APPENDIX B TO PART 136 - DEFINITION AND PROCEDURE FOR THE 

DETERMINATION OF THE METHOD DETECTION 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% confidence that the measured concentration is 

distinguishable from method blank results. 

Scope and Application 

The MDL procedure is designed to be a straightforward technique for estimation of the 

detection limit for a broad variety of physical and chemical methods. The procedure requires a 

complete, specific, and well defined analytical method. It is essential 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 set 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 experience 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 estimated MDL in section 1. 

Spiking levels in excess of 10 times the estimated 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 blank 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 least 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 across 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 establishing 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 deviation (S) of the replicate spiked blank 

measurements and the sample standard deviation of the replicate method blank measurements 

from all instruments. 

(ii) Compute the MDLs (MDL based on spiked blanks) as follows: 

ݏܮܦܯ ൌ ݐሺ௡ିଵ,			ଵି∝ୀ଴.ଽଽሻܵ௦

Where: 

MDLs = the method detection limit based on spiked blanks 

t(n-1, 1- = 0.99) = the Student’s t-value appropriate for a the single tailed 99th percentile t 

statistic and a standard deviation estimate with n-1 degrees of freedom. See Table 1. 

Ss = sample standard deviation of the replicate spiked blank sample analyses. 

(iii) Compute the MDLb (MDL based on method blanks) as follows: 

(A) If none of the method blanks give numerical results for an individual analyte, the 

MDLb 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 MDLb equal to the highest method blank result. If more than 100 method blanks 

are available, set MDLb to the level that is no less than the 99th percentile of the blank results. 

For "n" method blanks where n ≥ 100, sort the method blanks in rank order. The (n×0.99) ranked 

method blank result (round to the nearest whole number) is the MDLb. For example, to find 

MDLb 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 which rounds to the 162nd method blank 

result. Therefore, MDLb is 1.9 for n = 164 (10 is the 164th result, 5.0 is the 163rd result, and 1.9 
is the 162nd result). Alternatively, you may use spreadsheet algorithms 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 MDLb as: 

ܮܦܯ ௕ൌ ܺ ൅ ݐሺ௡ିଵ,ଵି∝ୀ଴.ଽଽሻܵ௕

Where: 

MDLb = 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 deviation estimate with n-1 degrees of freedom. See Addendum Table 1. 

Sb = sample standard deviation of the replicate blank sample analyses. 

(e) Set the greater of MDLs or MDLb as the initial MDL. 

(3) Ongoing Data Collection 

(a) During any quarter in which samples are 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 in Section 2. If any analytes are repeatedly not detected in the 

quarterly spike sample analysis, this is an indication that the spiking level 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-evaluate 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 following 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 MDLs and MDLb from the collected spiked 

blank and method blank results using 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 acceptable calibrations and batch QC. Include all 

routine data, with the exception of batches that 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 MDLs or MDLb. If the verified MDL is 

within a factor of 3 of the existing MDL, and fewer than 3% of the method blank results (for the 

individual analyte) have numerical results above the existing MDL, then the existing MDL may 

optionally be left unchanged. Otherwise, adjust the MDL to the new verification MDL. 

Addendum: Determination of the MDL For a Specific Matrix 

MDLs may be determined in specific sample matrices as well 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 signal to noise ratio of approximately 5-20, 

determine the matrix specific MDL according to Section 2, “Determine the initial MDL” without 

spiking additional analyte. 

(3) Calculate MDLb using method blanks, not the sample matrix. 
(4) If the signal to noise is less than 5, the 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 signal to noise greater than approximately 20, 

then the resulting MDL is likely to be biased high. 

TABLE 1: SINGLE TAILED 99TH 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 method 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]