The document discusses matrix effects in LC-MS and GC-MS analysis. In LC-MS, matrix effects occur due to competition between analytes and matrix components for ionization, which can suppress or enhance signals. Methods to evaluate and minimize effects include modified extraction, improved chromatography, and isotope-labeled internal standards. In GC-MS, matrix components can block or create active sites, affecting signals. Effects are addressed through calibration standards in matrix-matched solutions and internal standards. Relative quantification for metabolomics requires validation due to biological natural variation.

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

3. What are Matrix Effects? Matrix effect happens when co-eluting molecules alter the signal of the compound of Interest resulting in problems like, Inaccurate Quantitation Decreased method ruggedness Low analyte detectability Reporting even false positive or false negative results. Matrix effect is dependent on the particular compound and the matrix composition. 50/50 MeOH/H 2 O + 0.5% NH 4 OH 50/50 MeOH/H2O 50/50 MeOH/H 2 O + 0.5% TFA Terfenadine The mechanism of ‘matrix effect ‘ is different between LC/MS and GC/MS +41% -75% Rapid Commun. Mass Spectrom. 2004; 18: 49–58

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

9. How to minimize Matrix Effects? Two approaches to remove or minimize matrix effects are Modification to the Sample Extraction Methodology: However the success in a quantitative analysis varies depending on the natural variation among different individuals of the same organism 2. Improved Chromatographic Separation: e.g.. Column Switching, Solid Phase Extraction etc.. Matrix effects have predominantly been reported for clinical and pharmaceutical Applications. It is well known that retention on the column minimizes matrix effects. Good chromatographic separation allows one to reliably monitor relative changes in the abundance of several hundred compounds simultaneously

10. Alternative Calibration methods to compensate Matrix effects An effective elimination of the sources of the matrix effects is not likely in practice. The current compensation approaches include use of the following: Matrix-matched Standards : Calibration curves are prepared in matrix similar to matrix in which the analyte is present. Still accurate quantification is negatively affected by matrix variation from sample to sample. (ii) standard addition method : Analyzing extract solution added the known quantity standard solution; the calculation procedure is as follows: X = S I X / (I S - I X ). Where X is the amount of analyte in the extract solution; S is the amount of analyte spiked into the extract solution; I X is the signal intensity of analyte in the extract solution and I S is the signal intensity of the analyte in the spiked solution.. Still may lead to quantitation inaccuracies as the extent of effect depends on analyte concentration . (iii) Isotopically labeled internal standards : Isotopically labeled substances are is the best way to correct matrix effects because they have almost the same chemical properties and the same retention time as the non-labeled substance. However isotopically labeled Internal standards are not available for many analytes due to difficulties in synthesis And / or cost.

11. Matrix effects in Metabolic Profiling Using LC-ESI-QTOF-MS The compounds were spiked into any of the four matrixes and the signal strength was Compared to that in pure solvent. Absolute matrix effect = [1- (mean peak area in matrix / mean peak area in neat solvent)] For 2 , 4 , 5 , and 7 ion suppression was below 30% 6 was subjected to strong suppression in two Matrixes and massive ion suppression of >80% was found for 1. 3 was subjected to significant Ion enhancement in two matrixes Standards Anal. Chem. 2007, 79, 1507-1513

12. Relative Matrix effects are of Higher Relevance for Metabolomic studies the variation in matrix effect between samples derived from different experiments Or individuals that would in a scientific study be compared Evaluation of relative matrix effects ( Expressed as relative standard deviation of Peak area ) by Postextraction addition. The test set of seven reference compounds was added to pure solvent and to extracts derived from either eight different pools of Plants ( A. thaliana ) or eight individual Plants ( A. halleri ) Technical Variation (Blank): 3.4 -12.7% Relative matrix effects were practically zero for 2 -7 in A. thaliana root or leaf matrix . 5.6 – 25.1% variability in A. halleri matrix is due to growth of plants under less controlled conditions. Even for 1 , the variation was within acceptable limits of typical biological variation. Anal. Chem. 2007, 79, 1507-1513 n=8

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

14. Interference Experiments by Mixing Leaf and Root Extracts Anal. Chem. 2007, 79, 1507-1513 thaliana leaf and root extracts were diluted either with pure solvent or with the respective other sample in two different ratios (1:1, 1:3, v/v). Intensity ratios of matrix-diluted sample to solvent-diluted sample were determined for robust signals ( 93 in leaf extracts, 84 signals in root extracts ), log-transformed, and plotted against retention time (n = 4). Robust Signal Properties 1.S/N>6 2. t R > 10 mint. 3. Consistently absent in the other matrix 4. Signal strength varied as per dilution.

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

17. Internal Calibration – Calibration curve prepared in A. thaliana leaf extracts External Calibration – Calibration curve prepared in solvent Matrix Effects in GC-MS Analysis Data adopted from NATURE BIOTECHNOLOGY VOL 18 NOVEMBER 2000

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.