Application: The method was used to determine the content of paeoniflorin in Paeonia lactiflora Pall extract by HPLC.
Method Principle: The sample was extracted by ultrasonic extraction with a proper amount of dilute ethanol in a conical flask, cooled, shaken and filtered.
The filtrate was injected into a high performance liquid chromatograph for chromatographic separation. The absorption value of paeoniflorin was detected at
230nm wavelength by ultraviolet absorption detector, and its content was calculated.
Reagents:
1. Acetonitrile (chromatographic purity)
2. Phosphate
3. Ethanol
Instruments and equipments:
1. The instruments
1.1 High performance liquid chromatograph
1.2 The chromatographic column:Octadecyl bonded silica gel as filler (4.6mm×250mm)
1.3 Ultraviolet absorption detector
2. Chromatographic conditions
2.1 Mobile phase: acetonitrile 0.1% : phosphoric acid solution = 15:85
2.2 Detection wavelength: 230nm
Sample preparation:
1. Weighing and preparing test samples
Weigh about 10g of test sample, finely ground and evenly, and finely take 0.1g.
2. Preparation of reference solution
Paeoniflorin 1mg was accurately weighed and added with ethanol to make a reference solution of 0.1mg/mL.
3. Preparation of test solution
The test product was placed in a stopper conical flask, and 25ml diluted ethanol was added accurately.Mix, weigh, ultrasonic treatment for 30min, cool down,
then weigh the weight, dilute ethanol to make up for the lost weight, shake well, filter, take the filtrate as the test solution.
Note: "Precise weighing" means that the weighing should be accurate to one thousandth of the weight taken."Precise measuring" means that the accuracy of
measuring volume shall meet the accuracy requirements of the pipet of such volume in the CP standard.
Operation steps:
1. Standard curve drawing
Accurately weigh paeoniflorin solution 4,7,10,13,16 μL and inject into HPLC respectively. The peak area integral value of Paeoniflorin
was determined at 230nm wavelength by ultraviolet absorption detector. The standard curve was drawn by using the peak area integral value of injection volume
regression.
2. Test sample determination
Paeoniflorin was measured at 230nm by ultraviolet absorption detector. The content of Paeoniflorin was calculated by external standard method.