Method for determining content and content uniformity of menadione sodium bisulfite powder (for aquatic products) by RP-HPLC (Reverse Phase-High Performance Liquid Chromatography) method

Abstract

The invention provides a method for determining the content and the content uniformity of menadione sodium bisulfite powder (for aquatic products) by an RP-HPLC (Reverse Phase-High Performance Liquid Chromatography) method, and belongs to the technical field of veterinary drug detection. The method is high in precision, good in accuracy, strong in specificity, good in stability and simple and convenient to operate. Compared with the prior art, the detection method has the advantages that the content and the uniformity of menadione sodium hydrogen sulfite can be more accurately detected, the production quality and the stability of products can be effectively controlled, and the product quality and the market competitiveness are improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of veterinary drug detection, and relates to a determination method of content and content uniformity thereof. BACKGROUND

[0002] Sodium bisulfite menadione is a vitamin drug, mainly used for blocking jaundice, biliary fistula, chronic diarrhea, and absorption dysfunction caused by extensive intestinal resection, participating in the synthesis of thrombin in animal liver, promoting the formation of thromboplastin, accelerating blood clotting, and maintaining normal clotting time. It is used for auxiliary treatment of hemorrhage and septicemia of fish, carp, turtle and other aquaculture animals.

[0003] At present, the method for detecting the content and content uniformity of sodium bisulfite menadione powder (for aquaculture) is the Veterinary Drug Quality Standard 2017 edition (chemical medicine volume), the content determination is to extract menadione and then detect it by ultraviolet spectrophotometry. This method needs to extract menadione for many times, and the specification of sodium bisulfite menadione powder (for aquaculture) is only 1%, and the content uniformity needs to be determined. The detection steps of this method are relatively complicated, and the reproducibility and precision are relatively low. SUMMARY

[0004] In order to overcome the shortcomings of the above detection technology, the application provides a detection method for determining the content and content uniformity of sodium bisulfite menadione powder (for aquaculture) by RP-HPLC. Compared with the prior art, the content and content uniformity of sodium bisulfite menadione can be measured more quickly, simply and accurately. The production quality and stability of the product can be effectively controlled, and the product quality and market competitiveness can be improved.

[0005] The detection method and steps are as follows:

[0006] (1) Chromatographic conditions

[0007] Chromatographic column: C18 column, 4.6X250mm, particle size 5um

[0008] Mobile phase: 0.15mol / L sodium dihydrogen phosphate solution-methanol (70:30)

[0009] Flow rate: 1.0ml / min

[0010] Detection wavelength: 230nm

[0011] Column temperature: 30℃

[0012] Injection volume: 20ul

[0013] Detector: diode array detector

[0014] (2) Preparation of reference solution

[0015] Accurately weigh 10 mg of menadione reference substance into a 100-ml brown volumetric flask, dissolve in 5 ml of methanol, add 5 ml of 10% sodium bisulfite solution, shake, dilute with water to the mark, accurately pipette 5 ml into a 50-ml brown volumetric flask, dilute with water to the mark, shake well, and use as the reference substance solution.

[0016] (3) Preparation of the test solution

[0017] Accurately weigh 0.5 g of the test sample (equivalent to 5 mg of menadione sodium bisulfite) into a 200-ml brown volumetric flask, add an appropriate amount of water, shake to dissolve the menadione sodium bisulfite, add 1 ml of 10% sodium bisulfite solution, dilute with water to the mark, shake well, filter, and use as the test solution.

[0018] Content and content uniformity determination method

[0019] Accurately weigh 10 portions of the test sample to prepare the test solution according to the above method (3) with light protection. Accurately pipette 20 ul of the reference substance solution and the test solution into the liquid chromatograph, record the chromatogram, calculate the content of menadione sodium bisulfite in the test sample by the external standard method with peak area, multiply the result by 1.918, and the content of menadione sodium bisulfite in the test sample is obtained.

[0020] The methanol is chromatographically pure, the water is ultrapure water, and the sodium bisulfite is premium grade.

[0021] The method provided by the application for determining the content and content uniformity of menadione sodium bisulfite powder for aquatic use by RP-HPLC has the advantages of improved operation steps, simple operation, high precision, good reproducibility, strong specificity, good stability, maximum guarantee of product quality stability and safety of veterinary drugs, effective control of product production quality and stability, improvement of product quality and market competitiveness, and practical value for promotion in the veterinary industry. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The chromatogram of the menadione reference substance solution.

[0023] Figure 2 The chromatogram of the test solution of menadione sodium bisulfite powder.

[0024] Figure 3 The chromatogram of the blank preparation solution. DETAILED DESCRIPTION

[0025] The method for determining the content and content uniformity of menadione sodium bisulfite powder for aquatic use by RP-HPLC has the following steps:

[0026] (1) Chromatographic conditions.

[0027] Column: C18 column, 4.6X250mm, 5um

[0028] Mobile phase: 0.15mol / L sodium dihydrogen phosphate solution - methanol (70:30)

[0029] Flow rate: 1.0ml / min

[0030] Detection wavelength: 230nm

[0031] Column temperature: 30℃

[0032] Injection volume: 20ul

[0033] Detector: diode array detector

[0034] (2) Preparation of the control solution

[0035] Accurately weigh 10mg of menadione control, put it in a 100ml brown volumetric flask, dissolve it with 5ml of methanol, add 5ml of 10% sodium bisulfite solution, shake, dilute with water to the mark, accurately take 5ml, put it in a 50ml brown volumetric flask, dilute with water to the mark, shake well, and use it as the control solution.

[0036] (3) Preparation of the test solution

[0037] Accurately weigh 0.5g of the test product (equivalent to 5mg of sodium bisulfite menadione), put it in a 200ml brown volumetric flask, add water, shake to dissolve sodium bisulfite menadione, add 1ml of 10% sodium bisulfite solution, and dilute with water to the mark, shake well, filter, and use it as the test solution.

[0038] (4) Content and content uniformity determination method

[0039] Accurately weigh 10 portions of the test product and prepare the test solution according to the above (3) method, and operate in the dark. Accurately take 20ul of the control solution and the test solution, inject it into the liquid chromatograph, record the chromatogram, calculate the peak area by external standard method, multiply the result by 1.918, and the content of sodium bisulfite menadione in the test product is obtained.

[0040] The chromatogram of the menadione control solution at a wavelength of 230nm is shown in Figure 1 , and the chromatogram of the sodium bisulfite menadione powder test solution at 230nm is shown in Figure 2 .

[0041] Methodology:

[0042] 1. Specificity

[0043] Prepare a blank preparation without sodium bisulfite menadione, inject it according to the above chromatographic conditions, and the chromatogram at a wavelength of 230nm is shown inFigure 3 The results showed that the blank formulation did not cause any interference at the corresponding retention time of the menadione peak in sodium bisulfite.

[0044] 2. Linear relationship

[0045] Accurately weigh 100 mg of menadione sodium bisulfite raw material and place it in a 200 ml brown volumetric flask. Add 1 ml of 10% sodium bisulfite solution and dilute with water to the mark. Shake well to prepare the stock solution. Accurately measure appropriate amounts of the stock solution and dilute with water to obtain solutions with sodium menadione sodium bisulfite concentrations of 5, 10, 25, 50, 100, and 250 μg / ml. Inject 20 μl of each solution and record the chromatograms. Perform linear regression with concentration (Y) as the x-axis and peak area (X) as the y-axis. The results are shown in Table 1. Sodium menadione sodium bisulfite showed good linearity in the concentration range of 5–250 μg / ml.

[0046] Table 1 Results of linear relationship examination Drug Regression equation Linear range (ug / ml) [R 2 ]]> Sodium bisulfite menadione Y = 56624X - 5531.4 5～250 0.9998

[0047] 3. Precision

[0048] Take 20 μL of the above reference solution, operate and determine according to the above chromatographic conditions, inject the sample 6 times consecutively, record the chromatogram, and calculate the relative standard deviation (RSD) based on the peak areas of the 6 times.

[0049] The results showed that the RSD of the 6th peak area was 0.6%, which proves that the present invention has good precision.

[0050] 4. Recovery rate

[0051] Precisely weigh approximately 16 mg, 20 mg, and 24 mg of sodium menadione bisulfite raw material, add approximately 2 g of excipients, and place each in a 200 ml brown volumetric flask. Perform the determination under the chromatographic conditions described above, record the chromatogram, and calculate the content of sodium menadione bisulfite using the external standard method.

[0052] The results are shown in Table 2. The average recovery rate of the three batches with different amounts of raw materials added was ≥99.5%, and the RSD was ≤1.0%, which proves that the present invention has a high recovery rate.

[0053] Table 2 Results of Recovery Rate Study Amount of raw material added Average recovery (%) RSD (%) 16 mg 99.79 0.8 20 mg 100.82 0.9 24 mg 100.01 0.6

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above embodiments, those skilled in the art should understand that various changes can be made to it in form and detail, and all such changes should fall within the protection scope of the present invention without departing from the technical solutions of the present invention.

Claims

1. A method for determining the content and content uniformity of sodium menadione bisulfite powder (for aquaculture) by RP-HPLC, characterized in that, The detection method is as follows: (1) Chromatographic conditions Chromatographic column: C18 column, 4.6 x 250 mm, particle size 5 μm Mobile phase: 0.15 mol / L sodium dihydrogen phosphate solution — methanol (70:30) Flow rate: 1.0 ml / min Detection wavelength: 230nm Column temperature: 30℃ Injection volume: 20ul Detector: Diode array detector (2) Preparation of reference solution Accurately weigh 10 mg of menadione reference standard and place it in a 100 ml brown volumetric flask. Dissolve it in 5 ml of methanol, add 5 ml of 10% sodium bisulfite solution, shake, and dilute with water to the mark. Accurately measure 5 ml of this solution and place it in a 50 ml brown volumetric flask. Dilute with water to the mark and mix well to obtain the reference solution. (3) Preparation of the test solution Accurately weigh 0.5 g of the test sample (equivalent to 5 mg of sodium menadione bisulfite), place it in a 200 ml brown volumetric flask, add an appropriate amount of water, shake to dissolve sodium menadione bisulfite, add 1 ml of 10% sodium bisulfite solution, and dilute with water to the mark, shake well, filter, and obtain the test solution. (4) Method for determining content and content uniformity Accurately weigh 10 portions of the test sample and prepare the test sample solution according to the method described in (3) above, and operate in the dark. Accurately measure 20 μL each of the reference solution and the test sample solution, inject them into the liquid chromatograph, record the chromatogram, calculate the content of sodium menadione bisulfite in the test sample by peak area according to the external standard method, and multiply the result by 1.

918.

2. The method for detecting sodium bisulfite menadione powder (for aquaculture) according to claim 1, characterized in that... The menadione reference standard used to prepare the reference solution had a purity of ≥99.0%, the methanol was chromatographic grade, and the water was ultrapure water.

3. The method for detecting sodium bisulfite menadione powder (for aquaculture) according to claim 1, characterized in that... The sodium bisulfite in the added sodium bisulfite solution is of extremely high purity, with a concentration of 10%.

4. The method for detecting sodium bisulfite menadione powder (for aquaculture) according to claim 1, characterized in that... When preparing the reference solution, the amount of sodium bisulfite solution added should be 0.5% of the volume of the solution after dilution.

5. The method for detecting sodium bisulfite menadione powder (for aquaculture) according to claim 1, characterized in that... When preparing the test solution, the amount of sodium bisulfite solution added should be 0.5% of the volume of the solution after final volume adjustment.

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