UHPLC-ESI-HRMS Quantitation of Metabolites without Using Reference Standards: Impact of LC Flow Rate and Mobile Phase Composition on MS Responses


During LC-MS quantitation of drugs for pharmacokinetic assessment, usually metabolites are not quantified due to the unavailability of reference standards. If a metabolite is quantified without a reference standard, then it is assumed that the LC-MS response to a drug is similar to that of its metabolite and the standard curve, of the parent compound, is used to quantitate the metabolite. This approach could result in an over or underestimation of the metabolite. To evaluate the impact of mobile phase composition on LC-MS response, the mobile phases were interchanged between methanol, acetonitrile and a 50/50 mixture of methanol/acetonitrile. UHPLC flow rates were varied from 200-500 μL/min, with and without the addition of reverse composition of mobile phases, at the parent drug retention time. This change was necessary to achieve uniform MS responses for drugs and their metabolites. In this study, HRMS data, obtained using orbi-trap, resulted in a linear response over a wider dynamic range than that obtained using the linear ion trap. Overall, the parameters, required for achieving standard free quantitation, are dependent upon the mobile phase composition and LC flow rates.

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S. Katragadda, S. Mahmoud and D. Ramanathan, "UHPLC-ESI-HRMS Quantitation of Metabolites without Using Reference Standards: Impact of LC Flow Rate and Mobile Phase Composition on MS Responses," American Journal of Analytical Chemistry, Vol. 4 No. 10B, 2013, pp. 36-46. doi: 10.4236/ajac.2013.410A2005.

Conflicts of Interest

The authors declare no conflicts of interest.


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