Keller Jan-Henning, Karas Michael, Müller Walter E, Volmer Dietrich A, Eckert Gunter P, Tawab Mona Abdel, Blume Henning H, Dingermann Theodor, Schubert-Zsilavecz Manfred
Institute of Pharmaceutical Chemistry, Biocenter, Biocenter, JW Goethe-University, Marie-Curie-Strasse 9, 60439 Frankfurt, Germany.
Anal Chem. 2003 Nov 15;75(22):6084-8. doi: 10.1021/ac034520z.
Hyperforin is one of the essential active ingredients of St. John's wort extract, which is used as an antidepressant for mild to moderately severe depressions. In vitro and in vivo data as well as several clinical studies and meta analyses have confirmed the pharmacological effect of treatment with hyperforin-containing preparations. However, little is known about the brain availability of hyperforin until now. Accordingly, a highly sensitive and selective LC/MS method for this purpose was developed and validated. This method proved suitable for the determination of hyperforin in mouse brain, after oral administration of hyperforin sodium salt and St. John's wort extract. This method involves liquid-liquid extraction of hyperforin with ethyl acetate followed by separation with rapid reversed-phase high-performance liquid chromatography and tandem mass spectrometry detection using electrospray ionization. Excellent linearity was obtained for the entire calibration range from 0.25 to 10 ng/mL (corresponding to 2.5-100 ng/g brain tissue concentration, calculated with the factor derived from sample processing) with an average coefficient of correlation of 0.9992. The recovery of hyperforin from mouse brain homogenates was between 71.4 and 75.3% with a relative standard deviation of less than 3%. Validation assays for the lower limit of quantitation yielded an accuracy of 5.8%. Intraday accuracy and precision for the developed method were between 4.6 and 10.6% and 4.3-8.4%, respectively, while the interday parameters varied between 6.7 and 12.2% for accuracy and 2.0-5.0% for precision. After the method validation, hyperforin brain levels in mice, treated with 15 mg/kg hyperforin (either as the sodium salt or as 5% St. John's wort extract), were investigated. The average concentration of hyperforin found for the sodium salt group was 28.8+/-10.1 ng/g of brain (n = 8), which was somewhat higher than the hyperforin concentration of 15.8+/-10.9 ng/g of brain (n = 8), determined in the extract-treated group. This method is robust, selective, and highly sensitive and represents an appropriate tool to further prove the occurrence and distribution of hyperforin in mouse brain.
金丝桃素是圣约翰草提取物的重要活性成分之一，被用作治疗轻度至中度严重抑郁症的抗抑郁药。体外和体内数据以及多项临床研究和荟萃分析均证实了含金丝桃素制剂治疗的药理作用。然而，迄今为止，关于金丝桃素在脑内的可用性知之甚少。因此，开发并验证了一种用于此目的的高灵敏度和选择性的液相色谱/质谱方法。该方法经证明适用于在口服金丝桃素钠盐和圣约翰草提取物后测定小鼠脑中的金丝桃素。该方法包括用乙酸乙酯对金丝桃素进行液-液萃取，然后通过快速反相高效液相色谱分离，并使用电喷雾电离进行串联质谱检测。在0.25至10 ng/mL的整个校准范围内获得了出色的线性（对应于经样品处理得出的系数计算的脑组织浓度为2.5 - 100 ng/g），平均相关系数为0.9992。从小鼠脑匀浆中回收金丝桃素的回收率在71.4%至75.3%之间，相对标准偏差小于3%。定量下限的验证分析得出的准确度为5.8%。所开发方法的日内准确度和精密度分别在4.6%至10.6%和4.3 - 8.4%之间，而日间参数的准确度在6.7%至12.2%之间，精密度在2.0 - 5.0%之间。在方法验证后，对用15 mg/kg金丝桃素（以钠盐形式或5%圣约翰草提取物形式）处理的小鼠脑中的金丝桃素水平进行了研究。钠盐组中发现的金丝桃素平均浓度为28.8±10.1 ng/g脑（n = 8），略高于提取物处理组中测定的15.8±10.9 ng/g脑（n = 8）的金丝桃素浓度。该方法稳健、选择性高且灵敏度高，是进一步证明金丝桃素在小鼠脑中的存在和分布的合适工具。
