Clinical Laboratory News An AACC Publication August, 07 The CLN Focus on Mass Spectrometry is supported by Waters Corporation Matrix Experimental For Exogenous Analytes Collect six individual, drug free, native matrix samples Prepare to spike low and high concentration test samples for each of the six drug free native matrix samples using the standard sample preparation procedure in replicates of three; label tubes for low and tubes for high concentration aliquots for each test sample (n=; 8 low and 8 high concentration samples) Prepare low and high concentration control samples (substituting an equivalent volume of water or solvent mixture, dependent on analyte solubility for native matrix) in replicates of three (consider the final volume to allow reinjection for comparison with test samples). samples are prepared using the standard sample preparation procedure; label tubes for low and tubes for high concentration aliquots for each control sample (n=; low and high concentration samples). These samples are used as the reference i.e. there should be no ion suppression or enhancement since no residual native matrix is present. Following sample preparation, test and control samples are spiked with anaylte(s) at two concentrations (low and high). Samples may be spiked using a low volume of solution containing analyte(s) or a small volume of the final preparation is removed (typically %) and the same volume of a solution containing analyte(s) added. Prepare singlicate calibrators and duplicate quality control samples using the standard sample preparation procedure Analyze the prepared samples following the test order described: Calibrators, quality control set, solvent blank, low concentration test sample, low concentration control sample, low concentration test sample, low concentration control sample, low concentration test sample, low concentration control sample, high concentration test sample, high concentration control sample, high concentration test sample, high concentration control sample, high concentration test sample, high concentration control sample. quality control set
Process the data, establish that the calibration line and quality control samples meet acceptance criteria and determine analyte peak areas and internal standard peak areas for test and control samples Calculate the mean peak area, SD and %CV for test samples for each analyte Calculate the mean peak area, SD and %CV for control samples for each analyte Calculate the percent matrix effect for each analyte using the following equation: peakarea intest samples x00 peakarea incontrolsamples Calculate the mean response for each analyte using the following equation: Analytepeakarea Internalstandardpeakarea Additionally, calculate the normalized (internal standard adjusted) percent matrix effect for each analyte using the following equation: response intest samples % NormalizedMatrix x00 response incontrolsamples The percent matrix effect is a quantitative measure of how much the matrix influences response - Values <00% indicate suppression - Values >00% indicate enhancement Percent matrix effect may differ between the six matrices tested, the response %CV provides a measure of variability and should be less than % (as stated in CLSI Guideline C-A) Calculation of the normalized percent matrix effect should give confidence that the internal standard is compensating for matrix effects in the sample by giving a matrix effect value of close to 00%
Percent Matrix Example Calculations Analyte No internal standard adjustment, low concentration: Peak Area 00 8 99 9 07 9 7 7 0 00 97 9.7.9 00 99 9 00 9 08 8 7 0 0 Percent Matrix = 99% (n=, range 9-0%).
No internal standard adjustment, high concentration: Peak Area 990 80 88 77 97 90 80 009 9 08 879 907.9 90 99 0.7 890 97 8 897 99 99 07 79 987 77 8 98 7 08 Percent Matrix = 08% (n=, range 99-%)
Internal standard adjustment, low concentration: 0.088 0.09 0.08 99 0.08 0.0879 0.08 0.0900 0.088 0.089 0.0888 0 0.088 0.088 97 0.088 0.089. 0.0879 0.087. 0.088 0.0870 0.087 0.08 99 0.088 0.087 00 0.0907 0.087 0.087 0.08 0.08 0.088 98 Percent Normalized Matrix = 99% (n=, range 98-0%)
Internal standard adjustment, high concentration:..9.7 97...8.... 9.. 98.8.8.8.79...9..80.9 99..9 99..0.70... 00 Percent Normalized Matrix = 98% (n=, range 9-00%) In this example, there is little suggestion of a matrix effect, even without considering the internal standard, as matrix factors are in the region of 90-0%. Using internal standard adjusted matrix factor gives results close to 00%.
Analyte No internal standard adjustment, low concentration: Peak Area 09 07 07 9 0 0 9 0 00 00 0 9 88 0..9 9 99 0 0 9 08 09 07 9 08 9 Percent Matrix = % (n=, range -9%)
No internal standard adjustment, high concentration: Peak Area 7 79 7 8 77 07 70 77 9 9 7 70 0 8 8 08 0. 7 989. 9 8 80 7 8 79 0 9 8 9 9 Percent Matrix Factor = % (n=, range -7%)
Internal standard adjustment, low concentration: 0.0 0.00 0.08 97 0.009 0.0 0.00 0.00 0.00 0.0 0.08 97 0.0 0.0 9 0.08 0.0 7.0 0.0 0.09. 0.0 0.077 0.009 0.009 9 0.0 0.0 9 0.078 0.0 0.08 0.0 0.0 0.0 98 Percent Normalized Matrix = 9% (n=, range 9-98%)
Internal standard adjustment, high concentration:.0.. 0..7.7.7...9 0..9 0.7..7....... 00.0. 0.09.7..8.0. 9 Percent Normalized Matrix = 0% (n=, range 9-0%) In this example there is evidence of ion suppression, as the unadjusted matrix effects are well below 00%. However, using internal standard adjusted matrix effects gives results close to 00%.