1. A Brief Review of Literature on Matrix effects in LC and GC Coupled Mass-Spectrometric Analysis Benesh Joseph Kobayashi Lab Presentation as a part of Project Based Learning under the supervision of Prof. Ei’ichiro Fukusaki. 3 rd July 2007

2. Presentation Overview 1. Matrix Effects in LC-ESI-MS 2. Methods to determine the matrix effects in LC-ESI-MS 3. Sources of Matrix effects in LC-ESI-MS 5. Alternative Calibration methods to compensate Matrix effects 4. How to minimize Matrix effects? 6. Matrix effect in Metabolite Profiling Using LC-ESI-QTOF-MS 7. Matrix Effects in GC-MS 8. Conclusions Based on clinical/ Pharmaceutical research

4. Matrix Effect in LC-ESI-MS Matrix effects are the result of competition between nonvolatile matrix components and analyte ions for access to the droplet surface for transfer to the gas phase. Depending on the environment in which the ionization and ion evaporation processes take place, this competition may effectively decrease ( ion suppression ) or increase ( ion enhancement ) the efficiency of formation of the desired analyte ions.

5. Methods to Determine the Degree Matrix Effect in LC-ESI-MS 1. Postextraction addition of the Analyte Clinical Biochemistry 38 (2005) 328– 334

6. Methods to Determine the Degree Matrix Effect in LC-ESI-MS 2. Postcolumn Infusion Different Extraction/separation Methods Continuous infusion of the analyte of interest Results of postcolumn infusion experiments enable evaluation on the influence of different sample extraction techniques on matrix effects, the appropriate analytical column, where matrix effects occur and are absent during a chromatographic run, the mechanistic aspect of matrix effects, and the influence of mobile additives on response Clinical Biochemistry 49 (2003) 1041– 1044 Mobile Phase Serum liquid-liquid extract Serum protein precipitation extract Minutes

7. Sources of Matrix Effects 1. Mobile Phase additives 2. Buffer Additives Drugs were postcolumn infused into 50/50 methanol/water containing the additive Rapid Commun. Mass Spectrom. 2004; 18: 49–58

8. Sources of Matrix Effects 3. Endogenous Impurities in the Sample. SIROLIMUS – An Immunosuppressant drug (50 µg/L) was postcolumn infused (10µL/min) and selected reactant monitored following the mass transition m/z 931.6 864.6 Mobile Phase Whole blood sample prepared by protein Precipitation with acetonitrile Whole blood sample prepared by SPE Clinical Biochemistry 38 (2005) 328– 334

13. Anal. Chem. 2007, 79, 1507-1513 Postcolumn Infusion of Kinetin (2) and Biochanin (7) Solvent A. thaliana leaf extract A. thaliana root extract There is no indication for matrix effects to an extent that would be unacceptable for metabolomics studies, which are inherently compromised with regard to quantification t R =15.4 t R =40.9

15. Summary of Interference Experiments Relative matrix effects are negligible unless extremely divergent matrixes are compared and do not compromise the relative quantification that is aimed for in nontargeted metabolomics studies. Yet rigorous validation is necessary. Summary Anal. Chem. 2007, 79, 1507-1513

16. Matrix Effects in GC-MS Analysis While the matrix effect occur in the ESI interface in LC-ESI-MS, it happens in the In the column inlet and column in GC-MS. Ion Enhancement When a real sample is injected, the matrix components tend to block active sites (mainly free silanol groups) in the GC inlet and column, thus reducing losses of susceptible analytes caused by adsorption or degradation on these active sites. This phenomenon results in higher analyte signals in matrix-containing versus matrix free solutions, thus precluding the convenient use of calibration standards in solvent only, which would lead to overestimations of the calculated concentrations in the analyzed samples. Ion Suppression Gradual accumulation of nonvolatile matrix components in the GC system, results in formation of new active sites and gradual decrease in analyte responses. This effect, sometimes called matrix-induced diminishment , negatively impacts ruggedness (i.e., long-term repeatability of analyte peak intensities, shapes, and retention times), which is a highly important factor in routine GC analysis Anal. Chem. 2005, 77, 8129-8137

18. Alternative Calibration methods to compensate Matrix effects in GC-MS All the three methods discussed for LC-ESI-MS can be used in case of GC-MS too. Calibration using labeled internal standards has the highest accuracy and precision. Extremely high ‘individual biological variation’ seen in plants limits the use of Either Standard Addition Method or Matrix Matched Standard Method for quantitation. Data adopted from NATURE BIOTECHNOLOGY VOL 18 NOVEMBER 2000 GC-MS Analysis

19. Analyte Protectants to Overcome Matrix Effect in Pesticide Residues Analysis compounds that would strongly interact with the active sites in the column, thus providing strong enhancement of analyte responses. When added such Compounds practically eliminate any difference between calibrations obtained in matrix versus matrix-free solutions. - + Anal. Chem.2005, 77, 8129-8137

20. Conclusions It is mandatory to address the question of matrix effects for every metabolite profiling, especially for targeted – quantitative metabolic profiling study which involves a new extraction scheme or samples of new biological origin. Absolute quantification clearly requires labeled internal standards Even with a labeled internal standard it is recommended to evaluate the matrix effects as large suppression can significantly reduce the signal of the analyte or Internal Standard to a point where accuracy and precision become negatively affected.