Development of a Selective, Sensitive, and Rapid LC‐MS/MS Assay for Studying the Effect of Concurrent Intake of Pepper and Turmeric on Curcumin Level in Healthy Human Urineμ

Hypothesis We hypothesized that co‐administration of turmeric with pepper through a meal increases the bioavailability of curcumin (CCM), one of the components of turmeric, which could enhance its beneficial effects. Methods Fasted individuals (n=3) consumed a standardized test meal on two occasions, each separated by one week. Meal 1 consisted of 120 ml egg white (cooked) mixed with 5 g powdered turmeric. Meal 2 contained the same ingredients plus 0.5 g black pepper. At baseline and 10 subsequent time points, participants collected batches of urine for 24 hrs. The volume of urine at each period was measured and a portion of it collected in a 50‐mL Falcon tube and stored in ‐80 o C until analysis. Samples were thawed and aliquoted while protected from light. Then, to 400 μL of the urine sample was added 160 μL of triple‐deionized water, and 50 μL of glibenclamide (20 ng/mL) as internal standard (IS) and extracted twice with 500 μL of Ethyl acetate: Methanol (95:5). After centrifugation at 13.2 x10 3 g for 5 min, the organic phase were separated. The extracted samples were dried, reconstituted with 50 μL of methanol:acetonitrile:water:formic acid (23:41:36:1) used for assay. Separation of analytes was achieved by a C18 column and detected by tandem mass‐spectrometry. The concentration of CCM in urine samples was calculated using standard curves constructed based on 7 standard solutions ranged from 0.625‐40 ng/mL and accordingly, the amount of excreted CCM in individuals 24‐hrs urine was estimated. Results A simple liquid chromatography‐mass spectrometry (LC‐MS/MS) method for quantification of CCM in human urine was successfully developed. The mass spectrometry was more sensitive when negative mode was used. The ion transitions of m/z 367.0 →133.9 and 367.0 →173.0 for CCM, and m/z 494.0 →369.1 and 494 →171.8 for IS were used in multiple reaction mode. The ion spectra of CCM and IS are shown in figure 1. Chromatographic peaks of all analytes were separated with no interference with substances. The retention time of CCM and IS were 3.81, and 3.93 minutes, respectively. The limit of detection and limit of quantification were 0.156 and 0.312 ng/mL respectively. The validated method was then successfully applied to determine the urine concentration of CCM in human. The results of this study show that the total urinary excreted amount of CCM after 24‐hrs in individuals concurrently receiving turmeric and pepper (7.53±0.94 μg /24 h vs 23.71 ± 7.84 μg/24 hr) were significantly higher than individual receiving only turmeric. Conclusion A simple, selective, sensitive, and rapid LC‐MS/MS method was developed and validated to simultaneously determine the concentration of CCM and glibenclamide in human urine samples. This method has many advantages in terms of simple extraction procedure, higher sensitivity, and shorter run time. Moreover, the total urinary execrated CCM significantly increased in the group receiving CCM concomitantly with pepper compared with the group received only turmeric. The observed higher urine concentrations reveal that pepper could promote a higher rate of absorption of and enhance bioavailability of CCM, and increase its beneficial effects.