Back
 AJAC  Vol.4 No.10 B , October 2013
UHPLC-ESI-HRMS Quantitation of Metabolites without Using Reference Standards: Impact of LC Flow Rate and Mobile Phase Composition on MS Responses
Abstract: 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.
Cite this paper: 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. 10, 2013, pp. 36-46. doi: 10.4236/ajac.2013.410A2005.
References

[1]   M. Jemal and Y. Q. Xia, “LC-MS Development Strategies for Quantitative Bioanalysis,” Current Drug Metabolism, Vol. 7, No. 5, 2006, pp. 491-502.
http://dx.doi.org/10.2174/138920006777697927

[2]   W. A. Korfmacher, “Mass Spectrometry in Early Pharmacokinetic Investigations,” Methods and Principles in Medicinal Chemistry, Vol. 36, 2007, pp. 401-428.

[3]   M. Holcapek, L. Kolárová and M. Nobilis, “High-Performance Liquid Chromatography-Tandem mass Spectrometry in the Identification and Determination of Phase I and Phase II Drug Metabolites,” Analytical and Bioanalytical Chemistry, Vol. 391, No. 1, 2008, pp. 59-78.
http://dx.doi.org/10.1007/s00216-008-1962-7

[4]   Y. Hsieh and W. A. Korfmacher, “Increasing Speed and Throughput When Using HPLC-MS/MS Systems for Drug Metabolism and Pharmacokinetic Screening,” Current Drug Metabolism, Vol. 7, No. 5, 2006, pp. 479-489.
http://dx.doi.org/10.2174/138920006777697963

[5]   W. Korfmacher, “High-Resolution Mass Spectrometry Will Dramatically Change Our Drug-Discovery Bioanalysis Procedures,” Bioanalysis, Vol. 3, No. 11, 2011, pp. 1169-1171.
http://dx.doi.org/10.4155/bio.11.98

[6]   W. A. Korfmacher, “Principles and Applications of LC-MS in New Drug Discovery,” Drug Discovery Today, Vol. 10, No. 20, 2005, pp. 1357-1367.
http://dx.doi.org/10.1016/S1359-6446(05)03620-2

[7]   R. Ramanathan, M. Jemal, S. Ramagiri, Y. Q. Xia, W. G. Humpreys, T. Olah and W. A. Korfmacher, “It Is Time for a Paradigm Shift in Drug Discovery Bioanalysis: From SRM to HRMS,” Journal of Mass Spectrometry, Vol. 46, No. 6, 2011, pp. 595-601.
http://dx.doi.org/10.1002/jms.1921

[8]   R. Ramanathan and W. Korfmacher, “The Emergence of High Resolution Mass Spectrometry as the Premier Analytical Tool in the Pharmaceutical Bioanalysis Arena,” Bioanalyis, Vol. 4, 2012, pp. 501-503.

[9]   R. Ramanathan, R. Zhong, N. Blumenkrantz, S. K. Chowdhury and K. B. Alton, “Response Normalized Liquid Chromatography Nanospray Ionization Mass Spectrometry,” Journal of the American Society for Mass Spectrometry, Vol. 18, No. 10, 2007, pp. 1891-1899.
http://dx.doi.org/10.1016/j.jasms.2007.07.022

[10]   L. K. Kamdem, D. A. Flockhart and Z. Desta, “In Vitro Cytochrome P450-Mediated Metabolism of Exemestane,” Drug Metabolism and Disposition, Vol. 39, No. 1, 2011, pp. 98-105.
http://dx.doi.org/10.1124/dmd.110.032276

[11]   D. Liu, X. Zheng, Y. Tang, J. Zi, Y. Nan, S. Wang, C. Xiao, J. Zhu and C. Chen, “Metabolism of Tanshinol Borneolester in Rat and Human Liver Microsomes,” Drug Metabolism and Disposition, Vol. 38, No. 9, 2010, pp. 1464-1470.
http://dx.doi.org/10.1124/dmd.110.033381

[12]   M. Shebley, U. M. Kent, D. P. Ballou and P. F. Hollenberg, “Mechanistic Analysis of the Inactivation of Cytochrome P450 2B6 by Phencyclidine: Effects on Substrate Binding, electron Transfer, and Uncoupling,” Drug Metabolism and Disposition, Vol. 37, No. 4, 2009, pp. 745-752. http://dx.doi.org/10.1124/dmd.108.024661

[13]   R. Ramanathan, J. L. Josephs, M. Jemal, M. Arnold and W. G. Humphreys, “Novel MS Solutions Inspired by MIST,” Bioanalyis, Vol. 2, No. 7, 2010, pp. 1291-1313.
http://dx.doi.org/10.4155/bio.10.83

[14]   Y. Yang, M. F. Grubb, C. E. Luk, W. G. Humphreys and J. L. Josephs, “Quantitative Estimation of Circulating Metabolites without Synthetic Standards by Ultra-High-Performance Liquid Chromatography/High Resolution Accurate Mass Spectrometry in Combination with UV Correction,” Rapid Communications in Mass Spectrometry, Vol. 25, No. 21, 2011, pp. 3245-3251.
http://dx.doi.org/10.1002/rcm.5220

[15]   USFDA, “Guidance for Industry: Safety Testing of Drug Metabolites,” 2008, pp. 1-25.
http://www.fda.gov/cder/guidance/6897fnl.pdf.

[16]   F. Cuyckens, L. I. Balcaen, K. De Wolf, B. De Samber, C. Van Looveren, R. Hurkmans and F. Vanhaecke, “Use of the Bromine Isotope Ratio in HPLC-ICP-MS and HPLC-ESI-MS Analysis of a New Drug in Development,” Analytical and Bioanalytical Chemistry, Vol. 390, No. 7, 2008, pp. 1717-1729.
http://dx.doi.org/10.1007/s00216-007-1761-6

[17]   U. P. Dahal, J. P. Jones, J. A. Davis and D. A. Rock, “Small Molecule Quantification by Liquid Chromatography-Mass Spectrometry for Metabolites of Drugs and Drug Candidates,” Drug Metabolism and Disposition, Vol. 39, No. 12, 2011, pp. 2355-2360.
http://dx.doi.org/10.1124/dmd.111.040865

[18]   R. Espina, L. Yu, J. Wang, Z. Tong, S. Vashishtha, R. Talaat, J. Scatina and A. Mutlib, “Nuclear Magnetic Resonance Spectroscopy as a Quantitative Tool To Determine the Concentrations of Biologically Produced Metabolites: Implications in Metabolites in Safety Testing,” Chemical Research in Toxicology, Vol. 22, No. 2, 2009, pp. 299-310. http://dx.doi.org/10.1021/tx800251p

[19]   S. Schadt, L. Z. Chen and D. Bischoff, “Evaluation of Relative LC/MS Response of Metabolites to Parent Drug in LC/Nanospray Ionization Mass Spectrometry: Potential Implications in MIST Assessment,” Journal of Mass Spectrometry, Vol. 46, No. 12, 2011, pp. 1281-1286.
http://dx.doi.org/10.1002/jms.2018

[20]   D. A. Smith and R. S. Obach, “Metabolites: Have We MIST out the Importance of Structure and Physicochemistry?” Bioanalysis, Vol. 2, No. 7, 2010, pp. 1223-1233.
http://dx.doi.org/10.4155/bio.10.92

[21]   K. Vishwanathan, K. Babalola, J. Wang, R. Espina, L. Yu, A. Adedoyin, R. Talaat, A. Mutlib and J. Scatina, “Obtaining Exposures of Metabolites in Preclinical Species through Plasma Pooling and Quantitative NMR: Addressing Metabolites in Safety Testing (MIST) Guidance without Using Radiolabeled Compounds and Chemically Synthesized Metabolite Standards,” Chemical Research in Toxicology, Vol. 22, No. 2, 2009, pp. 311-322.
http://dx.doi.org/10.1021/tx8003328

[22]   P. Wright, Z. Miao and B. Shilliday, “Metabolite Quantitation: Detector Technology and MIST Implications,” Bioanalyis, Vol. 1, No. 4, 2009, pp. 831-845.
http://dx.doi.org/10.4155/bio.09.63

[23]   C. Yu, C. L. Chen, F. L. Gorycki and T. G. Neiss, “A Rapid Method for Quantitatively Estimating Metabolites in Human Plasma in the Absence of Synthetic Standards Using a Combination of Liquid Chromatography/Mass Spectrometry and Radiometric Detection,” Rapid Communications in Mass Spectrometry, Vol. 21, No. 4, 2007, pp. 497-502. http://dx.doi.org/10.1002/rcm.2863

[24]   D. Zhang, N. Raghavan, T. Chando, J. Gambardella, Y. Fu, D. Zhang, S. Unger and W. G. Humphreys, “LC-MS/MS-Based Approach for Obtaining Exposue Estimates of Metabolites in Early Clinical Trials Using Radioactive Metabolites as Reference Standards,” Drug Metabolism Letters, Vol. 1, No. 4, 2007, pp. 293-298.
http://dx.doi.org/10.2174/187231207783221411

[25]   M. Zhu, H. Zhang and W. G. Humphreys, “Drug Metabolite Profiling and Identification by High-Resolution Mass spectRometry,” The Journal of Biological Chemistry, Vol. 286, 2011, pp. 25419-25425.
http://dx.doi.org/10.1074/jbc.R110.200055

[26]   R. Ramanathan, N. Raghavan, S. N. Comezoglu and W. G. Humphreys, “A Low Flow Ionization Technique to Integrate Quantitative and Qualitative Small Molecule Bioanalysis,” International Journal of Mass Spectrometry, Vol. 301, No. 1-3, 2011, pp. 127-135.
http://dx.doi.org/10.1016/j.ijms.2010.07.027

[27]   A. Ranasinghe, R. Ramanathan, M. Jemal, C. J. D’Arienzo, W. G. Humphreys and T. Olah, “Integrated Quantitative and Qualitative Workflow for In-Vivo Bioanalytical Support in Drug Discovery using Hybrid Q-TOF MS,” Bioanalyis, Vol. 4, No. 5, 2012, pp. 511-528.
http://dx.doi.org/10.4155/bio.12.13

[28]   J. Schmidt, M. M. Meyer, I. Spector and S. R. Kass, “Infrared Multiphoton Dissociation Spectroscopy Study of Protonated p-Aminobenzoic Acid: Does Electrospray Ionization Afford the Aminoor Carboxy-Protonated ion?” The Journal of Physical Chemistry, Vol. 115, No. 26, 2011, pp. 7625-7632.
http://dx.doi.org/10.1021/jp203829z

[29]   M. Chang, Y. Li, R. Angeles, S. Khan, L. Chen, J. Kaplan and L. Yang, “Development of Methods to Monitor Ionization Modification from Dosing Vehicles and Phospholipids in Study Samples,” Bioanalysis, Vol. 3, No. 15, 2011, pp. 1719-1739.
http://dx.doi.org/10.4155/bio.11.164

[30]   Y. Q. Xia, J. Lau, T. Olah and M. Jemal, “Targeted Quantitative Bioanalysis in Plasma Using Liquid Chromatography/High-Resolution Accurate Mass Spectrometry: An Evaluation of Global Selectivity as a Function of Mass Resolving Power and Extraction Window, with Comparison of Centroid and Profile moDes,” Rapid Communications in Mass Spectrometry, Vol. 25, No. 19, 2011, pp. 2863-2878.
http://dx.doi.org/10.1002/rcm.5178

[31]   G. A. Valaskovic, L. Utley, M. S. Lee and J. T. Wu, “Ultra-Low Flow Nanospray for the Normalization of Conventional Liquid Chromatography/Mass Spectrometry through Equimolarrsesponse: Standard-Free Quantitative Estimation of Metabolite Levels in Drug Discovery,” Rapid Communications in Mass Spectrometry, Vol. 20, No. 7, 2006, pp. 1087-1096.
http://dx.doi.org/10.1002/rcm.2414

 
 
Top