An improved high performance liquid chromatography–photodiode array detection–atmospheric pressure chemical ionization–mass spectrometry method for determination of chlorophylls and their derivatives in freeze-dried and hot-air-dried Rhinacanthus nasutus

Rhinacanthus nasutus (L.) Kurz, a traditional Chinese herb possessing antioxidant and anti-cancer activities, has been reported to contain functional components like carotenoids and chlorophylls. However, the variety and amount of chlorophylls remain uncertain. The objectives of this study were to develop a high performance liquid chromatography–photodiode array detection–atmospheric pressure chemical ionization–mass spectrometry (HPLC–DAD–APCI–MS) method for determination of chlorophylls and their derivatives in hot-air-dried and freeze-dried R. nasutus. An Agilent Eclipse XDB-C18 column and a gradient mobile phase composed of methanol/N,N-dimethylformamide (97:3, v/v), acetonitrile and acetone were employed to separate internal standard zinc-phthalocyanine plus 12 cholorophylls and their derivatives within 21 min, including chlorophyll a, chlorophyll a′, hydroxychlorophyll a, 15-OH-lactone chlorophyll a, chlorophyll b, chlorophyll b′, hydroxychlorophyll b, pheophytin a, pheophytin a′, hydroxypheophytin a, hydroxypheophytin a′ and pheophytin b in hot-air-dried R. nasutus with flow rate at 1 mL/min and detection at 660 nm. But, in freeze-dried R. nasutus, only 4 chlorophylls and their derivatives, including chlorophyll a, chlorophyll a′, chlorophyll b and pheophytin a were detected. Zinc-phthalocyanine was found to be an appropriate internal standard to quantify all the chlorophyll compounds. After quantification by HPLC–DAD, both chlorophyll a and pheophytin a were the most abundant in hot-air-dried R. nasutus, while in freeze-dried R. nasutus, chlorophyll a and chlorophyll b dominated.