Development of an HPLC/Diode-Array Detector Method for Simultaneous Determination of Trigonelline, Nicotinic Acid, and Caffeine in Coffee

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Abstract
This paper describes an adequate procedure of reversed-phase HPLC/diode-array detector to be used in quality control to simultaneously quantify three nitrogen compounds: trigonelline, nicotinic acid, and caffeine, in coffee samples either in the green or roasted states. The chromatographic separation was achieved using a reversed-phase column (Spherisorb ODS2) with gradient elution of 0.01M phosphate buffer pH 4.0 (A) and methanol (B). The effluent was monitored by a diode-array detector and the chromatograms were recorded at 265 nm. The sample preparation was quite simple involving only boiling water extraction and filtration. A linear relationship was found between peak area and concentration range of 0.15–450 μg/mL, 0.10–500 μg/mL, and 0.05–500 μg/mL for trigonelline (at 268 nm), nicotinic acid (at 264 nm), and caffeine (at 276 nm), respectively. Extensive quality assurance of the proposed method was performed by the standard addition method in both green and roasted coffee. The precision in green coffee samples was better than 1.3, 5.8, and 1.1% and, for roasted coffee, better than 0.5, 2.4, and 1.2% for trigonelline, nicotinic acid, and caffeine, respectively. Also, for green coffee, the mean recovery values were 98 ± 1%, 84 ± 5%, and 99 ± 1% and for roasted coffee, these were 101 ± 1%, 98 ± 1%, and 99 ± 1% for trigonelline, nicotinic acid, and caffeine, respectively. The proposed method appears to be an adequate method for quality control in the coffee industry.