Elsevier

Journal of Chromatography B

Volume 796, Issue 2, 5 November 2003, Pages 439-444
Journal of Chromatography B

Short communication
Determination of scutellarin in rat plasma by high-performance liquid chromatography with ultraviolet detection

https://doi.org/10.1016/j.jchromb.2003.08.002Get rights and content

Abstract

A validated high-performance liquid chromatography method is described for the determination of scutellarin in rat plasma using a liquid–liquid extraction and ultraviolet (UV) absorbance detection. The separation used a Diamonsil ODS column (250mm×4.6 mm i.d., 5 μm particle size) with an isocratic mobile phase consisting of methanol–acetonitrile–50 mM dihydrogen ammonium phosphate buffer (22:15:63 (v/v/v), adjusted to pH 2.5 with 1 M phosphoric acid). The ultraviolet detector operated at 335 nm. Plasma samples were extracted with ethyl acetate after acidification. The extraction recovery of scutellarin ranged from 68.1 to 80.5%. High selectivity and a low quantitation limit (0.050 μg/ml) were achieved. The linear range was 0.050–12.5 μg/ml, correlation coefficient r=0.9981. The method has a good reproducibility, R.S.D. values were below 7.9% for within-day and between-day precision. The method is simple, rapid, and applicable to preliminary pharmacokinetic studies of scutellarin in rats.

Introduction

Flavonoids are widely occurring in the plant kingdom and contained in the common human diet, and comprise of flavones, flavonols, flavanones, flavanonols, isoflavone, flavanol, anthocyanidins. Many of these compounds exist as sugar conjugates [1]. Scutellarin, a flavone glucuronide, extracted from a Chinese herb Erigero breviscapus (Vant.) Hand.-Mazz [2], is not only an important component of a Chinese herb, but also a major constituent of Skullcap, a popular western herb. It has been used in the treatment of occlusive cerebral vascular diseases. It is efficacious in the treatment of cerebral infarction, coronary heart disease, angina pectoris [3], [4], [5]. For a better understanding of its pharmacokinetics, it is essential to use a sensitive and precise analytical method to determine the concentration of scutellarin in biological fluids.

There has been active interest in recent years in developing and optimizing analytical methods for detection of flavonoids in foods [6], [7], [8] and biological matrix. A number of methods have been described for flavonoids in biological fluids [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], including the determination of baicalin and baicalein in rat plasma by HPLC with electrochemical detection [9], and the quantitation of wogonin and its major metabolite wogonin-7β-d-glucuronide in rat plasma by LC–MS/MS [10]. Tsuchiya analyzed polyhydroxyflavones in human plasma using HPLC with diode array detection and solid-phase borate-complex extraction [11]. Several recent reports described the analysis of quercetin in human plasma using HPLC with electrochemical detection [12] and the identification of quercetin glucuronides in human plasma by LC–MS/MS [13]. Nielsen et al. determined the apigenin and acacetin in human urine by column-switching HPLC–UV [14]. However, to our knowledge, there is still no method described for the determination of scutellarin in biological fluids.

In the present study, we firstly established an HPLC–UV method that is sensitive, simple and suitable for determining scutellarin in rat plasma.

Section snippets

Materials

Scutellarin was from the Delta Information Center for Natural Organic Compounds (99.5%, Hong Kong, China). Injectio Breviscapini, which is an injection solution of scutellarin (20 mg/5 ml) was produced by Gejiu Bio-Medicine Industry Ltd. (Yunnan, China). 2-(4-Hydroxy-phenyl)-7-(3-morpholin-4-yl-propoxy)-chromen-4-one (internal standard, IS, see Fig. 1) was a kind of gift from Prof. Chun Hu (Shenyang Pharmaceutical University, Shenyang, China). Methanol and acetonitrile were of HPLC-grade, and

Optimization of HPLC conditions

In optimizing the chromatographic conditions, the pH of the mobile phase and organic modifier were explored. The mobile phases of pH 6.5 or 4.0 yielded tailing peaks. When the pH value of the mobile phase decreased from 6.5 to 4.0, the peaks became sharper. The peaks were sharp and symmetric when the pH value of the mobile phase was adjusted to 2.5. The use of an acid modifier is important to suppress ionization of the weak acidic phenolic group and interactions of these groups with residual

Conclusion

In this study, a specific and sensitive assay for the determination of scutellarin in rat plasma was presented. The method is simple, rapid, and applicable to preliminary pharmacokinetic studies of scutellarin in rats.

Acknowledgements

This work was supported by grants 39930180 and 30271525 of the National Natural Science Foundation of China. We are thankful to Professor Chun Hu for the supply of 2-(4-hydroxy-phenyl)-7-(3-morpholin-4-yl-propoxy)-chromen-4-one.

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