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Biologically Active Molecules
Various bioactive molecules are today isolated and/or synthesized to be used in products for food and drug industry. Our work involves development of selective isolation methods, preferentially using nontoxic solvents such as carbon dioxide and water, for antioxidants, vitamins, essential oils and other important plant components. Identification of those species is performed using chromatographic techniques with mass spectrometric detection.
Determination of resveratrol and its metabolites in serum and liver samples
Trans-resveratrol (RES), a naturally occurring molecule found in many foods, has been associated with a variety of beneficial biological activities, including anti-cancer, analgesic, and cardio- and neuro-protective processes. Methods using liquid chromatography-mass spectrometry with electrospray ionization (ESI) have previously been employed to detect and quantify RES in blood and tissue samples; however, the ESI conditions were not optimized for the highest sensitivity. The goal is to present an optimization of chromatographic and ESI conditions using design of experiments (DOE) with liquid chromatography-high resolution time of flight mass spectrometry (ESI-LC-HR-TOF MS) to measure levels of RES and two metabolites in rat serum and liver samples.
Determination of Celecoxib in human plasma samples
A sensitive method for the determination of Celecoxib (CXB) in human plasma samples was developed using liquid chromatography coupled to electrospray ionization and time of flight mass spectrometry (LC-ESI-TOF-MS). A full factorial design of experiments (FF-DOE) methodology was applied to optimize the ESI conditions for CXB determination and also to predict the effects of interactions of multiple parameters affecting ionization (i.e., capillary voltage, fragmentor voltage, electrolyte and electrolyte concentration). The optimum ionization voltages were 4500V and 220V for capillary and fragmentor, respectively. Even though the highest ESI efficiency was obtained without electrolytes, the addition of 1.0mM ammonium acetate was shown to be essential to buffer the matrix effect and ensure a consistent response. In contrast to previous studies, deuterated CXB was used as a recovery (surrogate) standard, which enabled the correction of CXB loss during sample preparation. The extraction recovery using solid phase extraction was 87-98%. The instrumental limit of detection of CXB (LOD), 0.33ng/mL, and matrix affected LOD, 0.55ng/mL, were similar and comparable to the previously reported LC-MS/MS LODs. This method was employed to determine CXB concentrations in human plasma samples. Upon administration of 400mg CXB to the healthy women, the concentrations found in the plasma were 440-3300ng/mL. The inter-day repeatability was less than 4% RSD.
Determination of lignans in flaxseed
At present, the methods such as LC/MS/MS and GC/MS in selected ion monitoring mode (SIM) employed for characterization of lignans are focused on the determination of known species. We have developed a new method using HPLC with high resolution time of flight MS (TOF MS) for the determination of flaxseed-derived lignans. The analytical method was optimized, and compared to two existing methods (HPLC/MS/MS and GC/MS). For the HPLC/TOF MS method, we have obtained comparable limits of detection (LODs) of 0.002–0.043 pg (injected) to the those acquired with optimized and improved HPLC/MS/MS (0.001–0.015 pg). As expected the LODs for the optimized GC/MS (SIM) were higher (0.02–3.0 pg), nevertheless lower than those reported previously. Besides the optimization of determination method, several key flaxseed sample preparation parameters (including extraction, hydrolysis, and sample purification) were evaluated resulting in the development of efficient protocol for lignan quantification from flaxseed hulls and embryos. The results confirmed the importance of simultaneous quantification of both aglycones and unhydrolyzed glucosides in order to obtain accurate total lignan concentrations. The high resolution TOF MS enabled a confirmation of identity of suspected but unknown species. This works was published by Popova et al. J. Chromatogr. A 2009, 1216 (2), 217–229.
Disclaimer: Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation (NSF), Department of Energy (DOE) or any other funding agency mentioned above.