphenol S, Journal of Southwest Normal University (Natural Science Edition), Vol. 19, No. 3, June 1994: 260-264]. The detection wavelength of aluminum derivatives in this method is 570nm, which is in the visible light region. In the routine high performance liquid chromatography-ultraviolet detection method, the detection wavelength is generally in the ultraviolet region within the range of 200-400nm. The sensitivity of the visible light region is low. For the detection of trace aluminum, it is difficult to avoid the interference of the complex matrix of the derivative solution at low concentrations, which leads to inaccurate determination.

[0009] There are literature reports that 8-hydroxyquinoline is used as a derivatization reagent to react with aluminum in water, then extracted with chloroform, and then the aluminum salt content in water is detected by high performance liquid chromatography-ultraviolet detection [Document 1: Extraction of aluminum in drinking water-high performance liquid chromatography analysis, Yunnan Environmental Science, Volume 19 Supplement, August 2000: 259-261; Document 2: Extraction and high performance liquid chromatography analysis of tris (8-hydroxyquinoline root) aluminum (III) complex, Journal of Inorganic Chemistry, Volume 16, No. 4, July 2000: 637-640]. The literature reports that this method has been tested for the interference of calcium (Ca), and the interference factor of calcium on the detection of aluminum is 50 times, and this method uses highly toxic chloroform as an extractant.

[0010] Generally speaking, there are no literature reports on the determination of aluminum content in calcium gluconate and sodium chloride injection. There are also almost no reports on the determination of aluminum by HPLC-UV after the derivatization reaction between the derivatization agent and aluminum in the pharmaceutical field. Currently, there are only a few literature reports on the determination of aluminum by HPLC-UV after the derivatization reaction between the derivatization agent and aluminum in other fields (such as water and ore). However, there are various problems in these methods. Either the aluminum derivatives are unstable; or the detection wavelength is in the visible light region, resulting in low sensitivity and large interference; or even if the aluminum derivative formed by 8-hydroxyquinoline as a derivatization agent is stable and the absorption wavelength is in the ultraviolet region, it is easily interfered by calcium. The test results show that the interference of calcium can be as high as 50 times, and the highly toxic extraction agent chloroform is used. Summary of the invention

[0011] The purpose of the present invention is to provide a method for detecting the aluminum content in calcium gluconate sodium chloride injection in view of the above problems, wherein the detection method adopts sample pretreatment-derivation-high performance liquid chromatography-ultraviolet method for detection; the detection method has the advantages of high sensitivity, almost no detection interference, good accuracy, stable aluminum derivatives formed, no highly toxic reagents during the test, short analysis time, etc., which can effectively solve the above-mentioned deficiencies existing in the prior art. Aluminum in calcium gluconate sodium chloride injection can be effectively controlled to ensure the quality of calcium gluconate sodium chloride injection.

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

[0013] The inventors have conducted a large number of experiments, and the experimental data show that calcium gluconate and sodium chloride injection needs to be pre-treated with incineration ashing, wet digestion, and microwave digestion, and the results obtained have better accuracy, less interference, and better specificity.

[0014] The inventors screened the derivatization reaction conditions. The experimental data showed that by adding 10 ml of acetic acid-ammonium acetate buffer (pH 4.5), 10 ml of a derivatizing agent solution (1% 3-amino-5-hydroxypyrazole) and a sample to be derivatized, diluting the mixture to 50 ml with water, shaking the mixture, and leaving the mixture for 15 minutes, the derivatization reaction was completed, and the resulting derivative product had better stability, less interference, and better specificity.

[0015] The inventors screened multiple brands, different models of chromatographic columns and different chromatographic conditions. The test data showed that when the chromatographic column was ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm) and the mobile phase was a mixed solution of methanol and a buffer solution, the detection method had better sensitivity and specificity, wherein the buffer solution was 0.1% formic acid aqueous solution, 0.1% formic acid aqueous solution containing 10mM ammonium acetate, and 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 the aluminum content in calcium gluconate sodium chloride injection, wherein the detection method adopts sample pretreatment-derivation-high performance liquid chromatography-ultraviolet method for detection.

[0017] In an embodiment of the present invention, the detection method of the present invention, wherein the sample pretreatment is to take 200 ml of calcium gluconate and sodium chloride injection and heat and concentrate it; the concentrate is further processed, wherein the further processing method is wet digestion, incineration ashing, microwave digestion, preferably incineration ashing, microwave digestion, and more preferably microwave digestion.

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

[0019] In the embodiment of the present invention, the detection method described in the present invention, wherein the detailed steps of the incineration ashing are to transfer the concentrate to a crucible, slowly incinerate until it is completely carbonized, cool it, add sulfuric acid to moisten it, heat it at low temperature until the sulfuric acid vapor is completely removed, and incinerate it at 500-550°C to completely ash it.

[0020] In the embodiment of the present invention, the detection method of the present invention, wherein the detailed steps of the microwave digestion are to transfer the concentrate to a digestion tube, add 5 ml of nitric acid, and perform microwave digestion for 1 hour at a temperature of 200° C., a power of 1200 W, and a pressure of 10 MPa. Then place it on a hot plate, drive out the acid at 150° C. for 1 hour, cool it, and set it aside.

[0021] In an embodiment of the present invention, the detection method described in the present invention, wherein the derivatization is to carry out a derivatization reaction of the pretreated sample with a 3-amino-5-hydroxypyrazole derivatization reagent, so that the aluminum in the sample and the 3-amino-5-hydroxypyrazole derivatization reagent form an aluminum derivative.

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

[0023] In an embodiment of the present invention, the detection method described in the present invention, wherein the high performance liquid chromatography-ultraviolet method is to use a high performance liquid chromatograph, a ultraviolet-visible spectrometer (including a diode array detector) as a detector, a chromatographic column with octadecylsilane bonded silica gel as a filler, and a mixed solution of methanol and buffer as a mobile phase for separation and detection.

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

[0025] In an embodiment of the present invention, the detection method described in the present invention, wherein the buffer solution is 0.1% formic acid aqueous solution, 0.1% formic acid aqueous solution containing 10mM ammonium acetate, 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, in the detection method of the present invention, the mobile phase is a mixed solution of methanol-buffer (35-45:65-55), preferably a mixed solution of methanol-buffer (40:60).

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

[0028] (1) Solution preparation

[0029] Acetic acid-ammonium acetate buffer (pH 4.5): Take 77 g of ammonium acetate, add 500 ml of water to dissolve it, then add 60 ml of glacial acetic acid and an appropriate amount of water to make 1000 ml, shake well, and you have it.

[0030] Diluent (diluent required for preparation of derivatization agent solution): Take 500 ml of methanol, add 100 ml of acetic acid-ammonium acetate buffer (pH 4.5), then dilute to 1000 ml with water, shake well, and obtain.

[0031] Derivatization agent solution (1% 3-amino-5-hydroxypyrazole): Take 5 g of 3-amino-5-hydroxypyrazole, accurately weigh it, put it in a 500 ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute it to the scale with the diluent, shake well, and you have it.

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

[0033] (2) Preparation of blank solution

[0034] Blank solution: Accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and leave for 15 minutes.

[0035] (3) Preparation of reference solution

[0036] Reference solution: Accurately pipette 1 ml of 5 μg / ml aluminum standard solution, 10 ml of acetic acid-ammonium acetate buffer (pH 4.5), and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and let stand for 15 minutes.

[0037] (4) Sample pretreatment

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

[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 on a heating plate, and heat at 150°C for 2 hours.

[0040] (5) Preparation of test solution

[0041] Test solution: Transfer the above pretreated sample to a 50 ml volumetric flask, then accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute to the scale with water, shake well, and leave for 15 minutes.

[0042] (6) Determination

[0043] Chromatographic column model: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); detection wavelength: 254nm; injection volume: 20μl; column temperature: 35℃; flow rate: 0.8ml / min; running time: isocratic running for 30 minutes. Calculated by peak area according to the external standard method.

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

[0045] (1) Solution preparation

[0046] Acetic acid-ammonium acetate buffer (pH 4.5): Take 77 g of ammonium acetate, add 500 ml of water to dissolve it, then add 60 ml of glacial acetic acid and an appropriate amount of water to make 1000 ml, shake well, and you have it.

[0047] Diluent (diluent required for preparation of derivatization agent solution): Take 500 ml of methanol, add 100 ml of acetic acid-ammonium acetate buffer (pH 4.5), then dilute to 1000 ml with water, shake well, and obtain.

[0048] Derivatization agent solution (1% 3-amino-5-hydroxypyrazole): Take 5 g of 3-amino-5-hydroxypyrazole, accurately weigh it, put it in a 500 ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute it to the scale with the diluent, shake well, and you have it.

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

[0050] (2) Preparation of blank solution

[0051] Blank solution: Accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and leave for 15 minutes.

[0052] (3) Preparation of reference solution

[0053] Reference solution: Accurately pipette 1 ml of 5 μg / ml aluminum standard solution, 10 ml of acetic acid-ammonium acetate buffer (pH 4.5), and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and let stand for 15 minutes.

[0054] (4) Sample pretreatment

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

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

[0057] (5) Preparation of test solution

[0058] Test solution: Transfer the above pretreated sample to a 50 ml volumetric flask, then accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute to the scale with water, shake well, and leave for 15 minutes.

[0059] (6) Determination

[0060] Chromatographic column model: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); detection wavelength: 254nm; injection volume: 20μl; column temperature: 35℃; flow rate: 0.8ml / min; running time: isocratic running for 30 minutes. Calculated by peak area according to the external standard method.

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

[0062] (1) Solution preparation

[0063] Acetic acid-ammonium acetate buffer (pH 4.5): Take 77 g of ammonium acetate, add 500 ml of water to dissolve it, then add 60 ml of glacial acetic acid and an appropriate amount of water to make 1000 ml, shake well, and you have it.

[0064] Diluent (diluent required for preparation of derivatization agent solution): Take 500 ml of methanol, add 100 ml of acetic acid-ammonium acetate buffer (pH 4.5), then dilute to 1000 ml with water, shake well, and obtain.

[0065] Derivatization agent solution (1% 3-amino-5-hydroxypyrazole): Take 5 g of 3-amino-5-hydroxypyrazole, accurately weigh it, put it in a 500 ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute it to the scale with the diluent, shake well, and you have it.

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

[0067] (2) Preparation of blank solution

[0068] Blank solution: Accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and leave for 15 minutes.

[0069] (3) Preparation of reference solution

[0070] Reference solution: Accurately pipette 1 ml of 5 μg / ml aluminum standard solution, 10 ml of acetic acid-ammonium acetate buffer (pH 4.5), and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and let stand for 15 minutes.

[0071] (4) Sample pretreatment

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

[0073] Microwave digestion: transfer the above concentrate to a digestion tube, add 5 ml of nitric acid, and perform microwave digestion for 1 hour at a temperature of 200°C, a power of 1200W, and a pressure of 10MPa. Then place it on a hot plate and remove the acid at 150°C for 1 hour, cool it, and set it aside.

[0074] (5) Preparation of test solution

[0075] Test solution: Transfer the above pretreated sample to a 50 ml volumetric flask, then accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute to the scale with water, shake well, and leave for 15 minutes.

[0076] (6) Determination

[0077] Chromatographic column model: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); detection wavelength: 254nm; injection volume: 20μl; column temperature: 35℃; flow rate: 0.8ml / min; running time: isocratic running for 30 minutes. Calculated by peak area according to the external standard method. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 It shows the spectrum of the blank auxiliary material solution under the item of "Specificity" in the method verification described in Example 1.

[0079] Figure 2 It shows the spectrum of the reference solution under the item of "Specificity" in the method validation described in Example 1.

[0080] Figure 3 It shows the spectrum of 100% spiked test solution under "Specificity" in the method validation described in Example 1.

[0081] Figure 4 It shows the spectrum of the quantitative limit solution under the item of "Linearity and Range" in the method validation described in Example 1. DETAILED DESCRIPTION

[0082] The present invention is further described by the following examples, but it should be understood that the following examples are not limited to the scope of the present invention.

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

[0084] Embodiment 1

[0085] 1.1 Detection method

[0086] 1.1.1 Solution preparation

[0087] Acetic acid-ammonium acetate buffer (pH 4.5): Take 77 g of ammonium acetate, add 500 ml of water to dissolve it, then add 60 ml of glacial acetic acid and an appropriate amount of water to make 1000 ml, shake well, and you have it.

[0088] Diluent (diluent required for preparation of derivatization agent solution): Take 500 ml of methanol, add 100 ml of acetic acid-ammonium acetate buffer (pH 4.5), then dilute to 1000 ml with water, shake well, and obtain.

[0089] Derivatization agent solution (1% 3-amino-5-hydroxypyrazole): Take 5 g of 3-amino-5-hydroxypyrazole, accurately weigh it, put it in a 500 ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute it to the scale with the diluent, shake well, and you have it.

[0090] 1.1.2 Blank solution

[0091] Blank solution: Accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and leave for 15 minutes.

[0092] 1.1.3 Reference solution

[0093] Reference solution: Accurately pipette 1 ml of 5 μg / ml aluminum standard solution, 10 ml of acetic acid-ammonium acetate buffer (pH 4.5), and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and let stand for 15 minutes.

[0094] 1.1.4 Sample pretreatment

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

[0096] Microwave digestion: transfer the above concentrate to a digestion tube, add 5 ml of nitric acid, and perform microwave digestion for 1 hour at a temperature of 200°C, a power of 1200W, and a pressure of 10MPa. Then place it on a hot plate and remove the acid at 150°C for 1 hour, cool it, and set it aside.

[0097] 1.1.5 Test solution

[0098] Test solution: Transfer the above pretreated sample to a 50 ml volumetric flask, then accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute to the scale with water, shake well, and leave for 15 minutes.

[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 in a mobile phase bottle, shake well, and sonicate.

[0101] Chromatographic column model: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); detection wavelength: 254nm; injection volume: 20μl; column temperature: 35℃; flow rate: 0.8ml / min; running time: isocratic run for 30 minutes.

[0102] 1.1.7 Determination

[0103] Determination: Take 20μl of the blank solution, reference solution, and test solution (1 injection each) and inject them into the HPLC. Inject according to the above chromatographic conditions and record the chromatogram. Calculate by peak area according to the external standard method. The aluminum content shall not exceed 25μg / L.

[0104] 1.2 Method validation

[0105] 1.2.1 Exclusivity

[0106] 1.2.1.1 Blank solution 1

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

[0108] 1.2.1.2 Blank solution 2

[0109] Blank solution 2: Accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatizing agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and leave for 15 minutes.

[0110] 1.2.1.3 Blank excipient solution

[0111] Preparation of blank auxiliary materials: Take 450 mg of calcium glucarate and 337.5 mg of sodium chloride, weigh accurately, place them in a 500 ml volumetric flask, add appropriate amount of water to dissolve, dilute with water to the scale, shake well, and obtain.

[0112] Pretreatment of blank auxiliary materials: Take 200 ml of blank auxiliary materials and place them in a beaker. Heat and concentrate them on an electric stove to about 5 ml. Transfer the above concentrate to a digestion tube, add 5 ml of nitric acid, and perform microwave digestion for 1 hour at a temperature of 200°C, a power of 1200W, and a pressure of 10MPa. Then place it on a hot plate and remove the acid at 150°C for 1 hour, cool it, and set it aside.

[0113] Blank auxiliary material solution: Transfer the above pretreated sample to a 50 ml volumetric flask, then accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute to the scale with water, shake well, and leave for 15 minutes.

[0114] 1.2.1.4 Reference solution

[0115] Reference solution: prepare the reference solution as in "1.1.3" of Example 1.

[0116] 1.2.1.5 Test solution

[0117] Test solution: prepare the test solution as in "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, and then accurately pipette 1ml of 5μg / ml aluminum single element standard solution, 10ml of acetic acid-ammonium acetate buffer (pH4.5), and 10ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute with water to the scale, shake well, and let it stand for 15 minutes.

[0120] 1.2.1.7 Determination

[0121] Determination: Take 20 μl of the above blank solution 1, blank solution 2, blank auxiliary material solution, reference solution, test solution, and 100% spiked test solution (1 injection each) and inject into the high performance liquid chromatograph, inject according to the above chromatographic conditions, and record the chromatogram. Among them, blank solution 1, blank solution 2, blank auxiliary material 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 reference solution as in "1.1.3" of Example 1.

[0125] 1.2.2.2 100% spiked test solution

[0126] 100% spiked test solution: Prepare the same 100% spiked test solution as in "1.2.1.6" of Example 1.

[0127] 1.2.2.3 Determination

[0128] Determination: Take 20 μl of the above-mentioned reference solution (5 injections) and 100% spiked test solution (1 injection) and inject them into the high performance liquid chromatograph, inject according to the above-mentioned chromatographic conditions, and record the chromatogram. The RSD of the peak area of ​​the aluminum derivative of the reference solution injected 5 times in a row shall not exceed 2%, and the separation degree of the aluminum derivative peak and the adjacent peak in the 100% spiked test solution shall be greater than 1.5.

[0129] 1.2.3 Limit of quantification and limit of detection

[0130] 1.2.3.1 Quantitative limit solution

[0131] Quantitation limit solution: Take an appropriate amount of reference solution, add an appropriate amount of acetic acid-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 reference solution, add an appropriate amount of acetic acid-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 Determination

[0135] Determination: Take 20μl of the above-mentioned quantitative limit solution (6 injections) and detection limit solution (3 injections) and inject them into the high performance liquid chromatograph, inject according to the above-mentioned chromatographic conditions, and record the chromatogram. The quantitative limit solution is injected 6 injections continuously, 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 quantitative limit solution shall not be greater than 10% of the limit concentration. The detection limit solution is injected 3 injections continuously, and 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 pipette 0.3 ml of 5 μg / ml aluminum standard solution, 10 ml of acetic acid-ammonium acetate buffer (pH 4.5), and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and leave for 15 minutes.

[0139] 50% linear solution: Accurately pipette 0.5 ml of 5 μg / ml aluminum standard solution, 10 ml of acetic acid-ammonium acetate buffer (pH 4.5), and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and leave for 15 minutes.

[0140] 100% linear solution: Accurately pipette 1 ml of 5 μg / ml aluminum standard solution, 10 ml of acetic acid-ammonium acetate buffer (pH 4.5), and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and leave for 15 minutes.

[0141] 150% linear solution: Accurately pipette 1.5 ml of 5 μg / ml aluminum standard solution, 10 ml of acetic acid-ammonium acetate buffer (pH 4.5), and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and leave for 15 minutes.

[0142] 200% linear solution: Accurately pipette 2 ml of 5 μg / ml aluminum standard solution, 10 ml of acetic acid-ammonium acetate buffer (pH 4.5), and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and leave for 15 minutes.

[0143] 1.2.4.2 Determination

[0144] Determination: Take 20 μl of each linear solution (1 needle each) and inject it into the high performance liquid chromatograph, sample according to the above chromatographic conditions, and record the chromatogram. The chromatogram is linear in the range of quantification limit concentration to 200% of the limit concentration, the regression linear correlation coefficient (r) should be ≥ 0.999, and the ratio of the absolute value of the Y-axis intercept to the 100% limit concentration response value should be within 25%.

[0145] 1.2.5 Accuracy

[0146] 1.2.5.1 Reference solution

[0147] Reference solution: prepare the reference solution as in "1.1.3" of Example 1.

[0148] 1.2.5.2 Background solution

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

[0150] 1.2.5.3 Recovery solution

[0151] 50% spiked test solution: Take the sample obtained by the same operation as the pre-treated sample under "1.4" in Example 1, place it in a 50ml volumetric flask, then accurately pipette 0.5ml of 5μg / ml aluminum single element standard solution, 10ml of acetic acid-ammonium acetate buffer (pH4.5), and 10ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute to the scale with water, shake well, and let it stand for 15 minutes. (Prepare 3 copies in parallel)

[0152] 100% spiked test solution: Prepare the same 100% spiked test solution as in "1.2.1.6" of Example 1. (Prepare 3 copies in parallel)

[0153] 150% spiked test solution: Take the sample obtained by the same operation as the pre-treated sample under "1.4" in Example 1, place it in a 50ml volumetric flask, and then accurately pipette 1.5ml of 5μg / ml aluminum single element standard solution, 10ml of acetic acid-ammonium acetate buffer (pH4.5), and 10ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute with water to the scale, shake well, and let it stand for 15 minutes. (Prepare 3 copies in parallel)

[0154] 1.2.5.4 Determination

[0155] Determination: Take 20 μl of the above-mentioned reference solution, background solution, and each test sample spiked solution (recovery solution) (1 injection each) and inject it into the high performance liquid chromatograph, inject according to the above-mentioned chromatographic conditions, and record the chromatogram. The recovery rates of the limit concentration 150%, 100%, and 50% recovery rate solutions are all between 80% and 120%, and the RSD of the recovery of 9 recovery rate solutions is not greater than 20%.

[0156] 1.2.6 Precision-Repeatability (Operator A)

[0157] 1.2.6.1 Blank solution

[0158] Blank solution: prepare the same blank solution as in "1.1.2" in Example 1.

[0159] 1.2.6.2 Reference solution

[0160] Reference solution: prepare the reference solution as in "1.1.3" of Example 1.

[0161] 1.2.6.3 100% spiked test solution

[0162] 100% spiked test solution: Prepare the same 100% spiked test solution as in "1.2.1.6" of Example 1. (Prepare 6 portions in parallel)

[0163] 1.2.6.4 Determination

[0164] Determination: Take 20 μl of the blank solution, reference solution, and each 100% spiked test solution (1 injection each) and inject it into the HPLC, inject according to the above chromatographic conditions, and record the chromatogram. Operator A prepares 6 100% spiked test solutions in parallel, and the RSD of the aluminum salt content in them is not greater than 20%.

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

[0166] 1.2.7.1 Blank solution

[0167] Blank solution: prepare the same blank solution as in "1.1.2" in Example 1.

[0168] 1.2.7.2 Reference solution

[0169] Reference solution: prepare the reference solution as in "1.1.3" of Example 1.

[0170] 1.2.7.3 100% spiked test solution

[0171] 100% spiked test solution: Prepare the same 100% spiked test solution as in "1.2.1.6" of Example 1. (Prepare 6 portions in parallel)

[0172] 1.2.7.4 Determination

[0173] Determination: Take 20 μl of the blank solution, reference solution, and each 100% spiked test solution (1 injection each) and inject it into the HPLC, sample according to the above chromatographic conditions, and record the chromatogram. Operator B prepared 6 100% spiked test solutions in parallel, and the RSD of the aluminum salt content in the solution was no more than 20%; the RSD of the aluminum salt content in the 12 solutions was no more than 20%.

[0174] 1.2.8 Solution stability

[0175] 1.2.8.1 Reference solution

[0176] Reference solution: prepare the reference solution as in "1.1.3" of Example 1.

[0177] 1.2.8.2 100% spiked test solution

[0178] 100% spiked test solution: Prepare the same 100% spiked test solution as in "1.2.1.6" of Example 1.

[0179] 1.2.8.3 Determination

[0180] Determination: At room temperature, take 20 μl of the reference solution and each 100% spiked test solution (1 injection each) and inject them into the high performance liquid chromatograph at about 0h, 2h, 4h, 8h, 16h, 24h, 48h, and 72h respectively, and inject the sample according to the above chromatographic conditions and record the chromatogram. The recovery rate of the reference solution measured at each time point is within the range of 90-110%; the relative deviation of the content of aluminum salt in the 100% spiked test solution measured at each time point and 0h shall not exceed 20%.

[0181] 1.2.9 Durability

[0182] 1.2.9.1 Blank solution

[0183] Blank solution: prepare the same blank solution as in "1.1.2" in Example 1.

[0184] 1.2.9.2 Reference solution

[0185] Reference solution: prepare the reference solution as in "1.1.3" of Example 1.

[0186] 1.2.9.3 100% spiked test solution

[0187] 100% spiked test solution: Prepare the same 100% spiked test solution as in "1.2.1.6" of Example 1.

[0188] 1.2.9.4 Determination

[0189] Determination: Take 20 μl of the blank solution, reference solution, and each 100% spiked test solution (1 injection each) and inject into the high performance liquid chromatograph, and inject according to each durable chromatographic condition (durability condition 1: column temperature 40°C, durability condition 2: column temperature 30°C; durability condition 3: flow rate 0.7 ml / min; durability condition 4: flow rate 0.9 ml / min), and record the chromatogram. The RSD of the aluminum salt content of the 100% spiked test solution under each durability condition and the normal condition shall not exceed 20%.

[0190] 1.3 Method validation results statistics

[0191] Embodiment 2

[0192] 2.1 Detection Method

[0193] 2.1.1 Solution preparation

[0194] Acetic acid-ammonium acetate buffer (pH 4.5): Take 77 g of ammonium acetate, add 500 ml of water to dissolve it, then add 60 ml of glacial acetic acid and an appropriate amount of water to make 1000 ml, shake well, and you have it.

[0195] Diluent (diluent required for preparation of derivatization agent solution): Take 500 ml of methanol, add 100 ml of acetic acid-ammonium acetate buffer (pH 4.5), then dilute to 1000 ml with water, shake well, and obtain.

[0196] Derivatization agent solution (1% 3-amino-5-hydroxypyrazole): Take 5 g of 3-amino-5-hydroxypyrazole, accurately weigh it, put it in a 500 ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute it to the scale with the diluent, shake well, and you have it.

[0197] 2.1.2 Blank solution

[0198] Blank solution: Accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and leave for 15 minutes.

[0199] 2.1.3 Reference solution

[0200] Reference solution: Accurately pipette 1 ml of 5 μg / ml aluminum standard solution, 10 ml of acetic acid-ammonium acetate buffer (pH 4.5), and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and let stand for 15 minutes.

[0201] 2.1.4 Sample pretreatment

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

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

[0204] 2.1.5 Test solution

[0205] Test solution: Transfer the above pretreated sample to a 50 ml volumetric flask, then accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute to the scale with water, shake well, and leave for 15 minutes.

[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 in a mobile phase bottle, shake well, and sonicate.

[0208] Chromatographic column model: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); detection wavelength: 254nm; injection volume: 20μl; column temperature: 35℃; flow rate: 0.8ml / min; running time: isocratic run for 30 minutes.

[0209] 2.1.7 Determination

[0210] Determination: Take 20μl of the above blank solution, reference solution, and test solution (1 injection each) and inject them into the high performance liquid chromatograph. Inject according to the above chromatographic conditions and record the chromatogram. Calculate by peak area according to the external standard method. The content of aluminum salt shall not exceed 25μg / L.

[0211] 2.2 Method validation

[0212] The method validation was performed in accordance with the method validation method under item "1.2" in Example 1, wherein only the indicators involving the test sample were performed, and the validation results under item "1.2" were taken for the items not involving the test sample.

[0213] 2.3 Method validation results statistics

[0214] Embodiment 3

[0215] 3.1 Detection method

[0216] 3.1.1 Solution preparation

[0217] Acetic acid-ammonium acetate buffer (pH 4.5): Take 77 g of ammonium acetate, add 500 ml of water to dissolve it, then add 60 ml of glacial acetic acid and an appropriate amount of water to make 1000 ml, shake well, and you have it.

[0218] Diluent (diluent required for preparation of derivatization agent solution): Take 500 ml of methanol, add 100 ml of acetic acid-ammonium acetate buffer (pH 4.5), then dilute to 1000 ml with water, shake well, and obtain.

[0219] Derivatization agent solution (1% 3-amino-5-hydroxypyrazole): Take 5 g of 3-amino-5-hydroxypyrazole, accurately weigh it, put it in a 500 ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute it to the scale with the diluent, shake well, and you have it.

[0220] 3.1.2 Blank solution

[0221] Blank solution: Accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and leave for 15 minutes.

[0222] 3.1.3 Reference solution

[0223] Reference solution: Accurately pipette 1 ml of 5 μg / ml aluminum standard solution, 10 ml of acetic acid-ammonium acetate buffer (pH 4.5), and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and let stand for 15 minutes.

[0224] 3.1.4 Sample pretreatment

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

[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 on a heating plate, and heat at 150°C for 2 hours.

[0227] 3.1.5 Test solution

[0228] Test solution: Transfer the above pretreated sample to a 50 ml volumetric flask, then accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute to the scale with water, shake well, and leave for 15 minutes.

[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 in a mobile phase bottle, shake well, and sonicate.

[0231] Chromatographic column model: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); detection wavelength: 254nm; injection volume: 20μl; column temperature: 35℃; flow rate: 0.8ml / min; running time: isocratic run for 30 minutes.

[0232] 3.1.7 Determination

[0233] Determination: Take 20μl of the above blank solution, reference solution, and test solution (1 injection each) and inject them into the high performance liquid chromatograph. Inject according to the above chromatographic conditions and record the chromatogram. Calculate by peak area according to the external standard method. The content of aluminum salt shall not exceed 25μg / L.

[0234] 3.2 Method validation

[0235] The method validation was performed in accordance with the method validation method under item "1.2" in Example 1, wherein only the indicators involving the test sample were performed, and the validation results under item "1.2" were taken for the items not involving the test sample.

[0236] 3.3 Method validation results statistics

[0237] Embodiment 4

[0238] 4.1 Detection Method

[0239] 4.1.1 Solution preparation

[0240] Acetic acid-ammonium acetate buffer (pH 4.5): Take 77 g of ammonium acetate, add 500 ml of water to dissolve it, then add 60 ml of glacial acetic acid and an appropriate amount of water to make 1000 ml, shake well, and you have it.

[0241] Diluent (diluent required for preparation of derivatization agent solution): Take 500 ml of methanol, add 100 ml of acetic acid-ammonium acetate buffer (pH 4.5), then dilute to 1000 ml with water, shake well, and obtain.

[0242] Derivatization agent solution (1% 3-amino-5-hydroxypyrazole): Take 5 g of 3-amino-5-hydroxypyrazole, accurately weigh it, put it in a 500 ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute it to the scale with the diluent, shake well, and you have it.

[0243] 4.1.2 Blank solution

[0244] Blank solution: Accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and leave for 15 minutes.

[0245] 4.1.3 Reference solution

[0246] Reference solution: Accurately pipette 1 ml of 5 μg / ml aluminum standard solution, 10 ml of acetic acid-ammonium acetate buffer (pH 4.5), and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and let stand for 15 minutes.

[0247] 4.1.4 Sample pretreatment

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

[0249] Microwave digestion: transfer the above concentrate to a digestion tube, add 5 ml of nitric acid, and perform microwave digestion for 1 hour at a temperature of 200°C, a power of 1200W, and a pressure of 10MPa. Then place it on a hot plate and remove the acid at 150°C for 1 hour, cool it, and set it aside.

[0250] 4.1.5 Test solution

[0251] Test solution: Transfer the above pretreated sample to a 50 ml volumetric flask, then accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute to the scale with water, shake well, and leave for 15 minutes.

[0252] 4.1.6 Chromatographic conditions

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

[0254] Chromatographic column model: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); detection wavelength: 254nm; injection volume: 20μl; column temperature: 35℃; flow rate: 0.8ml / min; running time: isocratic run for 30 minutes.

[0255] 4.1.7 Determination

[0256] Determination: Take 20μl of the above blank solution, reference solution, and test solution (1 injection each) and inject them into the high performance liquid chromatograph. Inject according to the above chromatographic conditions and record the chromatogram. Calculate by peak area according to the external standard method. The content of aluminum salt shall not exceed 25μg / L.

[0257] 4.2 Method validation

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

[0259] 4.3 Method validation results statistics

[0260] Embodiment 5

[0261] 5.1 Detection Method

[0262] 5.1.1 Solution preparation

[0263] Acetic acid-ammonium acetate buffer (pH 4.5): Take 77 g of ammonium acetate, add 500 ml of water to dissolve it, then add 60 ml of glacial acetic acid and an appropriate amount of water to make 1000 ml, shake well, and you have it.

[0264] Diluent (diluent required for preparation of derivatization agent solution): Take 500 ml of methanol, add 100 ml of acetic acid-ammonium acetate buffer (pH 4.5), then dilute to 1000 ml with water, shake well, and obtain.

[0265] Derivatization agent solution (1% 3-amino-5-hydroxypyrazole): Take 5 g of 3-amino-5-hydroxypyrazole, accurately weigh it, put it in a 500 ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute it to the scale with the diluent, shake well, and you have it.

[0266] 5.1.2 Blank solution

[0267] Blank solution: Accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and leave for 15 minutes.

[0268] 5.1.3 Reference solution

[0269] Reference solution: Accurately pipette 1 ml of 5 μg / ml aluminum standard solution, 10 ml of acetic acid-ammonium acetate buffer (pH 4.5), and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and let stand for 15 minutes.

[0270] 5.1.4 Sample pretreatment

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

[0272] Microwave digestion: transfer the above concentrate to a digestion tube, add 5 ml of nitric acid, and perform microwave digestion for 1 hour at a temperature of 200°C, a power of 1200W, and a pressure of 10MPa. Then place it on a hot plate and remove the acid at 150°C for 1 hour, cool it, and set it aside.

[0273] 5.1.5 Test solution

[0274] Test solution: Transfer the above pretreated sample to a 50 ml volumetric flask, then accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute to the scale with water, shake well, and leave for 15 minutes.

[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 in a mobile phase bottle, shake well, and sonicate to obtain.

[0277] Chromatographic column model: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); detection wavelength: 254nm; injection volume: 20μl; column temperature: 35℃; flow rate: 0.8ml / min; running time: isocratic run for 30 minutes.

[0278] 5.1.7 Determination

[0279] Determination: Take 20μl of the above blank solution, reference solution, and test solution (1 injection each) and inject them into the high performance liquid chromatograph. Inject according to the above chromatographic conditions and record the chromatogram. Calculate by peak area according to the external standard method. The content of aluminum salt shall not exceed 25μg / L.

[0280] 5.2 Method validation

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

[0282] 5.3 Method validation results statistics

[0283] Embodiment 6

[0284] 6.1 Detection Method

[0285] 6.1.1 Solution preparation

[0286] Acetic acid-ammonium acetate buffer (pH 4.5): Take 77 g of ammonium acetate, add 500 ml of water to dissolve it, then add 60 ml of glacial acetic acid and an appropriate amount of water to make 1000 ml, shake well, and you have it.

[0287] Diluent (diluent required for preparation of derivatization agent solution): Take 500 ml of methanol, add 100 ml of acetic acid-ammonium acetate buffer (pH 4.5), then dilute to 1000 ml with water, shake well, and obtain.

[0288] Derivatization agent solution (1% 3-amino-5-hydroxypyrazole): Take 5 g of 3-amino-5-hydroxypyrazole, accurately weigh it, put it in a 500 ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute it to the scale with the diluent, shake well, and you have it.

[0289] 6.1.2 Blank solution

[0290] Blank solution: Accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and leave for 15 minutes.

[0291] 6.1.3 Reference solution

[0292] Reference solution: Accurately pipette 1 ml of 5 μg / ml aluminum standard solution, 10 ml of acetic acid-ammonium acetate buffer (pH 4.5), and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and let stand for 15 minutes.

[0293] 6.1.4 Sample pretreatment

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

[0295] Microwave digestion: transfer the above concentrate to a digestion tube, add 5 ml of nitric acid, and perform microwave digestion for 1 hour at a temperature of 200°C, a power of 1200W, and a pressure of 10MPa. Then place it on a hot plate and remove the acid at 150°C for 1 hour, cool it, and set it aside.

[0296] 6.1.5 Test solution

[0297] Test solution: Transfer the above pretreated sample to a 50 ml volumetric flask, then accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute to the scale with water, shake well, and leave for 15 minutes.

[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 in a mobile phase bottle, shake well, and sonicate.

[0300] Chromatographic column model: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); detection wavelength: 254nm; injection volume: 20μl; column temperature: 35℃; flow rate: 0.8ml / min; running time: isocratic run for 40 minutes.

[0301] 6.1.7 Determination

[0302] Determination: Take 20μl of the above blank solution, reference solution, and test solution (1 injection each) and inject them into the high performance liquid chromatograph. Inject according to the above chromatographic conditions and record the chromatogram. Calculate by peak area according to the external standard method. The content of aluminum salt shall not exceed 25μg / L.

[0303] 6.2 Method Validation

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

[0305] 6.3 Method validation results statistics

[0306] Embodiment 7

[0307] 7.1 Detection Method

[0308] 7.1.1 Solution preparation

[0309] Acetic acid-ammonium acetate buffer (pH 4.5): Take 77 g of ammonium acetate, add 500 ml of water to dissolve it, then add 60 ml of glacial acetic acid and an appropriate amount of water to make 1000 ml, shake well, and you have it.

[0310] Diluent (diluent required for preparation of derivatization agent solution): Take 500 ml of methanol, add 100 ml of acetic acid-ammonium acetate buffer (pH 4.5), then dilute to 1000 ml with water, shake well, and obtain.

[0311] Derivatization agent solution (1% 3-amino-5-hydroxypyrazole): Take 5 g of 3-amino-5-hydroxypyrazole, accurately weigh it, put it in a 500 ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute it to the scale with the diluent, shake well, and you have it.

[0312] 7.1.2 Blank solution

[0313] Blank solution: Accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and leave for 15 minutes.

[0314] 7.1.3 Reference solution

[0315] Reference solution: Accurately pipette 1 ml of 5 μg / ml aluminum standard solution, 10 ml of acetic acid-ammonium acetate buffer (pH 4.5), and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and let stand for 15 minutes.

[0316] 7.1.4 Sample pretreatment

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

[0318] Microwave digestion: transfer the above concentrate to a digestion tube, add 5 ml of nitric acid, and perform microwave digestion for 1 hour at a temperature of 200°C, a power of 1200W, and a pressure of 10MPa. Then place it on a hot plate and remove the acid at 150°C for 1 hour, cool it, and set it aside.

[0319] 7.1.5 Test solution

[0320] Test solution: Transfer the above pretreated sample to a 50 ml volumetric flask, then accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute to the scale with water, shake well, and leave for 15 minutes.

[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 in a mobile phase bottle, shake well, and sonicate.

[0323] Chromatographic column model: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); detection wavelength: 254nm; injection volume: 20μl; column temperature: 35℃; flow rate: 0.8ml / min; running time: isocratic run for 40 minutes.

[0324] 7.1.7 Determination

[0325] Determination: Take 20μl of the above blank solution, reference solution, and test solution (1 injection each) and inject them into the high performance liquid chromatograph. Inject according to the above chromatographic conditions and record the chromatogram. Calculate by peak area according to the external standard method. The content of aluminum salt shall not exceed 25μg / L.

[0326] 7.2 Method Validation

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

[0328] 7.3 Method validation results statistics

[0329] Comparative Example 1

[0330] The aluminum salt in this product was 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 acetic acid-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 with 5 ml of chloroform three times, filter the extract through filter paper into a 25 ml volumetric flask, and dilute to the scale with chloroform.

[0332] Reference solution: accurately pipette 1 ml of 5 μg / ml aluminum standard solution, dilute to 160 ml with water, transfer to a separatory funnel, add 1.0 ml of acetic acid-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 with 5 ml of chloroform three times, filter the extract through filter paper into a 25 ml volumetric flask, and dilute to the mark with chloroform.

[0333] Test solution: Accurately pipette 160 ml of calcium gluconate and sodium chloride injection, place in a separatory funnel, add 1.0 ml of acetic acid-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 with 5 ml of chloroform three times, filter the extract through filter paper into a 25 ml volumetric flask, and dilute to the scale with chloroform.

[0334] 100% spiked test solution: Accurately pipette 1 ml of 5 μg / ml aluminum standard solution and 160 ml of calcium gluconate sodium chloride injection, place in a separatory funnel, add 1.0 ml of acetic acid-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 with 5 ml of chloroform three times, filter the extract through filter paper into a 25 ml volumetric flask, and dilute to the scale 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: chromatographic column model: ZORBAX Eclipse Plus (C8) (250*4.6mm, 5μm); detection wavelength: 390nm; injection volume: 10μl; column temperature: 30℃; flow rate: 1ml / min; running time: isocratic run for 30 minutes.

[0337] Determination: Take 10 μl of the above blank solution, reference solution, test solution, and 100% spiked test solution (1 injection each) and inject them into the high performance liquid chromatograph, inject according to 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%, and the matrix seriously interfered with the detection.

[0339] Comparative Example 2

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

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

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

[0346] Chromatographic conditions: chromatographic column model: Shim-pack ODS (C18) (250*4.6mm, 5μm); detection wavelength: 570nm; injection volume: 20μl; column temperature: 30℃; flow rate: 1ml / min; running time: isocratic run for 30 minutes.

[0347] Determination: Take 20 μl of the blank solution, reference solution, test solution, and 100% spiked test solution (1 injection each) and inject them into the high performance liquid chromatograph, inject according to 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, while 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 and sodium chloride injection, characterized in that The detection method adopts sample pretreatment-derivation-high performance liquid chromatography-ultraviolet detection method; it includes the following steps: Sample pretreatment: Take 200 ml of calcium gluconate and sodium chloride injection, heat and concentrate; further process the concentrate, wherein the further processing method is wet digestion, incineration ashing, microwave digestion; Derivatization: the pretreated sample is subjected to a derivatization reaction with a 3-amino-5-hydroxypyrazole derivatization reagent, wherein aluminum in the sample reacts with the 3-amino-5-hydroxypyrazole derivatization reagent to form an aluminum derivative; High performance liquid chromatography-ultraviolet detection: using a high performance liquid chromatograph, with an ultraviolet-visible spectrophotometer (including a diode array detector) as the detector, a chromatographic column with octadecylsilane bonded silica gel as the filler, and a mixed solution of methanol and buffer as the mobile phase for separation and detection; The buffer solution is 0.1% formic acid aqueous solution, 0.1% formic acid aqueous solution containing 10 mM ammonium acetate, and 0.1% formic acid aqueous solution containing 10 mM potassium dihydrogen phosphate.

2. The detection method according to claim 1, wherein: The buffer solution is preferably a 0.1% formic acid aqueous solution containing 10 mM ammonium acetate.

3. The detection method according to claim 1, wherein: The chromatographic column with octadecylsilane bonded silica gel as filler is ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm).

4. The detection method according to claim 1, wherein: The mobile phase is a mixed solution of methanol-buffer (35-45:65-55), preferably a mixed solution of methanol-buffer (40:60).

5. The detection method according to claim 1, wherein: The pretreatment method in step (2) is preferably incineration or microwave digestion, and more preferably microwave digestion.

6. The detection method according to claim 1, comprising the steps of: (1) Solution preparation Acetic acid-ammonium acetate buffer (pH 4.5): Take 77 g of ammonium acetate, add 500 ml of water to dissolve it, then add 60 ml of glacial acetic acid and an appropriate amount of water to make 1000 ml, shake well, and you have it. Diluent (diluent required for preparation of derivatization agent solution): Take 500 ml of methanol, add 100 ml of acetic acid-ammonium acetate buffer (pH 4.5), then dilute to 1000 ml with water, shake well, and obtain. Derivatization agent solution (1% 3-amino-5-hydroxypyrazole): Take 5 g of 3-amino-5-hydroxypyrazole, accurately weigh it, put it in a 500 ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute it to the scale with the diluent, shake well, and you have it. Mobile phase: Take 1600ml of acetonitrile and 2400ml of 0.1% formic acid aqueous solution containing 10mM ammonium acetate, put them in a mobile phase bottle, shake well, and ultrasonicate to obtain. (2) Preparation of blank solution Blank solution: Accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and leave for 15 minutes. (3) Preparation of reference solution Reference solution: Accurately pipette 1 ml of 5 μg / ml aluminum standard solution, 10 ml of acetic acid-ammonium acetate buffer (pH 4.5), and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and let stand for 15 minutes. (4) Sample pretreatment Concentration: Take 200 ml of calcium gluconate and sodium chloride injection and place it in a beaker. Heat it on an electric stove and concentrate it to about 5 ml. 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 above pretreated sample to a 50 ml volumetric flask, then accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute to the scale with water, shake well, and leave for 15 minutes. (6) Determination Chromatographic column model: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); detection wavelength: 254nm; injection volume: 20μl; column temperature: 35℃; flow rate: 0.8ml / min; running time: isocratic running for 30 minutes. Calculated by peak area according to the external standard method.

7. The detection method according to claim 1, comprising the following steps: (1) Solution preparation Acetic acid-ammonium acetate buffer (pH 4.5): Take 77 g of ammonium acetate, add 500 ml of water to dissolve it, then add 60 ml of glacial acetic acid and an appropriate amount of water to make 1000 ml, shake well, and you have it. Diluent (diluent required for preparation of derivatization agent solution): Take 500 ml of methanol, add 100 ml of acetic acid-ammonium acetate buffer (pH 4.5), then dilute to 1000 ml with water, shake well, and obtain. Derivatization agent solution (1% 3-amino-5-hydroxypyrazole): Take 5 g of 3-amino-5-hydroxypyrazole, accurately weigh it, put it in a 500 ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute it to the scale with the diluent, shake well, and you have it. Mobile phase: Take 1600ml of acetonitrile and 2400ml of 0.1% formic acid aqueous solution containing 10mM ammonium acetate, put them in a mobile phase bottle, shake well, and ultrasonicate to obtain. (2) Preparation of blank solution Blank solution: Accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and leave for 15 minutes. (3) Preparation of reference solution Reference solution: Accurately pipette 1 ml of 5 μg / ml aluminum standard solution, 10 ml of acetic acid-ammonium acetate buffer (pH 4.5), and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and let stand for 15 minutes. (4) Sample pretreatment Concentration: Take 200 ml of calcium gluconate and sodium chloride injection and place it in a beaker. Heat it on an electric stove and concentrate it to about 5 ml. Incandescence: Transfer the above concentrate to a crucible, slowly incinerate until completely carbonized, cool, add sulfuric acid to moisten, heat at low temperature until the sulfuric acid vapor is completely removed, and incinerate at 500-550℃ to completely incense. (5) Preparation of test solution Test solution: Transfer the above pretreated sample to a 50 ml volumetric flask, then accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute to the scale with water, shake well, and leave for 15 minutes. (6) Determination Chromatographic column model: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); detection wavelength: 254nm; injection volume: 20μl; column temperature: 35℃; flow rate: 0.8ml / min; running time: isocratic running for 30 minutes. Calculated by peak area according to the external standard method.

8. The detection method according to claim 1, comprising the steps of: (1) Solution preparation Acetic acid-ammonium acetate buffer (pH 4.5): Take 77 g of ammonium acetate, add 500 ml of water to dissolve it, then add 60 ml of glacial acetic acid and an appropriate amount of water to make 1000 ml, shake well, and you have it. Diluent (diluent required for preparation of derivatization agent solution): Take 500 ml of methanol, add 100 ml of acetic acid-ammonium acetate buffer (pH 4.5), then dilute to 1000 ml with water, shake well, and obtain. Derivatization agent solution (1% 3-amino-5-hydroxypyrazole): Take 5 g of 3-amino-5-hydroxypyrazole, accurately weigh it, put it in a 500 ml volumetric flask, add an appropriate amount of diluent to dissolve it, and dilute it to the scale with the diluent, shake well, and you have it. Mobile phase: Take 1600ml of acetonitrile and 2400ml of 0.1% formic acid aqueous solution containing 10mM ammonium acetate, put them in a mobile phase bottle, shake well, and ultrasonicate to obtain. (2) Preparation of blank solution Blank solution: Accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and leave for 15 minutes. (3) Preparation of reference solution Reference solution: Accurately pipette 1 ml of 5 μg / ml aluminum standard solution, 10 ml of acetic acid-ammonium acetate buffer (pH 4.5), and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole), place them in a 50 ml volumetric flask, dilute to the scale with water, shake well, and let stand for 15 minutes. (4) Sample pretreatment Concentration: Take 200 ml of calcium gluconate and sodium chloride injection and place it in a beaker. Heat it on an electric stove and concentrate it to about 5 ml. Microwave digestion: transfer the above concentrate to a digestion tube, add 5 ml of nitric acid, and perform microwave digestion for 1 hour at a temperature of 200°C, a power of 1200W, and a pressure of 10MPa. Then place it on a hot plate and remove the acid at 150°C for 1 hour, cool it, and set it aside. (5) Preparation of test solution Test solution: Transfer the above pretreated sample to a 50 ml volumetric flask, then accurately pipette 10 ml of acetic acid-ammonium acetate buffer (pH 4.5) and 10 ml of derivatization agent solution (1% 3-amino-5-hydroxypyrazole) into it, dilute to the scale with water, shake well, and leave for 15 minutes. (6) Determination Chromatographic column model: ZORBAX Eclipse Plus (C18) (250*4.6mm, 5μm); detection wavelength: 254nm; injection volume: 20μl; column temperature: 35℃; flow rate: 0.8ml / min; running time: isocratic running for 30 minutes. Calculated by peak area according to the external standard method.

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