Ultra-high-performance liquid chromatography-based identification and quantification of thymoquinone in Nigella sativa extract from different geographical regions

Abstract

Background: Thymoquinone (THQ) is a major bioactive constituent of traditional medicinal plant such as Black seed (Nigella sativa, Family: Ranunculaceae). Objective: The objective of this study was to identify and quantify THQ in Black seeds from different geographical regions and to develop a new ultra-high-performance liquid chromatography-photodiode array detector (UHPLC-PDA) method. Materials and Methods: Black seeds were collected in the end of autumn season. The mobile phase was used in this study [Acetonitrile (2 mM): Ammonium Formate at 50:50 proportions, with a flow rate of 0.200 mL min-1 and also with Isocratic elution on the Accucoro Vanguish C18 UHPLC column (1.5 μm; 100 × 2.1 mm)]. Results: Retention and total run times were 3.147 and 6.0 min, respectively, with injection volume of 5 μL at 254 nm. The method was validated for linearity (r2 ≥ 0.9987), accuracy (≥92.90%), and precision (coefficient of variance [CV] ≤2.54%) with a calibration curve range of 100.00–2000.00 ng/mL. THQ was degraded under ultra-violet-light, basic, acidic, and oxidation stress conditions during forced degradation studies. Our developed UHPLC method reported the degradation peaks of the final product of THQ. UHPLC-PDA analysis showed large variation (0.01%–3.03% w/w) of THQ in the samples of different regions in respect to their concentration and occurrence of metabolite. Conclusions: Turkey has the maximum quantity of THQ throughout the world. The authors suggested that Turkey is the best region for cultivating Black seed plant of superior variety. One more important study performed, i.e., the gas chromatography and mass spectrometry study, reported THQ (2.863%) to be the primary constituent in the extract of N. sativa. Abbreviations used: THQ: Thymoquinone; UHPLC-PDA: Ultra-high-performance liquid chromatography-photodiode array detector; ACN: Acetonitrile; UV: Ultraviolet; GC-MS: Gas chromatography and mass spectrometry; NSO: Nigella sativa extracted oil; TLC: Thin-layer chromatography; IR: Infrared spectroscopy; DSC: Differential scanning calorimetry; QC: Quality control; HQC: High-quality control; MQC: Middle-quality control' LQC: Low-quality control; LLOQQC: Lower limit of quality control; US-FDA: United States of Food and Drug Administration; PSS: Primary stock solution; SS: System suitability; LOD: Limit of detection; LOQ: Limit of quantification.