Compound detection method based on ultraviolet spectrophotometry

Abstract

The invention discloses a compound detection method based on ultraviolet spectrophotometry (hereinafter referred to as UV method), which comprises the following steps: step 1, sample pretreatment: taking methanol as a solvent, fully dissolving a sample, and carrying out rotary evaporation until the sample is dry to obtain a sample with volatile impurities removed, a sample preparation solution with volatile impurities removed, a preparation curve and a to-be-detected product; 2, precisely weighing a sample to be detected, and preparing solutions with different concentrations in a concentration range of 0.25 mg / ml-1. 5mg / ml by taking methanol as a solvent; 3, the wavelength of the ultraviolet spectrophotometer is set to be 258 nm, the absorbance of the sample solutions with different concentrations is measured, and a standard curve of the concentration of the sample solutions and the absorbance is established; 4, under the wavelength of 258 nm, the absorbance of a compound with the American chemical abstract number (CAS number) of 111549-97-4 is measured through an ultraviolet spectrophotometer, and the content of the compound is calculated with the standard curve as the benchmark. The quantitative detection method has a good linear relationship, is simple, convenient and rapid to operate, and is easy to popularize and promote.

Description

Technical Field

[0001] The invention discloses a technical field related to compound detection, and in particular to a compound detection method based on ultraviolet spectrophotometry. Background Art

[0002] The compound with CAS number 111549-97-4 is used as an important chemical raw material for the production of Haemophilus influenzae type b (Hib) conjugate vaccine. The vaccine is a new vaccine made by chemical synthesis. The compound constitutes the skeleton of the antigen part of the Hib vaccine. During the production process, the compound is produced by weighing its weight and then feeding it. The feeding coefficients of other materials are also calculated based on the mass of this compound. Therefore, its quantitative detection can contribute to the accuracy of feeding.

[0003] At present, all manufacturers on the market use the area normalization method of high performance liquid chromatography (HPLC) to detect the content of the compound with CAS number 111549-97-4. The area normalization method is only the percentage value of the sum of the area under the curve of the product and the area under the curve of the impurity with a measurable value of the detector under the detection conditions and the area under the curve of the product. The measured content is not the weight percentage of the content (see Note 1 below); it is not accurate enough as a method for content detection (see Note 2 below), but it is widely used in the market because of its simple and quick operation (dissolve the sample into the needle). If this method is used to detect the content of a product, it should be combined with more rigorous methodological verification to confirm that the area normalization method is applicable to the sample to be measured. Therefore, it is inaccurate to use the area normalization method alone to detect the content of the compound with CAS number 111549-97-4. In addition, the compound with CAS number 111549-97-4 will produce two peaks during the HPLC measurement process, resulting in the HPLC method being unsuitable for the detection of this compound.

[0004] Note 1: The 2020 edition of the Pharmacopoeia of the People's Republic of China (current edition, hereinafter referred to as the Chinese Pharmacopoeia) stipulates that the "content (%) of raw materials and pharmaceutical excipients, unless otherwise specified, is measured by weight... The symbol '%' indicates percentage, which refers to the proportion by weight."

[0005] Note 2: For example, if the detector is a UV-visible spectrophotometer and the detection wavelength is set to 220nm, then this method actually measures the ratio of the area under the curve of the product to the sum of the areas under the curve of the impurities and the product at 220nm, that is, it only detects the ratio of the area under the curve of the product to the sum of the areas under the curve of the impurities with UV absorption and the product at 220nm. If the impurities cannot be quantitatively analyzed, their UV absorption area does not correspond to their mass. For example, assuming that the purity of the sample measured is 90%, and the remaining 10% are all the same impurities; and the area under the curve of the pure sample of the same mass at 220nm is 1, while the area under the curve of the impurity at 220nm is 5. If the detection is carried out at 220nm, the area normalization method will result in the sample content being 90×1 / (90×1+10×5)=64.3%, which is far from the actual content of the sample. Or if we assume that the area under the sample curve is 1 and the impurities have no absorption, we will think that the sample purity is 100%, and we will be unaware of the existence of impurities that may affect subsequent reactions. Such content test results will result in us not knowing how much raw material has actually been put into the feeding process. In particular, the feeding amount of the compound with CAS number 111549-97-4 is still defaulted to 100% pure substance, which is used to guide the weight / volume of other raw materials to be weighed for production. Failure to know its actual input amount will lead to uncontrollable production.

[0006] Therefore, developing a method for detecting the content of the compound with CAS number 111549-97-4 has become an urgent problem to be solved. Summary of the invention

[0007] The invention provides a compound detection method based on ultraviolet spectrophotometry, which is used for quantitative detection of a compound with CAS number of 111549-97-4.

[0008] The present invention provides a compound detection method based on ultraviolet spectrophotometry, comprising the following steps:

[0009] Step 1: Sample pretreatment: using methanol as solvent, fully dissolving the sample and rotary evaporating to dryness to obtain a sample with volatile impurities removed. Similarly, the sample with volatile impurities removed is used as the sample to be tested;

[0010] Step 2: Accurately weigh the sample to be tested, use methanol as solvent, and prepare solutions of different concentrations, ranging from 0.25 mg / ml to 1.5 mg / ml;

[0011] Step 3: Set the wavelength of the UV spectrophotometer to 258 nm, measure the absorbance of samples of different concentrations, and establish a standard curve of sample concentration and absorbance;

[0012] Step 4: At a wavelength of 258 nm, the absorbance of the compound with CAS number 111549-97-4 is measured by an ultraviolet spectrophotometer, and the content of the sample compound is calculated by the weight loss ratio measured in step 1 based on the standard curve.

[0013] Furthermore, the method is applicable to the compound with CAS No. 111549-97-4.

[0014] Furthermore, in step 2, samples of different concentrations were prepared, including sample solutions of 1.5 mg / ml, 1.0 mg / ml, 0.75 mg / ml, 0.50 mg / ml and 0.25 mg / ml.

[0015] The invention discloses a quantitative detection method based on ultraviolet spectrophotometry, comprising the steps of sample preparation, curve preparation, quantitative detection and methodology verification, and based on the fact that the mass of a compound with CAS No. 111549-97-4 and its absorbance value measured on an ultraviolet spectrophotometer are linearly related within a certain range, quantitative detection can be performed on it.

[0016] The quantitative detection method provided by the present invention corrects the error of the HPLC area normalization method, which is an original detection method for the compound on the market. The equipment involved (balance, ultraviolet spectrophotometer) is easily available and easy to operate. The method itself has a good linear relationship and accurate quantitative analysis, meets the requirements of the "Pharmacopoeia of the People's Republic of China", is relatively simple and quick to operate, and is easy to popularize and promote.

[0017] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 The nuclear magnetic hydrogen spectra of the compound with CAS number 111549-97-4 of three manufacturers A Aladdin Company, B J&K Company, and C Caberson Company recorded in the specific implementation manner of the present invention;

[0021] Figure 2The HPLC area normalization method result of Aladdin Company recorded in the specific implementation mode of the present invention;

[0022] Figure 3 The HPLC area normalization method result recorded in the specific embodiment of the present invention by J&K Company;

[0023] Figure 4 A photograph of a thin layer chromatography plate obtained by spotting the sample corresponding to the peak with the largest area under the curve and the sample corresponding to the second largest peak in the HPLC spectrum of the product of J&K Company recorded in the specific embodiment of the present invention by thin layer chromatography;

[0024] Figure 5 This is a schematic diagram of the results of preliminary verification of whether the UV method can be used for quantitative detection of samples in Example 1 of the present invention;

[0025] Figure 6 This is a schematic diagram of the first test results of the methodological verification of the linearity and range of the compound with CAS No. 111549-97-4 by UV quantitative detection in Example 4 of the present invention;

[0026] Figure 7 This is a schematic diagram of the second test results of the methodological verification of linearity and range for quantitative detection of the compound with CAS No. 111549-97-4 by UV method in Example 4 of the present invention;

[0027] Figure 8 This is the preliminary back-calculation result of the first test recovery of linearity and range of Example 4 of the present invention;

[0028] Fig. 9 This is the preliminary back-calculation result of the linearity and range of the second test recovery of Example 4 of the present invention;

[0029] Fig.10 This is a schematic diagram of the accuracy and repeatability results of Example 4 of the present invention;

[0030] Fig.11 This is a schematic diagram of the intermediate precision results of Example 4 of the present invention;

[0031] Fig.12 This is a schematic diagram of the results of the detection method for confirming the sample in Example 5 of the present invention. DETAILED DESCRIPTION

[0032] The present invention is further explained below in conjunction with specific implementation schemes, but it is not intended to limit the protection scope of the present invention.

[0033] Among the commercial products of the compound with CAS No. 111549-97-4, several important manufacturers, including Aladdin, J&K and Cabosons, all use the HPLC area normalization method to test the content of their products, and all set the content range of their products to ≥90%. Judging from the NMR test spectrum, all the peaks of the products of the above manufacturers are clearly traceable. Based on experience, it can be judged that the content of this product is definitely more than 90%, but should be at least above 95%, and should be roughly ≥97-98%. Figure 1 ; Figure 1 The ones ending with A come from Aladdin, those ending with B come from Braunschweig, and those ending with C come from Caberson.

[0034] According to the method provided by the manufacturer, the content was determined by HPLC and calculated by area normalization method. It can be seen that the area under the curve of the largest peak in the spectrum is about 90%. Figure 2 , 3 The HPLC area normalization result of Aladdin's product was 90.84%, and the HPLC area normalization result of J&K's product was 88.71%, which was inconsistent with the nuclear magnetic resonance detection results.

[0035] The results were analyzed by taking the sample corresponding to the peak with the largest area under the curve in the HPLC spectrum and the sample corresponding to the second largest peak, and performing spot plate analysis by thin layer chromatography (TLC). The results are shown in Figure 4 . It can be seen that although the retention time on HPLC is different, the compounds corresponding to the first and second largest peaks of the area under the curve separated by HPLC are both compounds with CAS number 111549-97-4. It is speculated that the reason for this phenomenon is that the reagent used to dissolve the sample in the HPLC method is a pure organic reagent, and the mobile phase is an organic reagent: water. The mobile phase cannot dissolve the sample, resulting in partial sample separation. Adding the areas under the curve of the two peaks, the product contents of the two manufacturers detected by HPLC are 97.06% and 96.82% respectively, which is roughly consistent with our content range judgment through nuclear magnetic resonance spectra.

[0036] It can be seen from this that the HPLC area normalization method currently used by various manufacturers of the compound with CAS No. 111549-97-4 on the market is not suitable for the content detection of this compound, and a new method needs to be developed to detect the content of this compound.

[0037] To this end, the present embodiment provides a compound detection method based on ultraviolet spectrophotometry for quantitative detection of a compound with CAS number 111549-97-4; because the compound has ultraviolet absorption, the UV method also has the advantages of good linearity, mature equipment used (analytical balance and ultraviolet spectrophotometer are the most commonly used equipment in laboratories), and relatively simple operation, so a methodology for ultraviolet spectrophotometry is developed for it.

[0038] Example 1: Preliminary confirmation of whether the UV method can be used for quantitative detection of samples

[0039] Weigh the compounds with CAS number 111549-97-4 from two manufacturers respectively, prepare a 10 mg / ml solution, and then dilute it in multiples to prepare 7.5, 5, 2.5, 1.25... mg / ml solutions in turn, and perform preliminary UV testing until the absorbance value of the UV method reaches about 0.3 (according to the 2020 edition of the Chinese Pharmacopoeia, Part IV, p39, Ultraviolet-Visible Spectrophotometry, "The absorbance reading of the general test solution should be between 0.3 and 0.7").

[0040] Since the purpose of this step is only to preliminarily confirm whether the UV method is applicable to the sample, if there are four points in the dilution gradient, including the points with absorbance values ​​between 0.3 and 0.7, that are linear, it can be preliminarily confirmed that the UV method can be used for quantitative detection of the compound with CAS number 111549-97-4. The judgment standard for this test is set as when the correlation coefficient r ≥ 0.99 (degrees of freedom n-2 = 2, α = 0.01), that is, R 2 When ≥0.98, it is considered linear. The experiment was performed by two people in parallel, and the results are as follows Figure 5 As shown, the R 2 All of them are 0.999≥0.98, that is, all of them are linear.

[0041] Conclusion: When diluted to a concentration of 0.3125 mg / ml, the absorbance value measured by the UV method is around 0.3, and the four points including this concentration are linear. It can be preliminarily confirmed that the UV method can be used for quantitative detection of the compound with CAS number 111549-97-4.

[0042] Example 2: Confirmation of sample pretreatment method

[0043] Since the production process used by each manufacturer is unknown, the impurities contained in the product are unknown. Figure 1 ) and combined with experience, most of the impurities should be volatile impurities. Such impurities are likely to have ultraviolet absorption, which will lead to inaccurate quantitative detection results (see the background technology section, explanation 2), so the sample needs to be pre-treated to remove volatile impurities.

[0044] Methanol can dissolve the compound with CAS number 111549-97-4, and its boiling point is 64.7℃, which is relatively high among organic reagents, so it can better remove volatile impurities. Methanol has no absorption in the main ultraviolet detection band, but has a characteristic peak at 3.4ppm in the nuclear magnetic hydrogen spectrum, so any residue can be easily found. Methanol is also easy to obtain, so methanol was selected as the sample pretreatment reagent.

[0045] Combined with the conclusions obtained from the aforementioned NMR spectrum and HPLC area normalization method, if the sample mass decreases by about 2-3% after pretreatment, it can be determined that most of the impurities in the sample are volatile impurities, and the selected reagent methanol can effectively remove the impurities in the sample.

[0046] Use a 1 / 10,000 balance within the validity period of calibration to weigh about 5g of sample, add about 25ml of methanol to fully dissolve the sample, and evaporate it to dryness; add about 25ml of methanol again to fully dissolve the sample, and evaporate it to dryness; add about 25ml of methanol for the third time to fully dissolve the sample, and evaporate it to dryness. The third rotary evaporation time should be more than 12 hours to ensure that all methanol is removed.

[0047] Weigh the sample after rotary evaporation and calculate the weight loss. The weight loss should be about 2-3%. According to the description of "about" in the 2020 edition of the Chinese Pharmacopoeia, "when the amount taken is 'about' a certain amount, it means that the amount taken shall not exceed ±10% of the prescribed amount". Take 10% of 3% to set the judgment range, that is, the range of this test is set to 1.7-3.3%. That is, when the weight loss of all samples is within the range of 1.7-3.3%, it is determined that the above treatment method can be used as the sample pretreatment method.

[0048] Three samples each from Aladdin (ending with number A) and Braunwell (ending with number B) were tested in parallel. The results are shown in the table below.

[0049]

[0050]

[0051] Conclusion: Using methanol to fully dissolve the sample and then performing three rotary evaporations can be used as a pretreatment method for sample detection.

[0052] Example 3: Confirming the detection wavelength of the sample

[0053] 1. Confirm whether using methanol as a reagent will not interfere with the test results

[0054] Wash the cuvette 3 times with methanol and evaporate to dryness;

[0055] According to the requirements of the Pharmacopoeia of the People's Republic of China, use methanol to scan 200nm→400nm on the UV spectrophotometer. When measuring, only put the cuvette in the blank tank without adding reagents, that is, use air as the blank to determine whether the methanol solvent has no interference with the test. Determine according to the standards of the Chinese Pharmacopoeia.

[0056] Blank absorbance of methanol reagent:

[0057] 220-240(230)nm maximum value: -0.197(should be ≤0.40), Meets □Does not meet the requirements;

[0058] 241-250(245)nm maximum value: -0.192 (should be ≤0.20), Meets □Does not meet the requirements;

[0059] 251-300(275)nm Maximum value: -0.138(should be ≤0.10), Meets □Does not meet the requirements;

[0060] 301-400(350)nm maximum value: -0.087(should be ≤0.05), Meets □Does not meet the requirements.

[0061] Conclusion: Using methanol as a reagent has no interference with the test results.

[0062] 2. Confirm the detection wavelength of the sample

[0063] Prepare sample solutions of 0.75, 0.5, and 0.25 mg / ml respectively, scan from 200 nm to 400 nm on a UV spectrophotometer, use the above methanol solution as a blank, and measure the absorbance peak of each solution. Select the UV nm number where the peak is located, round it up, and use it as the detection wavelength for subsequent experiments.

[0064] UV absorption peak

[0065]

[0066]

[0067] Conclusion: 258nm was selected as the UV detection wavelength in subsequent experiments.

[0068] Example 4: Methodological verification of the quantitative detection of the compound with CAS number 111549-97-4 by UV method

[0069] Methodological validation was carried out in accordance with the requirements of the Chinese Pharmacopoeia (General Chapter 9101 of Part IV of the 2020 edition of the Pharmacopoeia of the People's Republic of China, "Guidelines for Validation of Analytical Methods"). When all items met the requirements, the method was judged to have passed the methodological validation and could be used for the quantitative detection of the compound with CAS number 111549-97-4.

[0070] 1. Linearity and range: confirm that the UV method can quantitatively detect the range of the compound with CAS number 111549-97-4.

[0071] Based on the preliminary study in Example 1, the linear range of this method was explored. The upper limit of the range should ensure that there are 5 points below the linear relationship, and the absorbance value is in the range of 0.3-0.7 or close to the point in the range at the middle of the 5 points; the lower limit should be a point higher than the minimum weighing value of the balance used, which can be directly weighed and fixed, and a one-step dilution preparation can be performed to further explore whether the value further down is still linear.

[0072] The upper and lower limits that meet the above requirements can be defined as the scope of this method.

[0073] After that, preliminary optimization is carried out within this range, that is, preliminary back-replacement of samples is carried out to confirm whether the recovery rate of this method is within the range of 98-101% (9101 "Guidelines for Validation of Analytical Methods", General Chapter 4 of the 2020 edition of the Pharmacopoeia of the People's Republic of China), and to preliminarily observe whether the products of different manufacturers do not affect their respective recoveries after rotary evaporation to remove volatile impurities, so as to confirm the curve range for the next step of specificity research.

[0074] Specific operations:

[0075] First, determine the minimum weighing value (minimum weighing) of the one-hundred-thousandth analytical balance to be used for weighing in the test. According to the provisions of the Metrology Verification Regulations of the People's Republic of China JJG1036-2022 "Electronic Balance", when the precision weighing (according to the requirements of the Chinese Pharmacopoeia, "'precision weighing' means that the weight should be weighed accurately to one thousandth of the weight taken", take U = 0.1%, k = 2), the minimum weighing value of the balance is 22.27 mg. That is, in this experiment, the samples used to prepare the points on the standard curve and quantitative detection can only weigh a mass not less than (≥) this value.

[0076] Accordingly, accurately weigh 400, 300, 200, 150, 100, 75, 50, and 25 mg of samples A and B after rotary evaporation, dissolve them in a 100 ml volumetric flask with methanol, and make up to volume;

[0077] Accurately weigh 25 mg of each sample A and B after rotary evaporation, dissolve them in methanol into a 200 ml volumetric flask and make up to volume;

[0078] Then use a calibrated 5ml pipette to take 5ml of the sample from the 200ml volumetric flask and transfer it to a 10ml volumetric flask to make up the volume.

[0079] That is, 10 samples of A and B were prepared at 4, 3, 2, 1.5, 1, 0.75, 0.5, 0.25, 0.125, and 0.0625 mg / ml, respectively.

[0080] Adjust the UV spectrophotometer to maximum accuracy, that is, the absorbance value is displayed to 3 decimal places.

[0081] Using methanol as a blank, measure the UV absorption of the above prepared samples at 258nm and calculate the linearity and range of each. That is, measure the UV absorption of 4, 3, 2, 1.5, 1, 0.75, 0.5, 0.25, 0.125, 0.0625 mg / ml samples and calculate their R 2 , to determine whether it is linear. This time, 5 points are taken, and the degree of freedom n-2 = 3. When α = 0.01, the significance critical value of linear correlation is 0.959, but the stricter r = 0.990 in Example 1 is still used as the limit, that is, when R 2 When ≥0.98, it was judged to be linear.

[0082] The test results are shown in the table below. According to GB5750.3-2023, "the correlation coefficient r is rounded down and retained to one decimal place other than 9". 2 This method is also adopted, and the table is retained to 3 decimal places.

[0083] Linearity and range first test, see Figure 6 , 8

[0084]

[0085] R 2 (Should be ≥0.98)

[0086]

[0087]

[0088] Conclusion: Any 5 points in the table meet the linearity requirements and can be selected arbitrarily for further verification of the analytical method.

[0089] Linearity and range second test, see Figure 7 , 9

[0090]

[0091] R 2 (Should be ≥0.98)

[0092]

[0093] like Figure 7 As shown, R 2 It should be ≥0.98. The points of 4mg / ml and 3mg / ml are obviously not on the line, so the R related to these two points was not calculated. 2 .

[0094] Conclusion: Any 5 points in the table meet the linearity requirements and can be selected arbitrarily for further verification of the analytical method.

[0095] In summary, in the actual detection process, in the upper limit of the detection range, when ① the concentration is 4 mg / ml, the detection upper limit 5.000 is reached, or it is not on the line; ② when the concentration is 3 mg / ml, it is not on the line; ③ when the concentration is 2 mg / ml and above, the absorbance value jumps and is unstable; in the lower limit of the detection range, ④ when the sample concentration is 0.0625 mg / ml, it can still highly meet the linear requirements, but this concentration is obtained by multiple dilution, not by direct weighing. Introducing one more step of operation will lead to possible error accumulation, and at this time the effective number of the measured result is already 2, so no further dilution is required to calculate the linearity according to R 2 Further seek the lower limit of the linear range.

[0096] The conclusion about the linearity test can be obtained: within the detection range of 0.0625-2mg / ml, the UV method meets the linearity requirements.

[0097] Based on this, the curves in the middle two ranges are selected, and the middle points, i.e. 0.75 and 0.5 mg / ml, are used as the detection concentrations of the samples. Two points are taken above and below, i.e., the 1.5-0.25 and 1-0.125 mg / ml curves in the above table are selected as the detection concentrations for the following verification items.

[0098] At this time, a preliminary back calculation of the recovery rate is performed, such as Figure 8 and Fig. 9 As shown; to confirm whether the recovery rate of this method is within the range of 98-101%, and to preliminarily observe whether the products of different manufacturers do not affect their respective recovery rates after the volatile impurities are removed by rotary evaporation, so as to carry out the next step of specificity research.

[0099] The recovery rate was initially calculated by back substitution. Two points were taken above and below the detection value to make a standard curve. The concentrations of 0.25, 0.5 and 0.75 mg / ml can basically reach the requirement of 98-101% (2 tests, only 1 of 24 values ​​was slightly out of range, see Figure 8 , 9 ).

[0100] Conclusion: The curves made by products from different manufacturers basically do not affect the calculation of their respective recovery rates.

[0101] In summary, the five-point curve of 1.5-0.25 mg / ml and the five-point curve of 1-0.125 mg / ml were selected as the sample preparation concentrations of the standard curve in the following specificity study; correspondingly, 0.75 mg / ml and 0.5 mg / ml were selected as the detection concentrations of the samples.

[0102] 2. Specificity: confirm the specificity of the UV quantitative detection of the compound with CAS number 111549-97-4.

[0103] Compare the same samples produced by two different companies (Aladdin and Bailingwei), prepare standard curves according to the detection range determined in Example 4, 1, and perform mathematical statistical tests on the variance, slope and intercept of the standard curves of the two companies' products, with a confidence interval of 95% or 99%. If there is no significant difference after mathematical statistical analysis, it is confirmed that the specificity of the UV method meets the requirements. If both meet the requirements, select the concentration with absorbance value in the range of 0.3-0.7 as the sample content detection concentration for the following test.

[0104] Accurately weigh 150, 100, 75, 50, and 25 mg of samples A and B after rotary evaporation, dissolve them in methanol in a 100 ml volumetric flask, and adjust the volume; accurately weigh 25 mg of samples A and B after rotary evaporation, dissolve them in methanol in a 200 ml volumetric flask, and adjust the volume. That is, prepare 6 samples of 1.5, 1, 0.75, 0.5, 0.25, and 0.125 mg / ml of A and B respectively.

[0105] Adjust the UV spectrophotometer to the maximum precision, that is, the absorbance value shows 3 decimal places. Use methanol as a blank and measure the UV absorption of the above prepared sample at 258nm. That is, measure the UV absorption of 1.5, 1, 0.75, 0.5, 0.25, and 0.125 mg / ml samples, and calculate the R of the two curves of 1.5-0.25 and 1-0.125 mg / ml. 2 , and conduct mathematical statistical tests on the variance, slope and intercept of the standard curves of the two products.

[0106] Exclusive first test

[0107]

[0108] R 2 (Should be ≥0.98)

[0109]

[0110] Under the condition of 6 degrees of freedom (n1-2+n2-2), the variance of the two curves was tested by F test, and the slope and intercept were tested by T test. The results showed that when the two curves were tested at 0.5 mg / ml as the sample detection point (center point), the confidence interval was 99%, and there was no significant difference when the confidence interval was 95% with 0.75 mg / ml as the sample detection point (center point).

[0111] The 0.75 mg / ml curve has an advantage.

[0112] Second test of specificity

[0113]

[0114] R 2 (Should be ≥0.98)

[0115]

[0116] Under the condition of 6 degrees of freedom (n1-2+n2-2), the variance of the two curves was tested by F test, and the slope and intercept were tested by T test. The results showed that when the two curves were tested at 0.5 mg / ml as the sample detection point (center point), the confidence interval was 99%, and there was no significant difference when the confidence interval was 95% with 0.75 mg / ml as the sample detection point (center point).

[0117] Again the curve at 0.75 mg / ml has the advantage.

[0118] However, according to the 2020 edition of the Chinese Pharmacopoeia, Part IV, p39, UV-Vis Spectrophotometry, "The absorbance reading of the general test solution is preferably between 0.3 and 0.7", the absorbance reading of the above 0.5mg / ml concentration sample is around 0.6, and the absorbance reading of the 0.75mg / ml concentration sample is around 0.9. Therefore, the 0.5mg / ml curve has an advantage. Therefore, the maximum deviation that the equipment may bring when the absorbance reading is 0.9 is calculated.

[0119] According to the technical parameter T% (±0.3%) of the ultraviolet spectrophotometer Shimadzu UV2450 used in this test, 0.6 and 0.9 were taken as the absorbance values, and the maximum deviation that may be caused by using 0.5 and 0.75 mg / ml as the sample detection point (center point) was calculated:

[0120]

[0121] That is, when the absorbance of the sample to be measured is close to 0.9, the deviation caused by the ultraviolet spectrophotometer Shimadzu UV2450 is within the range of ±1.15%, which is still approximately 1%, which is acceptable compared to ±0.86% of 0.5 mg / ml concentration.

[0122] Therefore, after comprehensive consideration, the curve with 0.75 mg / ml as the sample detection point (center point) was selected for subsequent experiments.

[0123] Conclusion: The UV method has very good specificity for quantitative detection of the compound with CAS number 111549-97-4. That is, samples produced by different manufacturers can be used to prepare standard curves after pretreatment for the detection of this compound.

[0124] 3. Accuracy and repeatability: confirm the accuracy and repeatability of the UV method for quantitative detection of the compound with CAS number 111549-97-4.

[0125] Take the processed samples from Bailingwei Company, prepare the standard curve with 0.75 mg / ml as the center point, prepare 6 samples of 0.75 mg / ml for content determination. Evaluate the determination results. According to the requirements of the 2020 edition of the Chinese Pharmacopoeia Part IV Analytical Method Validation Guidelines, if the recovery rates of the 6 samples are all within the range of 98-101%, it can be confirmed that the accuracy of the UV method meets the requirements; if the RSD% of the 6 samples is ≤1%, it can be confirmed that the repeatability of the UV method meets the requirements.

[0126] Weigh 150, 100, 75, 50, and 25 mg of sample B after rotary evaporation accurately, dissolve in methanol in a 100 ml volumetric flask, and adjust to volume; Weigh 6 portions of sample B after rotary evaporation accurately, each 75 mg, dissolve in methanol in a 100 ml volumetric flask, and adjust to volume. That is, prepare 5 samples with concentrations of sample B of 1.5, 1, 0.75, 0.5, and 0.25 mg / ml to make a standard curve, and use the standard curve to determine the content of 6 samples with a concentration of 0.75 mg / ml.

[0127] The UV spectrophotometer was adjusted to the maximum precision, that is, the absorbance value was displayed to 3 decimal places. Using methanol as a blank, the UV absorption of the above prepared samples was measured at 258nm. That is, the UV absorption of 1.5, 1, 0.75, 0.5, and 0.25 mg / ml samples was measured, and their R 2 . R 2 After meeting the requirements, measure the UV absorption of 6 0.75 mg / ml samples, and calculate the respective recoveries and the RSD% of the 6 samples.

[0128] Accuracy and repeatability (recovery rate should be within the range of 98% ≤ recovery rate ≤ 101%, RSD% should be ≤ 1%)

[0129]

[0130]

[0131] R 2 =0.998≥0.98, the curve is available;

[0132] The recoveries of the six 0.75 mg / ml samples were all in the range of 98-101%, and the accuracy of the UV quantitative detection of the compound with CAS number 111549-97-4 met the requirements;

[0133] The RSD% of 6 0.75 mg / ml samples was ≤1%, and the repeatability of the UV quantitative detection of the compound with CAS number 111549-97-4 met the requirements.

[0134] Conclusion: The accuracy and repeatability of the UV quantitative detection of compound CAS No. 111549-97-4 meet the requirements.

[0135] 4. Intermediate precision: confirm the intermediate precision of the UV quantitative detection of the compound with CAS number 111549-97-4.

[0136] The test was conducted on different days, i.e. over 3 days.

[0137] Take the processed samples from Bailingwei Company, prepare the standard curve with 0.75 mg / ml as the center point, prepare 2 samples of 0.75 mg / ml, and perform content determination. Evaluate the determination results. According to the requirements of the 2020 edition of the Chinese Pharmacopoeia Part IV Analytical Method Validation Guidelines, if the RSD% of the sample content is ≤1%, it can be confirmed that the intermediate precision of the UV method meets the requirements.

[0138] Weigh at least 2 portions of sample B after rotary evaporation, 75 mg each, dissolve in methanol in a 100 ml volumetric flask and make up to volume. That is, prepare 5 samples with concentrations of sample B of 1.5, 1, 0.75, 0.5, and 0.25 mg / ml to make a standard curve, and determine the content of the sample with a concentration of 0.75 mg / ml by using the standard curve.

[0139] The UV spectrophotometer was adjusted to the maximum precision, that is, the absorbance value was displayed to 3 decimal places. Using methanol as a blank, the UV absorption of the above prepared samples was measured at 258nm. That is, the UV absorption of 1.5, 1, 0.75, 0.5, and 0.25 mg / ml samples was measured, and their R 2 . R 2 After meeting the requirements, measure the UV absorption of two 0.75 mg / ml samples and calculate the corresponding sample content. Calculate the RSD% of the three tests.

[0140] Intermediate precision (RSD% should be ≤1)

[0141] Intermediate precision day 1 test (R 2 Should be ≥0.98, RSD% between samples should be ≤1)

[0142]

[0143] Intermediate precision day 2 test (R 2 Should be ≥0.98, RSD% between samples should be ≤1)

[0144]

[0145] Intermediate precision test on day 3 (R 2 Should be ≥0.98, RSD% between samples should be ≤1)

[0146]

[0147] The mean recovery rate (%) obtained on three different dates was 100.237, the standard deviation was 0.5058, and RSD%=0.505%≤1%.

[0148] Conclusion: The intermediate precision of the UV quantitative detection of compound with CAS number 111549-97-4 meets the requirements.

[0149] The overall conclusion of this step (Example 4) is shown in the following table:

[0150] Methodology Validation Project in conclusion Linear Meet the requirements scope 0.0625mg / ml-2mg / ml Exclusive Meet the requirements Accuracy Meet the requirements Repeatability Meet the requirements Intermediate precision Meet the requirements

[0151] Conclusion: The UV method for quantitative detection of the compound with CAS number 111549-97-4 has passed the methodology verification, meets the requirements of the Chinese Pharmacopoeia, and can be used for the quantitative detection of the compound with CAS number 111549-97-4.

[0152] Example 5: Confirmation of sample detection method

[0153] This Example 5 will confirm the sample detection method of the compound with CAS No. 111549-97-4 by UV quantitative detection. That is, the recovery rate of the pre-treated sample and the unpre-treated sample is compared to confirm whether the compound can be directly sampled for detection after the standard curve is prepared.

[0154] Note that the recovery rate of this step should meet the range requirement of 98-101% in the pharmacopoeia, but the conclusion does not affect the establishment of this method.

[0155] Take samples from Bailingwei Company to carry out the test in this step. According to the data obtained in the sample pretreatment in Example 2, the recovery rate of the sample after pretreatment should be in the range of 98-101%. Since the average value of the rotary evaporation weight loss of Bailingwei (B) sample is 1.990%, the recovery rate of the sample without pretreatment should be in the range of 96.04-98.99%.

[0156] Accurately weigh 150, 100, 75, 50, and 25 mg of sample B after rotary evaporation, dissolve it in a 100 ml volumetric flask with the above methanol, and make up to volume; accurately weigh 25 mg of sample B after rotary evaporation, dissolve it in a 200 ml volumetric flask with the above methanol, and make up to volume.

[0157] That is, six samples with concentrations of 1.5, 1, 0.75, 0.5, 0.25, and 0.125 mg / ml of B were prepared as standard curves, and two parallel samples with concentrations of 0.75 and 0.5 mg / ml were prepared from the pretreated sample B and the unpretreated sample B0, for a total of eight samples.

[0158] Adjust the UV spectrophotometer to the maximum precision, that is, the absorbance value shows 3 decimal places. Use methanol as a blank and measure the UV absorption of the above prepared sample at 258nm. That is, measure the UV absorption of 1.5, 1, 0.75, 0.5, 0.25, and 0.125 mg / ml samples, and calculate the R of the two curves of 1.5-0.25 and 1-0.125 mg / ml. 2 . R 2 After meeting the requirements, measure the UV absorption of 2 parallel samples with and without pretreatment at concentrations of 0.75 and 0.5 mg / ml, i.e. a total of 8 samples, respectively, and calculate the recovery of each corresponding sample to confirm whether it is within the expected range.

[0159] Confirm the sample detection method, see Fig.12

[0160] (R 2 Should be ≥0.98, 98%≤pre-treatment sample recovery rate≤101%, 96.04%≤unpre-treatment sample recovery rate≤

[0161] 98.99%)

[0162]

[0163] R 2 (Should be ≥0.98)

[0164]

[0165] That is, according to the method of the present invention, the content of the compound with CAS No. 111549-97-4 was quantitatively detected, and the result obtained in this test was 98.19%, which was consistent with the speculation of nuclear magnetic hydrogen spectrum; but without pre-treatment, directly using the commercial product as a sample would lead to a higher detection content. It is speculated that this is because the volatile impurities contained in the product have higher ultraviolet absorption than products of the same quality.

[0166] Conclusion: The sample recovery after pretreatment meets the requirements of the pharmacopoeia; however, commercially available samples cannot be measured directly and should be tested after pretreatment (rotary evaporation).

[0167] Those skilled in the art will readily appreciate other embodiments of the present invention after considering the specification and practicing the invention disclosed herein. This application is intended to cover any variations, uses or adaptations of the present invention that follow the general principles of the present invention and include common knowledge or customary techniques in the art that are not disclosed by the present invention. The specification and examples are to be considered exemplary only, and the true scope and spirit of the present invention are indicated by the following claims.

[0168] It should be understood that the present invention is not limited to what has been described above and that various modifications and changes may be made without departing from its scope. The scope of the present invention is limited only by the appended claims.

Claims

1. A method for detecting compounds based on ultraviolet spectrophotometry, characterized by the following: Step 1: Sample pretreatment: using methanol as solvent, fully dissolving the sample and rotary evaporating to dryness to obtain a sample with volatile impurities removed. Similarly, the sample with volatile impurities removed is also used as the sample to be tested; Step 2: Accurately weigh the sample to be tested, use methanol as solvent, and prepare solutions of different concentrations, ranging from 0.25 mg / ml to 1.5 mg / ml; Step 3: Set the wavelength of the UV spectrophotometer to 258 nm, measure the absorbance of samples of different concentrations, and establish a standard curve of sample concentration and absorbance; Step 4: At a wavelength of 258 nm, the absorbance of the compound with CAS number 111549-97-4 is measured by an ultraviolet spectrophotometer, and the content of the sample compound is calculated by the weight loss ratio measured in step 1 based on the standard curve.

2. The method for detecting compounds based on ultraviolet spectrophotometry according to claim 1, characterized in that: The method is applicable to the compound with CAS number 111549-97-4.

3. The method for detecting compounds based on ultraviolet spectrophotometry according to claim 1, characterized in that: In step 2, sample solutions of different concentrations are prepared, including 1.5 mg / ml, 1.0 mg / ml, 0.75 mg / ml, 0.50 mg / ml and 0.25 mg / ml sample solutions.

Images