APPENDIX B TO PART 136 - DEFI NITION AND PROCEDURE FOR THE DETERMINATION OF THE METHOD D ETECTION LIMIT - REVISION 2 Definition The method detection limit (MDL) is defined as the minimum measured concentration of a substance that can be reported with 99% c onfidence that the measured concentration is distinguishable from method blank results. Scope and Application The MDL procedure is designed to be a stra ightforward technique for estimation of the detection limit for a broad variety of physical an d chemical methods. The procedure requires a complete, specific, and well define d analytical method. It is essent ial that all sample processing steps used by the laboratory be included in the determination of the method detection limit. Procedure (1) Estimate the Initial MDL using one of the following: (a) The mean plus three times the standard deviation of a se t of method blanks. (b) The concentration value that corresponds to an instrument signal/noise in the range of 3 to 5. (c) The concentration equivalent of three times the standard deviation of replicate instrumental measurements of spiked blanks. (d) That region of the standard curve where there is a significant change in sensitivity, i.e., a break in the slope of the standard curve. (e) Instrumental limitations. (f) Previously determined MDL. It is recognized that the experi ence of the analyst is important to this process. However, the analyst should include some or all of the above considerations in the initial estimate of the MDL.(2) Determine the Initial MDL (a) Select a spiking level, typically 2–10 times the es timated MDL in section 1. Spiking levels in excess of 10 times the estima ted detection limit may be required for analytes with very poor recovery (e.g., an analyte with 10% recovery, spiked at 100 micrograms/L, mean recovery, 10 micrograms/L; MDL may calculate at 3 micrograms /L. So, in this case the spiking level is 33×MDL, but spiking lower may result in no recovery at all). (b) Process a minimum of 7 spiked bl ank samples and 7 method blank samples through all steps of the method, including any sample preservation. Both preparation and analysis of these samples must include at leas t three batches on three separate calendar dates. Existing data may be used if compliant with the requirements for at least 3 batches and generated within the last 2 years. (i) If there are multiple instruments that will be assigned the same MDL, then the samples must be distributed ac ross all of the instruments. (ii) A minimum of two spiked samples and two method blank samples prepared and analyzed on different calendar dates is required for each instrument. (c) Evaluate the spiking level: If any result for any individual analyte from the spiked blank samples does not meet the method qualitative identification criteria or does not provide a numerical result greater than zero then repeat the spikes at a higher concentration. Qualitative identification criteria are a set of rules or guidelines for establis hing the identification or presence of an analyte using a measurement system. Qualitative identification does not ensure that quantitative results for the analyte can be obtained. (d) Make all computations according to the defined method with final results in the method reporting units.(i) Calculate the sample standard deviat ion (S) of the replicate spiked blank measurements and the sample standard deviati on of the replicate method blank measurements from all instruments. (ii) Compute the MDL s (MDL based on spiked blanks) as follows:
ܮܦܯ ݏ ൌ ݐ ሺ௡ିଵ,			ଵି∝ୀ଴.ଽଽሻ ܵ ௦ Where:
MDL s = the method detection limit based on spiked blanks

t (n-1, 1-  = 0.99) = the Student’s t-value appropriate fo r a the single tailed 99th percentile t
statistic and a standard devi ation estimate with n-1 degr ees of freedom. See Table 1. S s = sample standard deviation of the re plicate spiked blank sample analyses. (iii) Compute the MDL b (MDL based on method blanks) as follows: (A) If none of the method blanks give numeri cal results for an individual analyte, the
MDL b does not apply. A numerical result includes both positive and negative results, including results below the current MDL, but not results of ND (not detected) commonly observed when a peak is not present in chromatographic analysis. (B) If some (but not all) of the method blanks for an individual analyte give numerical results, set the MDL b equal to the highest method blank re sult. If more than 100 method blanks are available, set MDL b to the level that is no less than the 99 thpercentile of the blank results. For "n" method blanks where n ≥ 100, sort the method blanks inrank order. The (n×0.99) ranked method blank result (round to the nearest whole number) is the MDL b . For example, to find
MDL b from a set of 164 method blanks where the highest ranked method blank results are ... 1.5, 1.7, 1.9, 5.0, and 10, then 164×0.99 = 162.36 whic h rounds to the 162nd method blank result. Therefore, MDL b is 1.9 for n = 164 (10 is the 164th result, 5.0 is the 163rd result, and 1.9is the 162nd result). Alternativ ely, you may use spreadsheet al gorithms to calculate the 99th percentile to interpolate between the ranks more precisely. (C) If all of the method blanks for an individual analyte give numerical results, calculate the MDL b as:
ܮܦܯ ௕ ൌ ܺ ൅ ݐ ሺ ௡ିଵ,ଵି∝ୀ଴.ଽଽ ሻ ܵ ௕ Where:
MDL b = the MDL based on method blanks
ܺ ത = mean of the method blank results

t (n-1, 1-  = 0.99) = the Student’s t-value appropriate for the single tailed 99th percentile t
statistic and a standard deviat ion estimate with n-1 degrees of freedom. See Addendum Table 1. S b = sample standard deviation of th e replicate blank sample analyses. (e) Set the greater of MDL s or MDL b as the initial MDL. (3) Ongoing Data Collection (a) During any quarter in which samples ar e being analyzed, prepare and analyze a minimum of two spiked blanks on each instrument , in separate batches if available, using the same spiking concentration used inSection 2. If any analytes are repeatedly not detected in the quarterly spike sample analysis, this is an indi cation that the spiking le vel is not high enough and should be adjusted upward. (b) Ensure that at least 7 spiked blanks and 7 method blanks are completed for the annual verification. (c) At least once per year, re-e valuate the spiking level. (i) If more than 5% of the spiked blanks do not return positive numerical results that meet all method qualitative identification criteria, then the spiking level must be increased and the initial MDL re-determined follo wing the procedure in Section 2.(d) If the method is altered in a way that can be reasonably expected to change the detection limit, then re-determine the initial MDL according to Section 2, and the ongoing data collection restarted. (4) Ongoing Annual Verification (a) At least once per year, re-calculate MDL s and MDL b from the collected spiked
blank and method blank results usin g the equations in section 2. (b) Include data generated within the last 2 years, but only data with the same spiking level. (c) Include the initial MDL spiked blanks if within two years. (d) Only use data associated with acceptabl e calibrations and batch QC. Include all routine data, with the exception of batches th at are rejected and the associated samples reanalyzed. If the method has been altered in a way that can be reasonably expected to change the detection limit, use only data collected after the change. (e) The verified MDL is the greater of the MDL s or MDL b . If the verified MDL is
within a factor of 3 of the exis ting MDL, and fewer than 3% of the method blank results (for the individual analyte) have nume rical results above the existing MDL, then the existing MDL may optionally be left unchanged. Otherwise, adju st the MDL to the new verification MDL. Addendum: Determination of the MDL For a Specific Matrix MDLs may be determined in sp ecific sample matrices as we ll as in reagent water. (1) Analyze the sample matrix to determine the native concentration of the analyte(s) of interest. (2) If the native concentration is at a si gnal to noise ratio of approximately 5-20, determine the matrix specific MD L according to Section 2, “Determine the initial MDL” without spiking additional analyte. (3) Calculate MDL b using method blanks, notthe sample matrix.(4) If the signal to noise is less than 5, th e analyte(s) should be spiked to obtain a concentration that will give results with a signal to noise of approximately 10-20. (5) If the analytes(s) of interest have si gnal to noise greater than approximately 20, then the resulting MDL is likely to be biased high. TABLE 1: SINGLE TAILED 99 TH PERCENTILE t STATISTIC Number of replicates Degrees of freedom (n-1) t (n-1, 0.99) 7 6 3.143 8 7 2.998 9 8 2.896 10 9 2.821 11 10 2.764 16 15 2.602 21 20 2.528 26 25 2.485 31 30 2.457 61 60 2.390 100 100 2.326

Documentation The analytical method used must be specifically identified by number or title and the MDL for each analyte expressed in the appropriate me thod reporting units. Data and calculations used to establish the MDL must be able to be reconstructed upon request. The sample matrix used to determine the MDL must also be identified with MDL value. Document the mean spiked and recovered analyte levels with the MDL. [49 FR 43430, Oct. 26, 1984; 50 FR 694, 696, Jan. 4, 1985, as amended at 51 FR 23703, June 30, 1986]