Single-drop liquid-phase microextraction for the determination of hypericin, pseudohypericin and hyperforin in biological fluids by high performance liquid chromatography.

ABSTRACT

The analysis of hypericin, pseudohypericin (collectively called in this study hypericins) and hyperforin in biological fluids is reported using single-drop liquid-phase microextraction in conjunction with HPLC-UV-fluorescence detection. A new option for analysis of the active principle constituents in biological samples is proposed, reducing the steps required prior to analysis. There are several parameters which determine the mass transfer such as the extraction solvent, drop and sample volumes, extraction time and temperature, pH and ionic strength, stirring rate and depth of needle tip in the bulk solution. These parameters were chosen to optimize the performance in the current study. The method was validated with respect to precision, accuracy and specificity. The intra-day precision values were below 2.3% for the high concentration level of control samples and 6.2% for the low level. The respective inter-day precision values were calculated to be below 4.4 and 7.1%, respectively, for the two concentration levels. Accuracy of the method, calculated as relative error, ranged from -2.6 to 7.0%. It was demonstrated that as long as the extraction procedure is consistently applied, quantitative analysis is performed accurately and reproducibly in human urine and plasma samples. Limits of quantitation (LOQs) in urine were calculated to be 3, 6 and 12 ng/ml for pseudohypericin, hypericin and hyperforin, respectively. Slightly higher limits were measured in plasma, i.e. 5, 12 and 20 ng/ml, for the respective analytes.