Value determination method of human angiotensin II purity standard substance

Abstract

The invention discloses a method for valuing a human angiotensin II purity standard substance. The method comprises the following steps: (1) selecting human angiotensin II with the purity of more than or equal to 99% as the purity standard substance; (2) determining the value of the human angiotensin II purity standard substance by adopting a mass balance method; (3) hydrolyzing the human angiotensin II purity standard substance, taking isotope labeled phenylalanine, isoleucine and valine as internal standards, and determining the value of the human angiotensin II purity standard substance by liquid chromatography-isotope dilution mass spectrometry; and (4) calculating the average value of the fixed value results of the mass balance method and the isotope dilution mass spectrometry to obtain the fixed value result of the human angiotensin II purity standard substance. According to the method for determining the value of the human angiotensin II purity standard substance provided by the invention, the value of the human angiotensin II purity standard substance is determined by adopting a mass balance method and a liquid chromatography-isotope dilution mass spectrometry, and the finally obtained value determination result of the Ang II purity standard substance is 0.8734 g / g, so that the method has traceability.

Description

Technical Field

[0001] The invention relates to the technical field of measurement, and in particular to a method for determining the purity of a human angiotensin II standard substance. Background Art

[0002] Human angiotensin Ⅱ (Ang Ⅱ) is an octapeptide with an amino acid sequence of Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, a CAS number of 4474-91-3, and a molecular formula of C 50 H 71 N 13 O 12 , the relative molecular mass is 1046.18, and the chemical structure is as follows:

[0003]

[0004] Ang Ⅱ is very active in the body. It can not only contract arteriolar smooth muscle and increase peripheral vascular resistance, but also stimulate the adrenal glands to secrete aldosterone in a part called the zona glomerulosa, causing an increase in blood volume. It can also excite the sympathetic nervous system, increase norepinephrine secretion, and constrict blood vessels. The above three effects all lead to increased blood pressure. Therefore, measuring the content of Ang Ⅱ is an important indicator for the prevention, diagnosis, control and treatment of hypertension.

[0005] In the existing technology, the determination of the relative molecular mass standard material of human angiotensin II (NIM-RM3622) in my country is based on electrospray ionization mass spectrometry-linear ion trap mass spectrometry and electrospray ionization mass spectrometry-quadrupole-time of flight mass spectrometry, which has a narrow scope of application. Therefore, it is of research significance to develop a new method for determining the purity standard material of human angiotensin II. Summary of the invention

[0006] The purpose of the present invention is to provide a method for determining the purity of human angiotensin II standard substance to solve at least one of the above technical problems.

[0007] The first aspect of the present invention provides a method for determining the purity of a human angiotensin II standard substance, comprising the following steps:

[0008] Human angiotensin II with a purity of ≥99% was selected as the purity standard substance;

[0009] The mass balance method is used to determine the purity of human angiotensin II standard substances. The mass balance method value result can be calculated by the following formula:

[0010] P=P o (1-X w -X a -Xe -X v ),

[0011] Where, P is the purity of human angiotensin II purity standard substance, P o The main component content of human angiotensin II purity standard material, X w is the moisture content, X a is the content of ash and anions, X e is the inorganic element content, X v is the content of volatile organic components;

[0012] The purity standard substance of human angiotensin II was hydrolyzed, and the purity standard substance of human angiotensin II was determined by liquid chromatography-isotope dilution mass spectrometry using isotope-labeled phenylalanine, isoleucine and valine as internal standards.

[0013] Calculate the average value of the mass balance method valuation results and the liquid chromatography-isotope dilution mass spectrometry method valuation results to obtain the valuation results of the human angiotensin II purity standard substance.

[0014] According to the method for determining the purity of human angiotensin II standard substance of the present invention, the value is determined by liquid chromatography-isotope dilution mass spectrometry and mass balance method. Based on the above two methods, the final value result of the purity of Ang II standard substance is 0.8734 g / g, which provides a technical reference for the value of the purity of human angiotensin II standard substance and has traceability.

[0015] In some embodiments, liquid chromatography-isotope dilution mass spectrometry is high performance liquid chromatography-isotope dilution mass spectrometry.

[0016] In some embodiments, the main component content of the human angiotensin II purity standard substance is determined by high performance liquid chromatography, and the chromatographic detection conditions are as follows: the chromatographic column is Agilent Poroshell 120EC-C18, 4.6mm×50mm, 4μm; the injection concentration is 1mg / mL; the injection volume is 5μL; the column temperature is 25°C; the flow rate is 0.5mL / min; the detection wavelength is 230nm; the detection time is 30min; the mobile phase includes mobile phase A and mobile phase B, mobile phase A is pH=3.0, 20mmol / L sodium dihydrogen phosphate buffer, and mobile phase B is 50% acetonitrile.

[0017] In other embodiments, the mobile phase gradient conditions, measured by volume fraction, can be: (1) at 0 min, mobile phase A is 67% and mobile phase B is 33%; (2) at 2 min, mobile phase A is 67% and mobile phase B is 33%; (2) at 2 min, mobile phase A is 67% and mobile phase B is 33%; (3) at 25 min, mobile phase A is 57% and mobile phase B is 43%; (4) at 30 min, mobile phase A is 57% and mobile phase B is 43%; (5) at 35 min, mobile phase A is 67% and mobile phase B is 33%; (6) at 40 min, mobile phase A is 67% and mobile phase B is 33%.

[0018] In some embodiments, the moisture content is determined using a Karl Fischer titrator, comprising the following steps: accurately weighing 6 mg of human angiotensin II purity standard material, determining it using a Karl Fischer titrator to obtain a determination result, performing six parallel determinations, and then determining without adding a sample to obtain a blank value of moisture in the air, and obtaining a final result after deducting the blank value from the determination result.

[0019] In some embodiments, the ash content is determined by an ignition residue test method, comprising the following steps: placing a precisely measured human angiotensin II purity standard substance in a crucible that has been burned to a constant weight, then placing the crucible at 700-800°C and burning until the purity standard substance is completely incinerated, transferring it to a dry environment for cooling, and after precise weighing, placing the crucible again at 700-800°C and burning to a constant weight, and calculating the ash content based on the residual mass after burning.

[0020] In some embodiments, the anion content is determined by ion chromatography, and the detection conditions adopted are as follows: the chromatographic column is an AS16 anion exchange column, 4mm×250mm; the column temperature is 35°C, the injection volume is 10μL, the flow rate is 0.7mL / min, the current is 44mA, isocratic elution is performed in 35mmol / LNaOH solution, and the detection time is 0-15min.

[0021] In some embodiments, when the inorganic element content is determined by inductively coupled plasma mass spectrometry, the following steps are included: weigh more than 3 mg of human angiotensin II purity standard material into a digestion tank, add 6 mL of concentrated nitric acid, mix well and perform digestion treatment, then cool the digestion solution to below 70°C, dissolve the digestion solution with water and transfer it to a 25 mL volumetric flask to make up the volume, use 2% HNO3 as a blank reagent, and use a single calibration point in semi-quantitative mode, with a concentration of 10 μg / L at the calibration point.

[0022] In some embodiments, the inorganic element content is determined by inductively coupled plasma mass spectrometry, and the detection conditions adopted are as follows: ion gas flow rate is 15.00 L / min, the number of purges is 45 times, the auxiliary gas flow rate is 0.80 L / min, the carrier gas flow rate is 0.80 L / min, and the nebulization chamber temperature is 2°C.

[0023] In other embodiments, the digestion treatment conditions may be: (1) temperature of 100°C, pressure of 15.0 atm, maintained for 2 min; (2) temperature of 120°C, pressure of 20.0 atm, maintained for 4 min; (3) temperature of 150°C, pressure of 25.0 atm, maintained for 4 min; (4) temperature of 180°C, pressure of 25.0 atm, maintained for 4 min; (2) temperature of 120°C, pressure of 20.0 atm, maintained for 4 min; (2) temperature of 120°C, pressure of 20.0 atm, maintained for 4 min.

[0024] In some embodiments, the volatile organic component content is determined by gas chromatography, and the detection conditions are as follows: maintain at 30°C for 10 min, increase the temperature to 140°C at a rate of 7°C / min, maintain for 2 min, then increase the temperature to 220°C at a rate of 15°C / min, maintain for 10 min, the transfer line temperature is 240°C, the gas chromatography column is DB-17MS 30m×0.250mm, the carrier gas is helium, the carrier gas flow rate is 1.0mL / min, the injection volume is 1000mL, and the split ratio is 1:30.

[0025] In some embodiments, the process of hydrolyzing the human angiotensin II purity standard material comprises the following steps:

[0026] Dissolve the human angiotensin II purity standard substance in 0.01 mol / L hydrochloric acid to prepare a 0.1 mg / g human angiotensin II solution;

[0027] Dissolve isotope-labeled phenylalanine, isoleucine and valine in 0.01 mol / L hydrochloric acid to prepare isotope-labeled amino acid mixed standard solutions with the contents of isotope-labeled phenylalanine, isoleucine and valine being 0.1 mg / g respectively;

[0028] Take 18 μL of human angiotensin II solution and mix it with 100 μL of isotope-labeled amino acid mixed standard solution, dry it, add 800 μL of 6 mol / L hydrochloric acid, remove oxygen and seal it, and hydrolyze it in an oven at 109.5-110.5°C for 36-120h.

[0029] In some embodiments, in the liquid chromatography-isotope dilution mass spectrometry, the chromatographic detection conditions used are as follows: in terms of volume fraction, the mobile phase consists of 95% mobile phase C and 5% mobile phase D, mobile phase C is 0.1% formic acid aqueous solution, and mobile phase D is acetonitrile; the elution condition is isocratic elution, and the elution time is 26 min; the chromatographic column is Agilent ZORBAX EclipsePlus 5μm C18, 4.6mm×150mm; the injection volume is 2μL; the flow rate is 0.2mL / min, and the column temperature is 30°C.

[0030] In some embodiments, in the liquid chromatography-isotope dilution mass spectrometry, the mass spectrometry detection conditions used are as follows: when determining phenylalanine, the cone voltage is 75-80V, and the collision energy is 10-15V; when determining isotope-labeled phenylalanine, the cone voltage is 95-100V, and the collision energy is 10-15V; when determining isoleucine, the cone voltage is 70-75V, and the collision energy is 5-10V; when determining isotope-labeled isoleucine, the cone voltage is 70-75V, and the collision energy is 5-10V; when determining valine, the cone voltage is 60-65V, and the collision energy is 10-15V; when determining isotope-labeled valine, the cone voltage is 65-70V, and the collision energy is 10-15V. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the chromatogram of the Agilent Poroshell 120EC-C18 column;

[0032] Figure 2 This is an enlarged chromatogram of the Agilent Poroshell 120EC-C18 column;

[0033] Figure 3 This is the chromatogram of the Agilent ZORBAX Eclipse XDB-C18 column.

[0034] Figure 4 This is an enlarged chromatogram of the Agilent ZORBAX Eclipse XDB-C18 column;

[0035] Figure 5 This is the UV spectrum of the purity standard substance of human angiotensin II;

[0036] Figure 6 This is the liquid chromatogram of the purity standard substance of human angiotensin II at 210nm. Figure 6 (a) is the original spectrum, Figure 6 (b) Figure 6 (a) is an enlarged view of;

[0037] Figure 7This is the liquid chromatogram of the purity standard substance of human angiotensin II at 230nm. Figure 7 (a) is the original spectrum, Figure 7 (b) Figure 7 (a) is an enlarged view of;

[0038] Figure 8 This is the liquid chromatogram of the purity standard substance of human angiotensin II at 254nm. Figure 8 (a) is the original spectrum, Figure 8 (b) Figure 8 (a) is an enlarged view of;

[0039] Fig. 9 This is the liquid chromatogram of the purity standard substance of human angiotensin II at 280nm. Fig. 9 (a) is the original spectrum, Fig. 9 (b) Fig. 9 (a) is an enlarged view of;

[0040] Fig.10 This is a liquid chromatogram of the purity standard substance of human angiotensin II in a water-50% acetonitrile system;

[0041] Fig.11 This is a liquid chromatogram of the purity standard substance of human angiotensin II in a 0.1% formic acid aqueous solution-50% acetonitrile system;

[0042] Fig.12 It is a liquid chromatogram of the purity standard substance of human angiotensin II in 20mmol / L sodium dihydrogen phosphate buffer (pH=3.0)-50% acetonitrile system;

[0043] Fig.13 This is the liquid chromatogram of the purity standard substance of human angiotensin II in the mobile phase with a volume ratio of 60:40. Fig.13 (a) is the original spectrum, Fig.13 (b) Fig.13 (a) is an enlarged view;

[0044] Fig.14 This is the liquid chromatogram of the purity standard substance of human angiotensin II in the mobile phase with a volume ratio of 65:35. Fig.14 (a) is the original spectrum, Fig.14 (b) Fig.14 (a) is an enlarged view;

[0045] Fig.15 This is the liquid chromatogram of the purity standard substance of human angiotensin II in the mobile phase with a volume ratio of 70:30. Fig.15 (a) is the original spectrum, Fig.15 (b) Fig.15 (a) is an enlarged view;

[0046] Fig.16 This is the liquid chromatogram of the purity standard substance of human angiotensin II under the mobile phase gradient elution conditions in Table 1. Fig.16 (a) is the original spectrum, Fig.16 (b) Fig.16 (a) is an enlarged view;

[0047] Fig.17 The effect of different pH values ​​on the separation of human angiotensin II purity standard substances, pH = 2.5 in Figure (a), pH = 3.0 in Figure (b), and pH = 3.5 in Figure (c);

[0048] Fig.18 The effect of pH=2.8 on the separation of human angiotensin II purity standard material;

[0049] Fig.19 The effect of 10mmol / L sodium dihydrogen phosphate on the separation of human angiotensin II purity standard material;

[0050] Fig. 20 The effect of 20mmol / L sodium dihydrogen phosphate on the separation of human angiotensin II purity standard material;

[0051] Fig.21 The effect of 30mmol / L sodium dihydrogen phosphate on the separation of human angiotensin II purity standard material;

[0052] Fig. 22 The effect of column temperature on the separation of human angiotensin II purity standard material is shown in Figure 2. Fig. 22 The column temperature of (a) is 20°C, Fig. 22 The column temperature of (b) is 25°C, Fig. 22 (c) The column temperature is 30°C;

[0053] Fig.23 This is a typical chromatogram of purity analysis of human angiotensin II purity standard material. Fig.23 (a) is the original spectrum, Fig.23 (b) Fig.23 (a) is an enlarged view;

[0054] Fig.24 For the linearity evaluation of reversed-phase HPLC method;

[0055] Fig.25 This is the optimization diagram of the hydrolysis time of the purity standard substance of human angiotensin II;

[0056] Fig.26 To confirm the purity of isotope-labeled phenylalanine;

[0057] Fig. 27To confirm the purity of isotope-labeled isoleucine;

[0058] Fig.28 To confirm the purity of isotope-labeled valine;

[0059] Fig.29 The total ion current of four amino acids is shown in Figure 2. Fig.29 The chromatographic column of (a) is a Phenomenex KINETEX C18 chromatographic column. Fig.29 The chromatographic column of (b) is Agilent ZORBAX Eclipse Plus C18;

[0060] Fig.30 The total ion current of four amino acids in 0.1% formic acid aqueous solution-acetonitrile system with different mobile phase volume ratios is shown in Figure 2. Fig.30 The volume ratio of the mobile phase in (a) is 90:10, Fig.30 The mobile phase volume ratio of (b) is 93:7, Fig.30 (c) The mobile phase volume ratio is 95:5;

[0061] Fig.31 for Fig.30 Enlarged views of the figures in Fig.31 (a) Fig.30 (a) is an enlarged view of Fig.31 (b) Fig.30 (b) is an enlarged view. Fig.31 (c) Fig.30 (c) an enlarged view;

[0062] Fig.32 This is the QQ diagram of the mass balance legal value data of the purity standard material of human angiotensin II;

[0063] Fig.33 This is the QQ graph of the isotope dilution mass spectrometry data of the purity standard material of human angiotensin II;

[0064] Fig.34 This is the QQ graph of the combined data of the two methods for the purity standard substance of human angiotensin II. DETAILED DESCRIPTION

[0065] The present invention is further described in detail below with reference to specific examples. The examples are only for explanation and are not intended to limit the present invention in any way. Unless otherwise specified, the raw materials and reagents used in the examples are conventional products that can be obtained commercially; the experimental methods in the examples where specific conditions are not specified are usually carried out according to conventional conditions in the art or according to the conditions recommended by the manufacturer.

[0066] The method for determining the purity of human angiotensin II standard substance of the present invention adopts mass balance method and liquid chromatography-isotope dilution mass spectrometry, which is described in detail as follows:

[0067] 1.1 Principle of the method

[0068] Human angiotensin II with a purity of ≥99% was selected as the purity standard material. The mass balance method was used to comprehensively examine the composition of the sample human angiotensin II (Ang II) purity standard material. Assuming that the target sample consists of only the main component, moisture, ash, volatile components and inorganic elements, the purity value can be defined as the value of the main component determination result minus the percentage of moisture, ash, inorganic elements and volatile components, thereby calculating the purity value of the Ang II purity standard material. The main component determination method of the mass balance method is generally gas chromatography or high performance liquid chromatography, the moisture determination is generally carried out by Karl Fischer titration or thermogravimetric analysis, the non-volatile component determination is carried out by high temperature calcination, and the volatile component determination is generally carried out by thermogravimetric analysis or gas chromatography.

[0069] The content of Ang II purity standard substance was calculated according to the following formula:

[0070] P=P o (1-X w -X a -X e -X v ).

[0071] Where, P is the purity of Ang II purity standard substance, P o is the main component content of Ang Ⅱ purity standard substance, X w is the moisture content, X a is the ash content and anion content, X e is the inorganic element content, X v It is the volatile organic content.

[0072] Among them, the main component content of human angiotensin II purity standard material was determined by high performance liquid chromatography, the water content was determined by Karl Fischer moisture meter, the ash content was determined by ignition residue test method, the anion content was determined by ion chromatography, the inorganic element content was determined by inductively coupled plasma mass spectrometry, and the volatile organic component content was determined by gas chromatography.

[0073] Ang Ⅱ purity standard material was hydrolyzed into amino acids under the action of 6 mol / L hydrochloric acid, and the amino acid content in the hydrolyzate was determined by liquid chromatography-isotope dilution mass spectrometry. The purity of Ang Ⅱ purity standard material was calculated based on the amino acid composition of Ang Ⅱ purity standard material. By using amino acid national standard materials and measurement methods and measuring instruments that meet the requirements of metrological characteristics, its traceability is guaranteed, and the fixed value results can ultimately be traced back to SI units.

[0074] 1.2 Experimental instruments

[0075] Electronic balance CPA225D (Sartotius, Germany), ultra-high performance liquid chromatography tandem mass spectrometer 1290infinityⅡ-6470 (Agilent, USA), liquid chromatograph 1260 (Agilent, USA), ion chromatograph Metrohm 940Professional IC Vario, quadrupole inductively coupled plasma mass spectrometer 7900 (ICP-MS, Agilent, USA), Karl Fischer titrator C20 (Mettler Toledo, Switzerland), and gas chromatography-mass spectrometer 7890A-5975C (Agilent, USA).

[0076] 1.3 Experimental reagents and standard substances

[0077] Acetonitrile (Merck, Germany, chromatography grade), formic acid (Shanghai MacLean Biochemical Technology Co., Ltd., chromatography grade), phosphoric acid (Shanghai MacLean Biochemical Technology Co., Ltd., chromatography grade), sodium dihydrogen phosphate (Shanghai Test, analytical grade), L-phenylalanine ( 13 C9, Phe, isotope-labeled purity 99%) (Cambridge Isotope Laboratory, USA), isotope-labeled L-valine ( 13 C5, Val, isotope-labeled purity 99%) (Cambridge Isotope Laboratory, USA), isotope-labeled L-isoleucine ( 13 C5, Ile, isotope labeled purity 99%) (Cambridge Isotope Laboratory, USA), GBW09292 Insulin (Human) Purity Standard Material (China Institute of Metrology), GBW(E)091097 Novel Coronavirus Nucleocapsid Protein Solution Standard Material (COVID-19) (China Institute of Metrology). The experimental water was distilled water.

[0078] The purity standard material of human angiotensin II comes from the high-purity human angiotensin II sold by Bid Pharmaceuticals, with CAS number 4474-91-3 and nominal purity ≥99%. A total of 7 bottles were purchased, each with 100 mg, all from the same batch, and stored in a low temperature environment of -20°C.

[0079] 1.4 Establishment and optimization of reversed-phase high performance liquid chromatography

[0080] 1.4.1 Initial experimental conditions

[0081] According to literature research, a phosphate buffer system was used as the mobile phase for HPLC, and the initial experimental conditions were as follows:

[0082] Chromatographic column: C18 reversed phase chromatography column; injection concentration: 1 mg / mL; injection volume: 5 μL; column temperature: 25°C; flow rate: 0.5 mL / min; detection wavelength: 230 nm; running time: 30 min; mobile phase A: 10 mmol / L sodium dihydrogen phosphate buffer, pH = 2.5; mobile phase B: 50% acetonitrile; mobile phase gradient selection is shown in Table 1.

[0083] Table 1 Mobile phase gradient elution conditions

[0084] Time / min A％ B％ 0 67 33 2 67 33 25 57 43 30 57 43 35 67 33 40 67 33

[0085] The conditions of the HPLC-area normalization method in the mass balance method were optimized. In order to clearly determine the separation degree of the main component and impurities to be measured, the optimization and final determination of the chromatographic column, wavelength, mobile phase, column temperature, injection concentration were all based on the selected Ang Ⅱ purity standard substance.

[0086] 1.4.2 Optimization of chromatographic columns

[0087] Using the initial experimental conditions, the effects of Agilent Poroshell 120EC-C18 (4.6mm×150mm, 4μm) and Agilent ZORBAX Eclipse XDB-C18 (4.6mm×150mm, 5μm) columns on the separation of Ang Ⅱ purity standard substances were compared. Figures 1 to 4 shown.

[0088] The results showed that the Agilent Poroshell 120EC-C18 column could fully separate the Ang Ⅱ purity standard substance and other impurities, so the Agilent Poroshell 120EC-C18 (4.6mm×150mm, 4μm) column was selected for subsequent optimization.

[0089] 1.4.3 Optimization of wavelength conditions

[0090] The wavelength of the selected Ang II purity standard substance was scanned to obtain the UV spectrum. The results are as follows: Figure 5 As shown, from Figure 5It can be seen that the maximum absorption wavelength of the sample is between 210 and 230 nm. Next, the effects of 210 nm, 230 nm, 254 nm, and 280 nm wavelengths on the peak area normalization test results were investigated. Figures 6 to 9 And as shown in Table 2.

[0091] The results showed that when the wavelength was 254nm or 280nm, the response value was too low, and the types of impurities detected were not as good as those at 210nm and 230nm. When the wavelength was 210nm and 230nm, a total of 5 impurity peaks were detected, indicating that the wavelength can fully detect the impurity peaks, and the measured purity of the main components of the Ang Ⅱ purity standard substance is equivalent. Considering that there may be strong solvent interference at 210nm, 230nm was selected as the detection wavelength on the premise of ensuring sufficient signal response of organic matter.

[0092] Table 2 Effect of different detection wavelengths on peak area normalization detection results

[0093]

[0094]

[0095] 1.4.4 Optimization of mobile phase

[0096] Using the initial experimental conditions, the effects of the mobile phase (AB) of water-50% acetonitrile (volume ratio 60:40), 0.1% formic acid aqueous solution-50% acetonitrile (volume ratio 60:40), and 20mmol / L sodium dihydrogen phosphate buffer (pH=3.0)-50% acetonitrile (volume ratio 60:40) on the detection results were investigated. The results are as follows Figures 10-12 The results showed that the peak shape of Ang Ⅱ purity standard substance in water-50% acetonitrile system was poor, and the main component and impurities could not be separated; in 0.1% formic acid aqueous solution-50% acetonitrile system, the main peak tailed seriously, and the number of impurities detected was 4. The gradient optimization of the mobile phase ratio was subsequently attempted, but no better results were screened; in 20mmol / L sodium dihydrogen phosphate buffer (pH=3.0)-50% acetonitrile system, the peak shape was better, and the number of impurities detected was 5, so the subsequent optimization was based on this system.

[0097] On the basis of the above results, 20mmol / L sodium dihydrogen phosphate buffer (pH=3.0) was used as mobile phase A and 50% acetonitrile was used as mobile phase B. The effects of mobile phase volume ratios of 60:40, 65:35, 70:30 and the gradient elution conditions in Table 1 on the detection results were investigated. The results are shown in FIG. Figures 13-16The results show that when the volume ratio of the mobile phase is 60:40 and 65:35, the separation between the main component and the impurities or between the impurities is not complete; when the volume ratio is 70:30, the analysis time is too long and the response value is too low; the main component and the impurities can be well separated by gradient elution under the gradient elution conditions in Table 1, and the response value meets the requirements. Therefore, Table 1 is selected as the gradient elution conditions for subsequent optimization.

[0098] 1.4.5 Optimization of mobile phase pH

[0099] Based on the gradient elution conditions in Table 1, the effect of the pH value of the mobile phase A20 mmol / L sodium dihydrogen phosphate buffer on the separation effect was analyzed. The effects of the pH values ​​of 20 mmol / L sodium dihydrogen phosphate buffer at 2.5, 3.0, and 3.5 on the separation effect of Ang Ⅱ purity standard substances were tried respectively. The results are shown in Table 1. Fig.17 As shown. The results show that when the pH value is 3.5, there are only two impurity peaks behind the main peak, and there is overlap of impurity peaks. The separation effect is not as good as the separation effect of pH 3.0. When the pH value of sodium dihydrogen phosphate buffer is 2.5, multiple impurity peaks appear in the chromatogram, which is inconsistent with the aforementioned multiple optimization conditions, and a shoulder peak appears after the main peak, which cannot be completely separated. It is speculated that during the separation process, the pH value is too low, resulting in partial hydrolysis of the Ang Ⅱ purity standard substance, resulting in additional impurity peptides.

[0100] In order to verify the effect of pH value on the separation of Ang II purity standard substances, we continued to try the effect of 20mmol / L sodium dihydrogen phosphate buffer at a pH value of 2.8 on the separation of Ang II purity standard substances. The results are as follows Fig.18 As shown in the figure, the results show that the separation effect of Ang II purity standard substance at pH 2.8 is basically the same as that at pH 3.0, indicating that at a relatively high pH value, Ang II purity standard substance is relatively stable and no additional impurities are generated. Therefore, 20mmol / L sodium dihydrogen phosphate buffer with pH = 3.0 was selected for subsequent optimization.

[0101] 1.4.6 Optimization of mobile phase ionic strength

[0102] In order to analyze the effect of mobile phase ionic strength on the retention time and separation of Ang Ⅱ purity standard substances, the concentration of sodium dihydrogen phosphate buffer in the mobile phase was optimized, and the effects of 10mmol / L, 20mmol / L, and 30mmol / L sodium dihydrogen phosphate buffer on the separation effect of Ang Ⅱ purity standard substances were tried respectively. The results are shown in Figure 2. Figures 19 to 21The results showed that with the increase of sodium dihydrogen phosphate concentration, the separation effect of human angiotensin II purity standard material did not change significantly, so the sodium dihydrogen phosphate buffer with an intermediate concentration of 20mmol / L was selected as the mobile phase.

[0103] 1.4.7 Optimization of column temperature

[0104] In order to analyze the effect of column temperature on the retention time and separation degree of Ang Ⅱ purity standard substance, the influence of column temperature conditions of 20℃, 25℃ and 30℃ on the separation effect was tried respectively. The results are as follows Fig. 22 The results show that at a column temperature of 20°C, the separation effect of Ang Ⅱ purity standard substance is not as good as that at 25°C and 30°C. At column temperatures of 25°C and 30°C, the separation effects of the samples are basically the same. Therefore, 25°C was selected as the column temperature.

[0105] 1.4.8 Optimization of injection concentration

[0106] The impurity conditions of Ang Ⅱ purity standard substances at concentrations of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL and 2.0 mg / mL were tested respectively, and the results are shown in Table 3. The results show that when the injection concentration is 0.1 mg / mL, the response value is low and the impurities detected are incomplete; when the injection concentration is 0.5 mg / mL, 1 mg / mL, and 2 mg / mL, the peak area normalization detection results are basically the same, so the middle concentration of 1 mg / mL is selected as the injection concentration.

[0107] Table 3 Effect of different injection concentrations on peak area normalization detection results

[0108]

[0109]

[0110] 1.4.9 Optimize the test conditions

[0111] After optimizing the chromatographic column, wavelength, mobile phase, column temperature, and injection concentration, the chromatographic test conditions finally optimized and determined for the determination of the main component content of the human angiotensin II purity standard substance were the same as the initial experimental conditions in 1.4.1.

[0112] The final optimized chromatographic conditions are as follows:

[0113] Chromatographic column: Agilent Poroshell 120EC-C18 (4.6 mm × 150 mm, 4 μm)

[0114] Injection concentration: 1 mg / mL

[0115] Injection volume: 5 μL

[0116] Column temperature: 25°C

[0117] Flow rate: 0.5mL / min

[0118] Detection wavelength: 230nm

[0119] Running time: 30 minutes

[0120] Mobile phase A: 20mmol / L sodium dihydrogen phosphate buffer, pH = 3.0

[0121] Mobile phase B: 50% acetonitrile

[0122] Mobile phase gradient:

[0123] Table 4 Optimized mobile phase gradient conditions

[0124] Time / min A％ B％ 0 67 33 2 67 33 25 57 43 30 57 43 35 67 33 40 67 33

[0125] 1.5 Methodological parameters for purity determination by reversed-phase HPLC

[0126] The methodological parameters of the optimized reversed-phase high performance liquid chromatography purity determination were investigated.

[0127] 1.5.1 Method reproducibility

[0128] Under the test conditions, 3 mg of Ang II purity standard substance was used as the sample, 5 parallel samples were taken, and each sample was analyzed 3 times. The chromatogram of the typical analysis result is shown in Fig.23 The results of 5 parallel samples are shown in Table 5.

[0129] Table 5 RP-HPLC purity of Ang Ⅱ purity standard substance (%)

[0130]

[0131] It can be seen from Table 5 that the reversed-phase HPLC purity analysis method has good method repeatability and can meet the requirements of the mass balance legal value.

[0132] 1.5.2 Method Linearity

[0133] 2.013 mg / g, 1.005 mg / g, 0.506 mg / g, 0.252 mg / g, 0.126 mg / g, 0.063 mg / g, 0.031 mg / g, 0.016 mg / g, and 0.008 mg / g of Ang II purity standard substance solutions were prepared respectively. The prepared samples were analyzed by reverse phase high performance liquid chromatography using the optimized test conditions. The peak areas and concentrations of the response samples were linearly fitted. The results are shown in Fig.24Calculate the linear correlation coefficient r 2 =0.9999, which shows that the reversed-phase HPLC method has good linearity.

[0134] 1.5.3 Limit of detection and limit of quantification of the method

[0135] Substitute 3 times the noise peak area and 10 times the noise peak area into Fig.24 In the obtained standard curve, the detection limit and quantification limit of the main component content of Ang Ⅱ purity standard substance determined by reversed-phase high performance liquid chromatography were 4.3×10 - 3 mg / g and 5.3×10 -3 mg / g.

[0136] 1.6 Establishment and optimization of isotope dilution mass spectrometry method

[0137] 1.6.1 Hydrolysis of human angiotensin II purity standard material

[0138] Weigh about 1 mg of Ang Ⅱ purity standard material using a balance with a minimum division of 0.01 mg, dissolve it in 10 mL of 0.01 mol / L hydrochloric acid, accurately weigh its mass, and prepare a 0.1 mg / g Ang Ⅱ solution. Take 18 μL of Ang Ⅱ solution and add it to a 2 mL ampoule, then add 100 μL of isotope labeled amino acid mixed standard solution (including equal concentrations of Phe, Ile and Val, 0.1 mg / g), and accurately weigh their masses. Place it in a vacuum centrifugal dryer and centrifuge for 70 minutes. After all the water in the sample is removed, take it out, add 800 μL of 6 mol / L hydrochloric acid, pass nitrogen for 2 minutes to deoxygenate, seal it, and hydrolyze it in an oven at 109.5-110.5 ° C. After hydrolysis, blow dry with nitrogen, re-dissolve it with 1 mL of 0.1% formic acid aqueous solution, filter it through a 0.22 μm filter membrane, and then measure it on the machine.

[0139] 1.6.2 High Performance Liquid Chromatography-Isotope Dilution Mass Spectrometry (HPLC-IDMS) Test Conditions

[0140] Chromatographic conditions: mobile phase A / B = 0.1% formic acid aqueous solution / acetonitrile = 95 / 5 (V / V), isocratic elution; running time is 26 min; chromatographic column is Agilent ZORBAX Eclipse Plus C18 (4.6 mm×150 mm, 5 μm); injection volume is 2 μL, flow rate is 0.2 mL / min, and column temperature is 30°C.

[0141] Mass spectrometry conditions: ion pair mass-to-charge ratio: phenylalanine: 166.2→120 (Phe) and 175.2→128 (labeled with Phe); isoleucine: 132.19→86.1 (Ile) and 138.18→91.1 (labeled with Ile); valine: 118.15→72.1 (Val) and 123.15→76.1 (labeled with Val).

[0142] 1.6.3 Optimization of hydrolysis time

[0143] In order to ensure that the Ang Ⅱ purity standard substance can be quantitatively hydrolyzed into amino acids, the hydrolysis time needs to be optimized. The peak area ratio of each amino acid to each labeled amino acid in the mass spectrometer is used as a reference value. The hydrolysis is completed when the peak area ratio is the largest or tends to a stable plateau. This time point is the optimal hydrolysis time. The relative proportions of Phe, Ile and Val in the hydrolyzate with hydrolysis time of 12h, 24h, 36h, 48h, 72h, 96h and 120h were investigated. The results are shown in the figure. Fig.25 The results showed that when the hydrolysis time reached 72h, the relative proportions of the three amino acids reached a plateau. Therefore, the plateau period of 96h was selected as the hydrolysis time of the Ang Ⅱ purity standard material and used for subsequent isotope dilution mass spectrometry (IDMS) quantitative analysis.

[0144] 1.6.4 Optimization of mass spectrometry conditions

[0145] The cone voltage (Fragmentor) and collision energy (CE) of phenylalanine: 166.2→120 (Phe) and 175.2→128 (labeled Phe); isoleucine: 132.19→86.1 (Ile) and 138.18→91.1 (labeled Ile); valine: 118.15→72.1 (Val) and 123.15→76.1 (labeled Val) were optimized, wherein the optimization range of the cone voltage was 0-250 V, and the optimization range of the collision energy was 0-80 V. The automatic optimization function of the instrument was used. According to the optimization results, the experimental conditions were selected as follows: phenylalanine: cone voltage of 75-80 V, collision energy of 10-15 V, isotope-labeled phenylalanine: cone voltage of 95-100 V, collision energy of 10-15 V; isoleucine: cone voltage of 70-75 V, collision energy of 5-10 V; isotope-labeled isoleucine: cone voltage of 70-75 V, collision energy of 5-10 V; valine: cone voltage of 60-65 V, collision energy of 10-15 V; isotope-labeled valine: cone voltage of 65-70 V, collision energy of 10-15 V.

[0146] 1.6.5 Confirmation of isotope internal standard

[0147] As the internal standard of isotope dilution mass spectrometry, non-labeled compounds should not be mixed, otherwise the accuracy of quantification will be affected. In order to confirm that the isotope-labeled phenylalanine, isoleucine and valine used for quantification do not contain non-labeled amino acids, a stock solution of isotope-labeled amino acids was prepared separately, and LC-MS analysis was performed. EIC graphs were extracted in the channels of labeled amino acids and non-labeled amino acids, respectively. The results are shown in Figure 2. Figures 26-28 The analysis results show that the isotope-labeled amino acids used as internal standards do not contain non-labeled amino acids.

[0148] 1.6.6 Optimization of liquid phase conditions

[0149] The effects of Agilent ZORBAX Eclipse Plus C18 columns (4.6 mm × 150 mm, 5 μm) and Phenomenex KINETEX C18 columns (2.1 mm × 150 mm, 2.6 μm) on the separation of Ang Ⅱ purity standard substances after hydrolysis were compared. Chromatographic conditions: mobile phase was 0.1% formic acid aqueous solution-acetonitrile (volume ratio was 95:5), isocratic elution, injection volume was 2 μL, flow rate was 0.2 mL / min, column temperature was 30 ° C, and the separation effect of four amino acids, Val, Ile, Phe and leucine (Leu), was tested. The results are shown in Figure 2. Fig.29 The results show that the Agilent ZORBAX Eclipse Plus C18 column was selected for subsequent optimization.

[0150] The effects of the volume ratios of mobile phases A and B on the detection results were investigated as follows: Fig.30 and Fig.31 As shown. The results show that when the volume ratio of mobile phase A and B is 90:10 and 93:7 respectively, the separation of Ile and Leu is not complete and there is partial overlap; when the volume ratio of mobile phase A and B is 95:5, Ile and Leu can be better separated. Therefore, mobile phase A / B = 0.1% formic acid aqueous solution / acetonitrile = 95 / 5 (V / V) was selected as the subsequent liquid chromatography quantitative analysis condition.

[0151] 1.7 Methodological parameters of isotope dilution mass spectrometry

[0152] The methodological parameters of the optimized isotope dilution mass spectrometry method were investigated.

[0153] 1.7.1 Correctness of the method

[0154] The isotope dilution mass spectrometry method was used to verify the accuracy of the GBW09292 insulin (human) purity standard material and the GBW (E) 091097 novel coronavirus nucleocapsid protein solution standard material (novel coronavirus). Taking the national amino acid standard material as the standard, the above two standard materials were measured by isotope dilution mass spectrometry based on amino acid analysis, and 3 sub-samples were taken respectively. The results are shown in Table 6. The results show that the average values ​​of the measurement results of the GBW09292 insulin (human) purity standard material and the GBW (E) 091097 novel coronavirus nucleocapsid protein solution standard material (novel coronavirus) are both within the range of the standard material determination results, so the determination using isotope dilution mass spectrometry is reliable.

[0155] Table 6 Verification results of standard substances

[0156]

[0157] 1.7.2 Reproducibility of the method

[0158] The HPLC-IDMS method was used to determine the content of 5 units of Ang Ⅱ purity standard substance. Each unit was analyzed 3 times. The mass of each unit of Ang Ⅱ purity standard substance was 3 mg. The results are shown in Table 7.

[0159] Table 7 Ang Ⅱ purity standard substance content determination results (g / g)

[0160]

[0161] As can be seen from Table 7, the total average of the determination results of the Ang Ⅱ purity standard substance content is 0.8757 g / g, and the relative standard deviation is 0.88%, indicating that the HPLC-IDMS purity analysis method has good method repeatability and can meet the determination requirements of isotope dilution mass spectrometry.

[0162] 1.7.3 Evaluation of detection limit and quantification limit

[0163] The detection limit and quantification limit of the HPLC-IDMS method were calculated according to formula (1-1):

[0164]

[0165] In the formula, R m-STD is the mass ratio of amino acid to isotope-labeled amino acid in the standard solution; R h-STD is the peak height ratio of the amino acid in the standard solution to the isotope-labeled amino acid; M sample is the mass of isotope-labeled amino acid in the sample solution (g); m sample is the mass of amino acids in the sample solution (g); R h-sampleis the ratio of 3 or 10 times the noise signal intensity in the non-labeled amino acid channel to the corresponding labeled amino acid signal intensity. After calculation, the detection limit and quantification limit of Ang Ⅱ standard purity material are 6.5×10 -7 g / g and 2.2×10 -6 g / g.

[0166] Example 1

[0167] This embodiment provides a method for determining the purity of Ang II standard substances, comprising the following steps:

[0168] (1) Human angiotensin II with a purity of ≥99% was selected as the purity standard substance;

[0169] (2) The purity of human angiotensin II standard substance was determined by mass balance method. The mass balance method value result was calculated by the following formula:

[0170] P=P o (1-X w -X a -X e -X v ),

[0171] Where, P is the purity of human angiotensin II purity standard substance, P o The main component content of human angiotensin II purity standard material, X w is the moisture content, X a is the ash content and anion content, X e is the inorganic element content, X v is the content of volatile organic components;

[0172] (3) The purity standard material of human angiotensin II was hydrolyzed, and isotope-labeled phenylalanine, isoleucine and valine were used as internal standards. The purity standard material of human angiotensin II was determined by isotope dilution mass spectrometry;

[0173] (4) Calculate the average of the results of mass balance method and isotope dilution mass spectrometry method to obtain the value of the purity standard substance of human angiotensin II.

[0174] 1. Analysis of mass balance method value results

[0175] (1) Analysis of the results of the main component content of Ang Ⅱ purity standard material by reversed-phase high performance liquid chromatography

[0176] Under the test conditions optimized and determined by the HPLC method (see 1.4.9 Optimized test conditions), 3 mg of Ang II purity standard substance was used as the sample, 5 parallel samples were taken, and each sample was analyzed 3 times as the purity result of the Ang II purity standard substance. The results are shown in Table 8.

[0177] Table 8 Purity results of human angiotensin II purity standard substances (%)

[0178]

[0179] (2) Determination of water content in Ang Ⅱ purity standard material

[0180] According to GB / T 606-2003 "General Method for Determination of Water in Chemical Reagents - Karl Fischer Method", approximately 6 mg of Ang Ⅱ purity standard material was accurately weighed each time, and the determination was performed using a Karl Fischer moisture analyzer, and the determination was repeated 6 times. In order to eliminate the influence of moisture in the air on the determination results, the sample addition process, mass and time were simulated, but no sample was added for determination, the blank value of moisture in the air was calculated, and the final result was obtained after deducting the blank value. The results are shown in Table 9.

[0181] Table 9 Karl Fischer method to determine the moisture content of Ang Ⅱ purity standard substances

[0182]

[0183] It can be seen from Table 9 that in this measurement, the average value of the water content of the Ang Ⅱ purity standard material determined by the Karl Fischer method is 12.24%. Therefore, the absolute water content ≈6mg×12.24%=0.43mg, which is within the range (10μg~10mg) that the Karl Fischer coulometric method can accurately measure water.

[0184] (3) Determination of ash content in Ang Ⅱ purity standard material

[0185] Refer to the 2020 edition of the Chinese Pharmacopoeia → Part III → General Rules of Part III → 0841 Ignition Residue Inspection Method for determination. First, burn the clean crucible in a muffle furnace to constant weight, then add about 5 mg of Ang Ⅱ purity standard material accurately weighed, accurately weighed, ignited at 700-800℃ to completely ash, moved to a dryer, cooled, accurately weighed, and ignited again at 700-800℃ to constant weight. Calculate the ash content based on the residual mass after ignition. Three parallel determinations were performed, and the ash content of the Ang Ⅱ purity standard material was not detected. Therefore, ion chromatography and other methods were used to determine the inorganic impurity components in the Ang Ⅱ purity standard material.

[0186] (4) Determination of anion content in Ang Ⅱ purity standard material

[0187] Ion chromatography was used to determine the anions that may be contained in the Ang II purity standard material, such as phosphate, sulfate, nitrate, chloride, and fluoride. The standard curve method was used for quantification, and the specific experimental conditions are shown in Table 10.

[0188] Table 10 Ion chromatography analysis experimental conditions

[0189]

[0190] The methodological parameters of ion chromatography were investigated, the method repeatability, method linearity and method detection limit (LOD) were analyzed, and the anions in the Ang Ⅱ purity standard material were determined. The results are shown in Table 11.

[0191] Table 11 Methodological parameters of ion chromatography and analysis results of anion concentration in Ang Ⅱ purity standard substances

[0192] Repeatability Linear LOD Concentration in sample Fluoride ion 1.45％ 0.997 0.005mg / g N / A Chloride ion 2.31％ 0.999 0.0008mg / g <![CDATA[6.703×10 -5 g / g]]> Sulfate ion 2.94％ 0.999 0.0039mg / g <![CDATA[5.190×10 -5 g / g]]> Nitrate 2.41％ 0.999 0.0037mg / g <![CDATA[1.754×10 -5 g / g]]> Phosphate ion 2.93％ 0.999 0.005mg / g N / A

[0193] The anion content in the Ang II purity standard material was calculated from the data in Table 11 to be 0.014%.

[0194] (5) Determination of inorganic element content in Ang Ⅱ purity standard material

[0195] The verification was carried out by testing common inorganic elements. The determination method was inductively coupled plasma mass spectrometry (ICP-MS). No less than 3 mg of Ang Ⅱ purity standard substance was accurately weighed in parallel as a sample. The sample was added to a digestion tank. Then 6 mL of concentrated nitric acid was added to the digestion tank. After gently shaking and mixing, the solution was placed in a microwave digester for digestion. The microwave digestion conditions are shown in Table 12.

[0196] Table 12 Microwave digestion conditions

[0197]

[0198]

[0199] After microwave digestion, the digestion solution was cooled to below 70°C, a small amount of pure water was added to dissolve and transferred to a 25mL volumetric flask to obtain the test solution, and 2% HNO3 was used as a blank reagent. Instrument conditions: plasma gas flow rate is 15.00L / min; purge times are 45 times; auxiliary gas flow rate is 0.80L / min; carrier gas flow rate is 0.80L / min; atomization chamber temperature is 2°C. In semi-quantitative mode, a single calibration point is used, and the concentration of the calibration point is 10μg / L. As a result, the Ba element was detected, and its concentration was 0.14mg / kg. According to the test results, the inorganic element content of the Ang Ⅱ purity standard substance residue was finally calculated to be 0.035%, of which the main element was Ba.

[0200] (6) Determination of volatile organic components in Ang II purity standard substances

[0201] The volatile organic components that may exist in the purity standard substance of Ang Ⅱ were determined by headspace injection-gas chromatography-mass spectrometry and quantified by external standard method. The headspace gas chromatography of the purity standard substance of Ang Ⅱ dissolved in DMSO and water was tested respectively.

[0202] The experimental conditions were as follows: the gas chromatography column was DB-17MS 30m×0.250mm (Agilent, USA); the carrier gas was helium; the carrier gas flow rate was 1.0mL / min; the injection volume was 1000mL; the split ratio was 1:30; the programmed temperature conditions were: initially at 30°C for 10min, then heated to 140°C at a rate of 7°C / min, held for 2min, then heated to 220°C at a rate of 15°C / min, held for 10min; the transfer line temperature was 240°C.

[0203] First, the solvent DMSO was selected, and the results showed that no other solvents were detected except DMSO. Then 3 mg of Ang II purity standard material was weighed, and the volume was adjusted to 5 mL sample tube with DMSO, and the gas chromatography-mass spectrometry analysis was performed. The results showed that no other solvents were detected except DMSO.

[0204] At the same time, in order to exclude the residual solvent of DMSO, 3 mg of Ang II purity standard substance was weighed and diluted to a 5 mL sample tube with H2O. To increase the stability of the solution, 0.01 mol / L HCl was added. First, the solvent was analyzed by headspace GC-MS with 0.01 mol / L HCl aqueous solution. Due to the small molecular weight of H2O and HCl, the remaining solvents were not detected.

[0205] Finally, the headspace quality analysis results of Ang Ⅱ purity standard substance in 0.01mol / L HCl were tested, and no solvent residue was detected. The external standard method was used for quantitative analysis, and the methodological parameters and measurement results are shown in Table 13. The results show that the 30 solvent residues in the table were not detected in the Ang Ⅱ purity standard substance.

[0206] Table 13 Analysis results of volatile organic components in Ang Ⅱ purity standard substances

[0207]

[0208]

[0209] (7) Analysis of mass balance method results

[0210] The test results of moisture content, ash content, anion content and residual solvent content in Ang Ⅱ purity standard material are shown in Table 14.

[0211] Table 14 Quantitative results of impurities in Ang Ⅱ purity standard substances

[0212] Impurity ingredients content(%) Moisture 12.24 cation 0.035 Anions 0.014 Solvent residue /

[0213] The purity results of the Ang Ⅱ purity standard substance determined by HPLC in Table 8 were deducted from the moisture content, cation content, anion content and solvent residue to obtain the mass balance method value results, and the results are shown in Table 15.

[0214] Table 15 Ang Ⅱ purity standard material mass balance method statutory value results (g / g)

[0215]

[0216] The normality test of the fixed value results was performed using IBM SPSS20 software, and the results are shown in Table 16.

[0217] Table 16 Normality test of Ang Ⅱ purity standard material value data (Shapiro-Wilk method)

[0218] Value determination method Statistics Degrees of Freedom Significance Mass Balance Method 0.903 15 0.107

[0219] From Table 16, we can see that the calculated significance is greater than 0.05 and obeys the normal distribution. Fig.32 ) further confirms that the expected standard is basically distributed around the straight line and can be considered to obey the normal distribution.

[0220] The mass balance legal value data of Ang Ⅱ purity standard material in Table 16 were tested for outliers using Grubbs and Dixon outlier test methods, respectively. The results are shown in Table 17.

[0221] Table 17 Outlier test of mass balance method data

[0222]

[0223] It can be seen from Table 17 that the average value of each standard substance unit was tested by the Grubbs and Dixon outlier test methods, and no outliers were found. Therefore, the arithmetic mean of the five units was taken as the fixed value result of the mass balance method of Ang Ⅱ purity standard substance, that is, 0.8735 g / g.

[0224] 2. Analysis of isotope dilution mass spectrometry results

[0225] Under the optimized conditions of the isotope dilution mass spectrometry method, 3 mg of human angiotensin II purity standard substance was used as the sample, 5 parallel samples were taken, and each sample was analyzed 3 times as the determination results of the isotope dilution mass spectrometry method. The results are shown in Table 18.

[0226] Table 18 Ang Ⅱ purity standard material isotope dilution mass spectrometry value results (g / g)

[0227]

[0228] The normality test of the isotope dilution mass spectrometry results was performed using IBM SPSS20 software, and the results are shown in Table 19.

[0229] Table 19 Normality test of Ang Ⅱ purity standard material value data (Shapiro-Wilk method)

[0230] Value determination method Statistics Degrees of Freedom Significance IDMS Act 0.962 15 0.722

[0231] As can be seen from Table 19, the calculated significance is greater than 0.05, which obeys the normal distribution. Checking the QQ graph (Figure 35) for further confirmation, it can be seen that the expected standard is basically distributed around the straight line, which can be considered to obey the normal distribution.

[0232] The outlier test methods of Grubbs and Dixon were used to test the isotope dilution mass spectrometry data of Ang II purity standard material in Table 19, and the results are shown in Table 20.

[0233] Table 20 Outlier test of isotope dilution mass spectrometry data

[0234]

[0235] It can be seen from Table 20 that the average value of each standard substance unit was tested by the Grubbs and Dixon outlier test methods, and no outliers were found. Therefore, the arithmetic mean of the five units was used as the fixed value result of the isotope dilution mass spectrometry method for the Ang II purity standard substance, which is 0.8733 g / g.

[0236] 3. Statistical analysis of data from two valuation methods

[0237] Cochran's rule was used to test the homogeneity of variance of the mass balance method and the isotope dilution mass spectrometry method. The results are shown in Table 21.

[0238] Table 21 Variance homogeneity test of two fixed value methods (Cochran method)

[0239] <![CDATA[s MB ]]> <![CDATA[s IDMS ]]> <![CDATA[s max ]]> ∑s C C(0.05，2，15) Discrimination Ang II <![CDATA[1.94×10 -5 ]]> <![CDATA[5.51×10 -5 ]]> <![CDATA[5.51×10 -5 ]]> <![CDATA[7.45×10 -5 ]]> 0.740 0.745 Equal precision

[0240] After analysis, it was found that the C values ​​between the two determination methods for purity standard substances were both less than the C critical value, that is, the determination results of the two methods had homogeneity of variance and the same accuracy.

[0241] IBM SPSS25 software was used to perform an independent sample t-test on the data measured by the two methods. The results are shown in Table 22.

[0242] Table 22 Results of variance homogeneity test and independent sample t-test for two fixed value methods

[0243]

[0244] After analysis, for the Ang II purity standard substance, the p-value between the two determination methods is less than 0.05, that is, there is a statistically significant difference in the determination results between the two methods. The difference between the two methods is generally taken as an uncertainty component and synthesized into the uncertainty of the determination result.

[0245] According to JJF1006-1994 "Technical Specifications for Primary Reference Materials", the fixed value data of the two methods were combined, and the combined measurement data of the reference materials were tested for normality using IBM SPSS 25 software. The analysis results are shown in Table 23.

[0246] Table 23 Normality test of combined data of two fixed value methods

[0247]

[0248] It can be seen from Table 9 that the measurement results of the combined data of the two setting methods are in line with the normal distribution assumption. Checking the QQ diagram (Figure 36) for further confirmation, it can be seen that the expected standards are basically distributed around the straight line, and can be considered to obey the normal distribution.

[0249] The original data table 23 was tested for outliers using the Grubbs and Dixon outlier test methods, respectively. The results are shown in Table 24.

[0250] Table 24 Combined data of two fixed value methods, fixed value data outlier test

[0251]

[0252] The results showed that no outliers were found after Dixon and Grubbs tests.

[0253] Finally, the average value of the mass balance method and the isotope dilution mass spectrometry method was calculated as the value of the Ang II purity standard substance in this example, namely:

[0254]

[0255] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for determining the purity of human angiotensin II standard substance, characterized in that: The following steps are involved: Human angiotensin II with a purity of ≥99% was selected as the purity standard substance; The purity standard substance of human angiotensin II was determined by mass balance method. The value of mass balance method was calculated by the following formula: P=P o (1-X w -X a -X e -X v ), Where, P is the purity of human angiotensin II purity standard substance, P o The main component content of human angiotensin II purity standard material, X w is the moisture content, X a is the ash and anion content, X e is the inorganic element content, X v is the content of volatile organic components; The purity standard substance of human angiotensin II was hydrolyzed, and the purity standard substance of human angiotensin II was determined by liquid chromatography-isotope dilution mass spectrometry using isotope-labeled phenylalanine, isoleucine and valine as internal standards. The average value of the mass balance method valuation result and the liquid chromatography-isotope dilution mass spectrometry method valuation result was calculated to obtain the valuation result of the purity standard substance of human angiotensin II.

2. The value setting method according to claim 1, characterized in that: The main component content of the human angiotensin II purity standard substance is determined by high performance liquid chromatography, and the chromatographic detection conditions are as follows: the chromatographic column is Agilent Poroshell 120EC-C18, 4.6mm×50mm, 4μm; the injection concentration is 1mg / mL, the injection volume is 5μL, the column temperature is 25°C, the flow rate is 0.5mL / min, the detection wavelength is 230nm, and the detection time is 30min; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is a sodium dihydrogen phosphate buffer with a pH value of 3.0 and a concentration of 20mmol / L, and the mobile phase B is a 50% acetonitrile solution; In terms of volume fraction, the gradient conditions of the mobile phase are: (1) at 0 min, the mobile phase A is 67% and the mobile phase B is 33%; (2) at 2 min, the mobile phase A is 67% and the mobile phase B is 33%; (2) at 2 min, the mobile phase A is 67% and the mobile phase B is 33%; (3) at 25 min, the mobile phase A is 57% and the mobile phase B is 43%; (4) at 30 min, the mobile phase A is 57% and the mobile phase B is 43%; (5) at 35 min, the mobile phase A is 67% and the mobile phase B is 33%; (6) at 40 min, the mobile phase A is 67% and the mobile phase B is 33%.

3. The value setting method according to claim 1, characterized in that: The moisture content is measured by a Karl Fischer moisture meter, comprising the following steps: accurately weighing 6 mg of human angiotensin II purity standard material, measuring with the Karl Fischer moisture meter to obtain a measurement result, performing parallel measurement 6 times, and then measuring without adding a sample to obtain a blank value of moisture in the air, and obtaining the moisture content after deducting the blank value from the measurement result.

4. The value setting method according to claim 1, characterized in that: The ash content is determined by an ignition residue inspection method, which includes the following steps: placing a precisely measured human angiotensin II purity standard substance in a crucible that has been ignited to a constant weight, then igniting the crucible at 700-800° C. until the purity standard substance is completely ashed, transferring it to a dry environment for cooling, and after precise weighing, igniting the crucible again at 700-800° C. to a constant weight, and calculating the ash content based on the residual mass after ignition.

5. The value determination method according to claim 1, characterized in that: The anion content was determined by ion chromatography, and the detection conditions adopted were as follows: the chromatographic column was an AS16 anion exchange column, 4mm×250mm; the column temperature was 35°C, the injection volume was 10μL, the flow rate was 0.7mL / min, the current was 44mA, the elution was isocratic in 35mmol / LNaOH solution, and the detection time was 0-15min.

6. The value determination method according to claim 1, characterized in that: The inorganic element content is determined by inductively coupled plasma mass spectrometry. The method comprises the following steps: weighing more than 3 mg of human angiotensin II purity standard substance into a digestion tank, adding 6 mL of concentrated nitric acid, mixing and performing digestion treatment, then cooling the digestion solution to below 70°C, dissolving the digestion solution with water and transferring it to a 25 mL volumetric flask for constant volume, using a 2% HNO3 solution as a blank reagent, and using a single calibration point in a semi-quantitative mode, with the concentration of the calibration point being 10 μg / L.

7. The value setting method according to claim 6, characterized in that: When the inorganic element content was determined by inductively coupled plasma mass spectrometry, the detection conditions adopted were as follows: ion gas flow rate was 15.00 L / min, purge times were 45 times, auxiliary gas flow rate was 0.80 L / min, carrier gas flow rate was 0.80 L / min, and atomization chamber temperature was 2°C; The digestion treatment conditions are as follows: (1) temperature is 100°C, pressure is 15.0 atm, and maintained for 2 min; (2) temperature is 120°C, pressure is 20.0 atm, and maintained for 4 min; (3) temperature is 150°C, pressure is 25.0 atm, and maintained for 4 min; (4) temperature is 180°C, pressure is 25.0 atm, and maintained for 4 min; (2) temperature is 120°C, pressure is 20.0 atm, and maintained for 4 min; (2) temperature is 120°C, pressure is 20.0 atm, and maintained for 4 min.

8. The value setting method according to claim 1, characterized in that: The volatile organic component content is determined by gas chromatography, and the detection conditions are as follows: the gas chromatography column is DB-17MS 30m×0.250mm, the carrier gas is helium, the carrier gas flow rate is 1.0mL / min, the injection volume is 1000mL, the split ratio is 1:30, maintained at 30°C for 10min, heated to 140°C at a rate of 7°C / min, maintained for 2min, then heated to 220°C at a rate of 15°C / min, maintained for 10min, and the transmission line temperature is 240°C.

9. The value determination method according to claim 1, characterized in that: The hydrolysis of the human angiotensin II purity standard substance comprises the following steps: Dissolve the human angiotensin II purity standard substance in 0.01 mol / L hydrochloric acid to prepare a 0.1 mg / g human angiotensin II solution; Dissolve isotope-labeled phenylalanine, isoleucine and valine in 0.01 mol / L hydrochloric acid to prepare an isotope-labeled amino acid mixed standard solution with the content of isotope-labeled phenylalanine, isoleucine and valine being 0.1 mg / g respectively; Take 18 μL of human angiotensin II solution and mix it with 100 μL of isotope-labeled amino acid mixed standard solution, dry it, add 800 μL of 6 mol / L hydrochloric acid, remove oxygen and seal it, and hydrolyze it in an oven at 109.5-110.5°C for 36-120h.

10. The value determination method according to claim 1, characterized in that: In the liquid chromatography-isotope dilution mass spectrometry, the chromatographic detection conditions used are as follows: in terms of volume fraction, the mobile phase is composed of 95% mobile phase C and 5% mobile phase D, the mobile phase C is a 0.1% formic acid aqueous solution, and the mobile phase D is acetonitrile; the elution condition is isocratic elution, the elution time is 26 minutes, the chromatographic column is Agilent ZORBAX Eclipse Plus 5μm C18, 4.6mm×150mm; the injection volume is 2μL, the flow rate is 0.2mL / min, and the column temperature is 30°C; The mass spectrometry detection conditions adopted are as follows: when determining phenylalanine, the cone voltage is 75-80V, and the collision energy is 10-15V; when determining isotope-labeled phenylalanine, the cone voltage is 95-100V, and the collision energy is 10-15V; when determining isoleucine, the cone voltage is 70-75V, and the collision energy is 5-10V; when determining isotope-labeled isoleucine, the cone voltage is 70-75V, and the collision energy is 5-10V; when determining valine, the cone voltage is 60-65V, and the collision energy is 10-15V; when determining isotope-labeled valine, the cone voltage is 65-70V, and the collision energy is 10-15V.

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