Abstract: A method for determining the concentration of asarone in human serum by reversed-phase high-performance liquid chromatography is established. This method is simple, sensitive, and accurate in measurement results.
To establish a method for the determination of asarone concentration in human serum by reversed-phase high-performance liquid chromatography for bioavailability research: Method: Use Shimpack CLCODS (150mm × 6.0mm, 10μm) as the chromatography column, and use methanol-water 80:20) is the mobile phase, the flow rate is 1 ml / min, the fluorescence excitation wavelength is 265 nm, the emission wavelength is 365 nm, and the column temperature is 40 ° C. Results: The lowest detection concentration of Asarum was 0.2ng / ml, with a linear range of 1 ～ 200ng / ml; intraday RSD≤6.74% (n = 3), and intraday RSD≤4.95% (n = 5). Conclusion: This method is simple, sensitive and accurate. It is suitable for the study of asarum pharmacokinetics and bioavailability
Key words: reversed-phase high performance liquid chromatography; asarone; blood drug concentration
Asarum is based on the main active ingredient in the Chinese medicine Shichangpu, a-Asarum (2,4,5-trimethoxy-1-enylbenzene, also known as a-Asarone), which is synthetically Antiepileptic drugs. Since the blood concentration of the drug after entering the human body is very low, it is difficult to study its pharmacokinetics and bioavailability, and it is necessary to find a quantitative detection method with high sensitivity and specificity. In this article, the author established a reversed-phase high-performance liquid chromatography fluorescence detection quantitative method to meet the research requirements of Asarum pharmacokinetics and bioavailability.
l Experimental method
1. Reagents and instruments
Xixinnao reference substance (provided by Guilin Pharmaceutical No. 2 Factory, batch number: 981006); Xixinnao tablet 1, Xixinnao tablet 2 (both developed by Hubei Provincial School of Pharmaceutical Inspection, batch number, 20000405); Xixinnao injection (Second Guilin Pharmaceutical Plant, batch number: 981006); methanol (superior purity, Beijing Chemical Plant); other reagents are analytically pure. LC-6A high-performance liquid chromatography (HPLC) instrument, RF 535 type fluorescence detector (Shimadzn, Japan).
1.2 Chromatographic conditions
Chromatography column: Shimpack CLC ODS (150mm × 6.0mm. 10μm): guard column: Shimpack G ODS (10mm × 4.0mm, 10μm); mobile phase: methanol-water (80:20); flow rate: lml / min; column temperature : 40 ℃; fluorescence excitation wavelength: 265nm, emission wavelength: 365nm.
1. 3 blood sample processing
Take 0.2ml of serum, add 2.5ml of dichloromethane, vortex for 2min, then centrifuge at 2O00g for 10min, all the lower layer liquid was transferred to another sharp bottom test tube, placed in a 35 ° C water bath, and dried with nitrogen. After the residue was dissolved in 50 μl of methanol, 20 μl was injected.
1.4 Preparation of standard curve
Precisely weigh 50mg of Asarum reference substance, place it in a 50ml measuring flask, dissolve with methanol and dilute to the mark, prepare a standard stock solution with a concentration of 1mg / ml. Before use, dilute with methanol to a series of standard working solutions of a certain concentration. Take appropriate amounts of each, and add 0.2ml of blank human serum to prepare standard serum with a concentration of 1, 5, 25, 50, 100, 200ng / ml. , According to the method under "1.3".
1.5 Calculation
Calculate the content of asarum by external standard method.
2 results
2.1 Chromatographic behavior
Under the "1.2" chromatographic conditions, the peak shape of Asarum is symmetrical and steep, without interference from endogenous peaks, and the retention time is 7.68 min.
2.2 Standard curve and linear range Asarone concentration (Y) is in the range of 1 ~ 200ng / ml, and has a good linear relationship with the peak height (X), the regression equation is Y = 20.87X-7.98, r = 0.9998. The lowest detection concentration of serum is 0.2ng / ml.
2.3 Recovery rate and precision test
Within the concentration range of the standard curve, five concentrations of 2.5, 10, 40, 75, and 150 ng / ml were selected for the method recovery test. Each concentration was measured 3 times in parallel and 5 times in parallel during the day. It can be seen that the average recovery rate is (101.18 ± 1.77)%, RSD = 1.75%.
3 Application
Using the determination method established in this article, the author conducted a bioavailability study on Asarum Tablets using different production processes, that is, 18 healthy volunteers were given a single dose of Asarum Tablet 1 (120mg) Tablet 2 (120 mg) and Asarum injection (16 mg) were used to determine the concentration of Asarum in the serum of the subjects. After calculation, the absolute bioavailability of Xixinnao Tablet 1 was (5.4 ± 1.6)%, and that of Xixinnao Tablet 2 was (9.5 ± 2.5)%. Taking Xixinnao Tablet 1 as a reference preparation, the relative bioavailability of Xixinnao Tablet 2 was (181.9 ± 15.2)%. It is proved that the bioavailability of Asarum Tablet 2 (reformed by production process) has increased by 81.9%.
4 Discussion
Asarum has a maximum absorption at a wavelength of 258nm, but its concentration in human blood is extremely low, only reaching the level of ng / ml, and the ultraviolet detector cannot meet the measurement requirements. In this article, the author chooses the fluorescence detection method according to the characteristics of the chemical structure of Asarum (containing 3 methoxy and 1 propenyl benzene ring), the sensitivity is greatly improved, which is higher than that of thin layer chromatography and HPLC-UV method. After being absorbed, Xinnao can quickly enter the red blood cells, and its concentration in whole blood, blood cells, and plasma is the highest in blood cells. "Therefore, when separating serum, special care should be taken not to cause hemolysis.
Asarum has a low melting point. When dichloromethane is heated and dried, the temperature should preferably not exceed 35 ° C. In addition, the concentration range measured by this method is wide, and the low concentration quantification often has large errors. Therefore, the standard curve can be divided into two concentration ranges during operation to reduce the measurement error.
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