Shenkang Injection is made of 4 traditional Chinese medicines such as Astragalus and Salvia miltiorrhiza. It is a new drug used for the treatment of chronic renal failure. The method of administration is intravenous drip. It has been reported in the literature that the content of protocatechuic aldehyde in Salvia miltiorrhiza was determined by TLC [1,2] or HPLC [3 ~ 5]. In this paper, the TLC method was used as a purification method for sample preparation, and the content of protocatechuic aldehyde in Shenshen injection Shenkang injection was determined by HPLC. The method was accurate, sensitive and reproducible.
Organ package, also called transaction package, is a kind of document package that is often used in business and office.Because of its internal partition, pull open the same as the organ, so named for the organ package.The organ bag has various materials and sizes, which can be used to store all kinds of office documents in daily office, which is convenient to carry and easy to save.The PP organ bag produced by our company is made of high quality environmental protection and durable PP raw material. Generally, a transparent name card bag is pressed on the front, and the button switch or hand carry is provided for easy carrying.
Expanding Folders,Expanding File Folder,Pp Expanding File Folder,Custom Expanding Folders shaoxing chaofeng stationery manufacturing CO.,LTD. , https://www.chaofengstationery.com
1 Drugs and reagents Protocatechualdehyde reference product is provided by China National Institute for the Control of Pharmaceutical and Biological Products; Shenkang Injection was developed by the Preparation Research Office of Chengdu Institute of Traditional Chinese Medicine, batch number: 910314, 920815, 931014, specifications: 100 mL per bottle; The experimental water is double-distilled water; the remaining reagents are of analytical grade.
2 Instrument and chromatographic conditions
STI 5000 liquid chromatograph, ultraviolet detector. Analysis column: Shimpack ODS column (5 μm, 6 mm × 150 mm); pre-column: YWG-C18 column (7 μm, 4.6 mm × 150 mm); mobile phase: methanol-water-glacial acetic acid (24: 75: 1 ); Flow rate: 1.0 mL.min-1; column temperature: 30 ℃; detection wavelength: 328 nm.
3 Determination of the linear regression equation Take the original catechol reference substance, add methanol to dissolve and dilute to 1 mg * mL-1 reference substance stock solution, put it in the refrigerator at 4 ℃ and store it for use; precisely measure the appropriate amount of the stock solution and mix with methanol A control solution containing protocatechuic aldehydes of 20, 40, 60, 70, 80, and 120 μg. ML-1 was injected, each with 10 μL, and measured according to chromatographic conditions. The area formed by the baseline of protocatechualdehyde is called the peak area. A = × σ × h = 2.507σh = 1.064 Wh / 2h> Peak area (Y) returns to the reference solution concentration (X) to obtain a linear equation:
Y = 177029 + 70651X r = 0.9996
The results showed that the detection limit of protocatechualdehyde was 0.9 μg.mL-1 (S / N = 3: 1), and the linear range was 20 ～ 120 μg.mL-1.
4 Determination method
4.1 Preparing the test solution, accurately weigh 20 mL of the sample, place it in a 60 mL separatory funnel wetted with water, add 5 mL of saturated sodium chloride solution, extract 3 times with ether, 20 mL each time, shake 1 min, combine the extracts, evaporate to dryness in a water bath (60-65 ℃), dissolve the residue in absolute ethanol, quantitatively transfer to a 1 mL volumetric flask, and dilute to the mark as a test solution for TLC separation.
4.2 TLC Separation and HPLC Measurement Precisely weigh 200 μL of the above test solution, and spot it on a thin layer plate containing 0.5% CMC silica gel G (10 cm × 20 cm, layer thickness 0.5 mm, activated at 105 ℃ for 30 min, spot 5 cm strip), another point 1 mg.mL-1 protocatechualdehyde reference substance anhydrous ethanol solution 2 μL on the other side as a control, using the developing agent chloroform-benzene-ethyl acetate-carboxylic acid (15: 10: 8 ： 1.8) Unfold, remove and evaporate the solvent, dry for 30 min, under 360 nm ultraviolet light (see Figure 1), scratch the scratches with the bright yellow fluorescent strips before and after the original catechol reference product Inside the silica gel powder, place in a centrifuge tube with a stopper, add 10 mL of absolute ethanol, shake vigorously for 1 min, centrifuge (3000 r.min-1, 5 min), and pour out the supernatant. Repeat the elution three times in this way, combine the eluents, evaporate to dryness, and dissolve the residue in a few times with an appropriate amount of methanol and transfer to a 1 mL volumetric flask. (See Figure 2-A), use the two-point interpolation method to calculate the content of protocatechuic aldehyde.
4.3 Blank check Take 20 mL of blank sample (940624) without Salvia miltiorrhiza, according to the items "4.1" and "4.2", draw 10 μL into the liquid chromatograph (see Figure 2-B), it can be seen that the blank sample came out of the original catechol There is no interference peak at the peak.
5 Determination of sample recovery rate Accurately measure the sample with known content, quantitatively add protocatechualdehyde reference substance solutions of different concentration levels, and determine according to the above measurement
6 Repeatability measurement Take 10 μL of 40 μg.mL-1 reference solution and inject 6 times in succession. The RSD of the peak area measured is 0.11%.
7 Sample determination Take 20 mL of sample, process according to item "4.1", "4.2" method, inject 10 μL, determine the content of batch 910314, 920815, 931014 batch samples are 3.191, 3.063, 3.108 μg.mL-1 (n = 3 ), RSD are 0.22%, 0.31%, 0.19%.
8 Discussion
8.1 After extracting samples with different solvents, dissolve the sample directly, the separation effect is not ideal, and the spectrum is messy. Column chromatography and thin layer method were used to compare the pretreatment results. The thin layer method was simple and easy to use, and some impurities were discarded.
8.2 Under 360 nm ultraviolet rays, the protocatechuic aldehyde spots are not fluorescent, so the ellipse is eluted with the bright yellow fluorescent stripe spots before and after the protocatechuic control spots to ensure that all protocatechuic aldehydes in the sample are scraped off.
8.3 During the sample pretreatment, chloroform, ethyl formate, benzene and ether were used for extraction. The result was better with ether extraction, and the recovery rate after three extractions was 99%. Saturated sodium chloride is added to prevent emulsification during extraction.
Determination of protocatechuic aldehyde in Shenkang injection by liquid chromatography