Impact of protein binding on thymoquinone's analytical detection

Background and Aims: Much data is reported for the promising anticancer activity of Thymoquinone (TQ), an active component of Nigella sativa L. (Ranunculaceae). However, no analytical methods have been reported for its quantification from blood or serum. The aims of this study are: 1) to develop a method for the isolation and detection of TQ from spiked serum, and 2) to determine the impact of protein binding on TQ's analytical detection. Methods: Solid phase extraction/liquid-liquid extraction/protein precipitation, ultracentrifugation-HPLC, and Mass Spectrometry were used. Results: In vitro , TQ prepared in ACN or PBS was detected by a C 18 reversed-phase HPLC-UV assay using an isocratic mobile phase of water: ACN (45:55% v/v) at a flow rate of 1ml/min. The limit of detection was 0.05µg/ml. Interestingly, in spiked serum the average recovery of TQ was 3.25% when 10µg/ml TQ was used and 75% when 100µg/ml TQ was used. The low recovery was due to high protein binding. The percentage of binding was >95% within 10min of incubation. Further investigation showed that bovine serum albumin (BSA) and alpha-acid glycoprotein play a major role in this binding. Binding studies showed that TQ bind covalently to cystein-34 of the amino acid sequence of BSA. Conclusion: This data provides evidence that due to the high percentage of binding (covalent/non-covalent) HPLC methods using unlabeled TQ cannot be used for TQ detection in serum. This finding should be taken in consideration when further pharmacokinetic/pharmacodynamic profiling of TQ is to be performed.