Method for detecting free bilirubin in Wanshi calculus bovis heart-clearing pill

Abstract

The method comprises the following steps: preparing a test solution, taking a bezoar Wanshi heart-clearing pill sample, grinding until the powder is free of particles, the color is not deepened any more and the color of the medicine powder is uniform, adding a solvent for dissolving, then carrying out ice-bath ultrasonic treatment, centrifuging, taking supernate, and filtering to obtain the test solution. According to the method, the sample inspection accuracy and reliability are improved, the controllability of each factor is mastered, the connection of various production environments is facilitated, the cost is greatly saved, and positive economic benefits are generated.

Description

Technical Field

[0001] The invention relates to the detection of bilirubin, in particular to a method for detecting free bilirubin in Wan's Niuhuang Qingxin Pills. Background Art

[0002] The first supplement to the 2015 edition of the Chinese Pharmacopoeia added an inspection of free bilirubin in Wan's Niuhuang Qingxin Pills. Currently, this inspection has the problem of low one-time success rate, and the test results of different people on the same batch of samples vary greatly. The same inspector has a large deviation in the same operation between two samples, which affects the judgment of product quality and the progress of production tasks.

[0003] After investigation, the Shanghai Institute for Drug Control, the drafting unit of the standard, found that the positive factors affecting free bilirubin are: moisture, temperature, pH, etc. However, it is still unclear which factor is the main factor causing the large test deviation. Summary of the invention

[0004] The invention provides a method for detecting free bilirubin in Wanshi Niuhuang Qingxin Pills, which controls the sample grinding time and environmental humidity and the sample moisture so as to improve the one-time success rate of the inspection.

[0005] Specifically, the method for detecting free bilirubin in Wan's Niuhuang Qingxin Pills includes preparing a test solution, taking a sample of Wan's Niuhuang Qingxin Pills, grinding it into a fine powder without particles and the color no longer deepens, and the powder has a uniform color, adding a solvent to dissolve it, then ultrasonically treating it in an ice bath, centrifuging it, taking the supernatant, filtering it, and obtaining the test solution.

[0006] The grinding time is preferably 2 h.

[0007] During grinding, add anhydrous calcium carbonate to disperse the sample so that the sample can be fully extracted.

[0008] Anhydrous calcium carbonate needs to be dried at 105°C for 6 hours before use.

[0009] Furthermore, during the grinding process, a dehumidifier is used to control humidity and maintain the humidity of the environment at 40%.

[0010] The present invention also includes the preparation of a reference substance solution: taking a bilirubin reference substance and adding dichloromethane to prepare a solution containing 6.5 μg of the bilirubin reference substance per 1 ml.

[0011] During ultrasonic treatment, the water temperature was controlled at about 0°C.

[0012] Advantages of the present invention:

[0013] 1. Improve the accuracy and reliability of sample inspection, master the controllability of various factors, and help connect various production environments.

[0014] 2. Generate positive economic benefits

[0015] At present, the unit price of free bilirubin test of Wanshi Niuhuang Qingxin Pills is 400 yuan / time. For each batch of free bilirubin test of Wanshi Niuhuang Qingxin Pills, the existing technology needs to complete 3.8 experiments, and the one-time success rate is only 68.2%, while the present invention only needs one test, and the cost saved = the test fee of the existing technology - the test fee of the present invention = 400×3.8-400×1=1120 yuan. If the free bilirubin test of 20 batches of Wanshi Niuhuang Qingxin Pills and intermediate products is completed in one year, the total cost saved is 1120×20=22400 yuan. DETAILED DESCRIPTION

[0016] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0017] 1. Factor analysis

[0018] Table 1 Current situation survey

[0019]

[0020] Five batches of samples were inspected, with an average experimental number of 3.8 times and a one-time success rate of 68.2%.

[0021] The 11 factors listed in Table 2 were analyzed and verified to be the main factors affecting the one-time success rate of free bilirubin test of Wanshi Niuhuang Qingxin Pills.

[0022] Table 2 Factor confirmation plan

[0023]

[0024]

[0025] 2.2 Confirmation of factors

[0026] 2.2.1 Factor 1-2: Irregular operation and differences in operation by personnel

[0027] The team members conducted a comparison experiment between two inspectors A and B, in which inspector A conducted two tests and inspector B conducted one test, to check and test the free bilirubin on the same batch of Wan's Niuhuang Qingxin Pills.

[0028] Preparation of test solution: Take the product under the weight difference item, cut into pieces, take about 2g, accurately weigh, accurately add anhydrous calcium carbonate in an appropriate amount, specifically add 1 to 2 times the amount according to the water content of the sample, mix thoroughly and grind into powder, take an appropriate amount of powder (equivalent to taking 413mg of the product), accurately weigh, put in a stoppered conical flask, accurately add 20ml of dichloromethane, stopper, weigh the weight, vortex until fully mixed, ultrasonically treat in an ice bath for 30 minutes at a power of 500W and a frequency of 53kHz, weigh the weight again, make up the lost weight with dichloromethane, shake well, centrifuge at a speed of 4000 revolutions per minute, take the dichloromethane solution, filter, and take the filtrate to obtain.

[0029] Result judgment: In the chromatogram of the test sample, the chromatographic peak that appears at the position corresponding to the retention time of the chromatographic peak of the reference sample should be smaller than the chromatographic peak of the reference sample, or no chromatographic peak should appear.

[0030] Table 3 Research results on irregular operation and differences in operation of personnel

[0031]

[0032] After on-site assessment, the operating skills of experimenters A and B meet the requirements. However, judging from the test results in the above table, there is a large deviation in the test results for the same person and for different people. The results cannot be used to judge the product quality.

[0033] Conclusion: Not a major cause.

[0034] 2.2.2 Factor 3: Instability of HPLC

[0035] Confirmation process: The team members conducted two tests by inspectors A and B, respectively, to check the free bilirubin of the same batch of Wanshi Niuhuang Qingxin Pills:

[0036] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel is used as filler; acetonitrile-1% glacial acetic acid (95:5) is used as mobile phase; detection wavelength is 450nm. The theoretical plate number calculated based on the bilirubin peak should be no less than 3000.

[0037] Preparation of reference solution: Take an appropriate amount of bilirubin reference substance, weigh accurately, and add dichloromethane to make a solution containing 6.5 μg per 1 ml.

[0038] Determination method: Accurately pipette 5μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0039] In the chromatogram of the test sample, the chromatographic peak that appears at the position corresponding to the retention time of the chromatographic peak of the reference sample should be smaller than the chromatographic peak of the reference sample, or no chromatographic peak should appear.

[0040] batch Theoretical plate number (standard greater than 3000) Repeatability (less than 2.0%) Separation (less than 1.5) 1 11948 0.98 No impurity peaks around 2 17319 0.62 No impurity peaks around 3 12482 0.72 No impurity peaks around 4 13210 0.83 No impurity peaks around 5 12561 0.90 No impurity peaks around 6 11989 0.56 No impurity peaks around 7 13412 0.75 No impurity peaks around 8 18618 0.94 No impurity peaks around 9 17522 0.64 No impurity peaks around 10 13012 0.55 No impurity peaks around 11 12357 0.71 No impurity peaks around

[0041] The free bilirubin was determined 11 times, and the suitability of the system met the requirements. After the system suitability of the HPLC was investigated, it met the requirements of the pharmacopoeia, indicating that the HPLC was stable and was not the main factor affecting the results.

[0042] Conclusion: Not a major factor.

[0043] 2.2.3 Free bilirubin content of raw material in vitro cultured bezoar

[0044] Confirmation method: Determine the influence of the free bilirubin content of the raw material in vitro cultivated bezoar through comparative experiments.

[0045] Confirmation process: For the four batches of raw materials of in vitro cultured bezoar, the free bilirubin test method of Wanshi Niuhuang Qingxin Pills was compared with the free bilirubin test method of in vitro cultured bezoar:

[0046] Wanshi Niuhuang Qingxin Pills free bilirubin test:

[0047] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; acetonitrile-1% glacial acetic acid (95:5) was used as mobile phase; detection wavelength was 450nm. The theoretical plate number calculated based on the bilirubin peak should not be less than 3000.

[0048] The test solution was prepared by converting the sample amount according to the amount of Wan's Niuhuang Qingxin Pills (equivalent to 413 mg of Wan's Niuhuang Qingxin Pills), accurately weighing, placing in a stoppered conical flask, accurately adding 20 ml of dichloromethane, sealing, weighing, vortexing to mix thoroughly, ultrasonically treating in an ice bath (power 500 W, frequency 53 kHz) for 30 minutes, weighing again, making up the lost weight with dichloromethane, shaking, centrifuging (speed of 4000 revolutions per minute), taking the dichloromethane solution, filtering, and taking the filtrate to obtain the solution.

[0049] Determination method: Accurately pipette 5μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0050] Judgment criteria: In the chromatogram of the test sample, at the position corresponding to the retention time of the chromatographic peak of the reference sample, the chromatographic peak that appears should be smaller than the chromatographic peak of the reference sample, or no chromatographic peak should appear.

[0051] In vitro cultured bezoar free bilirubin test: Free bilirubin Take 10 mg of fine powder of this product, accurately weigh, place in a 5 ml volumetric bottle, add 4 ml of chloroform, warm slightly, cool, add chloroform to the scale, shake well, filter, take the filtrate, and measure the absorbance at a wavelength of 453 nm according to the UV-visible spectrophotometry (General Rule 0401, i.e., Part 4 of the 2020 edition of the Chinese Pharmacopoeia "0401 UV-visible Spectrophotometry"). The absorbance shall not exceed 0.70.

[0052] Table 4 Research results of free bilirubin content of raw materials in vitro cultured bezoar

[0053]

[0054] The in vitro cultured bezoar was examined using two methods, and both methods met the requirements of the pharmacopoeia.

[0055] Conclusion: Not a major factor.

[0056] 2.2.4 Differences in water content and free bilirubin in the scented powder of Wanshi Niuhuang Qingxin Pills

[0057] Confirmation method: Through comparative experiments, the influence of different water contents of the fragrant powder of Wanshi Niuhuang Qingxin Pills and the difference in free bilirubin was determined.

[0058] Confirmation process: For the same batch of Wan's Niuhuang Qingxin Pills with fragrant powder, take 2 grams and dry them in a 90℃ oven for 4 hours, and take another 2 grams and add 40mg water for treatment; after completing the treatments respectively, they are tested according to the free bilirubin test method of Wan's Niuhuang Qingxin Pills.

[0059] Table 5 The results of the study on the difference of free bilirubin between different water content of Wanshi Niuhuang Qingxin Pills with fragrant powder

[0060]

[0061] As a powder, scented powder has better uniformity than pellets. Through this investigation, it can be seen that after adding water, the peak area of ​​free bilirubin increases significantly, and the influence of water is very large.

[0062] Conclusion: Factors.

[0063] 2.15 Wanshi Niuhuang Qingxin Pills with Fragrant Powder Preparation Process

[0064] Confirmation method: Determine the influence of the preparation process of Wanshi Niuhuang Qingxin Pills with fragrant powder through comparative experiments.

[0065] Confirmation process: The scented powder is prepared in the laboratory according to the preparation process, and samples are taken from five different parts for free bilirubin testing and compared with the actual production data.

[0066] Table 6. Research results of the preparation process of Wanshi Niuhuang Qingxin Pills with fragrant powder

[0067]

[0068] By conducting small batch preparations in the laboratory and comparing the free bilirubin test of scented powder with that of normal production batches, it was found that there was no significant difference in the test results of the scented powder production process.

[0069] Conclusion: Not a major factor.

[0070] 2.2.6 Effect of honey water content on free bilirubin after pelleting, and relationship between sample grinding time and sample water content and free bilirubin

[0071] Confirmation method: Through comparative experiments, determine the effect of honey water content on free bilirubin after pelleting, and the relationship between the effect of sample grinding time on sample moisture and free bilirubin.

[0072] Confirmation process:

[0073] 1) The honey was refined to different degrees to control its moisture content, and then the honey was mixed with scented powder prepared in the laboratory to make a medicine ball, which was ground for 1 to 2 hours and divided into two parts for the determination of free bilirubin. The other part was dried in an oven at 105°C for 5 hours to determine the water loss, and then the free bilirubin was determined.

[0074] 2) For the same batch of samples, under the condition of controlling the environmental humidity at about 40%, two different inspectors grind the samples for 20 minutes, 1 hour, and 2 hours, and then compare the free bilirubin measured.

[0075] Table 7 Research results of different treatment degrees of the same batch of honey

[0076]

[0077] Table 8 Research results of the same batch of samples

[0078]

[0079]

[0080] From Table 7, it can be seen that when the water content of honey is 11.6%-13.8%, the impact is not very obvious; the reason may be that after 1 to 2 hours of grinding, when the water content is measured, the difference in water content of each sample is small, so the results are similar. From the theoretical content of no loss of water content in the sample after grinding, the grinding process produces a certain amount of water loss. From Table 8, it can be seen that the results of the free bilirubin peak area of ​​samples with different grinding times for the two inspectors A and B show a downward trend, and when the grinding time is 2h, the sample is ground to powder without particles and the color no longer deepens, and the color of the powder is uniform. The results of the two inspectors are the lowest among the three time lengths. Considering that the grinding time causes water loss, it is the main factor.

[0081] Conclusion: The effect of honey water content on free bilirubin after pelleting was confirmed as a non-factor. The relationship between the effect of sample grinding time on sample moisture and free bilirubin was confirmed as a factor.

[0082] 2.2.7 Ice bath ultrasonic operation of samples

[0083] Confirmation method: Determine the relationship between ice bath ultrasonic operation of the sample and free bilirubin through comparative experiments.

[0084] Confirmation process: For the same batch of scented powder samples, other extraction conditions including the same operator were used, with the ultrasonic temperature set to 30°C for extraction and controlled at 0°C for ice bath extraction, and the peak areas of free bilirubin under the two conditions were compared to determine whether there was a significant impact.

[0085] Table 9 Ice bath ultrasonic operation of samples

[0086]

[0087] Through the test of the same batch of scented powder under the same conditions except ultrasonication, it is obvious that the peak area of ​​free bilirubin under ultrasonication at 30°C is higher than that under ice bath at 0°C.

[0088] Conclusion: Factors.

[0089] 2.2.8 Impact of environmental humidity

[0090] Confirmation method: Determine the relationship between the humidity effect of the environment and free bilirubin through comparative experiments.

[0091] Confirmation process:

[0092] The difference between the moisture content of the scented powder of Wanshi Niuhuang Qingxin Pills and the free bilirubin is affected by the moisture content of the scented powder; and the relationship between the influence of sample grinding time on sample moisture and free bilirubin. When grinding under 40% humidity, the sample moisture has a certain decrease. Through these two studies, it is speculated that the moisture in the air will have a certain impact on the examination of free bilirubin.

[0093] Refer to the data on the difference in water content of the fragrant powder of Wanshi Niuhuang Qingxin Pills and free bilirubin, the effect of sample grinding time on sample moisture and the relationship between free bilirubin.

[0094] Since the previous sample grinding operations were all carried out under 40% conditions, and from the results of the factor analysis in item 2.2.6, namely the different water contents of the fragrant powder of Wan's Niuhuang Qingxin Pills and the difference in free bilirubin, it can be determined that moisture has a greater impact on free bilirubin, so the environmental humidity can be determined as the factor.

[0095] Conclusion: Factors.

[0096] 2.2.9 Effect of ambient temperature on samples

[0097] Confirmation method: Determine the effect of ambient temperature on the sample and its relationship with free bilirubin through comparative experiments.

[0098] Confirmation process: From the results of the sample ice bath ultrasonic operation study, it can be determined that low temperature is conducive to the true detection of free bilirubin in the sample, so this study mainly focuses on the influence of temperature increase conditions. One part of the same bottle of bilirubin reference substance was treated in a 105℃ oven and then used to prepare the free bilirubin reference substance; the other part was directly used to prepare the free bilirubin reference substance; the F values ​​of the two reference substances were compared.

[0099] Table 10 Study results of the influence of ambient temperature on samples

[0100]

[0101] From the above results, it can be seen that high temperature will not affect free bilirubin. From the above 2.2.6 factor analysis, the free bilirubin did not increase significantly when the scented powder was dried at 90℃. In the 2.2.4 factor analysis, another sample was heated at 108℃, and the free bilirubin was lower than the unheated one. It can be seen that heating does not increase free bilirubin, but it can reduce the transfer of bound bilirubin to free bilirubin by reducing the moisture content, making the free bilirubin content closer to the true value of the sample content. However, since the adjustment of the ambient temperature can neither achieve the drying effect nor meet the statutory environmental requirements of the pharmacopoeia, it cannot be implemented as a measure.

[0102] Conclusion: Not a major factor.

[0103] a) Implementation of countermeasures

[0104] The moisture content of scented powder greatly affects the amount of free bilirubin, which indicates that the entire experiment has a significant correlation with the water content. In addition to the ultrasonic temperature required by the pharmacopoeia, controlling the environmental humidity and sample grinding time are all for the purpose of reducing the moisture content. Based on the research results, it is determined that the main reason for the experiment is to reduce the moisture content.

[0105] The sample is controlled to be ground for 1-2 hours under 40% ambient humidity conditions, until the sample is ground into powder without particles and the color no longer deepens, and the powder color is uniform. The ultrasonic temperature is maintained and ice is added in time to cool it down.

[0106] Table 11 Results of the implementation strategy research

[0107]

[0108] According to the above factors, four inspectors tested different batches of samples at the same time, and all the tests were successful at one time. The key factors are moisture and ultrasonic temperature.

[0109] Conclusion: Through the implementation of the above factors and measures, the goal can be achieved and the one-time success rate of the free bilirubin test item of Wanshi Niuhuang Qingxin Pills can be improved.

[0110] 3 Implementation Plan

[0111] Preparation of reference solution: Take the bilirubin reference substance and add dichloromethane to make a solution containing 6.5 μg of bilirubin reference substance per 1 ml.

[0112] Preparation of test solution: Take the product under the weight difference item, cut into pieces, take about 2g, accurately weigh 2.0000g~2.2000g, accurately add 1~2 times the amount of anhydrous calcium carbonate according to the water content of the sample, mix thoroughly and grind for 2h, grind until the powder has no particles and the color no longer deepens, and the color of the powder is uniform, take an appropriate amount of powder (equivalent to 413mg of this product (weighed sample amount)), accurately weigh, put it in a stoppered conical flask, accurately add 20ml of dichloromethane (solvent), plug it tightly, weigh the weight, vortex until fully mixed, power 500W, frequency 53kHz ice bath ultrasonic treatment for 30 minutes, weigh the weight again, make up the lost weight with dichloromethane, make up the lost weight with dichloromethane, shake well, centrifuge for 1min at a speed of 4000 revolutions per minute, take the dichloromethane solution, filter, and take the filtrate to obtain.

[0113] Anhydrous calcium carbonate needs to be dried at 105°C for 6 hours before use.

[0114] When grinding, a dehumidifier is required to control humidity and maintain the humidity at 40%.

[0115] Large honey pills need to be ground into powder without any pill particles, the color no longer deepens, and the color is uniform.

[0116] During ultrasonic treatment, the water temperature was controlled at about 0°C.

[0117] By strictly implementing the formulated countermeasures, controlling the environmental humidity and sample moisture through grinding, and strictly following the ice bath ultrasonic temperature required by the pharmacopoeia, the free bilirubin test applied to the fragrant powder and pellets of Wan's Niuhuang Qingxin Pills ran well and achieved the one-time success rate requirement.

[0118] Table 12 One-time success rate of free bilirubin test of Wanshi Niuhuang Qingxin Pills

[0119]

[0120] The unit price for the free bilirubin test of the Laboratory's Niuhuang Qingxin Pills is 400 yuan per time.

[0121] Table 13 Comparison of the number of completed experiments and one-time success rate

[0122]

[0123] Cost saved for each batch of free bilirubin test of Wanshi Niuhuang Qingxin Pills completed after the activity = pre-activity test fee - post-activity test fee = 400 × 3.8 - 400 × 1 = 1120 yuan;

[0124] From September 2020 to June 2021, a total of 20 batches of Wanshi Niuhuang Qingxin Pills and intermediate products were tested for free bilirubin, saving a total cost of 1120×20=22,400 yuan.

[0125] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for detecting free bilirubin in Wanshi Niuhuang Qingxin Pills, comprising preparing a test solution, characterized in that: Take a sample of Wanshi Niuhuang Qingxin Pills, grind it into a fine powder without particles and the color no longer deepens, and the powder is uniform in color, add a solvent to dissolve it, then ultrasonically treat it in an ice bath, centrifuge it, take the supernatant, filter it, and obtain the test solution.

2. The method for detecting free bilirubin in Wanshi Niuhuang Qingxin Pills according to claim 1, characterized in that: The grinding time is 2h.

3. The method for detecting free bilirubin in Wanshi Niuhuang Qingxin Pills according to claim 1, characterized in that: During the grinding, anhydrous calcium carbonate is added.

4. The method for detecting free bilirubin in Wanshi Niuhuang Qingxin Pills according to claim 3, characterized in that: The anhydrous calcium carbonate needs to be dried at 105° C. for 6 hours before use.

5. The method for detecting free bilirubin in Wanshi Niuhuang Qingxin Pills according to claim 1, characterized in that: During the grinding process, the environment was maintained at a humidity of 40%.

6. The method for detecting free bilirubin in Wanshi Niuhuang Qingxin Pills according to claim 1, characterized in that: It also includes the preparation of a reference solution: take a bilirubin reference substance and add dichloromethane to prepare a solution containing 6.5 μg of bilirubin reference substance per 1 ml.

7. The method for detecting free bilirubin in Wanshi Niuhuang Qingxin Pills according to claim 1, characterized in that: During the ultrasonic treatment, the water temperature is controlled at about 0°C.

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