6-[4-(diphenylamino) phenyl] coumarin-3-carboxylic acid ethyl ester purity standard substance as well as preparation method and application thereof

Abstract

The invention discloses a 6-[4-(diphenylamino) phenyl] coumarin-3-carboxylic acid ethyl ester purity standard substance, the purity of the purity standard substance is 99.6%, the extension uncertainty is 0.4%, and the relative extension uncertainty is 0.4%, and the purity standard substance is prepared by the following steps: selecting 6-[4-(diphenylamino) phenyl] coumarin-3-carboxylic acid ethyl ester with the nominal purity of greater than or equal to 98.0%, and adding a proper amount of water to the selected 6-[4-(diphenylamino) phenyl] coumarin-3-carboxylic acid ethyl ester; carrying out 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, determining a value by adopting a differential scanning calorimetry and a mass balance method, and performing uncertainty evaluation on the purity standard substance candidate to obtain the purity standard substance. The purity standard substance disclosed by the invention is accurate and reliable in valuing result and has traceability.

Description

Technical Field

[0001] The invention relates to the technical field of metrology, and in particular to a purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester and a preparation method and application thereof. Background Art

[0002] Ethyl 6-[4-(Diphenylamino)phenyl]coumarin-3-carboxylate (DPA), CAS No.: 1056693-13-0, relative molecular mass 465.1, molecular formula C 30 H 23 NO4, the structural formula is as follows:

[0003]

[0004] DPA is a D-π-A type coumarin compound, which is widely used in the detection of various ion-active substances and the labeling of biomacromolecules due to its excellent photophysical and chemical properties. There is no DPA purity standard material in the prior art. Summary of the invention

[0005] The first object of the present invention is to provide a purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester.

[0006] The second object of the present invention is to provide a method for preparing a purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester.

[0007] The third object of the present invention is to provide the application of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester purity standard material.

[0008] According to a first aspect of the present invention, a purity standard material of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester is provided, wherein the purity of the purity standard material is 99.6%, the expanded uncertainty is 0.4%, the relative expanded uncertainty is 0.4%, and the purity is 99.6%, and the purity of the purity standard material is 99.6%, the expanded uncertainty is 0.4%, and the relative expanded uncertainty is 0.4%, and the purity standard material is prepared by the following method:

[0009] 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester with a nominal purity of ≥98.0% is selected, and a purity standard substance candidate is obtained after qualitative identification and initial uniformity inspection; after the candidate purity standard substance is subjected to uniformity inspection and stability investigation, differential scanning calorimetry and mass balance method are used for value determination, and uncertainty assessment is performed on the candidate purity standard substance to obtain.

[0010] The 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester purity standard substance (DPA) of the present invention has been subjected to uniformity and stability inspections, value determination and uncertainty evaluations, and the purity, relative expanded uncertainty and expanded uncertainty of the DPA purity standard substance are measured. The measured values ​​are accurate and reliable, and have traceability, and can be used for content detection of DPA in related devices or laser materials and confirmation of detection methods.

[0011] According to a second aspect of the present invention, there is provided a method for preparing a purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester, comprising the following steps:

[0012] (1) Selecting 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester with a nominal purity of ≥98.0%, and obtaining a candidate purity standard material of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester through qualitative identification and preliminary homogeneity inspection;

[0013] (2) Conducting homogeneity testing and stability studies on the candidate purity standard material for ethyl 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylate;

[0014] (3) The candidate purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester is valuated by differential scanning calorimetry and mass balance method, and the average of the valuated results by differential scanning calorimetry and mass balance method is calculated to obtain the valuated result of the candidate purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester;

[0015] (4) An uncertainty evaluation is performed on the candidate purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester. The candidate purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester that meets the requirements of uniformity test, stability investigation and uncertainty is the purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester.

[0016] The method for preparing a purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester of the present invention uses commercially available high-purity DPA as a raw material, obtains a DPA purity standard substance candidate after qualitative identification and initial uniformity inspection, then performs uniformity inspection and stability inspection on the purity standard substance, and then respectively determines the purity standard substance candidate by using differential scanning calorimetry and mass balance method, so that the determination result of the DPA purity standard substance is more accurate and reliable, and finally obtains the DPA purity standard substance through uncertainty assessment, thereby providing a technical reference for the value of the DPA purity standard substance and having traceability.

[0017] In some embodiments, qualitative identification includes at least one of mass spectrometry, nuclear magnetic resonance hydrogen spectrum analysis, and elemental analysis.

[0018] In some embodiments, the homogeneity test, stability test and main component content determination of the candidate of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester purity standard material are carried out by high performance liquid chromatography, and the chromatographic detection conditions are as follows: the chromatographic column is a C18 chromatographic column, 5μm, 4.6mm×150mm; the wavelength is 316nm, the mobile phase is methanol, the flow rate of the mobile phase is 0.3mL / min, the column temperature is 35°C, and the injection concentration is 0.1mg / L. Preferably, the chromatographic column is an Agilent ZORBAX EclipsePlus C18 chromatographic column.

[0019] In some embodiments, the stability study includes a long-term stability study and a short-term stability study.

[0020] In some embodiments, when the purity standard substance candidate of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester is determined by differential scanning calorimetry, the detection conditions adopted are: the sample weight is 1 to 3 mg, the protective gas flow rate is 40 mL / min, the temperature is slowly increased at 170-220°C, and the slow heating rate is 1K / min.

[0021] In some embodiments, the purity standard substance candidate of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester is determined by mass balance method, and the test result can be calculated by the following formula:

[0022] P=P o ×(1-ABC),

[0023] Wherein, P is the purity of the candidate for 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester purity standard substance, P o is the main component content of the candidate purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester, A is the water content, B is the inorganic element content, and C is the residual solvent content.

[0024] In some embodiments, the moisture content is determined using a thermogravimetric analyzer, comprising the following steps: weighing 3 to 5 mg of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester purity standard material candidate and placing it in a thermogravimetric analyzer, heating it to 80 to 95° C. at a heating rate of 5 K / min, then heating it to 100 to 110° C. at a heating rate of 1 K / min, and then maintaining it at 100 to 110° C. for 50 to 70 min.

[0025] In some embodiments, the inorganic element content is determined by inductively coupled plasma mass spectrometry, comprising the following steps:

[0026] The candidate purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester was digested, and then the digestion solution was cooled to below 70°C, dissolved in pure water and transferred to a 100mL volumetric flask to make up the volume to obtain a test solution, and 2% HNO3 was used as a blank. An inductively coupled plasma mass spectrometer was used in a semi-quantitative mode with a single calibration point to perform determination at a calibration point of 10 μg / L.

[0027] In some embodiments, the detection conditions of inductively coupled plasma mass spectrometry are: plasma gas flow rate is 15.00 L / min, purge times are 45 times, auxiliary gas flow rate is 0.80 L / min, carrier gas flow rate is 0.80 L / min, and nebulization chamber temperature is 2°C.

[0028] In some embodiments, the digestion process includes the following steps: weighing 100 mg of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester purity standard material candidate into a digestion tank, adding 6 mL of concentrated nitric acid to the digestion tank, gently shaking and mixing, and placing in a microwave digestion instrument for digestion.

[0029] In some embodiments, the residual solvent content is determined by headspace injection-gas chromatography, and the detection conditions are: the chromatographic column is a DB-624 gas chromatograph column, 60m×0.25mm, 1.40μm; the carrier gas is helium, and the carrier gas flow rate is 10.0mL / min; the injection volume is 1000μL; the programmed temperature conditions are maintained at 45°C for 5min, heated to 120°C at a rate of 7°C / min, heated to 230°C at a rate of 15°C / min, and maintained for 15min.

[0030] In some embodiments, when performing uncertainty assessment, the uncertainty includes the uncertainty introduced when determining the value, the uncertainty caused by uniformity, and the uncertainty caused by instability.

[0031] In some embodiments, the uncertainty introduced when determining the value comes from the uncertainty introduced by differential scanning calorimetry purity measurement, the uncertainty component introduced by liquid chromatography-area normalization method purity measurement, the uncertainty component introduced by moisture test, the uncertainty component introduced by inorganic element test, and the uncertainty component introduced by residual solvent test.

[0032] The third aspect of the present invention provides the application of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester purity standard material, which can be used as a measuring tool for detecting DPA content in related devices (such as electronic components) or laser materials and confirming the detection method, in order to ensure that the values ​​are accurate and comparable and to unify the relevant values. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of DPA raw material;

[0034] Figure 2 The corresponding assignment of the H NMR spectrum of the DPA raw material;

[0035] Figure 3 is the mass spectrum of DPA raw material;

[0036] Figure 4 It is the ultraviolet wavelength scanning diagram of the candidate DPA purity standard material;

[0037] Figure 5 It is an enlarged view of the chromatographic extraction of the candidate DPA purity standard substance at 310 nm;

[0038] Figure 6 This is a chromatogram of a candidate DPA purity standard substance in the PAD detection method using methanol as the mobile phase;

[0039] Figure 7 This is a comparison chart of the methanol solution of the DPA purity standard substance candidate before and after adding deionized water. Figure 7 In the middle, the left tube is after adding deionized water, and the right tube is before adding deionized water;

[0040] Figure 8 The chromatogram of the DPA purity standard substance candidate in the mobile phase of methanol:n-hexane=95:5;

[0041] Fig. 9 It is an enlarged chromatogram of the DPA purity standard substance candidate in the mobile phase of methanol:n-hexane=95:5;

[0042] Fig.10 The results of the DPA purity standard material candidate test using the Agilent ZORBAX Eclipse Plus C18 chromatographic column;

[0043] Fig.11 This is an enlarged view of the test results of the DPA purity standard material candidate using the Agilent ZORBAX Eclipse Plus C18 chromatographic column;

[0044] Fig.12 The test results of the candidate DPA purity standard material with the chromatographic column of ODS-HC18 from Zhongpu Technology;

[0045] Fig.13 This is an enlarged view of the test results of the DPA purity standard substance candidate using the chromatographic column of Zhongpu Technology ODS-HC18;

[0046] Fig.14 The liquid chromatograms of DPA purity standard substance candidates at different flow rates;

[0047] Fig.15 The effect of different flow rates on the main component peak area of ​​DPA purity standard material candidates;

[0048] Fig.16 The liquid chromatograms of DPA purity standard substance candidates at different column temperatures;

[0049] Fig.17 The effect of different column temperatures on the main component peak area of ​​DPA purity standard material candidates;

[0050] Fig.18 The liquid chromatograms of DPA purity standard substance candidates at different injection concentrations;

[0051] Fig.19 is the methanol reagent chromatogram;

[0052] Fig. 20 This is the liquid chromatogram of the optimized DPA purity standard substance candidate;

[0053] Fig.21 The differential scanning calorimetry diagram of the candidate DPA purity standard material;

[0054] Fig. 22 The purity analysis results of the DPA purity standard material candidate;

[0055] Fig.23 This is the long-term stability trend chart of the DPA purity standard material candidate;

[0056] Fig.24 This is the short-term stability trend chart of the candidate DPA purity standard material (60±5℃);

[0057] Fig.25 This is the short-term stability trend chart of the candidate DPA purity standard material (4±1℃);

[0058] Fig.26 It is the gas chromatogram of the blank solvent dimethyl sulfoxide under headspace conditions;

[0059] Fig. 27 Gas chromatograms of standard solutions of methanol, ethanol, acetonitrile, dichloromethane, tetrahydrofuran and toluene under headspace conditions;

[0060] Fig.28 It is the gas chromatogram of the DPA purity standard substance candidate in dimethyl sulfoxide solution under headspace conditions;

[0061] Fig.29 QQ plot of differential scanning calorimetry data;

[0062] Fig.30 QQ plot for the mass balance method value data;

[0063] Fig.31 This is the QQ plot of the merged fixed value data. DETAILED DESCRIPTION

[0064] 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.

[0065] Example 1

[0066] The preparation method of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester purity standard material comprises the following steps:

[0067] (1) Selecting 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester with a nominal purity of ≥98.0%, performing qualitative identification through mass spectrometry analysis, nuclear magnetic resonance hydrogen spectrum analysis, and elemental analysis, and then performing a preliminary homogeneity inspection through high performance liquid chromatography to obtain a 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester purity standard substance candidate;

[0068] (2) Conducting homogeneity testing and stability studies on the candidate purity standard material for ethyl 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylate;

[0069] (3) The candidate purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester is valuated by differential scanning calorimetry and mass balance method, and the average of the valuated results by differential scanning calorimetry and mass balance method is calculated to obtain the valuated result of the candidate purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester;

[0070] (4) An uncertainty evaluation is performed on the candidate purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester. The candidate purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester that meets the requirements of uniformity test, stability investigation and uncertainty is the purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester.

[0071] The differential scanning calorimetry (DSC) principle used in the examples is described as follows:

[0072] Differential Scanning Calorimetry (DSC) measures the purity of a substance based on the principle that the melting point (freezing point) of a substance decreases due to the presence of impurities. It is an accurate, fast and convenient method for substances with higher purity. The more impurities there are in a substance, the greater the drop in the melting point (freezing point). When the molar fraction of impurities in a substance is less than 2%, the relationship between the drop in the substance's freezing point and the impurity content can be expressed by the Van't Hoff equation, as shown in the following formula:

[0073]

[0074] Where T0 is the melting point (freezing point) of an ideal pure substance, K; T m is the melting point (freezing point) of the substance, K; R is the gas constant, whose value is 8.314 J / (K·mol); X is the molar fraction of impurities in the substance being measured; △H is the molar melting enthalpy of the substance, J / mol.

[0075] T0-T m It is the amount of decrease in the melting point (freezing point) of a substance due to the presence of impurities. As can be seen from the above formula, the decrease in the melting point (freezing point) of a substance is linearly related to the molar fraction of the impurity. According to the above formula, at a selected heating rate, the melting DSC curve of the DPA purity standard substance candidate is measured, and after analysis, the molar melting enthalpy of the DPA purity standard substance candidate is obtained. The purity analysis software is then used to substitute the molar melting enthalpy change into the formula for fitting calculation, and after correction, the purity value of the DPA purity standard substance candidate is obtained.

[0076] The mass balance method (MB) principle used in the examples is described as follows:

[0077] The Mass Balance Method includes the determination of the main component, moisture, ash, volatile components and inorganic elements of the target substance (usually a sample with a high purity) to comprehensively examine the sample composition. The Mass Balance Method assumes that the target sample (pure product) consists of only the main component, moisture, ash, volatile components and inorganic elements, so the sum of their percentages is 100%. The purity value of the sample can be defined as the value of the percentage of moisture, ash, inorganic elements and volatile components removed from the result of the main component determination, supplemented by the uncertainty after the determination of various components, and comprehensively evaluated as the uncertainty of the Mass Balance Method.

[0078] The mass balance method is used to determine the purity standard substance candidate of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester, and the test result can be calculated by the following formula:

[0079] P=P o ×(1-ABC),

[0080] Wherein, P is the purity of the candidate for 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester purity standard substance, P o is the main component content of the candidate purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester, A is the water content, B is the inorganic element content, and C is the residual solvent content.

[0081] The main component determination method of the mass balance method is generally gas chromatography or high performance liquid chromatography, the moisture determination is generally carried out by Karl Fischer titration or thermogravimetric analysis, the ash content is determined by high temperature calcination, the volatile component is generally determined by thermogravimetry or gas chromatography, and the inorganic element determination is generally carried out by inductively coupled plasma mass spectrometry.

[0082] The preparation method of the purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester (DPA) in this embodiment is described in detail as follows:

[0083] 1. Materials and equipment

[0084] 1.1 Experimental container

[0085] 1L brown reagent bottle, 25mL Class A volumetric flask; the packaging container is a 2mL brown sample bottle, which is washed and dried for use.

[0086] 1.2 Raw material selection

[0087] The raw material of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester is a commercially available high-purity DPA product purchased from Tokyo Chemical Industry (TCI), with a specification of 200 mg and a marked purity of ≥98.0%. A total of 15 bottles were purchased, all from the same batch. All 15 bottles of purchased DPA raw materials were transferred to a 1L clean brown reagent bottle, shaken and mixed, and sealed and stored in a 4°C refrigerator away from light.

[0088] 1.3 Experimental reagents

[0089] Methanol (Merck, chromatography grade), hydrochloric acid, concentrated HNO3; the experimental water was purified three times by reverse osmosis, ion exchange, and quartz distillation.

[0090] 1.4 Experimental instruments

[0091] High performance liquid chromatograph (Agilent, USA, 1290 series, UV detector), differential scanning calorimeter (Netzsch 214), synchronous thermal analyzer (TA, SDT650), microwave digestion instrument (Guangdong Bojing Technology Co., Ltd., EXCEL), quadrupole inductively coupled plasma mass spectrometer (Agilent, 7900 series).

[0092] The weighing instrument was an electronic balance (CPA225D, d=0.01 mg) (Sartorius, Germany).

[0093] 2. Preparation and packaging of DPA purity standard material candidates

[0094] 2.1 Qualitative analysis of DPA raw materials

[0095] The premixed DPA raw material was subjected to qualitative experimental analysis such as mass spectrometry, nuclear magnetic resonance hydrogen spectrum, elemental analysis, etc. (all were sample tests).

[0096] 2.1.1 Experimental instruments

[0097] The mass spectrometer was a Bruker Solarix XR Fourier transform high-resolution mass spectrometer (a high-precision mass spectrometer equipped with an ESI ion source using an actively shielded 9.4 Tesla magnet), which can achieve a measurement error of less than 1ppm, a resolution of up to 1,000,000, and a single-charge mass range of 20-10,000Da. The low-resolution ESI positive ion mode was used for analysis, and the solvent condition was methanol to achieve accurate mass determination for determining the molecular formula.

[0098] The elemental analysis sample delivery instrument is ElementarAnalysensysteme GmbH company vario EL elemental analyzer, equipped with a precise one-millionth balance. The analysis accuracy: CHN reaches 0.1%, S reaches 0.3%. The samples are stored dry and away from light during delivery, and the percentage of CHNS in organic compounds is quantitatively determined.

[0099] The sample delivery instrument for H NMR spectrum was a 400MHz Bruker NMR instrument equipped with a broadband probe, a reverse probe, a variable temperature unit and a 5mmPABBO BB-1H / D Z-GRD. Resolution: H is less than or equal to 0.2Hz; sbr sensitivity: H: 390:1 (5mm0.1%EB); sbr line type: H: <10Hz at 0.55%, <5Hz at 0.11%. The solvent conditions for sample delivery were deuterated chloroform, dissolved in an NMR analysis tube, and the internal standard was TMS.

[0100] 2.1.2 H NMR spectroscopy analysis

[0101] According to the literature "Photoinduced electron transfer in photorobust coumarins linked with electron donors affording long lifetimes of triplet charge-separated states. [J]. Chemphyschem, 2010, 11 (12): 2594-2605", the qualitative results of the hydrogen nuclear magnetic resonance spectrum of DPA are as follows: 1 H-NMR (300MHz, CDCl3): δ = 8.58 (s, 1H), 7.83 (dd, 1H, J = 2.1, 8.4Hz), 7.75 (d, 1H, J = 2.1Hz), 7.43 (t, 2H, J = 8.7Hz), 7.29 (t, 5H, J =8.4Hz),7.15(dd,4H,J=2.7,8.1Hz),7.07(t,3H,J=7.2Hz),4.32(q,2H,J=6.9Hz),1.42(t,3H,J=7.2Hz),HRMS(FAB+):calcd for M+,461.1627; found,461.1629.

[0102] Using deuterated chloroform as solvent, DPA raw material and deuterated chloroform solvent were placed in a nuclear magnetic resonance analysis tube, and tetramethylsilane (TMS) was used as the internal standard for mass spectrometry analysis. The results are as follows: Figure 1The results show that the nuclear magnetic peak with a chemical shift of about 1.5 in the DPA raw material corresponds to the H on the -CH3 group, the nuclear magnetic peak with a chemical shift of about 4.5 corresponds to the H on the -CH2- group, and the nuclear magnetic peak with a chemical shift of 7.0 to 8.5 corresponds to the H on the aromatic ring skeleton. The number ratio of H atoms is consistent with that in the chemical structure. Therefore, the corresponding attribution of the nuclear magnetic resonance hydrogen spectrum of the DPA raw material corresponds to the above-mentioned literature report. The results are shown in Figure 2 shown.

[0103] 2.1.3 Electrospray ionization mass spectrometry (ESI-MS) analysis

[0104] Using methanol as solvent, the DPA raw material was analyzed by ESI-MS in low-resolution ESI positive ion mode. The results are as follows: Figure 3 As shown. Figure 3 The molecular ion peak at m / z = 461.16 and the isotope ion peak at m / z = 463.17 (6.0%) can be clearly observed. The relevant spectra are consistent with the data in the mass spectrum library in NIST Chemistry WebBook and also correspond to the literature reports.

[0105] 2.1.4 Element (EA) analysis

[0106] The DPA raw material stored in the dark was subjected to elemental analysis to quantitatively determine the percentage of C, H, N, and S in the DPA raw material. The results showed that among the relevant element contents, C was 78.0%, H was 5.02%, and N was 3.03%, which was consistent with the molecular formula of DPA C. 30 H 23 The NO4 theoretical calculation value is consistent.

[0107] Through mass spectrometry, nuclear magnetic resonance hydrogen spectrum and elemental analysis, it was verified that the purchased DPA raw material was DPA and could be directly used in subsequent experiments.

[0108] 2.2 Initial inspection of uniformity

[0109] The DPA raw material was preliminarily homogeneously tested by HPLC-PAD detection. Samples were randomly taken from the upper, middle and lower parts of the premixed DPA raw material bottle, dissolved with methanol to make a 0.1 mg / mL solution, and 1 mL was sampled from each bottle. The sample was injected after filtering through a 0.22 μm microporous membrane. The mobile phase was pure methanol, the flow rate was 0.3 mL / min, the column temperature was 35 °C, the detection wavelength was 316 nm, and the running time was 18 min. The results of the preliminary uniformity test are shown in Table 1.

[0110] Table 1 Preliminary inspection data of the uniformity of the raw materials of DPA purity standard substances

[0111]

[0112] It can be seen from Table 1 that the standard deviation of the initial inspection of DPA raw material uniformity is 0.022%, F = 1.42, F α =5.14, F <F α , indicating that the DPA raw materials in different positions of the same batch after premixing have no obvious inhomogeneity and can be packaged.

[0113] 2.3DPA purity standard material candidates and packaging

[0114] The DPA raw material that passed the qualitative test and the initial uniformity test was placed in a desiccator filled with phosphorus pentoxide and dried for 24 hours, and then packaged into 2 mL clean brown glass bottles in a clean and dry packaging environment, and marked as DPA purity standard substance candidate. The packaging unit of each bottle is 10 mg. After tightening the bottle cap, the bottle mouth is sealed with a sealing film. A total of 200 bottles are packaged and stored at a low temperature of 4°C, away from light. A total of 1 batch was prepared this time to obtain the DPA purity standard substance candidate.

[0115] 3. Establishment and optimization of high performance liquid chromatography and differential scanning calorimetry

[0116] 3.1 Establishment and optimization of HPLC method

[0117] First, the conditions of the HPLC-area normalization method were optimized. In order to clearly determine the separation degree of the main components and impurities of the DPA purity standard substance candidate, the optimization and final determination of the chromatographic column, mobile phase, concentration and wavelength were based on the final packaged DPA purity standard substance candidate.

[0118] 3.1.1 Initial experimental conditions

[0119] The detector is a high performance liquid chromatograph (PAD detector); the chromatographic column is a C18 column; the column temperature is 30°C; the mobile phase is methanol; the wavelength is 254nm; the injection concentration is 0.1mg / mL; and the flow rate is 0.2mL / min.

[0120] 3.1.2 Investigation of wavelength conditions

[0121] According to literature reports, DPA is separated and purified by column chromatography using methanol or a mixed solvent of methanol and cyclohexane. Therefore, the DPA purity standard substance candidate is first subjected to UV scanning in methanol solvent. The results are as follows: Figure 4 As shown. Figure 4 It can be seen that the DPA purity standard substance candidate has absorption peaks at 260-360nm and 220-245nm, and the maximum absorption wavelength is 332nm. At the same time, the DPA purity standard substance candidate also has ultraviolet absorption peaks at 316nm and 232nm, but because 232nm is close to the maximum ultraviolet absorption of methanol, 232nm is not considered as the wavelength for HPLC condition optimization.

[0122] 296nm, 306nm, 316nm, 332nm, 340nm and 254nm, a wavelength commonly used for organic matter, were selected as the wavelengths for DPA purity standard substance candidate selection. The experimental results are shown in Table 2. The experiment found that at 254nm, the response value of the test results of the DPA purity standard substance candidate was low and was not suitable for the analysis of the compound; at other wavelengths, the DPA purity standard substance candidate could be well detected with sharp peaks. Therefore, from the perspective of impurity detection, a total of 5 impurities with large peak areas were found at 5 different wavelengths, of which only at 316nm ( Figure 5 ) and all the main impurity peaks responded with high response values, so 316nm was selected as the optimized wavelength condition.

[0123] Table 2 Chromatographic peak area percentage of each impurity component in DPA purity standard substance candidate at different wavelengths

[0124]

[0125] 3.1.3 Investigation of mobile phase

[0126] First, the solubility of DPA in different solvents such as water, methanol, acetonitrile, n-propanol, isopropanol, ethanol, isooctane, o-xylene and tetrahydrofuran was investigated. It was found that DPA was almost insoluble in water, n-hexane and isooctane, while it had the highest solubility in tetrahydrofuran, o-xylene and ethyl acetate. However, considering the corrosion of tetrahydrofuran and o-xylene to the sealing ring in the liquid phase system, and the possible damage of ethyl acetate to the chromatographic column, tetrahydrofuran, o-xylene and ethyl acetate were not selected as mobile phases for the protection of instruments and chromatographic columns.

[0127] Among the remaining solvents, DPA has the highest solubility in methanol. Therefore, methanol was first used as the solvent and mobile phase, and isocratic elution was used to examine the chromatographic behavior of the DPA purity standard substance candidate. Figure 6 The results show that when pure methanol is used as the mobile phase, the chromatographic peak corresponding to the detection result of the DPA purity standard substance candidate is sharp, with a retention time of 7 to 8 minutes, a high impurity response before and after the main peak, and an obvious separation effect.

[0128] The effect of the mobile phase ratio on the detection results of the DPA purity standard substance candidate was investigated. When deionized water with a mass fraction of about 10% of the total mass was added to the methanol solution of the 0.1 mg / mL DPA purity standard substance candidate, the results were as follows: Figure 7As shown in the figure, solids can be observed to precipitate from the solution after adding deionized water, so water-containing mixed solvents cannot be selected as mobile phases. Methanol:n-hexane = 95:5 (V:V) was selected as the mobile phase to investigate its chromatographic behavior. The results are shown in the figure. Figure 8 and Fig. 9 The peak time and peak area percentage when methanol-n-hexane is the mobile phase are shown in Table 3. Figure 6 and Figure 8 The difference in the chromatographic test results indicates the influence of the composition of the mobile phase on the retention time of the DPA purity standard substance candidate. Figure 6 ,from Figure 8 No more impurity peaks were observed, and the normalized peak area percentages of the DPA purity standard substance candidate in pure methanol and methanol:n-hexane = 95:5 (V:V) were 99.604% and 99.694%, respectively, indicating that the peak area percentages of the main impurities of the DPA purity standard substance candidate in the methanol-n-hexane system were less than the peak area percentages of the main impurities in pure methanol. Finally, pure methanol was selected as the mobile phase for subsequent analysis.

[0129] Table 3 Peak elution time and peak area percentage when methanol-n-hexane mixed solvent is used as mobile phase

[0130] Peak time Peak area percentage (%) 6.8min 0.047 7.3min 0.246 8.4min 99.694 14.0min 0.013

[0131] 3.1.4 Selection of chromatographic columns

[0132] After calculation, the polarity index logP value of DPA is greater than 0, and the polarity is relatively small. Experiments have found that the DPA purity standard substance candidate is insoluble in normal phase solvents such as n-hexane. The use of normal phase columns for chromatographic analysis may result in the failure to detect the DPA purity standard substance candidate or precipitation during the detection process. Therefore, only the DPA purity standard substance candidate in different reverse phase chromatographic columns was examined.

[0133] The liquid phase experimental conditions for column optimization are: pure methanol as mobile phase; wavelength of 316 nm; injection concentration of 0.1 mg / mL; flow rate of 0.3 mL / min; column temperature of 35 °C; injection volume of 2 μL. The chromatographic columns selected were ODS-H column (250 mm × 4.6 mm, 5 μm) from Zhongpu Technology and ZORBAX Eclipse Plus C18 column (150 mm × 4.6 mm, 5 μm) from Agilent. The chromatographic peaks of the two columns for the DPA purity standard substance candidate and its impurities are shown in the following figure. Figures 10 to 13 The peak time and peak area percentage in the detection results are shown in Table 4.

[0134] The results showed that due to the long ODS-H column of Zhongpu Technology, the main peak and impurity elution time of the DPA purity standard substance candidate during the liquid phase detection was also long, but the peak shape and separation of impurity peaks and main peaks were similar when the DPA purity standard substance candidate was analyzed by liquid chromatography in the two columns. Among them, when the ODS-H column of Zhongpu Technology was used, the impurity detection content was small, and the main impurity peak that appeared at 15.4min could not be completely separated from the main peak; when the Agilent ZORBAX Eclipse Plus C18 column was used, the impurity detection content was large, and almost all impurities could be completely separated, so the Agilent ZORBAX Eclipse Plus C18 column was selected as the column condition for subsequent experiments.

[0135] Table 4 Peak elution time and peak area percentage of chromatographic analysis under different chromatographic columns

[0136]

[0137] 3.1.5 Investigation of flow rate

[0138] The effects of flow rates of 0.2 mL / min, 0.25 mL / min, 0.3 mL / min, and 0.35 mL / min on the detection of DPA purity standard substance candidates in liquid chromatography analysis were investigated. The results are as follows Fig.14 and Fig.15 The results show that there is no significant difference in the detection of the main components and impurities of the DPA purity standard material candidate at different flow rates. Taking into account the peak time and the pressure tolerance of the chromatographic column, 0.3mL / min was selected as the flow rate of the mobile phase.

[0139] 3.1.6 Investigation of column temperature

[0140] The effects of column temperatures of 25°C, 30°C, 35°C, and 40°C on the detection of DPA purity standard substance candidates were investigated. The results are as follows Fig.16 and Fig.17 The results showed that there was no significant difference in the peak time and component detection of the DPA purity standard substance candidates at different column temperatures, so the middle value of 35°C was selected as the temperature of the column oven.

[0141] 3.1.7 Investigation of concentration

[0142] The dissolution of methanol solutions of DPA purity standard substance candidates with different concentrations was investigated. It was found that the preparation of 0.1 mg / mL methanol solution of DPA purity standard substance candidate required ultrasonic dissolution. When preparing 0.3 mg / mL methanol solution of DPA purity standard substance candidate, ultrasound could not completely dissolve the DPA purity standard substance candidate in methanol. The DPA purity standard substance candidate could be dissolved in methanol solution by heating, but the mixture was left at room temperature to cool, resulting in precipitation of the methanol solution of the DPA purity standard substance candidate. Therefore, in terms of the impurity response, the chromatographic peaks of methanol solutions of three different concentrations of DPA purity standard substance candidate, 0.1 mg / mL, 0.08 mg / mL, and 0.06 mg / mL, were investigated. Fig.18 As shown. Fig.18 It can be seen that as the concentration of the DPA purity standard substance candidate increases, the detection of impurities is obvious. When the concentrations of the DPA purity standard substance candidate are 0.1 mg / mL, 0.08 mg / mL and 0.06 mg / mL, the HPLC normalized peak areas of the DPA purity standard substance candidate are 99.606%, 99.633% and 99.652%, respectively, and the chromatographic column is not overloaded, so the methanol solution of the DPA purity standard substance candidate with a concentration of 0.1 mg / mL is selected as the optimized experimental condition.

[0143] 3.1.8 Solvent blank test

[0144] The chromatographic experimental detection conditions are as follows: wavelength of 316 nm, methanol as the mobile phase, concentration of the DPA purity standard substance candidate of 0.1 mg / mL, flow rate of the mobile phase of 0.3 mL / min, column temperature of 35°C, and an Agilent ZORBAX Eclipse Plus C18 column (5 μm, 4.6 mm×150 mm) as the chromatographic column.

[0145] Under the above chromatographic experimental conditions, chromatographic grade methanol was used as the solvent to investigate the effect of the blank solvent. The results are as follows: Fig.19 The results show that no candidate DPA purity standard substance was detected in methanol solvent, so methanol solvent does not contain candidate DPA purity standard substance, and the solvent has no effect on the detection and determination of candidate DPA purity standard substance.

[0146] 3.1.9 Final experimental conditions

[0147] The final optimized conditions for liquid chromatography determination of DPA purity standard material candidates are shown in Table 5.

[0148] Table 5 Final experimental conditions of chromatography of DPA purity standard material candidates

[0149] Detector PAD wavelength 316nm Mobile phase Pure methanol Chromatographic columns C18 chromatographic column (5μm, 4.6mm×150mm) (or similar column) concentration 0.1mg / mL Column temperature 35℃ Flow rate 0.3mL / min

[0150] The liquid chromatography analysis results of the DPA purity standard substance candidate under the final experimental conditions are as follows Fig. 20 As shown. The separation degree can be used to determine the separation of the separated substances in the chromatographic column. It is represented by R, which is equal to the ratio of the difference in retention time between two adjacent peaks to the average peak width of the two chromatographic peaks. When R<1, the two peaks partially overlap. When R=1, the separation degree can reach 98%, and the two peaks are basically separated; when R=1.5, the separation degree can reach 99.7%, and the two peaks are completely separated.

[0151] Under the final experimental conditions, the separation degree of the main component of the DPA purity standard substance candidate is above 5, and the separation degree of all other impurities is above 1.5, indicating that the separation degree is good under the experimental conditions. Therefore, the experimental conditions meet the chromatographic measurement requirements of the DPA purity standard substance candidate.

[0152] 3.2 Establishment and optimization of differential scanning calorimetry

[0153] 3.2.1 Initial experimental conditions

[0154] If the sample weight exceeds 5 mg, the purity measurement result will decrease. Generally, a large sample weight affects the internal transfer speed and temperature gradient of the sample, resulting in reduced resolution. Under the premise of ensuring the measurement results, try to reduce the sample weight. After comprehensive consideration, the sample weight is 1 to 3 mg.

[0155] Take samples evenly from the upper, middle and lower parts of the DPA purity standard substance candidate bottle, accurately weigh 1-3 mg of the DPA purity standard substance candidate, and place it in a dry and clean crucible; set the protective gas flow rate to 30 mL / min; the slow heating range is from room temperature to 170°C; the slow heating rate is 5K / min. Cover and seal the bottle, and make sure that the protective gas is nitrogen with a flow rate of 50 mL / min.

[0156] 3.2.2 Investigation of heating rate

[0157] According to the requirements of GB / T 28723-2012 "Determination of Purity of Solid Organic Chemicals - Differential Scanning Calorimetry", 1-3 mg of DPA purity standard material candidate was weighed as a sample, and the sample was rapidly heated at a heating rate of 10 K / min to a temperature 25 °C lower than the estimated melting point of 195 °C, i.e., 170 °C, and then heated from 170 °C to 220 °C at heating rates of 0.3 K / min, 0.5 K / min, 1 K / min, 5 K / min, and 10 K / min, respectively, to obtain the purity values ​​of the DPA purity standard material candidate at different heating rates, as shown in Table 6. The results show that when the heating rate is 5 K / min and 10 K / min, the purity value data is significantly higher. When the heating rate is 0.3 K / min, the differential scanning calorimetry peak shape is poor.

[0158] Table 6 Purity values ​​of DPA purity standard material candidates at different heating rates

[0159] Heating rate (K / min) 0.3 0.5 1 5 10 Mass (mg) 1.41 1.58 1.60 1.32 1.30 purity(%) 99.533 99.535 99.560 99.810 99.963

[0160] At the heating rates of 0.5 K / min and 1 K / min, the above experimental conditions were used for 6 tests respectively, and the purity measurement data of the DPA purity standard substance candidate were recorded. The results are shown in Table 7.

[0161] Table 7 Purity values ​​of DPA purity standard material candidates at 0.5K / min and 1K / min

[0162] Heating rate (K / min) 1 2 3 4 5 6 average 0.5 99.512 99.623 99.495 99.503 99.523 99.554 99.535 1 99.534 99.514 99.626 99.612 99.457 99.617 99.560

[0163] A t-test was performed based on the data in Table 7, and the results are shown in Table 8. The results show that according to the t-test, there is no significant difference in the purity values ​​of the DPA purity standard material candidates when the heating rates are 0.5 K / min and 1 K / min, respectively. Considering the accuracy and experimental time consumption, 1 K / min is selected as the slow heating rate.

[0164] Table 8 Purity t-test results of DPA purity standard material candidates at 0.5K / min and 1K / min

[0165]

[0166]

[0167] 3.2.3 Investigation of protective gas flow rate

[0168] At a heating rate of 1 K / min, the effects of protective gas flow rates of 20 mL / min, 30 mL / min, 40 mL / min, and 50 mL / min on the purity values ​​of DPA purity standard substance candidates were investigated, and the results are shown in Table 9. It was found that there was no significant difference in the purity value detection of DPA purity standard substance candidates at different gas flow rates, and 40 mL / min was finally selected as the protective gas flow rate for subsequent experiments.

[0169] Table 9 Purity values ​​of DPA purity standard material candidates at different protective gas flow rates

[0170] Protective gas flow rate (mL / min) 20 30 40 50 Mass (mg) 1.44 1.51 1.30 1.57 purity(%) 99.525 99.512 99.563 99.501

[0171] 3.2.4 Final experimental conditions

[0172] The final experimental conditions for determining the purity of DPA standard material candidates by differential scanning calorimetry are shown in Table 10.

[0173] Table 10 Final experimental conditions for DSC analysis

[0174]

[0175] The DPA purity standard material candidate was subjected to DSC analysis according to the final experimental conditions. The results are as follows Fig.21 The measurement results were analyzed using the NEZSCH Proteus analysis software that comes with the instrument. The results are shown in Fig. 22 shown.

[0176] 4. Uniformity test

[0177] 4.1 Experimental methods

[0178] According to the technical specification of standard materials JJF1343-2012 "General Principles and Statistical Principles of Standard Material Value", the precision of the uniformity test method should not be lower than that of the value method. Therefore, the high performance liquid chromatograph (PAD detector) in the value method is selected for measurement. The chromatographic detection conditions are: wavelength of 316nm, mobile phase of pure methanol, chromatographic column of C18 column (5μm, 4.6mm×150mm), concentration of 0.1mg / mL, column temperature of 35℃, and flow rate of 0.3mL / min. 15 bottles of samples were randomly selected from the DPA purity standard material candidates that have been packaged and numbered according to the head, tail and middle numbers. Each bottle of purity standard material candidate was weighed and a suitable amount was prepared into a solution. Each sample was measured three times in parallel, and the purity values ​​of the three measurements were used as the results of uniformity evaluation, and variance analysis was performed on them. All instruments and measuring tools used were first calibrated to confirm that the instruments were in normal working condition and to ensure the traceability of the instruments and measuring tools.

[0179] According to the above experiment, 15 groups of data were obtained, and the uniformity tests within and between bottles were carried out and evaluated by F test method. This method is to judge whether there is systematic deviation between the measurement values ​​of each group by comparing the variance between groups and the variance within groups, that is, the method of variance analysis.

[0180] According to JJF1343-2012 "General Principles and Statistical Principles for the Determination of Standard Materials", the uniformity variance analysis method is calculated by the following formula:

[0181] set up

[0182]

[0183] The sum of variances between groups is:

[0184] The within-group variance is:

[0185] The degrees of freedom between groups are: ν1 = m-1 (4-5),

[0186] The degrees of freedom within the group are: ν2=Nm (4-6),

[0187] The between-group variance is:

[0188] The within-group variance is:

[0189] The statistic F is:

[0190] This statistic is an F-distributed variate with degrees of freedom (v1, v2).

[0191] According to the degrees of freedom (v1, v2) and the given significance level α, the critical F can be obtained from the F distribution critical value table. α If the F value satisfies F<F α , it is considered that there is no significant difference between the data groups and the samples are uniform.

[0192] 4.2 Uniformity test results

[0193] The homogeneity measurement data results of the DPA purity standard material candidates are listed in Table 11.

[0194] Table 11 Homogeneity evaluation measurement data of DPA purity standard material candidates

[0195]

[0196]

[0197] Then, the uniformity of the candidate DPA purity standard material was tested by variance analysis, and Q1, Q2, v1, v2, s1 were calculated according to formula (4-1) to formula (4-9). 2 s2 2 , F value, the results are shown in Table 12.

[0198] Table 12 Analysis of variance of homogeneity results of DPA purity standard material candidates

[0199] Source of Difference SS Df MS F P-value <![CDATA[F crit ]]> Between groups <![CDATA[0.066(Q1)]]> <![CDATA[14(v1)]]> <![CDATA[4.74E-03(s1 2 )]]> 1.115 0.385 2.037 Within group <![CDATA[0.128(Q2)]]> <![CDATA[30(v2)]]> <![CDATA[4.25E-03(s2 2 )]]> total 0.194 44

[0200] As can be seen from Table 12, the variance analysis results of the homogeneity of the DPA purity standard material candidates are F<F crit , it can be considered that there is no significant difference between the data groups, indicating that the candidates for DPA purity standard substances are uniform.

[0201] 5. Stability test

[0202] 5.1 Experimental Methods

[0203] The stability of a standard substance refers to the change of the characteristic quantity of the specified value 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 long-term regular inspections 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 characteristics of a standard substance under specified storage conditions. Short-term stability is the stability of a standard substance during transportation under transportation conditions. The DPA purity standard substance candidate of this embodiment is based on the technical specifications of standard substances. In accordance with the principle of dense first and sparse later, the long-term stability of the developed standard substance is carried out at 4°C for 12 months and the short-term stability under simulated transportation conditions for 7 days. Measured using a high-performance liquid chromatography instrument (PAD detector).

[0204] The experimental method for stability investigation is the same as that for uniformity, and comparative measurements are performed within time intervals. Referring to the technical specifications of the standard material, three bottles of samples are randomly selected from the candidate DPA purity standard material developed according to the head, tail and middle numbers each time, and the samples are measured according to the chromatographic detection conditions in Table 5. Each bottle of sample is measured three times, and the average value is taken, and then evaluated by regression analysis.

[0205] 5.2 Long-term stability test

[0206] The long-term stability of the DPA purity standard material candidate was tested according to the above experimental method, and the results are shown in Table 13.

[0207] Table 13 Long-term stability test results of DPA purity standard material candidates

[0208]

[0209] Since no physical / chemical model can truly describe the degradation mechanism of DPA purity standard material candidates, a straight line is used as an empirical model:

[0210] Y=β0+β1X(5-1),

[0211] In formula (5-1), β0 and β1 are regression coefficients, X is time, and Y is the characteristic value of the DPA purity standard substance candidate.

[0212] The regression parameters are calculated as follows:

[0213] The slope is:

[0214] 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.

[0215] The intercept is:

[0216] The standard deviation s(β1) of β1 can be calculated by formula (5-4):

[0217]

[0218] In formula (5-4), s is the standard deviation of each point on the straight line, calculated by formula (5-5):

[0219]

[0220] In formula (5-5), n is the number of measurements.

[0221] If |β1|<t 0.05,n-2 ·s(β1), indicating that the slope is not significant, that is, no instability is observed. Under the conditions of degrees of freedom n-2 = 6 and confidence level p = 0.95, we can get t 0.05,6 =2.45.

[0222] According to formula (5-1), a regression analysis was performed on the long-term stability test results of the DPA purity standard substance candidates in Table 13. The results are shown in Table 14.

[0223] Table 14 Results of regression analysis of long-term stability data of DPA purity standard substance candidates

[0224] Candidates <![CDATA[β0]]> <![CDATA[β1]]> <![CDATA[S(β1)]]> <![CDATA[t 0.05,n-2 ·s(β1)]]> in conclusion DPA 99.601 1.24E-03 9.77E-04 2.39E-03 <![CDATA[|β1|<t 0.05,6 ·s(β1)]]>

[0225] It can be seen from Table 14 that for the DPA purity standard material candidates,

[0226] t 0.05,6 s(β1)=2.45×9.77×10 -4 =2.39×10 -3 , |β1|=1.24×10 -3 ,

[0227] |β1| <t 0.05,6 ·s(β1), so no obvious instability is observed.

[0228] At the same time, the stability results of the DPA purity standard material candidates change over time ( Fig.23 ) shows that the purity value of the DPA purity standard substance candidate has no obvious upward or downward trend. It can be seen that the value of the developed DPA purity standard substance candidate has no obvious trend change, indicating that the preparation method and storage conditions of the DPA purity standard substance candidate within the 12-month validity period can effectively ensure the stability of the value of the DPA purity standard substance candidate.

[0229] 5.3 Short-term stability test

[0230] Taking into account the hot and humid weather in the south and the low temperature in the north, as well as the time limit for transportation, we stored the DPA purity standard substance candidate in an incubator at (60±5)°C and (60±5)%RH and in a refrigerator at (4±1)°C for one week, during which the purity of the DPA purity standard substance candidate was tested regularly. The results are shown in Tables 15 and 16.

[0231] Table 15 Short-term stability test results of DPA purity standard material candidates (60±5℃)

[0232]

[0233] Table 16 Short-term stability test results of DPA purity standard material candidates (4±1℃)

[0234]

[0235] The data in Table 15 and Table 16 are respectively fitted into a straight line with x representing time and y representing the purity value of the DPA purity standard substance candidate, as shown in FIG. Fig.24 and Fig.25As shown. The same analytical method as the long-term stability test was used to perform regression analysis on the data in Tables 15 and 16. Under the conditions of freedom n-2 = 4 and confidence level p = 0.95, the table showed that = 2.78 was obtained, and the calculation results were listed in Table 17. The results showed that the characteristics of the DPA purity standard material candidate did not change significantly under the simulated transportation conditions and maintained good stability.

[0236] Table 17 Regression analysis results of short-term stability test of DPA purity standard material candidates

[0237]

[0238] 6. Fixed value

[0239] In accordance with the requirements of JJF 1006-1994 "Technical Specifications for Primary Standard Materials", the purity of DPA purity standard material candidates is determined by an independent reference method using two or more different principles in one laboratory. The determination methods used are differential scanning calorimetry and mass balance method.

[0240] 6.1 Differential Scanning Calorimetry (DSC)

[0241] Take samples from the upper, middle and lower parts of the raw material bottle of the DPA purity standard substance candidate, accurately weigh about 1-3 mg of the DPA purity standard substance candidate, accurate to 0.01 mg, place it in a dry and clean aluminum crucible, cover it and press it tightly, the N2 flow rate is 40 mL / min, and the temperature is increased at a rate of 10K / min at 30-170°C, and at a rate of 1K / min at 170-220°C. According to the results of the previous investigation, the relative molecular weight of DPA is 461.508 g / mol, and the melting point of DPA is about 195°C. Therefore, during the experiment, a calibrated differential scanning calorimeter was used, and the standard substance indium (GBW (E) 130182) with a melting point close to it was selected to calibrate the melting enthalpy change. After the test is completed, the data is loaded into the Proteus purity analysis software for analysis. The results are shown in Table 18.

[0242] Table 18 Purity determination results of DPA purity standard material candidates (DSC method)

[0243]

[0244]

[0245] The average value of the results in Table 18 is the purity of the DPA purity standard substance candidate valued by DSC:

[0246] 6.2 Mass balance method

[0247] 6.2.1 Liquid Chromatography Area Normalization Method

[0248] The method used to determine the main components of the DPA purity standard material candidate is the liquid chromatography area normalization method. Ten packages of all DPA purity standard material candidates were randomly selected, and 1 to 3 sub-samples were randomly taken out from each bottle. The test solution was prepared using chromatography-grade methanol as the solvent, and a 0.1 mg / mL solution was prepared for testing. The test results are shown in Table 19.

[0249] Table 19 Purity test results of main components of DPA purity standard material candidates (HPLC method)

[0250] Serial number 1 2 3 4 5 purity(%) 99.595 99.665 99.633 99.678 99.654 Serial number 6 7 8 9 10 purity(%) 99.590 99.632 99.621 99.638 99.625

[0251] 6.2.2 Moisture test

[0252] A thermogravimetric analyzer was used to test the moisture content of the DPA purity standard substance candidate. About 3 to 5 mg of the DPA purity standard substance candidate was accurately weighed, and the temperature was raised from room temperature to 90°C at a heating rate of 5 K / min, and then to 105°C at a heating rate of 1 K / min and maintained for 60 min until all the moisture in the DPA purity standard substance candidate was evaporated. The measurement was repeated 6 times and the average value was taken as the moisture test result. The results are shown in Table 20.

[0253] Table 20 Moisture test results of DPA purity standard material candidates

[0254]

[0255] 6.2.3 Inorganic element test

[0256] 6.2.3.1 Experimental methods and conditions

[0257] The common inorganic elements of the DPA purity standard material candidate were determined by inductively coupled plasma mass spectrometry (ICP-MS). Ten bottles of DPA purity standard material candidate were randomly selected, the samples were mixed, and about 100 mg of DPA purity standard material candidate powder was accurately weighed in a digestion tank, 6 mL of concentrated nitric acid was added, and the mixture was gently shaken and then placed in a microwave digestion instrument for digestion. After completion, the digestion solution was cooled to below 70°C, a small amount of pure water was added to dissolve and transferred to a 100 mL volumetric flask to make up the volume to obtain the test solution, and 2% HNO3 was used as a blank.

[0258] The experimental conditions of the instrument are as follows: plasma gas flow rate of 15.00 L / min, purge times of 45 times, auxiliary gas flow rate of 0.80 L / min, carrier gas flow rate of 0.80 L / min, and nebulizer temperature of 2°C. In semi-quantitative mode, a single calibration point was used, and the concentration of the calibration point was 10 μg / L (using Agilent's Multi-element calibration standard 2A standard solution diluted 1000 times from 10 μg / mL to 10 μg / L), and the measurement was performed three times, and the average value was taken. The results are shown in Table 21.

[0259] Table 21 Test results of residual cations of DPA purity standard material candidates

[0260] element Test results (%) element Test results (%) element Test results (%) Be 0.9ng / L As 41.3ng / L Sm Not detected B Not detected Se 0 Eu Not detected N Not detected Br Not detected G Not detected Na 1.5 μg / L R 481.5ng / L Tb Not detected Mg 3.6 μg / L Sr 118.8ng / L Dy Not detected Al 18.7 μg / L Y Not detected Ho Not detected Si Not detected Zr Not detected Er Not detected P Not detected Nb Not detected Tm Not detected S Not detected Mo Not detected Yb Not detected Cl Not detected Ru Not detected Lu Not detected K 36.6 μg / L R Not detected Hf Not detected Ca 8.8 μg / L Pd Not detected Ta Not detected Sc Not detected Ag 0 W Not detected Ti Not detected Cd 0 Re Not detected V 24.4ng / L In Not detected Os Not detected Cr 1.6 μg / L Sn Not detected Ir Not detected Mn 175.5ng / L Sb Not detected Pt Not detected Fe 6.8 μg / L Te Not detected Au Not detected Co 1.9ng / L I Not detected Hg Not detected Ni 149.5ng / L Cs 14.5ng / L Tl 4.2ng / L Cu 183.9ng / L Ba 615.8ng / L Pb 139.1ng / L Zn 1.5 μg / L La Not detected Bi Not detected Ga Not detected Ce Not detected Th Not detected Ge Not detected Pr Not detected U 0 Li 54ng / L Nd Not detected

[0261] According to the test results in Table 21, the total content of common inorganic elements after digestion of the DPA purity standard substance candidate is calculated as follows:

[0262] ∑c i =3473.6ng / L+77.5μg / L=81.0μg / L,

[0263]

[0264] 6.3.4 Solvent Residue Test

[0265] During the processing of DPA purity standard substance candidates, residual solvents such as toluene, acetonitrile, tetrahydrofuran, ethanol, methanol, and dichloromethane may remain. According to "JY / T 021-1996 General Rules for Analytical Gas Chromatography Methods", the headspace injection-gas chromatography method is used to perform standard comparison between DPA purity standard substance candidates and the above solvents.

[0266] 6.3.4.1 Experimental conditions

[0267] The chromatographic column was a DB-624 (60m×0.25mm, 1.40μm) gas chromatographic column; the carrier gas was helium, and the carrier gas flow rate was 10.0mL / min; the programmed temperature conditions were: initially 45°C for 5min, then heated to 120°C at a rate of 7°C / min, then heated to 230°C at a rate of 15°C / min, and held for 15min; no split; the heating box was 70°C, the loop was 90°C, the transfer line was 110°C, and the injection volume was 1000μL.

[0268] 6.3.4.2 Solvent Residue Test Results

[0269] According to the above experiment, the test results of solvent residue of DPA purity standard material candidate are as follows: Figures 26-28As shown, under the headspace conditions corresponding to each solvent (detection limit of 100 mg / L), no solvent other than dimethyl sulfoxide was detected in the DPA purity standard substance candidate solution. Therefore, there is no residual solvent in the DPA purity standard substance candidate.

[0270] 6.3.5 Results of the mass balance method

[0271] After deducting moisture, inorganic elements, and solvent residues from the test results of the DPA purity standard substance candidates in Table 19, the fixed value results of the mass balance method are obtained, as shown in Table 22.

[0272] Table 22 Results of the mass balance legal value of DPA purity standard substance candidates

[0273] Serial number 1 2 3 4 5 purity(%) 99.595 99.665 99.633 99.678 99.654 After deduction (%) 99.541 99.611 99.579 99.624 99.600 Serial number 6 7 8 9 10 purity(%) 99.590 99.632 99.621 99.638 99.625 After deduction (%) 99.536 99.578 99.567 99.584 99.571

[0274] The average value of the results in Table 22 is taken as the purity of the DPA purity standard material candidate according to the mass balance legal value, that is:

[0275]

[0276] 6.4 Statistical analysis of fixed value data

[0277] According to JJF1343-2012 “General Principles and Statistical Principles for the Valuation of Reference Materials”, the value data of candidate DPA purity reference materials were subjected to normal distribution test, suspicious value analysis, equal precision test and consistency test.

[0278] 6.4.1 Within-group normal distribution test

[0279] First, the SPSS software was used to test the normality of the two value determination methods, DSC method and mass balance method (MB), respectively. The results are shown in Table 23. In Table 23, "*" is the lower limit of true significance, and "a" is the Rielly significance correction. Due to the small number of samples, the KS result is used as the standard. From the results of Table 23, it can be seen that the two value determination methods sig.=0.2>0.05 obey the normal distribution. View the QQ graph ( Fig.29 and Fig.30 ) is further confirmed. It can be seen from the figure that they are basically close to the straight line, and it can be considered that the two fixed value methods obey the normal distribution.

[0280] Table 23 Normal distribution test results of two fixed value methods

[0281]

[0282] 6.4.2 Analysis of suspicious values

[0283] Examine the data of the two calibration methods separately, and use the Dixon criterion and the Grubbs criterion for outlier analysis. The specific analysis process is as follows.

[0284] (1) Discrimination by Dixon criterion

[0285] Use the Dixon criterion to analyze the DSC calibration results. Arrange them in ascending order:

[0286] 99.560 < 99.561 < 99.588 < 99.601 < 99.605 < 99.607 < 99.612 < 99.623 < 99.628 < 99.637,

[0287] Since n = 10, look up the table to get D(0.05, 10) = 0.530,

[0288]

[0289] Therefore, r 10 > r1, r 10 < D(0.05, 10), indicating that there are no outliers in the DSC calibration data.

[0290] Use the Dixon criterion to analyze the calibration results of the mass balance method. Arrange them in ascending order:

[0291] 99.536 < 99.541 < 99.567 < 99.571 < 99.578 < 99.579 < 99.584 < 99.600 < 99.611 < 99.624,

[0292] Since n = 10, look up the table to get D(0.05, 10) = 0.530,

[0293]

[0294] Therefore, r 10 > r1, r 10 < D(0.05, 10), indicating that there are no outliers in the calibration data of the mass balance method.

[0295] (2) Discrimination by Grubbs criterion

[0296] Use the Grubbs criterion to analyze the DSC calibration results. The arithmetic mean

[0297] The experimental standard deviation is: s = 0.026%,

[0298] Look up the table to get the critical value of the Grubbs criterion: G(0.05, 10) = 2.290,

[0299] Therefore, it shows that there are no outliers in the DSC fixed-value data.

[0300] The Grubbs criterion is used to analyze the results of the mass balance method for value determination.

[0301] The arithmetic mean is:

[0302] The experimental standard deviation is: s = 0.028%,

[0303] Looking up the table, the critical value of the Grubbs criterion is: G(0.05,10) = 2.290.

[0304] Therefore, it shows that there are no outliers in the data determined by the mass balance method.

[0305] 6.4.3 Equal-precision test

[0306] First, perform statistical analysis on the data of the two value determination methods, calculate their respective standard deviations, and obtain s DSC = 0.026%, s MB = 0.040%. Use the Cochran criterion to judge the data of the above two value determination methods. According to the Cochran criterion, calculate the statistic according to the following formula:

[0307]

[0308] According to the critical value table of the Cochran test, the critical value C(0.05,2,10) = 0.8010. Since C(0.05,2,10) < C, the means of the data of the two value determination methods are of equal precision.

[0309] 6.4.4 Consistency test

[0310] The independent-samples t-test method is used to analyze the consistency of the two groups of value determination results. The results are shown in Table 24. As can be seen from Table 24, the significance of the two groups of fixed-value data results is 0.920. Therefore, the significance is greater than 0.05, indicating homogeneous variance. In the t-test result, p > 0.05, indicating that there is no significant difference between the two groups of fixed-value results.

[0311] Table 24 Statistical results of the independent-samples t-test after combining data

[0312]

[0313] 6.4.5 Inter-group normal distribution test

[0314] Combine the data results of the two value determination methods into a group and determine whether it conforms to the normal distribution. The results are shown in Table 25. In Table 25, "*" is the lower limit of true significance, and "a" is the Riley's significance correction. It can be seen from Table 25 that the sig. calculated by the two value determination methods is 0.2 > 0.05, indicating that it follows a normal distribution. Check the Q-Q plot ( Fig.31 ) for further confirmation. It can be considered that it follows a normal distribution.

[0315] Table 25 Normal distribution test results of two value determination methods

[0316]

[0317] 6.4.6 Analysis of suspicious values in combined data

[0318] Combine the data results of the two value determination methods and use the Dixon criterion and Grubbs criterion for the analysis of suspicious values. The specific analysis process is as follows.

[0319] (1) Discrimination by Dixon criterion

[0320] Use the Dixon criterion to analyze the combined value determination results. Arrange them in ascending order:

[0321] 99.536 < 99.541 < 99.560 < 99.561 < 99.567 < 99.571 < 99.578 < 99.579 < 99.584 < 99.588 < 99.600 < 99.601 < 99.605 < 99.607 < 99.611 < 99.612 < 99.623 < 99.624 < 99.628 < 99.637,

[0322] Since n = 20, look up the table to get D(0.05, 20) = 0.489,

[0323]

[0324] Therefore, r1 > r 20 , r1 < D(0.05, 20), indicating that there are no abnormal values in the combined value determination data.

[0325] (2) Discrimination by Grubbs criterion

[0326] Use the Grubbs criterion to analyze the combined value determination results.

[0327] The arithmetic mean is:

[0328] Calculate the experimental standard deviation as: s = 0.029%,

[0329] The critical value of the Grubbs criterion is: G(0.05,20)=2.709,

[0330] Therefore, it shows that there are no abnormal values ​​in the merged fixed value data.

[0331] 6.4.7 Final determination results

[0332] The determination results obtained by the DSC method and the mass balance method (MB) were combined, and the final determination result was obtained by calculating the average value, as shown in Table 26. The purity of the DPA purity standard substance candidate was 99.6%.

[0333] Table 26 Final determination results of DPA purity standard material candidates

[0334]

[0335]

[0336] 7. Uncertainty assessment

[0337] 7.1 Sources of uncertainty

[0338] According to JJG 1006-1994 "Technical Specifications for the Preparation of Primary Reference Materials" and JJF 1343-2012 "General Principles and Statistical Principles for the Determination of Reference Materials", the total uncertainty of the reference value is composed of three parts: char ; Uncertainty u caused by uniformity bb ; Uncertainty u caused by instability s .

[0339] 7.2 Uncertainty analysis

[0340] 7.2.1 Uncertainty introduced when determining the value

[0341] The uncertainty of the value of DPA purity standard material candidate comes mainly from: (1) the uncertainty component introduced by differential scanning calorimetry (DSC) purity measurement; (2) the uncertainty of the mass balance method, including: the uncertainty component introduced by liquid chromatography-area normalization method purity measurement; the uncertainty component introduced by moisture test; the uncertainty component introduced by residue test; the uncertainty component introduced by residual solvent test.

[0342] 7.2.1.1 Uncertainty components introduced by DSC purity measurement

[0343] The purity of the DPA purity standard material candidate measured by DSC is based on the principle that its melting point decreases due to the presence of impurities. The more impurities a substance has, the greater the melting point decreases. The relationship between the melting point decrease and the impurity content can be expressed by the Van't Hoff equation. During the measurement process, the factors that affect the accuracy of the measurement results include: environmental conditions, personnel operation, weighing balance, test instrument, molar melting enthalpy change analysis and software accuracy. According to the measurement method and mathematical model, the sources of uncertainty in the DSC method for measuring DPA purity mainly include the following six aspects: (1) The uncertainty generated when enthalpy change is calibrated with indium; (2) The uncertainty generated by various random factors (such as environment, personnel, etc.) during the measurement process; (3) The uncertainty generated by the balance weighing error during sample weighing; (4) The uncertainty generated by the temperature error of the instrument; (5) The uncertainty generated by the molar enthalpy change error of the instrument; (6) The uncertainty generated by selecting different upper and lower limits of the melting peak when performing sample purity analysis.

[0344] (1) Uncertainty when using indium to calibrate enthalpy change

[0345] The standard uncertainty produced by calibrating the enthalpy change with indium is the type A uncertainty, expressed as u A1(DSC) Under the same conditions, the melting enthalpy change of indium, a candidate for DPA purity standard material, was measured multiple times, and the measurement results are shown in Table 27.

[0346] Table 27 DSC determination of DPA purity standard material candidate indium melting enthalpy change results

[0347]

[0348] It can be seen from Table 27 that the standard uncertainty generated when indium is used to calibrate the enthalpy change is calculated by the following formula:

[0349] u A1(DSC) =R / c=0.03 / 2.53=0.012J / g

[0350] In the formula, R is the range and c is the range coefficient.

[0351] (2) Uncertainty caused by various random factors during the measurement process

[0352] The standard uncertainty caused by various random factors is Type A uncertainty, symbolized by u A2(DSC) It means that by analyzing the repeatability test results of the samples, it can be found that:

[0353]

[0354] (3) Uncertainty caused by balance weighing error during sample weighing

[0355] The molar melting enthalpy change of the sample (DPA purity standard substance candidate) is expressed as the heat absorbed by the unit mole of sample. The number of moles of the sample is the ratio of the mass of the sample to the molar mass. Therefore, the weighing error of the balance will bring uncertainty to the purity measurement result, which is expressed as u m(DSC) express.

[0356] The calculation formula for the molar melting enthalpy change of the sample is:

[0357]

[0358] Where Q is the heat absorbed by the sample, J; m is the mass of the sample, g; M is the molar mass of the sample, g / mol.

[0359] The deviation of the molar melting enthalpy change caused by the balance weighing error can be obtained by taking the partial derivative of ΔH with respect to m:

[0360]

[0361] The weighing error of the balance is dm=0.01 mg, and m=1.61 mg (average value of multiple measurements). From the experimental results, it can be seen that the ΔH of the DPA purity standard substance candidate is 48612.95 J / mol (average value of multiple measurements).

[0362] because The uncertainty caused by the balance weighing error is:

[0363]

[0364] According to the experimental results, we know that: T0 = 469.29K, T m =469.13K, then

[0365] The uncertainty distribution of this experiment is considered as uniform distribution, including factors The uncertainty caused by the balance weighing error is:

[0366]

[0367] (4) Uncertainty caused by temperature error of the instrument

[0368] The uncertainty caused by the temperature error of the instrument is expressed as u T(DSC) It is indicated that the uncertainty caused by temperature error is:

[0369]

[0370] Before the experiment, the instrument was calibrated with the standard substance indium (GBW(E)130182). The temperature deviation of the instrument to the sample was dT = 0.2K. Substituting each data into formula (7-1) and taking the absolute value, we get:

[0371]

[0372] Considering uniform distribution, the inclusion factor but:

[0373]

[0374] (5) Uncertainty caused by the molar enthalpy change error of the instrument

[0375] The standard uncertainty is expressed as u H(DSC) If it is expressed as: then the uncertainty caused by the error of the molar melting enthalpy change of the instrument is:

[0376]

[0377] The melting heat of the standard substance indium (GBW(E)130182) is (28.53±0.30) J / g (k=2), and the molar mass is 114.82 g / mol. The uncertainty of its molar melting enthalpy change is:

[0378]

[0379] Considering uniform distribution, substitute each data into formula (7-2):

[0380]

[0381] (6) Uncertainty caused by different upper and lower limits of melting peaks

[0382] When different upper and lower temperature positions of the melting peak are selected, the results of the purity analysis will be different, so uncertainty may also be generated. This uncertainty is a type A uncertainty, symbolized by u A3(DSC) The experiment was conducted by selecting different upper and lower temperature positions of the melting peak, and the results are shown in Table 28.

[0383] Table 28 Analysis results of variable limit purity of DPA purity standard material candidates

[0384]

[0385] From Table 28, we can see that the uncertainty caused by the upper and lower limits of different melting peaks is:

[0386] u A3(DSC) =R / c=0.00316.

[0387] (7) Composite uncertainty

[0388] The uncertainty assessment results of the purity of DPA purity standard material candidates measured by DSC are shown in Table 29.

[0389] Table 29 Uncertainty evaluation results of the purity of DPA purity standard material candidates measured by DSC

[0390]

[0391] In summary, the uncertainty introduced by DSC purity measurement is:

[0392]

[0393] 7.2.1.2 Uncertainty components introduced by the mass balance method for purity measurement

[0394] First, the uncertainty introduced by HPLC-area normalization method includes the uncertainty introduced by measurement repeatability, the uncertainty introduced by wavelength difference and the uncertainty of impurity analysis.

[0395] (1) Repeatability uncertainty of the measurement results of the method (u a )

[0396] The standard deviation of the mean value of the liquid chromatography method is s = 0.028%, and the uncertainty of the method repeatability is equal to the standard deviation of the mean value of the fixed value result:

[0397] (2) Uncertainty introduced by differences in liquid chromatography response factors

[0398] The uncertainty u introduced by the response difference of each component at different detection wavelengths b Taking into account the responses of the main components and impurity compounds at the corresponding detection wavelengths, the DPA purity standard material candidates were subjected to purity analysis at different wavelengths. The results are shown in Table 30.

[0399] Table 30 Purity data of DPA purity standard material candidates at different wavelengths

[0400]

[0401]

[0402]

[0403] Among them B imaxλ is the maximum percentage content of impurity component i at 5 detection wavelengths, B i定值λ It is the percentage content of the impurity component at a fixed wavelength. b-i is the uncertainty component of impurity component i, and the evaluation results are listed in Table 31.

[0404] Table 31 Chromatographic peak area percentage of each impurity component at different wavelengths

[0405]

[0406] Therefore, the uncertainty introduced by the liquid chromatography determination is:

[0407]

[0408] (3) Uncertainty introduced by moisture testing

[0409] According to the moisture measurement data, the measured R = 0.033%, n = 6 (look up the table c = 2.53), the uncertainty component introduced by moisture is calculated using the range method:

[0410] (4) Uncertainty introduced by residue testing (u 残渣 )

[0411] According to the method and data of residue measurement, due to the use of semi-quantitative mode, the relative uncertainty of the measurement results is generally 50% to 100%. In this experiment, 100% is used as the relative uncertainty of the test of inorganic elements in the residue. Because the impurity content is small, the impact on the final uncertainty of the purity of the standard substance is also small. Therefore, the uncertainty introduced by the residue test is:

[0412] u 残渣 =∑w i ×u i =0.0081%×100%=0.0081%.

[0413] In summary, the combined standard uncertainty of the mass balance method is:

[0414]

[0415] 7.2.1.3 Uncertainty introduced by differences between differential scanning calorimetry and mass balance method

[0416] The uncertainty introduced by the method difference is half of the difference between the DSC and mass balance method results, as shown in the following formula:

[0417]

[0418] 7.2.1.4 Total uncertainty introduced by the fixed value (u char )

[0419] The uncertainty introduced by the determination of the DPA purity standard material candidate is:

[0420]

[0421] 7.2.2 Standard uncertainty due to uniformity (u bb )

[0422] According to the calculation results of the uniformity part in Table 12, The variance between bottles is greater than the variance within the bottle, so the standard deviation between bottles is equivalent to the uncertainty component caused by the inhomogeneity between bottles, that is, the uncertainty of the inhomogeneity between samples is:

[0423]

[0424] u bb =0.013%.

[0425] 7.2.3 Uncertainty introduced by stability (u s )

[0426] According to the stability experimental data analysis results of DPA purity standard material candidates, the regression variance analysis table for evaluating stability was used to estimate the standard uncertainty.

[0427] The uncertainty contribution to long-term stability with a validity period of t = 12 months is:

[0428] u s,长期 =s(β1)·t=9.77×10 -4 ×12=0.012%,

[0429] The uncertainty contribution of short-term stability (60°C) is:

[0430] u s,60℃ =s(β1)·t=7.62×10 -3 ×7=0.053%,

[0431] Therefore, the uncertainty introduced by stability is:

[0432]

[0433] 7.3 Combined total uncertainty

[0434] In summary, the total uncertainty of the DPA purity standard material candidate is:

[0435]

[0436] The expanded uncertainty is: U 95 = k × u 总 =2×0.2%=0.4%,

[0437] The relative expanded uncertainty is:

[0438] The DPA purity standard substance candidate of this embodiment was subjected to uniformity test, stability test, value determination and uncertainty assessment, and it was found that the uniformity, stability and uncertainty of the DPA purity standard substance candidate met the requirements, the value was accurate and stable, and it met the metrological characteristics, that is, the DPA purity standard substance.

[0439] After uniformity test, stability test, value determination and uncertainty assessment, it can be known that the value determination result of DPA purity standard substance is: W = (99.6 ± 0.4)%, k = 2.

[0440] 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. 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester purity standard substance, characterized in that: The purity of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester purity standard material is 99.6%, the expanded uncertainty is 0.4%, the relative expanded uncertainty is 0.4%, and it is prepared by the following method: 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester with a nominal purity of ≥98.0% is selected, and a purity standard substance candidate is obtained after qualitative identification and initial uniformity inspection; after the candidate purity standard substance is subjected to uniformity inspection and stability investigation, differential scanning calorimetry and mass balance method are used for value determination, and uncertainty assessment is performed on the candidate purity standard substance to obtain the product.

2. The method for preparing the purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester according to claim 1, characterized in that: The following steps are involved: (1) Selecting 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester with a nominal purity of ≥98.0%, performing qualitative identification by mass spectrometry, nuclear magnetic resonance hydrogen spectrum analysis, and elemental analysis, and then performing a preliminary homogeneity inspection by high performance liquid chromatography to obtain a candidate purity standard substance for 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester; (2) Conducting homogeneity testing and stability studies on the candidate purity standard material for ethyl 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylate; (3) The candidate purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester is valuated by differential scanning calorimetry and mass balance method, and the average of the valuated results by differential scanning calorimetry and mass balance method is calculated to obtain the valuated result of the candidate purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester; (4) An uncertainty evaluation is performed on the candidate purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester. The candidate purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester that meets the requirements of uniformity test, stability investigation and uncertainty is the purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester.

3. The preparation method according to claim 2, characterized in that: When the mass balance method is used to determine the purity standard substance candidate of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester, the test result can be calculated by the following formula: P=P o ×(1-A-B-C), Wherein, P is the purity of the candidate for 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester purity standard substance, P o is the main component content of the candidate purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester, A is the water content, B is the inorganic element content, and C is the residual solvent content.

4. The preparation method according to claim 3, characterized in that: The uniformity test, the stability investigation and the main component content determination of the 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester purity standard material candidate are carried out by high performance liquid chromatography, and the chromatographic detection conditions are as follows: the chromatographic column is a C18 chromatographic column, 5μm, 4.6mm×150mm; the wavelength is 316nm, the mobile phase is methanol, the flow rate of the mobile phase is 0.3mL / min, the column temperature is 35°C, and the injection concentration is 0.1mg / L.

5. The preparation method according to claim 3, characterized in that: The moisture content is measured by a thermogravimetric analyzer, comprising the following steps: weighing 3 to 5 mg of a purity standard substance candidate of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester and placing it in the thermogravimetric analyzer, heating it to 80 to 95° C. at a heating rate of 5 K / min, then heating it to 100 to 110° C. at a heating rate of 1 K / min, and then maintaining it at 100 to 110° C. for 50 to 70 minutes.

6. The preparation method according to claim 3, characterized in that: The inorganic element content is determined by inductively coupled plasma mass spectrometry, comprising the following steps: The candidate purity standard substance of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester was digested, and then the digestion solution was cooled to below 70°C, dissolved in pure water and transferred to a 100mL volumetric flask to obtain a test solution, and 2% HNO3 was used as a blank, and an inductively coupled plasma mass spectrometer was used in a semi-quantitative mode, using a single calibration point, and the concentration was measured at a calibration point of 10 μg / L; The detection conditions of inductively coupled plasma mass spectrometry were as follows: plasma gas flow rate of 15.00 L / min, purge times of 45 times, auxiliary gas flow rate of 0.80 L / min, carrier gas flow rate of 0.80 L / min, and atomization chamber temperature of 2°C.

7. The preparation method according to claim 3, characterized in that: The residual solvent content is determined by headspace injection-gas chromatography, and the detection conditions are: the chromatographic column is a DB-624 gas chromatograph column, 60m×0.25mm, 1.40μm; the carrier gas is helium, and the carrier gas flow rate is 10.0mL / min; the injection volume is 1000μL; the programmed temperature conditions are maintained at 45°C for 5min, heated to 120°C at a rate of 7°C / min, heated to 230°C at a rate of 15°C / min, and maintained for 15min.

8. The preparation method according to claim 2, characterized in that: When the differential scanning calorimetry is used to determine the purity standard substance candidate of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester, the detection conditions adopted are: the sample weight of the purity standard substance candidate of 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester is 1 to 3 mg; the protective gas flow rate is 40 mL / min, the temperature is slowly increased at 170-220°C, and the slow heating rate is 1K / min.

9. The preparation method according to claim 2, characterized in that: The stability investigation includes long-term stability investigation and short-term stability investigation; when performing the uncertainty assessment, the uncertainty includes the uncertainty introduced when setting the value, the uncertainty caused by uniformity and the uncertainty caused by instability.

10. Use of the 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester purity standard substance according to claim 1 as a measuring tool for detecting the 6-[4-(diphenylamino)phenyl]coumarin-3-carboxylic acid ethyl ester content in electronic components or laser materials or for confirming the detection method.

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