Human angiotensin II purity standard substance as well as preparation method and application thereof

Abstract

The invention discloses a human angiotensin II purity standard substance, the mass concentration of which is 0.8734 g / g, the relative expansion uncertainty of which is 1.8% and the expansion uncertainty of which is 0.016 g / g. The purity standard substance is prepared by the following steps: selecting human angiotensin II of which the nominal purity is greater than or equal to 99%; carrying out sequence analysis, mass spectrum qualitative identification and primary homogeneity detection to obtain a purity standard substance candidate; and performing uniformity test and stability investigation on the purity standard substance candidate through a mass balance method, performing valuing by adopting the mass balance method and an isotope dilution mass spectrometry, and performing uncertainty evaluation on the purity standard substance candidate to obtain the purity standard substance. The human angiotensin II purity standard substance is accurate and reliable in valuing result, has traceability, and can be used for human angiotensin II quantity value traceability, measurement result quality control and standard substance evaluation.

Description

Technical Field

[0001] The invention relates to the field of measurement technology, and in particular to a human angiotensin II purity standard substance and a preparation method and application thereof. 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] AngⅡ 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 AngⅡ is an important indicator for the prevention, diagnosis, control and treatment of hypertension.

[0005] In the existing technology, my country has not yet released national certified standard materials related to AngⅡ purity. The only human angiotensin Ⅱ standard material is NIM-RM3622 human angiotensin Ⅱ relative molecular mass standard material. However, this standard material can only be used for relative molecular mass determination of peptides and proteins based on electrophoresis and high-performance gel exclusion chromatography, molecular weight distribution detection of peptide health products, and confirmation and evaluation of related analytical methods, quality control, and has a narrow scope of application. It cannot meet the quantitative requirements of angiotensin Ⅱ in actual testing work and customer production verification. Therefore, it is of research significance to develop new human angiotensin Ⅱ purity standard materials. Summary of the invention

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

[0007] The second object of the present invention is to provide a method for preparing a purity standard substance of human angiotensin II to solve at least one of the above technical problems.

[0008] The third object of the present invention is to provide an application of the above-mentioned human angiotensin II purity standard substance to solve at least one of the above-mentioned technical problems.

[0009] The first aspect of the present invention provides a human angiotensin II purity standard substance, the mass concentration of the human angiotensin II purity standard substance is 0.8734 g / g, the relative expanded uncertainty is 1.8%, the expanded uncertainty is 0.016 g / g, and it is prepared by the following method:

[0010] Human angiotensin II with a nominal purity ≥ 99% is selected, and a purity standard substance candidate is obtained after sequence analysis, mass spectrometry qualitative identification and initial uniformity inspection; the purity standard substance candidate is subjected to a uniformity inspection and stability investigation by the mass balance method, and then the mass balance method and isotope dilution mass spectrometry are used for value determination, and the purity standard substance candidate is subjected to uncertainty assessment.

[0011] According to the human angiotensin II purity standard substance of the present invention, the mass concentration, relative expanded uncertainty and expanded uncertainty are measured, the determination results are accurate and reliable, and the traceability of the purity or content-related values ​​of human angiotensin II is achieved.

[0012] The second aspect of the present invention provides a method for preparing the above-mentioned human angiotensin II purity standard substance, comprising the following steps:

[0013] (1) Select human angiotensin II with a nominal purity of ≥99%, and obtain a candidate human angiotensin II purity standard material after sequence analysis, mass spectrometry qualitative identification and initial homogeneity test;

[0014] (2) The homogeneity and stability of the candidate human angiotensin II purity standard material were tested by mass balance method;

[0015] (3) The candidate purity standard substance of human angiotensin II is quantified by mass balance method and isotope dilution mass spectrometry respectively, and the average of the quantification results of mass balance method and isotope dilution mass spectrometry method is calculated to obtain the quantification result of the candidate purity standard substance of human angiotensin II;

[0016] (4) The uncertainty of the candidate human angiotensin II purity standard substance was evaluated to obtain the human angiotensin II purity standard substance.

[0017] According to the preparation method of the purity standard substance of human angiotensin II of the present invention, commercially available high-purity human angiotensin II is used as a raw material, and a candidate for the purity standard substance of human angiotensin II is obtained after an initial uniformity test, and the candidate for the purity standard substance of human angiotensin II is qualitatively identified, and a uniformity test and a stability test are conducted by a mass balance method, and then the candidate for the purity standard substance of human angiotensin II is respectively determined by a mass balance method and an isotope dilution mass spectrometry method, so that the determination result of the purity standard substance of human angiotensin II is more accurate and reliable, and finally the purity standard substance of AngⅡ is obtained after uncertainty assessment, so as to provide a technical reference for the quantity value of the purity standard substance of human angiotensin II, and the method has traceability.

[0018] In some embodiments, the homogeneity test, stability test and value determination of the human angiotensin II purity standard material candidate are carried out using the mass balance method, and the result can be calculated by the following formula:

[0019] P=P o (1-X w -X a -X e -X v ),

[0020] Where, P is the purity of the candidate human angiotensin II purity standard substance, P o The main component content of the candidate for purity standard material of human angiotensin II, 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.

[0021] In some embodiments, the homogeneity initial test and the main component content of the human angiotensin II purity standard material candidate are determined by high performance liquid chromatography, and the chromatographic detection conditions for the homogeneity initial test are as follows: the chromatographic column is a C18 reverse phase chromatographic column, the mobile phase A is a pH=2.5, 10mmol / L sodium dihydrogen phosphate buffer, the mobile phase B is 50% acetonitrile, the flow rate is 0.5mL / min, the column temperature is 25°C, and the detection wavelength is 230nm;

[0022] The chromatographic detection conditions for the main component content of the candidate human angiotensin II purity standard substance 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℃; 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.

[0023] 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%.

[0024] In some embodiments, the moisture content is determined using a Karl Fischer titrator, comprising the following steps: accurately weighing 6 mg of a human angiotensin II purity standard material candidate, 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.

[0025] 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 candidate 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 incinerated, 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.

[0026] 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.

[0027] 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 candidate into a digestion tank, add 6 mL of concentrated nitric acid, mix and digest, 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 a fixed 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] In some embodiments, the stability study includes a long-term stability study, a short-term stability study, and an open-bottle stability study.

[0032] In some embodiments, the method of quantifying the purity of a human angiotensin II standard substance candidate by isotope dilution mass spectrometry comprises the following steps:

[0033] The purity of the candidate human angiotensin II purity standard material was determined by isotope dilution mass spectrometry using isotope-labeled phenylalanine, isoleucine and valine as internal standards after hydrolysis.

[0034] In some embodiments, the process of hydrolyzing the candidate human angiotensin II purity standard material comprises the following steps:

[0035] The candidate human angiotensin II purity standard substance is dissolved in 0.01 mol / L hydrochloric acid to prepare a 0.1 mg / g human angiotensin II solution;

[0036] 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;

[0037] 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.

[0038] In some embodiments, in the 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 Eclipse Plus 5μmC18, 4.6mm×150mm; the injection volume is 2μL; the flow rate is 0.2mL / min, and the column temperature is 30°C.

[0039] In some embodiments, in the isotope dilution mass spectrometry, the mass spectrometry detection conditions used are as follows: when measuring phenylalanine, the cone voltage is 75-80V, and the collision energy is 10-15V; when measuring isotope-labeled phenylalanine, the cone voltage is 95-100V, and the collision energy is 10-15V; when measuring isoleucine, the cone voltage is 70-75V, and the collision energy is 5-10V; when measuring isotope-labeled isoleucine, the cone voltage is 70-75V, and the collision energy is 5-10V; when measuring valine, the cone voltage is 60-65V, and the collision energy is 10-15V; when measuring isotope-labeled valine, the cone voltage is 65-70V, and the collision energy is 10-15V.

[0040] In some embodiments, when performing uncertainty assessment, the uncertainty includes the uncertainty introduced by the fixed value, the uncertainty introduced by the heterogeneity of the standard material, and the uncertainty introduced by the instability of the standard material.

[0041] In some embodiments, the uncertainty introduced by the valuation includes the uncertainty introduced by the valuation by mass balance method and the uncertainty introduced by the valuation by isotope dilution mass spectrometry method.

[0042] In some embodiments, the uncertainty introduced by the mass balance method includes the uncertainty introduced by the high performance liquid chromatography method, the uncertainty introduced by the water content measurement, the uncertainty introduced by the anion content measurement, and the uncertainty introduced by the inorganic element content measurement.

[0043] In some embodiments, the uncertainty introduced by the HPLC assay includes the uncertainty introduced by the measurement repeatability and the uncertainty introduced by the variability in the HPLC response factor.

[0044] In some embodiments, the uncertainty introduced by isotope dilution mass spectrometry valuation includes the uncertainty introduced by amino acid purity, the uncertainty introduced by the hydrolysis efficiency of the candidate human angiotensin II purity standard substance, the uncertainty introduced by weighing, the uncertainty introduced by the mass ratio, the uncertainty introduced by the peak area ratio, the uncertainty introduced by the molecular weight of human angiotensin II, and the uncertainty introduced by calculating the mass fraction of the candidate human angiotensin II purity standard substance from amino acids.

[0045] In some embodiments, the amino acids include phenylalanine, isoleucine, and valine.

[0046] The third aspect of the present invention provides the application of human angiotensin II purity standard substance in human angiotensin II value traceability, human angiotensin II measurement result quality control, and evaluation of human angiotensin II standard substance.

[0047] Specifically, human angiotensin II purity standard material can be used for mass value traceability of serum angiotensin II standard material, and can also be used for quality control, relevant method confirmation and evaluation of human angiotensin II measurement results in in vitro diagnosis or clinical testing, and can also be used for the evaluation of repeatability, linearity and cross-contamination rate in closed luminescence immunoassay analyzers. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is the mass spectrometry analysis diagram of human angiotensin II raw material;

[0049] Figure 2 , Figure 3 This is the sequencing map of the raw material of human angiotensin II;

[0050] Figure 4 This is the calibration test spectrum of human angiotensin II standard;

[0051] Figure 5 This is a picture of the candidate for purity standard substance of human angiotensin II after packaging;

[0052] Figure 6 This is the chromatogram of the Agilent Poroshell 120EC-C18 column;

[0053] Figure 7 This is an enlarged chromatogram of the Agilent Poroshell 120EC-C18 column;

[0054] Figure 8 This is the chromatogram of the Agilent ZORBAX Eclipse XDB-C18 column.

[0055] Fig. 9 This is an enlarged chromatogram of the Agilent ZORBAX Eclipse XDB-C18 column;

[0056] Fig.10 This is the UV spectrum of the candidate purity standard substance for human angiotensin II;

[0057] Fig.11 This is the liquid chromatogram of the candidate for purity standard substance of human angiotensin II at 210nm. Fig.11 (a) is the original spectrum, Fig.11 (b) Fig.11 (a) is an enlarged view of;

[0058] Fig.12 This is the liquid chromatogram of the candidate for purity standard substance of human angiotensin II at 230nm. Fig.12 (a) is the original spectrum, Fig.12 (b) Fig.12 (a) is an enlarged view of;

[0059] Fig.13 This is the liquid chromatogram of the candidate for purity standard substance of human angiotensin II at 254nm. Fig.13 (a) is the original spectrum, Fig.13 (b) Fig.13 (a) is an enlarged view of;

[0060] Fig.14 This is the liquid chromatogram of the candidate for purity standard substance of human angiotensin II at 280nm. Fig.14 (a) is the original spectrum, Fig.14 (b) Fig.14 (a) is an enlarged view of;

[0061] Fig.15 It is a liquid chromatogram of a candidate for purity standard substance of human angiotensin II in a mobile phase (AB) of water-50% acetonitrile system;

[0062] Fig.16 It is a liquid chromatogram of a candidate for purity standard substance of human angiotensin II in a mobile phase (AB) of 0.1% formic acid aqueous solution-50% acetonitrile system;

[0063] Fig.17 It is a liquid chromatogram of a candidate for purity standard substance of human angiotensin II in a mobile phase (AB) of 20mmol / L sodium dihydrogen phosphate buffer (pH=3.0)-50% acetonitrile system;

[0064] Fig.18 This is the liquid chromatogram of the candidate for purity standard substance of human angiotensin II in the mobile phase (AB) with a volume ratio of 60:40. Fig.18 (a) is the original spectrum, Fig.18 (b) Fig.18 (a) is an enlarged view;

[0065] Fig.19 This is the liquid chromatogram of the candidate for purity standard substance of human angiotensin II in the mobile phase (AB) with a volume ratio of 65:35. Fig.19 (a) is the original spectrum, Fig.19 (b) Fig.19 (a) is an enlarged view;

[0066] Fig. 20 This is the liquid chromatogram of the candidate for purity standard substance of human angiotensin II in the mobile phase (AB) with a volume ratio of 70:30. Fig. 20 (a) is the original spectrum, Fig. 20 (b) Fig. 20 (a) is an enlarged view;

[0067] Fig.21 This is the liquid chromatogram of the candidate for purity standard substance of human angiotensin II under the mobile phase gradient elution conditions in Table 1. Fig.21 (a) is the original spectrum, Fig.21 (b) Fig.21 (a) is an enlarged view;

[0068] Fig. 22 The effect of different pH values ​​on the separation of candidate 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);

[0069] Fig.23 The effect of pH = 2.8 on the separation of candidate purity standard substances for human angiotensin II;

[0070] Fig.24 The effect of 10mmol / L sodium dihydrogen phosphate on the separation of candidate purity standard substances for human angiotensin II;

[0071] Fig.25 The effect of 20mmol / L sodium dihydrogen phosphate on the separation of candidate purity standard substances for human angiotensin II;

[0072] Fig.26 The effect of 30mmol / L sodium dihydrogen phosphate on the separation of candidate purity standard substances for human angiotensin II;

[0073] Fig. 27 The effect of column temperature on the separation of candidate purity standard substances of human angiotensin II is shown in Figure 2. Fig. 27 The column temperature of (a) is 20°C, Fig. 27 The column temperature of (b) is 25°C, Fig. 27 (c) The column temperature is 30°C;

[0074] Fig.28 This is a typical chromatogram of the purity analysis of the candidate for the purity standard material of human angiotensin II. Fig.28 (a) is the original spectrum, Fig.28 (b) Fig.28 (a) is an enlarged view;

[0075] Fig.29 For the linearity evaluation of reversed-phase HPLC method;

[0076] Fig.30 This is an optimization diagram of the hydrolysis time of the candidate purity standard material of human angiotensin II;

[0077] Fig.31 To confirm the purity of isotope-labeled phenylalanine;

[0078] Fig.32 To confirm the purity of isotope-labeled isoleucine;

[0079] Fig.33 To confirm the purity of isotope-labeled valine;

[0080] Fig.34 The total ion current of four amino acids is shown in Figure 2. Fig.34 (a) The chromatographic column is a Phenomenex KINETEX C18 column, Fig.34 (b) The chromatographic column is Agilent ZORBAX Eclipse Plus C18;

[0081] Fig.35 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.35 The volume ratio of the mobile phase in (a) is 90:10, Fig.35 The volume ratio of the mobile phase in (b) is 93:7, Fig.35 (c) The mobile phase volume ratio is 95:5;

[0082] Fig.36 for Fig.35 Enlarged views of the figures in Fig.36 (a) Fig.35 (a) is an enlarged view of Fig.36 (b) Fig.35 (b) is an enlarged view. Fig.36 (c) Fig.35 (c) an enlarged view;

[0083] Fig.37 This is a long-term stability trend diagram of the main component in the candidate of human angiotensin II purity standard material;

[0084] Fig.38 This is a long-term stability trend chart of the water content in the candidate of human angiotensin II purity standard material;

[0085] Fig.39 This is a long-term stability trend chart of the candidate purity standard material for human angiotensin II;

[0086] Fig.40 This is the short-term stability trend diagram of the candidate purity standard material of human angiotensin II at 40°C;

[0087] Fig.41 This is the short-term stability trend diagram of the candidate purity standard material of human angiotensin II at 25°C;

[0088] Fig.42 This is the short-term stability trend chart of the candidate purity standard material of human angiotensin II at 4°C;

[0089] Fig.43 The stability results of the candidate human angiotensin II purity standard material at 40°C within 2 days;

[0090] Fig.44 The stability results of the candidate human angiotensin II purity standard material at 40°C within 3 days;

[0091] Fig.45 This is the QQ diagram of the mass balance legal value data of the candidate purity standard material of human angiotensin II;

[0092] Fig.46 This is the QQ graph of the isotope dilution mass spectrometry data of the candidate purity standard material for human angiotensin II;

[0093] Fig.47 QQ plot of the combined data of two methods for the candidate of human angiotensin II purity standard material. DETAILED DESCRIPTION

[0094] 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.

[0095] Example 1

[0096] This embodiment provides a purity standard substance of human angiotensin II, which is prepared by the following steps:

[0097] (1) Select human angiotensin II with a nominal purity of ≥99%, and obtain a candidate human angiotensin II purity standard material after sequence analysis, mass spectrometry qualitative identification and initial homogeneity test;

[0098] (2) The homogeneity and stability of the candidate human angiotensin II purity standard material were tested by mass balance method;

[0099] (3) The candidate purity standard substance of human angiotensin II is quantified by mass balance method and isotope dilution mass spectrometry respectively, and the average of the quantification results of mass balance method and isotope dilution mass spectrometry method is calculated to obtain the quantification result of the candidate purity standard substance of human angiotensin II;

[0100] (4) The uncertainty of the candidate human angiotensin II purity standard substance was evaluated to obtain the human angiotensin II purity standard substance.

[0101] Among them, the principle of the mass balance method (Mass Balance Method) used in the embodiment is as follows: Assuming that the target sample consists of and only consists of the main components, moisture, ash, volatile components and inorganic elements, the purity value can be defined as the numerical value of the main component determination result minus the percentage content of moisture, ash, inorganic elements and volatile components, thereby calculating the purity value of the AngⅡ purity standard substance candidate. The main component determination method of the mass balance method is generally gas chromatography or high performance liquid chromatography, the moisture determination generally adopts Karl Fischer titration or thermogravimetric analysis, the non-volatile component determination adopts high temperature calcination method, and the volatile component determination generally adopts thermogravimetric method or gas chromatography. The content of the AngⅡ purity standard substance candidate is calculated according to the following formula: P=P o (1-X w -X a -X e -X v ).

[0102] Where, P is the purity of the candidate AngⅡ purity standard substance, P ois the purity of the candidate AngⅡ 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.

[0103] The principle of the isotope dilution mass spectrometry method used in the embodiment is as follows: the AngⅡ purity standard substance candidate is hydrolyzed into amino acids under the action of 6mol / L hydrochloric acid, and the amino acid content in the hydrolyzate is determined by liquid chromatography-isotope dilution mass spectrometry. According to the amino acid composition of the AngⅡ purity standard substance candidate, the purity of the AngⅡ purity standard substance candidate is calculated. By using amino acid national standard substances and measurement methods and measuring instruments that meet the metrological characteristics requirements, the traceability is guaranteed, and the fixed value results can ultimately be traced back to SI units.

[0104] The preparation method of the purity standard substance of human angiotensin II in this embodiment is described in detail as follows: 1. Materials and Equipment

[0105] 1. Materials

[0106] AngⅡ raw material: high-purity human angiotensin II sold by a pharmaceutical intermediate company (Bidder Pharmaceuticals), CAS number 4474-91-3, nominal purity ≥99%, a total of 7 bottles were purchased, each bottle of 100 mg, all from the same batch, stored in a low temperature environment of -20°C.

[0107] Isotope-labeled amino acids were purchased from Cambridge Isotope Laboratory, USA, including L-phenylalanine ( 13 C9, Phe, purity 99%), L-valine ( 13 C5, Val, purity 99%), L-isoleucine ( 13 C6, Ile,, purity 99%). Amino acid purity standard materials were purchased from the National Institute of Metrology, China, including GBW09235 L-phenylalanine purity standard material (purity 99.8%, U = 0.4%, k = 2), GBW09236 L-valine purity standard material (purity 99.4%, U = 0.6%, k = 2), GBW09238 L-isoleucine purity standard material (purity 99.7%, U = 0.3%, k = 2).

[0108] Experimental reagents: acetonitrile (Merck, chromatography grade); formic acid (McLean, chromatography grade); phosphoric acid (McLean, chromatography grade); sodium dihydrogen phosphate (Shanghai Test, analytical grade); distilled water was used for the experiment.

[0109] 2. Equipment

[0110] The fixed-value equipment includes electronic balance (CPA225D, Sartotius), ultra-high performance liquid chromatography tandem mass spectrometer (1290infinityⅡ-6470, Agilent), liquid chromatograph (1260, Agilent), quadrupole inductively coupled plasma mass spectrometer (ICP-MS, 7900, Agilent), water card (C20, MettlerToledo), and gas chromatography-mass spectrometer (7890A-5975C, Agilent), all of which are fixed assets of the unit and provide calibration certificates.

[0111] Other equipment included a single-channel manually adjustable pipette (10 μL-100 μL, Eppendorf); a nitrogen blowdown apparatus (HGC-24A, Beijing Hengao), a vacuum drying oven (DZF-6034, Shanghai Yiheng), an ultrapure water system (Milli-Q, MILLIPORE), etc.

[0112] 2. Preparation of Candidate Purity Standards for Human Angiotensin II

[0113] 1. Raw material selection

[0114] Human angiotensin II with a nominal purity of ≥99% was purchased as the sample raw material, with a CAS number of 4474-91-3, and stored in a low temperature environment of -20°C.

[0115] 2. Qualitative identification of raw materials

[0116] The qualitative experimental analysis of AngⅡ sample raw materials, such as mass spectrometry and sequencing, was commissioned to the Analysis and Testing Center of Peking University.

[0117] Specifically, the mass spectrometry analysis instrument is a German Bruker Solarix XR Fourier transform high-resolution mass spectrometer equipped with an ESI ion source and an actively shielded 9.4 Tesla magnet. The measurement error is less than 1ppm, the resolution can reach 1000000, and the single charge mass range is 20-10000Da. From the mass spectrum ( Figure 1 ) an obvious molecular ion peak of m / z=1047.53018 can be observed, which is consistent with the theoretical molecular weight of AngⅡ.

[0118] The instrument for sequencing is Shimadzu's fully automatic protein peptide sequencer (PPSQ-33A). The Edman degradation method was used to analyze the N-terminal sequence of the AngⅡ sample. Take 5mg of AngⅡ sample raw material and dissolve it with 0.1mol / L ammonium bicarbonate solution to a volume of 2ml. Add 10mg DTT to the solution, fill it with nitrogen to react for 3 hours, and adjust the pH to neutral with acetic acid after the reaction. After the reaction is completed, the solution becomes turbid, and the liquid is centrifuged (3000rpm) for 2min. Take the supernatant for liquid separation. Use 30%CAN+70%ddH2O to dissolve and separate to obtain a pure product spot film. After the sample is spotted, place the target membrane in the reactor. After assembling the reactor, place it in a fixed position on the instrument. Use the software PPSQ-30Analysis to set the sample name, sample number, number of test cycles, and select the method file. After the settings are completed, start the test. The test results are as follows Figure 2 and Figure 3 As shown, compared with the standard calibration test spectrum ( Figure 4 ) By comparison, it can be seen that the N-terminal sequence of AngⅡ in the test sample is NH2-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, that is, DRVYIHPF, which is consistent with the theoretical sequence of AngⅡ.

[0119] 3. Initial inspection of uniformity

[0120] The sample raw materials after typing and identification were subjected to preliminary uniformity inspection by high performance liquid chromatography. Samples were randomly taken from the upper, middle and lower parts of the premixed angiotensin II (human) sample raw material bottle, and a 1 mg / g solution was prepared. The solution was filtered through a 0.22 μm microporous filter membrane and injected. The chromatographic column was a C18 reverse phase column. The mobile phase gradient was shown in Table 1. Mobile phase A was 10 mmol / L sodium dihydrogen phosphate buffer (pH 2.5), mobile phase B was 50% acetonitrile, the flow rate was 0.5 mL / min, the column temperature was 25°C, the detection wavelength was 230 nm, and the data of the preliminary uniformity inspection were shown in Table 2. As shown in Table 2, the standard deviation of the preliminary uniformity inspection of the human angiotensin II sample raw materials was 0.029%, F=3.09, and F α =5.14, F <F α This indicates that there is no obvious heterogeneity in the human angiotensin II sample raw materials at different locations of the same batch after premixing, and they can be packaged.

[0121] When packaging, transfer all the AngⅡ sample materials to the same sample bottle, turn it upside down, mix it thoroughly, and then dispense it into 2mL brown ampoules. Each bottle should be filled with no less than 3mg. A total of 210 bottles should be dispensed and stored in a refrigerator at -20℃. They should be marked as candidate for purity standard material of human angiotensin II. Their appearance should be as follows: Figure 5 As shown, it can be stably stored at -20℃ without freezing, cracking or leakage.

[0122] Table 1 Mobile phase gradient settings

[0123]

[0124]

[0125] Table 2 Preliminary homogeneity test data of angiotensin II (human) sample raw materials

[0126]

[0127] 3. Establishment and optimization of reversed-phase high performance liquid chromatography

[0128] 1. Initial experimental conditions

[0129] According to literature research, a phosphate buffer system was used as the mobile phase for HPLC, and the initial experimental conditions were as follows:

[0130] Chromatographic column: C18 reverse phase 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 3.

[0131] Table 3 Mobile phase gradient elution conditions

[0132] Time / min A％ B％ 0 67 33 2 67 33 25 57 43 30 57 43 35 67 33 40 67 33

[0133] In order to clearly determine the separation degree between the main components and impurities to be measured, the optimization and final determination of the chromatographic column, wavelength, mobile phase, column temperature, injection concentration were based on the selected AngⅡ purity standard substance candidate.

[0134] 2. Optimization of chromatographic columns

[0135] Using the initial experimental conditions, the effects of Agilent Poroshell 120EC-C18 (4.6 mm × 150 mm, 4 μm) and Agilent ZORBAX Eclipse XDB-C18 (4.6 mm × 150 mm, 5 μm) columns on the separation of AngⅡ purity standard substance candidates were compared. The results are shown in Figure 2. Figures 6 to 9 .

[0136] The results showed that the Agilent Poroshell 120EC-C18 column could fully separate the AngⅡ purity standard substance candidate and other impurities. Therefore, the Agilent Poroshell 120EC-C18 (4.6mm×150mm, 4μm) column was selected for subsequent optimization.

[0137] 3. Optimization of wavelength conditions

[0138] The wavelength of the selected AngⅡ purity standard substance candidate was scanned to obtain the UV spectrum. The results are as follows Fig.10 As shown, from Fig.10 It can be seen that the maximum absorption wavelength of the AngⅡ purity standard substance candidate 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 11 to 14 As shown in Table 4. The results show that when the wavelength is 254nm or 280nm, the response value is too low, and the types of impurities detected are not as good as those at 210nm and 230nm. When the wavelength is 210nm and 230nm, a total of 5 impurity peaks are detected, indicating that the wavelength can fully detect the impurity peaks, and the measured purity of the main components in the AngⅡ purity standard substance candidates is equivalent. Considering that there may be strong solvent interference at 210nm, 230nm is selected as the detection wavelength on the premise of ensuring sufficient signal response of organic matter.

[0139] Table 4 Effect of different detection wavelengths on peak area normalization detection results

[0140]

[0141] 4. Optimization of mobile phase

[0142] Using the initial experimental conditions, the effects of 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 of AngⅡ purity standard substance candidates were investigated. The results are as follows Figures 15 to 17 The results showed that the peak shape of the candidate AngⅡ purity standard substance in the water-50% acetonitrile system was poor, and the main component and impurities could not be separated; in the 0.1% formic acid aqueous solution-50% acetonitrile system, the main peak tailed severely, and the number of impurities detected was 4. The subsequent attempt to optimize the gradient of the mobile phase ratio failed to screen out a better result; in the 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.

[0143] 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 3 on the detection results were investigated. The results are as follows Figures 18 to 21 The 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 3, and the response value meets the requirements. Therefore, Table 3 is selected as the gradient elution conditions for subsequent optimization.

[0144] 5. Optimization of pH value of mobile phase A

[0145] Based on the gradient elution conditions in Table 3, 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 the AngⅡ purity standard substance candidate were tried respectively. The results are as follows: Fig. 22 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 AngⅡ purity standard substance candidate, resulting in additional impurity peptides.

[0146] In order to verify the effect of the pH value of mobile phase A on the separation of AngⅡ purity standard substance candidates, we continued to try the effect of the pH value of 20mmol / L sodium dihydrogen phosphate buffer at 2.8 on the separation of AngⅡ purity standard substance candidates. The results are as follows: Fig.23 As shown, the results show that the separation effect of the AngⅡ purity standard substance candidate at pH 2.8 is basically the same as that at pH 3.0, indicating that the AngⅡ purity standard substance candidate is relatively stable under relatively high pH values ​​and no additional impurities are generated. Therefore, 20mmol / L sodium dihydrogen phosphate buffer with pH = 3.0 was selected for subsequent optimization.

[0147] 6. Optimization of ionic strength of mobile phase A

[0148] In order to analyze the effect of the ionic strength of mobile phase A on the retention time and separation of the candidate AngⅡ purity standard substance, the concentration of sodium dihydrogen phosphate buffer of mobile phase A was optimized. The effects of 10mmol / L, 20mmol / L, and 30mmol / L sodium dihydrogen phosphate buffer on the separation effect of the candidate AngⅡ purity standard substance were tried respectively. The results are shown in Figure 2. Figures 24-26 The 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. Therefore, sodium dihydrogen phosphate buffer with an intermediate concentration of 20 mmol / L was selected as mobile phase A.

[0149] 7. Optimization of column temperature

[0150] In order to analyze the effect of column temperature on the retention time and separation of AngⅡ purity standard substance candidates, the effects of column temperatures of 20℃, 25℃, and 30℃ on the separation effect were tried respectively. The results are as follows: Fig. 27 The results show that at a column temperature of 20°C, the separation effect of the AngⅡ purity standard substance candidate 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 AngⅡ purity standard substance candidate are basically the same. Therefore, 25°C was selected as the column temperature.

[0151] 8. Optimization of injection concentration

[0152] The impurity conditions of the candidate AngⅡ purity standard substance 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 5. 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.

[0153] Table 5 Effect of different injection concentrations on peak area normalization detection results

[0154]

[0155]

[0156] 9. Optimize the test conditions

[0157] After optimizing the chromatographic column, wavelength, mobile phase, column temperature, and injection concentration, the chromatographic test conditions used for the determination of the main component content of the candidate human angiotensin II purity standard substance are finally optimized and determined as follows:

[0158] Chromatographic column: Agilent Poroshell 120 EC-C18 (4.6 mm × 150 mm, 4 μm);

[0159] Injection concentration: 1 mg / mL;

[0160] Injection volume: 5 μL;

[0161] Column temperature: 25°C;

[0162] Flow rate: 0.5 mL / min;

[0163] Detection wavelength: 230nm;

[0164] Running time: 30min;

[0165] Mobile phase A: 20 mmol / L sodium dihydrogen phosphate buffer, pH = 3.0;

[0166] Mobile phase B: 50% acetonitrile;

[0167] Mobile phase gradient:

[0168] Table 6 Optimized mobile phase gradient conditions

[0169] Time / min A％ B％ 0 67 33 2 67 33 25 57 43 30 57 43 35 67 33 40 67 33

[0170] 10. Methodological parameters for purity determination by reversed-phase high performance liquid chromatography

[0171] The methodological parameters of the optimized reversed-phase high performance liquid chromatography purity determination were investigated. In terms of method repeatability, under the detection conditions, 5 3mg AngⅡ purity standard substance candidates were taken as parallel samples, and each sample was analyzed 3 times. The chromatograms of typical analysis results are shown in Figure 2. Fig.28 The results of 5 parallel samples are shown in Table 7. As shown in Table 7, the reversed-phase HPLC purity analysis method has good method repeatability and can meet the requirements of uniformity inspection and determination of mass balance method.

[0172] Table 7 RP-HPLC purity of candidate AngⅡ purity standard substances (%)

[0173]

[0174] In terms of method linearity, solutions of candidate AngⅡ purity standard substances at concentrations of 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 were prepared, and the prepared samples were analyzed by reversed-phase HPLC using the optimized test conditions. The peak areas and concentrations of the response samples were linearly fitted. The results are shown in Figure 2. Fig.29 Calculate the linear correlation coefficient r 2 =0.9999, which shows that the reversed-phase HPLC method has good linearity.

[0175] For the detection limit and quantification limit of the method, 3 times the noise peak area and 10 times the noise peak area were substituted into Fig.29 In the obtained standard curve, the detection limit and quantification limit of the main component content of the candidate AngⅡ purity standard substance by reversed-phase high performance liquid chromatography were 4.3×10 -3 mg / g and 5.3×10 -3 mg / g.

[0176] 4. Establishment and optimization of isotope dilution mass spectrometry method

[0177] 1. Hydrolysis of Candidate Purity Standard Material of Human Angiotensin II

[0178] About 1 mg of AngⅡ purity standard material candidate was weighed on a balance with a minimum division of 0.01 mg, dissolved in 10 mL of 0.01 mol / L hydrochloric acid, and its mass was accurately weighed to prepare a 0.1 mg / g AngⅡ solution. 18 μL of AngⅡ solution was added to a 2 mL ampoule, and then 100 μL of isotope labeled amino acid mixed standard solution (including equal concentrations of Phe, Ile and Val, 0.1 mg / g) was added, and their masses were accurately weighed to obtain a sample solution, which was placed in a vacuum centrifugal dryer for centrifugal drying for 70 min. After all the water in the sample solution was removed, it was taken out, 800 μL of 6 mol / L hydrochloric acid was added, nitrogen was passed for 2 min to deoxygenate, and then sealed, and hydrolyzed in an oven at 109.5-110.5 ° C. After hydrolysis, it was dried with nitrogen, re-dissolved with 1 mL of 0.1% formic acid aqueous solution, filtered through a 0.22 μm filter membrane, and then measured on the machine.

[0179] 2. High performance liquid chromatography-isotope dilution mass spectrometry (HPLC-IDMS) test conditions

[0180] 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.

[0181] 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).

[0182] 3. Optimization of hydrolysis time

[0183] In order to ensure that the candidate AngⅡ 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 spectrum 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.30 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 candidate AngⅡ purity standard substance and used for subsequent quantitative analysis by isotope dilution mass spectrometry.

[0184] 4. Optimization of mass spectrometry conditions

[0185] 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.

[0186] 5. Confirmation of isotope internal standard

[0187] 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 31-33 The analysis results show that the isotope-labeled amino acids used as internal standards do not contain non-labeled amino acids.

[0188] 6. Optimization of liquid phase conditions

[0189] 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 candidate AngⅡ purity standard substances after hydrolysis were compared. Chromatographic conditions: mobile phase (AB) 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.34 The results showed that the Agilent ZORBAX EclipsePlus C18 column was selected for subsequent optimization.

[0190] The effects of the volume ratios of mobile phases A and B on the detection results were investigated as follows: Fig.35 and Fig.36 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.

[0191] 7. Methodological parameters of isotope dilution mass spectrometry

[0192] The methodological parameters of the optimized isotope dilution mass spectrometry method were investigated.

[0193] (1) Correctness of the method

[0194] 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 8. 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.

[0195] Table 8 Verification results of standard substances

[0196]

[0197] (2) Repeatability of the method

[0198] The HPLC-IDMS method was used to determine the content of AngⅡ purity standard substance candidates in 5 units (bottles), and each unit was analyzed 3 times. The results are shown in Table 9. The results showed that the total average content determination results of AngⅡ purity standard substance candidates were 0.8757 g / g, and the relative standard deviation was 0.88%, indicating that the HPLC-IDMS purity analysis method has good method repeatability and can meet the value determination requirements of isotope dilution mass spectrometry.

[0199] Table 9 Content determination results of candidate AngⅡ purity standard substances (g / g)

[0200]

[0201]

[0202] (3) Evaluation of detection limit and quantification limit

[0203] The detection limit and quantification limit of the HPLC-IDMS method were calculated according to formula (2-1):

[0204]

[0205] 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-sample is 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 the candidate AngⅡ standard purity substance are 6.5×10 -7 g / g and 2.2×10 -6 g / g.

[0206] 4. Uniformity Test

[0207] 1. Homogeneity test principles and statistical methods

[0208] In accordance with the technical requirements for the uniformity evaluation of standard materials in the national metrology technical specification JJF 1343-2022 "Value determination, uniformity and stability evaluation of standard materials", a certain number of minimum packaging units are randomly selected (sampling can be performed according to the method shown in the random number table), and a high-precision test method is used to measure each sample under the same experimental conditions, so that the differences between the samples are fully reflected by the heterogeneity of the samples.

[0209] ANOVA is the most commonly used method for statistical testing of homogeneity. This method determines whether there are systematic differences between the measured values ​​of each group by comparing the variance between groups and the variance within groups. If the ratio of the two is less than the critical value of the statistical test, the sample is considered to be homogeneous.

[0210] In order to test the homogeneity of the candidate AngⅡ purity standard substance, suppose a samples are drawn and n i For a simple balanced design, the number of repeated measurements ni for all units is the same, that is, n1 = n2 = ... = n a=n0. One-way ANOVA can be used for analysis. The statistical model is shown in formula (4-1):

[0211] x ij =μ+δ i +ε ij (4-1),

[0212] Among them, x ij is the jth observation value of the i-th unit; μ is the total number of all possible results (observation result x ij Assumed to be generated from it) (true) mean; δ i is the effect of unit i on the result, that is, the (true) deviation between unit i and μ; ε ij is the random error of the j-th observation of the ith unit, also known as the residual term.

[0213] Mean square between units M between and residual mean square M within From formula (4-2) and formula (4-3), we can get:

[0214]

[0215] in,

[0216]

[0217] The mean square between groups M between , within-group mean square M within and n0 to calculate the inter-unit standard deviation s bb , and as the uncertainty component u introduced by the inhomogeneity between units bb :

[0218]

[0219] in,

[0220]

[0221] 2. Uniformity inspection plan

[0222] (1) Sampling and measurement methods

[0223] A total of 210 units of this batch of AngⅡ purity standard substance candidates were developed, so 11 units were randomly selected from them for standard substance homogeneity test. Using the randbetween function of Excel software, 11 random numbers were generated, and 11 units were taken out from the standard substance candidates according to the random numbers. The mass balance method was used to repeat the measurement of each unit of AngⅡ purity standard substance candidate 3 times. Each time the analysis was performed, 0.5 mg of AngⅡ purity standard substance candidate solid was taken out from the sample unit, and the taken AngⅡ purity standard substance candidate solid was prepared into a 1 mg / g solution for analysis. In order to eliminate the error caused by instrument drift during the measurement process, the sample was analyzed three times in the order of 1→11, 11→1, and odd and even interlaced. Reverse phase high performance liquid chromatography was used for homogeneity test, and its experimental conditions were optimized conditions.

[0224] (2) Determination of impurity content

[0225] ① Determination of water content in candidate AngⅡ purity standard substances

[0226] According to GB / T 606-2003 "General Method for Determination of Water in Chemical Reagents - Karl Fischer Method", add a certain volume of methanol solvent specified in the product standard to the reaction bottle, immerse the platinum electrode, and titrate to the endpoint with Karl Fischer reagent under stirring. Quickly add the amount of sample specified in the product standard (about 6 mg), and titrate to the endpoint with Karl Fischer reagent.

[0227] Since each packaging unit only has 3 mg of AngⅡ purity standard substance candidate, it cannot meet the determination requirements of Karl Fischer method. Two units (bottles) of AngⅡ purity standard substance candidate are combined to determine the water content. Therefore, about 6 mg of AngⅡ purity standard substance candidate is accurately weighed each time, and the determination is performed using Karl Fischer moisture meter, and the determination is 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 are simulated, but the determination is performed without adding samples, and the blank value of moisture in the air is calculated. The final result is obtained after deducting the blank, and the results are shown in Table 10. The results show that in this measurement, the average value of the moisture content of AngⅡ purity standard substance candidate determined by Karl Fischer method is 12.24%. Therefore, the absolute moisture content ≈6 mg×12.24%=0.43 mg, which is within the range of moisture that can be accurately measured by Karl Fischer coulometric method (10 μg~10 mg).

[0228] Table 10 Water content of candidate AngⅡ purity standard substances determined by Karl Fischer method

[0229]

[0230] ② Determination of ash content in candidate AngⅡ purity standard substances

[0231] 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 accurately weighed AngⅡ purity standard substance candidate, accurately weigh, and burn at 700-800℃ to completely ash, move to a dryer, cool, accurately weigh, and burn again at 700-800℃ to constant weight. Calculate the ash content based on the residual mass after burning. The ash content of the AngⅡ purity standard substance candidate was not detected in three parallel measurements, so ion chromatography and other methods were used to determine the inorganic impurity components in the AngⅡ purity standard substance candidate.

[0232] ③Determination of anion content in candidate AngⅡ purity standard substances

[0233] Ion chromatography was used to determine the anions that may be contained in the candidate AngⅡ 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 11.

[0234] Table 11 Experimental conditions for ion chromatography analysis

[0235]

[0236] The methodological parameters of ion chromatography were investigated, the repeatability, linearity and detection limit (LOD) of the method were analyzed, and the anions in the candidate AngⅡ purity standard substance were determined, and the results are shown in Table 12. The anion content in the candidate AngⅡ purity standard substance was calculated to be 0.014% based on the data in Table 12.

[0237] Table 12 Analysis results of anion concentration in candidate AngⅡ purity standard substances

[0238] Repeatability Linear LOD concentration 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

[0239] ④ Determination of inorganic element content in candidate AngⅡ purity standard substances

[0240] Verification was carried out by testing common inorganic elements. The determination method was inductively coupled plasma mass spectrometry (ICP-MS). Not less than 3 mg of AngⅡ purity standard substance candidate 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 mixture was placed in a microwave digester for digestion. The microwave digestion conditions are shown in Table 13.

[0241] Table 13 Microwave digestion conditions

[0242] step Temperature(℃) Pressure(atm) Time (min) 1 100 15.0 2 2 120 20.0 4 3 150 25.0 4 4 180 30 6 5 200 35 10 6 220 40 10

[0243] 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 was used, and the concentration of the calibration point was 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 residual of the candidate AngⅡ purity standard substance was finally calculated to be 0.035%, of which the main element was Ba.

[0244] ⑤ Determination of volatile organic components in candidate AngⅡ purity standard substances

[0245] The volatile organic components that may exist in the candidate AngⅡ purity standard substance were determined by headspace injection-gas chromatography-mass spectrometry and quantified by external standard method. The headspace gas chromatography of the candidate AngⅡ purity standard substance dissolved in DMSO and water was tested respectively.

[0246] 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.

[0247] First, the solvent DMSO was selected, and the results showed that no other solvents were detected except DMSO. Then 3 mg of the AngⅡ purity standard substance candidate 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.

[0248] At the same time, in order to exclude the residual solvent of DMSO, 3 mg of the candidate AngⅡ 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.

[0249] Finally, the headspace quality analysis results of the AngⅡ purity standard substance candidate in 0.01mol / L HCl were tested, and no solvent residue was detected. The external standard method was used for quantification, and the methodological parameters and measurement results are shown in Table 14. The results show that the 30 solvent residues in the table were not detected in the AngⅡ purity standard substance candidate.

[0250] Table 14 Analysis results of volatile organic components in candidate AngⅡ purity standard substances

[0251]

[0252] Finally, the test results of moisture content, ash content, anion content, and residual solvent content in the AngⅡ purity standard substance candidate are shown in Table 15.

[0253] Table 15 Quantitative results of impurities in candidate AngⅡ purity standard substances

[0254] Impurity ingredients content(%) Moisture 12.24 cation 0.035 Anions 0.014 Solvent residue /

[0255] (3) Measurement results and statistical analysis

[0256] The reverse phase high performance liquid chromatography area normalization method was used for analysis, and then the data of moisture content, cation content and anion content in Table 15 were deducted using the mass balance method. The results are shown in Table 16.

[0257] Table 16 Homogeneity test data of AngⅡ purity standard substance candidates (g / g)

[0258] unit Times 1 Times 2 Times 3 Number of observations Sum average value variance 1 0.8731 0.8731 0.8726 3 2.6188 0.8730 8.333E-08 2 0.8726 0.8731 0.8727 3 2.6184 0.8728 7.000E-08 3 0.8726 0.8726 0.8731 3 2.6183 0.8728 8.333E-08 4 0.8732 0.8729 0.8728 3 2.6189 0.8730 4.333E-08 5 0.8729 0.8723 0.8720 3 2.6172 0.8724 2.100E-07 6 0.8734 0.8722 0.8727 3 2.6183 0.8728 3.633E-07 7 0.8735 0.8724 0.8727 3 2.6186 0.8729 3.233E-07 8 0.8741 0.8734 0.8728 3 2.6203 0.8734 4.233E-07 9 0.8727 0.8728 0.8727 3 2.6182 0.8727 3.333E-09 10 0.8728 0.8727 0.8732 3 2.6187 0.8729 7.000E-08 11 0.8729 0.8728 0.8728 3 2.6185 0.8728 3.333E-09

[0259] The homogeneity of the candidate human angiotensin II purity standard material was tested by variance analysis, and the results are shown in Table 17. From the results in Table 17, it can be seen that the calculated F value is less than F 0.05(10,22) This indicates that the candidate human angiotensin II purity standard substance has good uniformity.

[0260] Table 17 Analysis results of AngⅡ purity standard substance candidates based on variance analysis

[0261]

[0262] The mean square between groups M between , within-group mean square M within and n0 to calculate the inter-unit standard deviation s bb , and as the uncertainty component u introduced by the inhomogeneity between units bb :

[0263]

[0264] When conducting the homogeneity test, 0.5 mg of sample is taken out from each sample unit for analysis, so the minimum sampling amount of the AngⅡ purity standard material candidate is 0.5 mg.

[0265] 5. Stability inspection

[0266] The stability of a standard substance refers to how the characteristic quantity of a given value changes over time. The stability of a standard substance is affected by physical, chemical and storage conditions. It is necessary to use a high-precision analytical method to conduct regular inspections over a long period of time to determine the relative stability period. The stability of a standard substance includes long-term stability and short-term stability. Long-term stability refers to the stability of the characteristic quantity of a standard substance under specified storage conditions. Short-term stability refers to the stability of a standard substance during transportation under transportation conditions. This project is based on the technical specifications for standard substances and follows the principle of close proximity at the beginning and sparse proximity at the end. A 6-month long-term stability experiment at -20°C and a 7-day short-term stability experiment under simulated transportation conditions were conducted on the candidate AngⅡ purity standard substance.

[0267] The stability is evaluated by measuring the characteristic value of the AngⅡ purity standard substance candidate at different times, and plotting the relationship between the characteristic value and time with time as the X-axis and the characteristic value as the Y-axis.

[0268] The basic model of stability assessment can be expressed as:

[0269] Y=β0+β1X (5-1),

[0270] In formula (5-1), β0 and β1 are regression coefficients, X is time, and Y is the characteristic value of the candidate standard substance.

[0271] The regression parameters are calculated as follows:

[0272] The slope is calculated by formula (5-2):

[0273]

[0274] In formula (5-2), X i is the i-th time point; Y i is the observed value at the i-th time point; is the average of all time points; is the average of all observations.

[0275] The intercept is calculated by formula (5-3):

[0276]

[0277] The standard deviation s(β1) of β1 in formula (5-3) can be calculated by formula (5-4):

[0278]

[0279] In formula (5-4), s is the standard deviation of each point on the straight line, which can be calculated by formula (5-5):

[0280]

[0281] In formula (5-5), X i is the i-th time point; Y i is the observed value at the i-th time point; β0 and β1 are regression coefficients, and n is the number of measurements.

[0282] Use t-test to make the following judgment: If |β1|<t 0.05,n-2 ·s(β1), it indicates that the slope is not significant and no instability is observed.

[0283] According to formula (5-6), the uncertainty introduced by stability is calculated:

[0284] u s =s(β1)·X (5-6),

[0285] In formula (5-6), u s is the uncertainty introduced by stability; s(β1) is the standard deviation of β1; X is the given shelf life.

[0286] Considering that the candidate AngⅡ purity standard substance is packaged in brown ampoules, and the ampoules are sealed by melting to prevent the volatile substances such as water in the sample from changing during the storage period, the reversed-phase high-performance liquid chromatography results are used to deduct various impurities (i.e., mass balance method) to evaluate the possible changes in the main components of the standard substance during storage. At the same time, since the candidate AngⅡ purity standard substance has a high water content, the Karl Fischer titration method is also used to investigate the water content before and after 6 months of storage.

[0287] 1. Long-term stability study

[0288] At different time intervals, three samples of candidate angiotensin II purity standard materials were taken out each time to characterize the average results of stability investigation by mass balance method. The main component determination method was reversed phase high performance liquid chromatography, the water content was determined by Karl Fischer titration, the ash content was determined by high temperature calcination, and the volatile component was generally determined by gas chromatography. The original data of the long-term stability investigation of the main component are shown in Table 18, the original data of the long-term stability investigation of water content are shown in Table 19, and the analysis results are shown in Table 20. Since the final fixed value result is high, in addition to the main component result, the water stability result is also focused on.

[0289] Table 18 Long-term stability experimental data of the main components in the candidate AngⅡ purity standard substances

[0290]

[0291]

[0292] Table 19 Experimental data on long-term stability of water in candidate AngⅡ purity standard substances

[0293]

[0294] Table 20 Long-term stability test results of AngⅡ purity standard substance candidates

[0295]

[0296] Under the conditions of degrees of freedom n-2 = 6 and confidence level p = 0.95, we can get t by looking up the table. 0.05,6 =2.45.

[0297] According to the data in Tables 18 to 20, the relationship between the characteristic value and time is plotted with time as the X-axis and the characteristic value as the Y-axis. The results are as follows: Figures 37-39 As shown. Figures 37-39 It can be seen that there is no obvious upward or downward trend in the quantity of the standard substance.

[0298] according to Figures 37-39 The fitting straight line and equation (5-1) to equation (5-6) were used for regression analysis, and the regression analysis results are shown in Table 21.

[0299] Table 21 Regression analysis results of long-term stability experimental data of AngⅡ purity standard substance candidates

[0300]

[0301]

[0302] It can be seen from Table 21 that the conclusions obtained after calculation are all |β1| <t 0.05,6 ·s(β1), so no obvious instability was observed.

[0303] The above results indicate that within the 12-month shelf life, the preparation method and storage conditions adopted by the AngⅡ purity standard substance candidate can effectively ensure the quantitative stability of the AngⅡ purity standard substance candidate.

[0304] 2. Short-term stability study

[0305] The transport conditions at 40℃, 25℃ and 4℃ were simulated, and 6 groups of AngⅡ purity standard substance candidates were randomly selected as samples, with 3 tubes (bottles) in each group, and placed in incubators to investigate the changes of AngⅡ purity standard substance candidates over time during different transport times. The specific experimental steps are to take out the human angiotensin II purity standard substance candidates from -20℃ on days 0, 2, 4, 5, 6, and 7, and place them under simulated transport conditions, and take them out together after 7 days (the storage time of the samples under transport conditions is 7, 5, 3, 2, 1, and 0 days, respectively), and use the results measured by reversed-phase high performance liquid chromatography to deduct various impurities (i.e., mass balance method) for short-term stability investigation, and the results are shown in Tables 22 to 24.

[0306] Table 22 Short-term stability test data of AngⅡ purity standard substance candidates at 40℃ (g / g)

[0307] 1 2 3 average value 0 0.8726 0.8726 0.8731 0.8728 1 0.8724 0.8724 0.8722 0.8723 2 0.8722 0.8722 0.8722 0.8722 3 0.8718 0.8719 0.8717 0.8718 5 0.8703 0.8704 0.8703 0.8703 7 0.8703 0.8702 0.8703 0.8703

[0308] Table 23 Short-term stability test data of AngⅡ purity standard substance candidates at 25℃ (g / g)

[0309] 1 2 3 average value 0 0.8723 0.8722 0.8728 0.8724 1 0.8730 0.8728 0.8724 0.8727 2 0.8725 0.8724 0.8724 0.8724 3 0.8720 0.8725 0.8725 0.8723 5 0.8724 0.8726 0.8723 0.8724 7 0.8724 0.8722 0.8723 0.8723

[0310] Table 24 Short-term stability test data of AngⅡ purity standard substance candidates at 4°C (g / g)

[0311]

[0312]

[0313] According to the data in Tables 22 to 24, the relationship between the characteristic value and time is plotted with time as the X-axis and the characteristic value as the Y-axis. The results are as follows: Figures 40-42 shown.

[0314] according to Figures 40-42 The fitted straight line and equations (5-1) to (5-6) were used for regression analysis, and the short-term stability test results are shown in Table 24. The results showed that, according to the t test, under two different storage conditions of 25°C and 4°C, the value of the AngⅡ purity standard substance candidate was stable within 7 days, while the value was unstable when stored at 40°C for 7 days.

[0315] Table 25 Short-term stability qualitative test results of AngⅡ purity standard substance candidates (g / g)

[0316]

[0317] According to the data in Table 25, the stability test of the AngⅡ purity standard substance candidate at different storage times at 40°C was carried out. The results are as follows: Fig.43 , Fig.44 and as shown in Table 26. The results showed that, through the t-test, the AngⅡ purity standard material candidate was stable within 2 days of storage at 40°C, and was unstable after 3 days.

[0318] Table 26 Short-term stability test results of AngⅡ purity standard substance candidates (40℃)

[0319] time b s(b) s(b)·t in conclusion 2 days <![CDATA[-2.66×10 -4 ]]> <![CDATA[7.60×10 -5 ]]> <![CDATA[9.60×10 -4 ]]> Stablize 3 days <![CDATA[-2.92×10 -4 ]]> <![CDATA[3.72×10 -5 ]]> <![CDATA[1.60×10 -4 ]]> Unstable

[0320] 3. Opening stability test

[0321] The stability of the candidate AngⅡ purity standard substance after opening was tested by reverse phase high performance liquid chromatography. A unit of the candidate AngⅡ purity standard substance was randomly selected, and 0.5 mg of the candidate AngⅡ purity standard substance was taken out from the bottle each time after opening the bottle for analysis. After analysis, the bottle mouth was sealed with a sealing film and stored in a -20℃ refrigerator. The above process was repeated every 1 day for a total of 3 days. The results are shown in Table 27.

[0322] Table 27 Results of stability test of AngⅡ purity standard substance candidates after opening the bottle (g / g)

[0323]

[0324] According to the data in Table 27, the relationship between purity and time is plotted with time as the X-axis and purity as the Y-axis. The results are as follows: Fig.45 shown.

[0325] according to Fig.45 The fitting straight line and regression analysis were used to obtain the results of the stability test of the candidate AngⅡ purity standard substance after opening the bottle, as shown in Table 28. The results showed that the candidate AngⅡ purity standard substance could be stored at -20℃ for at least 4 days after opening the bottle, as shown in Table 28.

[0326] Table 28 Opening stability test results of AngⅡ purity standard substance candidates (-20℃)

[0327] Temperature / ℃ Day 0 1 day 2 days 4 days b s(b) s(b)·t in conclusion -20 0.8732 0.8726 0.8725 0.8722 <![CDATA[2.67×10 -4 ]]> <![CDATA[1.04×10 -4 ]]> <![CDATA[4.50×10 -4 ]]> Stablize

[0328] 6. Fixed value

[0329] This example uses the mass balance method and isotope dilution mass spectrometry to determine the purity of AngⅡ standard substance candidates.

[0330] 1. Analysis of mass balance method value results

[0331] The main component content of the candidate AngⅡ purity standard substance was determined by reversed-phase high performance liquid chromatography. The analysis test conditions were the same as those in “9. Optimized test conditions”.

[0332] Five portions (bottles) of the AngⅡ purity standard substance candidate were taken as parallel samples, and each sample was analyzed 3 times. The results were used as the purity results of the AngⅡ purity standard substance candidate. The results are shown in Table 29.

[0333] Table 29 Purity results of candidate human angiotensin II purity standard substances (%)

[0334]

[0335] According to the data in Table 15, the moisture content was finally measured to be 12.24%, the cation content was 0.035%, the anion content was 0.014%, and no solvent remained.

[0336] The purity value of the candidate human angiotensin II purity standard material determined by reversed-phase high performance liquid chromatography was deducted from the water content, cation content, anion content and solvent residue test results to finally obtain the mass balance method valuation result, as shown in Table 30.

[0337] Table 30 Mass balance legal value results of AngⅡ purity standard substance candidates (g / g)

[0338]

[0339] The normality test of the fixed value results was performed using IBM SPSS20 software, and the results are shown in Table 31. As can be seen from Table 31, the calculated significance is greater than 0.05 and obeys the normal distribution. View the QQ graph ( Fig.45 ) further confirms that the expected standard is basically distributed around the straight line and can be considered to obey the normal distribution.

[0340] Table 31 Normality test of the value data of candidate AngⅡ purity standard substances (Shapiro-Wilk method)

[0341] Value determination method Statistics Degrees of Freedom Significance Mass Balance Method 0.903 15 0.107

[0342] The mass balance legal value data of the AngⅡ purity standard substance candidate materials in Table 30 were tested for outliers using the Grubbs and Dixon outlier test methods, respectively. The results are shown in Table 32.

[0343] Table 32 Outlier test of mass balance method data

[0344]

[0345] As can be seen from Table 32, 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 mass balance method for the candidate AngⅡ purity standard substance, which is 0.8735 g / g.

[0346] 2. Analysis of isotope dilution mass spectrometry results

[0347] Under the optimized conditions of the isotope dilution mass spectrometry method, 5 portions (bottles) of human angiotensin II purity standard material candidate were taken as parallel samples, and each sample was analyzed 3 times as the determination result of the isotope dilution mass spectrometry method. The results are shown in Table 33.

[0348] Table 33 Isotope dilution mass spectrometry results of AngⅡ purity standard material candidates (g / g)

[0349]

[0350] The normality test of the isotope dilution mass spectrometry results was performed using IBM SPSS20 software, and the results are shown in Table 34.

[0351] Table 34 Normality test of the value data of Ang II purity standard substance candidates (Shapiro-Wilk method)

[0352] Value determination method Statistics Degrees of Freedom Significance IDMS Act 0.962 15 0.722

[0353] From Table 34, we can see that the calculated significance is greater than 0.05 and obeys the normal distribution. Fig.46 ) further confirms that the expected standard is basically distributed around the straight line and can be considered to obey the normal distribution.

[0354] The isotope dilution mass spectrometry data of the Ang II purity standard material candidate in Table 34 were tested for outliers using the Grubbs and Dixon outlier test methods, respectively. The results are shown in Table 35.

[0355] Table 35 Outlier test of isotope dilution mass spectrometry data

[0356]

[0357] It can be seen from Table 35 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 candidate, which is 0.8733 g / g.

[0358] 3. Statistical analysis of data from two valuation methods

[0359] 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 36.

[0360] Table 36 Variance homogeneity test of two fixed value methods (Cochran method)

[0361] <![CDATA[s MB ]]> <![CDATA[s IDMS ]]> <![CDATA[s max ]]> ∑s C C(0.05,2,15) Discrimination AngII <![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

[0362] After analysis, it was found that the C values ​​between the two determination methods for the candidate 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 precision.

[0363] The data obtained by the two methods were subjected to an independent sample t-test using IBM SPSS25 software. The results are shown in Table 37.

[0364] Table 37 Results of variance homogeneity test and independent sample t-test for two fixed value methods

[0365]

[0366] After analysis, for the candidate AngⅡ purity standard material, 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.

[0367] 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 SPSS25 software. The analysis results are shown in Table 38.

[0368] Table 38 Normality test of combined data of two fixed value methods

[0369]

[0370] It can be seen from Table 38 that the results of the combined data of the two valuation methods are consistent with the normal distribution assumption. Fig.47 ) further confirms that the expected standard is basically distributed around the straight line and can be considered to obey the normal distribution.

[0371] The original data table 38 was tested for outliers using the Grubbs and Dixon outlier test methods, respectively. The results are shown in Table 39.

[0372] Table 39 Combined data of two fixed value methods, fixed value data outlier test

[0373]

[0374] The results showed that no outliers were found after Dixon and Grubbs tests.

[0375] Finally, the average value of the mass balance method and the isotope dilution mass spectrometry method was calculated as the value result of the AngⅡ purity standard substance candidate in this example, that is:

[0376]

[0377] 7. Uncertainty Analysis

[0378] According to the provisions of JJG 1006-1994 "Technical Specifications for the Preparation of Primary Reference Materials" and JJF 1343-2022 "Value Determination and Uniformity and Stability Evaluation of Reference Materials", the total uncertainty of the standard value is composed of three parts, including: (1) the uncertainty introduced by the value determination; (2) the uncertainty introduced by the heterogeneity of the reference material; (3) the uncertainty introduced by the instability of the reference material.

[0379] 1. Uncertainty assessment of mass balance method value results

[0380] The uncertainty of the mass balance method valuation results includes the uncertainty component introduced by the HPLC area normalization method, the uncertainty component introduced by the moisture content determination, the uncertainty component introduced by the anion content determination, the uncertainty component introduced by the inorganic element content determination and the uncertainty component introduced by the volatile organic solvent content determination.

[0381] (1) Uncertainty components introduced by HPLC area normalization method

[0382] The uncertainty introduced by HPLC area normalization measurement includes the uncertainty component introduced by measurement repeatability and the uncertainty component introduced by wavelength difference.

[0383] ① Uncertainty component introduced by measurement repeatability (u a )

[0384] The standard deviation of the average value of the HPLC results is s = 0.44%, and the uncertainty of the method repeatability is calculated by the following formula:

[0385]

[0386] ② Uncertainty component introduced by the difference in liquid chromatography response factor (u b )

[0387] The uncertainty u introduced by the response difference of each component at different detection wavelengthsb Taking into account the responses of the main components and impurity compounds at the corresponding detection wavelengths, it is calculated by the following formula:

[0388]

[0389] In the formula, B imaxλ is the maximum percentage content of impurity component i at the four detection wavelengths, B iminλ It is the minimum percentage content of impurity components. b-i is the uncertainty component of impurity component i. The evaluation results are shown in Table 40.

[0390] Table 40 Chromatographic peak area percentage of each impurity component at different wavelengths

[0391]

[0392] In Table 40, “---” means not detected.

[0393] Therefore, the relative uncertainty introduced by HPLC determination is calculated by the following formula:

[0394]

[0395] (2) The uncertainty introduced by the moisture content measurement u(X W )

[0396] According to the moisture measurement data, the range R = 1.06%, n = 6 (look up the table c = 2.53), the relative uncertainty component introduced by the moisture content measurement is calculated using the range method, as shown in the following formula:

[0397]

[0398] (3) Uncertainty introduced by anion content determination u(X 1n )

[0399] According to the method and data of anion measurement, the measured R = 0.0060 mg / g, n = 3 (look up c = 1.69), the uncertainty component introduced by the anion content determination is calculated using the range method, as shown in the following formula:

[0400] (4) Uncertainty u(X) introduced by inorganic element content 2n )

[0401]

[0402] The inorganic element content was detected by Angilent 7700x inductively coupled plasma mass spectrometer. According to the calibration certificate of the instrument, its expanded uncertainty is U = 2.7 ng / kg, k = 2, so the relative uncertainty component introduced by the determination of inorganic element content is calculated by the following formula:

[0403]

[0404] (5) Uncertainty of the results of mass balance method

[0405] In summary, the combined standard uncertainty of the mass balance method is:

[0406]

[0407] 2. Uncertainty assessment of isotope dilution mass spectrometry results

[0408] The calculation model of isotope dilution mass spectrometry is:

[0409]

[0410] Wherein, C is the mass fraction of the candidate AngⅡ purity standard substance (unit: g / g); M is the mass of the candidate AngⅡ purity standard substance used for hydrolysis (unit: g); R1 is the peak area ratio of the amino acid in the high-standard solution to the labeled amino acid; R2 is the peak area ratio of the amino acid in the low-standard solution to the labeled amino acid; P is the purity of the amino acid (unit: g / g); m 标 is the mass of the labeled amino acid in the hydrolyzed sample (unit: g); R 样 is the peak area ratio of the amino acid after sample hydrolysis to the labeled amino acid; W1 is the mass ratio of the amino acid in the high-standard solution to the labeled amino acid; W2 is the mass ratio of the amino acid in the low-standard solution to the labeled amino acid; P H is the hydrolysis efficiency of the candidate AngⅡ purity standard substance; R AA is the amino acid purity ratio factor (unit: g / g); M Ang is the molecular weight of the candidate AngⅡ purity standard substance; N AA is the number of amino acids in AngⅡ; M AA is the molecular weight of the amino acid.

[0411] According to the mathematical model of the measurement results of AngⅡ purity standard substance candidates, the uncertainty mainly comes from the uncertainty components of weighing, peak area ratio measurement repeatability, hydrolysis efficiency, standard substance purity, AngⅡ molecular weight, amino acid molecular weight, and amino acid purity proportional factor.

[0412] During the experiment, Val, Ile and Phe were used to measure the content of the candidate AngⅡ purity standard substance, and the average value was taken. The uncertainty of the quantitative result of each amino acid on the candidate AngⅡ purity standard substance was calculated. The following takes Phe as an example to calculate the measurement uncertainty.

[0413] (1) Uncertainty u(P) introduced by amino acid purity

[0414] GBW09235 L-phenylalanine purity standard material was used in the experiment. According to the standard material certificate, the purity value result was (0.998±0.004) g / g, k=2. Therefore, the expanded uncertainty introduced by the purity of the standard material is:

[0415]

[0416] (2) Uncertainty introduced by hydrolysis efficiency u(P H )

[0417] According to the results of 2022-B-03 "Comparison of the measurement capability of isotope dilution mass spectrometry of the novel coronavirus N protein", the hydrolysis efficiency is estimated to be 1%. The isotope dilution mass spectrometry measurement result of the AngⅡ purity standard material candidate is 0.8733g / g, so the relative uncertainty introduced by the hydrolysis efficiency is calculated according to the following formula:

[0418]

[0419] (3) Uncertainty introduced by weighing

[0420] The steps involved in weighing in this experiment include:

[0421] ① The process of preparing the initial solution: weighing three amino acids, weighing three isotope-labeled amino acids, weighing water when dissolving three amino acids, and weighing water when dissolving three isotope-labeled amino acids;

[0422] ② Preparation of stock solution: weigh the amino acid solution when preparing the stock solution, weigh the isotope labeled amino acid solution when preparing the stock, and weigh the water when preparing the stock;

[0423] ③ When preparing the isotope dilution method standard solution, weigh the amino acid solution when preparing the high standard, weigh the isotope labeled amino acid solution when preparing the high standard, weigh the amino acid solution when preparing the low standard, and weigh the isotope labeled amino acid solution when preparing the low standard.

[0424] ④ When processing samples, weigh the candidate AngⅡ purity standard substance, weigh water when preparing AngⅡ stock solution, weigh AngⅡ stock solution when preparing candidate AngⅡ purity standard substance, and weigh the isotope-labeled amino acid solution when preparing candidate AngⅡ purity standard substance.

[0425] The above weighing process is recorded as two times, one for peeling and one for weighing. The sum of squares is taken as the uncertainty introduced by weighing.

[0426] The uncertainty component introduced by the balance weighing is approximately rectangularly distributed. Since a balance with an accuracy of 0.01 mg is used in the analysis process, the uncertainty introduced by the weighing of the Phe purity standard substance is:

[0427]

[0428] The relative standard uncertainty is:

[0429]

[0430] The uncertainty analysis results introduced by the rest of the balance weighing process are synthesized.

[0431] (4) Uncertainty introduced by mass ratio

[0432] The mass ratio is the ratio of the masses of two weighings. Therefore, the uncertainty introduced by the mass ratio is calculated as follows:

[0433]

[0434] The formula for calculation is:

[0435] (5) Uncertainty introduced by peak area ratio

[0436] The uncertainty introduced by the peak area ratio is evaluated according to the uncertainty type A method. A standard or sample is measured 6 times to obtain the area A of the labeled amino acid and the non-labeled amino acid. The relative standard deviation of the area ratio of the 6 repeated measurements is calculated. The uncertainty introduced by the peak area ratio is calculated according to the following formula:

[0437]

[0438] (6) Uncertainty introduced by molecular weight

[0439] According to the molecular formula of AngⅡ 50 H 71 N 13 O 12 , calculate the uncertainty introduced by molecular weight according to the following formula:

[0440]

[0441] Where N i is the number of atoms of the element; u i is the uncertainty of the relative atomic mass of the element; M Ang is the molecular weight of AngⅡ.

[0442] According to the IUPAC International Atomic Mass Table, the relative atomic masses and uncertainties of the five atoms are: C: 12.0107±0.0008; H: 1.00794±0.00007; O: 15.9994±0.0003; N: 14.00674±0.00007; S: 32.065±0.005.

[0443] For each element, the standard uncertainty is converted from the reference uncertainty according to a uniform distribution.

[0444] Therefore, the uncertainty introduced by the molecular weight of AngⅡ is:

[0445]

[0446] Similarly, the uncertainty introduced by the molecular weight of Phe is:

[0447]

[0448] (7) Uncertainty in calculating the mass fraction of AngⅡ from phenylalanine

[0449] The uncertainty introduced when calculating the mass fraction of AngⅡ from phenylalanine was calculated according to the following formula:

[0450]

[0451] (8) Standard uncertainty of AngⅡ mass fraction isotope dilution mass spectrometry determination results

[0452] Similarly, the uncertainties introduced when calculating the mass fraction of AngⅡ by valine and isoleucine are:

[0453] u(c,Val)=1.13%, u(c,Ile)=1.10%,

[0454] Since the isotope dilution mass spectrometry results of the AngⅡ purity standard material candidate are obtained by taking the arithmetic mean of the results of the three amino acids, the standard uncertainty of the isotope dilution mass spectrometry results of the AngⅡ mass fraction is:

[0455]

[0456] (9) Uncertainties introduced by the two methods in the process of setting the value

[0457] The uncertainty introduced by the difference between the mass balance method and the isotope dilution mass spectrometry is introduced as half of the difference in the fixed value results.

[0458] The uncertainties obtained by the two setting methods are synthesized according to formula (7-3) to obtain the uncertainty of the setting process.

[0459]

[0460] In formula (7-3), u char The uncertainty introduced by the setting process; u(P MB ) is the uncertainty introduced by the mass balance method; u(P IDMS ) is the uncertainty introduced by isotope dilution mass spectrometry; u(P MB-IDMS ) is the uncertainty introduced by the difference between the two determination methods.

[0461] Therefore, the uncertainty calculated by the two fixed value methods according to formula (7-3) is:

[0462]

[0463] 3. Uncertainty introduced by uniformity

[0464] The standard deviation caused by the inhomogeneity is s bb =1.07×10 -4 , therefore, the relative standard uncertainty introduced by sample homogeneity is:

[0465]

[0466] 4. Uncertainty introduced by stability

[0467] According to the stability test data analysis results in Table 21 and Table 25, the regression variance analysis table for evaluating stability is used to estimate the standard uncertainty.

[0468] The uncertainty contribution to long-term stability with a validity period of t = 12 months is:

[0469] u lts =s(β1)×t=1.44×10 -5 ×12=1.73×10 -4 g / g

[0470] The uncertainty contribution of short-term stability at 4°C is:

[0471] u sts =s(β1)·t=1.89×10 -5 ×7=1.32×10 -4 g / g

[0472] Therefore, the uncertainty introduced by stability is:

[0473]

[0474] The relative standard uncertainty is:

[0475]

[0476] 5. Total uncertainty of AngⅡ purity standard material candidates

[0477] The total relative standard uncertainty of the candidate AngⅡ purity standard substance was synthesized according to the following formula:

[0478]

[0479] Taking the coverage factor k equal to 2, at the 95% confidence level, the relative expanded uncertainty of the candidate AngⅡ purity standard substance is obtained as:

[0480] U rel = k × u r =1.8%, k=2.

[0481] In summary, in this example, through the preparation of AngⅡ purity standard substance candidate, the establishment and optimization of mass balance method and isotope mass spectrometry dilution method, qualitative inspection, initial uniformity inspection, stability investigation, fixed value and uncertainty assessment, AngⅡ purity standard substance was developed, and its quantitative summary results are shown in Table 41.

[0482] Table 41 Summary of mass values ​​of AngⅡ purity standard substances

[0483]

[0484] 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. Human angiotensin II purity standard substance, characterized in that: The human angiotensin II purity standard substance has a mass concentration of 0.8734 g / g, a relative expanded uncertainty of 1.8%, and an expanded uncertainty of 0.016 g / g, and is prepared by the following method: Human angiotensin II with a nominal purity ≥ 99% is selected, and a purity standard substance candidate is obtained after sequence analysis, mass spectrometry qualitative identification and initial uniformity inspection; the purity standard substance candidate is subjected to a uniformity inspection and stability investigation by the mass balance method, and then the mass balance method and isotope dilution mass spectrometry are used for value determination, and the purity standard substance candidate is subjected to uncertainty assessment.

2. The method for preparing the purity standard substance of human angiotensin II according to claim 1, characterized in that: The following steps are involved: (1) Selecting human angiotensin II with a purity of ≥99%, and obtaining a candidate human angiotensin II purity standard material after sequence analysis, mass spectrometry qualitative identification and initial homogeneity test; (2) The homogeneity and stability of the candidate human angiotensin II purity standard material were tested by mass balance method; (3) The candidate purity standard substance of human angiotensin II is quantified by mass balance method and isotope dilution mass spectrometry respectively, and the average of the quantification results of mass balance method and isotope dilution mass spectrometry method is calculated to obtain the quantification result of the candidate purity standard substance of human angiotensin II; (4) The uncertainty of the candidate human angiotensin II purity standard substance was evaluated to obtain the human angiotensin II purity standard substance.

3. The preparation method according to claim 2, characterized in that: The homogeneity test, stability test and value determination of the candidate human angiotensin II purity standard substance are carried out by mass balance method, and the test result is 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 the candidate for purity standard material of human angiotensin II, 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, Among them, the moisture content is measured by a Karl Fischer moisture meter, the ash content is measured by an ignition residue test method, the anion content is measured by ion chromatography, the inorganic element content is measured by inductively coupled plasma mass spectrometry, and the volatile organic component content is measured by gas chromatography.

4. The preparation method according to claim 3, wherein the homogeneity initial inspection and the main component content of the human angiotensin II purity standard substance candidate are determined by high performance liquid chromatography, and the chromatographic detection conditions for the homogeneity initial inspection are as follows: the chromatographic column is a C18 reverse phase chromatographic column, the mobile phase A is a pH=2.5, 10mmol / L sodium dihydrogen phosphate buffer, the mobile phase B is 50% acetonitrile, the flow rate is 0.5mL / min, the column temperature is 25°C, and the detection wavelength is 230nm; The chromatographic detection conditions of the main component content of the human angiotensin II purity standard substance candidate 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; In terms of volume fraction, the mobile phase gradient conditions are as follows: (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%.

5. The preparation method according to claim 2, characterized in that: The stability study includes long-term stability study, short-term stability study and bottle opening stability study.

6. The preparation method according to claim 2, characterized in that: When determining the purity standard material candidate of human angiotensin II by the isotope dilution mass spectrometry, the method comprises the following steps: The candidate purity standard substance of human angiotensin II was hydrolyzed, and isotope-labeled phenylalanine, isoleucine and valine were used as internal standards. The purity standard substance candidate of human angiotensin II was determined by isotope dilution mass spectrometry to obtain the result of isotope dilution mass spectrometry on the candidate purity standard substance of human angiotensin II.

7. The preparation method according to claim 6, characterized in that: The process of hydrolyzing the candidate human angiotensin II purity standard substance comprises the following steps: The candidate human angiotensin II purity standard substance is dissolved 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 isotope-labeled amino acid mixed standard solutions with the contents 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.

8. The preparation method according to claim 6, characterized in that: In the 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, and 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.

9. The preparation method according to claim 2, characterized in that: When performing the uncertainty assessment, the uncertainty includes the uncertainty introduced by the fixed value, the uncertainty introduced by the heterogeneity of the standard material, and the uncertainty introduced by the instability of the standard material.

10. Use of the human angiotensin II purity standard substance according to claim 1 in tracing the value of human angiotensin II, quality control of human angiotensin II measurement results, and evaluation of human angiotensin II standards.

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