Determination of Trace Levels of Hydroxyhydroquinone in Coffee by High-Performance Liquid Chromatography

A method using high-performance liquid chromatography (HPLC) has been developed for the determination of trace levels of hydroxyhydroquinone (HHQ) in coffee, which inhibits the effects of chlorogenic acids on improving human hypertension. In reversed-phase HPLC coupled to ultraviolet detection (UVD), the use of a 0.1% wt phosphoric acid 0.1 mmol/L 1-hydroxyethane-1, 1-diphosphonic acid (HEDP) 5% methanol aqueous solution (v/v) (pH 2.1) as a preparation solution and a mobile phase enabled a stable measurement of standard HHQ without any time-dependent changes after the preparation. To stably measure HHQ existing in coffee, a pretreatment using a cationic ion-exchange solid-phase extraction was effective. The HPLC coupled to electrochemical detection (ECD) was constructed for a highly sensitive detection of HHQ (the limit of detection was 0.001 mu g/ml, for the HPLC/ECD, compared with 0.05 mu g/mL for the HPLC/LTVD). In the HPLC/UVD and HPLC/ECD, HHQ was detected without any interference from co-existing components in the coffee, and the reproducibility of repeated analyses and the linearity of calibration lines were acceptable. When the HPLC/UVD was applied to a commercial canned coffee containing a relatively high level of HHQ, the quantitative value was 6.03 +/- 0.31 mu g/g, and the recovery of standard HHQ spiked was 99.0 +/- 2.5%. In the analysis of low levels of HHQ in two kinds of canned coffees prepared in our laboratory by HPLC/ECD, the quantitative values were 0.28 +/- 0.01 and 0.65 +/- 0.02 mu g/g, and the recoveries were 101.2 +/- 0.3 and 98.4 +/- 0.7%. This method is useful for the quantitative analysis of HHQ in coffee, and can be an effective tool to study the effects of chlorogenic acids in coffee on improving human hypertension.