A BETTER APPROACH TO

CALCULATING LIMITS OF DETECTION

AND LIMITS OF QUANTITATION

by Will McHale - Life Sciences

Market Manager, ERA

Part of almost every instrument validation effort, analytical

laboratories are continually challenged with appropriately
determining Limits of Detection (LOD) and Limits of Quantitation
(LOQ). An LOD is the point at which a true signal is detectable from the
noise of the detector being employed, where the ratio has historically
been a three to one signal to noise ratio. Few laboratories employ a
scientifically sound determination of LODs and LOQs, electing rather
to examine a single low level standard and consider data precision.
By example, a series of standards are analyzed in triplicate. The data are collected and the mean and standard deviation are calculated
from the results, and then multiplied by three to provide the laboratory with an LOD. In the table below, a laboratory has analyzed six
different concentrations of standards for Total Organic Carbon (TOC).

Prepared
50 60 70 80 90 100
Concentration (ppb)

Rep 1 42 62 66 68 80 100
Rep 2 47 65 71 70 81 89
Rep 3 45 65 75 72 81 94
Mean 45 64 71 70 81 94
Standard Deviation 2.52 1.73 4.51 2.00 0.577 5.51

Given the analytical data collected, the LOD using any one of these single concentrations individually would result in an LOD anywhere
between 1.67 and 16.5 ppb.

The data suggests that the variability of standards impacts the standard deviation, given the wide range observed. If the only variable
was based on laboratory or analytical variability, then the standard deviations would be of a smaller and similar in magnitude across the
concentration range, especially over the short time frame required to analyze the samples. The increase in variability of the data results
in artificial inflation of the LOD and LOQ.

If the data are pooled, and the LOD and LOQ are determined based not on the standard deviation at a single concentration, but rather a
linear regression across a concentration range, a much more realistic LOD and LOQ can be determined. Using the same analytical data
above, graphing the standard deviation compared to the theoretical concentration the data yields the below regression equation.

102987 LOD Vial 1 y = 0.0412x - 0.1053

R2 = 0.1373
6
5
Standard Deviation

4
3
2
1
0
0 20 40 60 80 100
Concentration

Figure 1.

2
In the regression equation the Y intercept is the S 0 for the calculation of the LOD, which is now based on six analytical data points
across a concentration range as opposed to three data points at a single concentration. T he LOD calculated is now based on the
S 0 = 0.1 ppb (y-intercept = 0.1053) to produce an LOD of 0.3 ppb. T he LOQ is calculated as 3.0 ppb. By using this method of
determining the laboratory has lowered their LOD by a factor of 5 to 50, as well as, more accurately portraying their capability
across the working range of the instrument.

To determine the working range of the instrument, we graph the measured values against the theoretical values as below. Agreement for the
concentration range of 50 ppb to 100 ppb is quite acceptable.

102987 Linearity Vial 1 y = 0.8505x - 6.9365

R2 = 0.9166
100

80
Measured [C]

60

40

20

0
0 20 40 60 80 100 120
Theoretical [C]

Figure 2.

The LOD and LOQ are now based on the entire working range of the instrument and accurately reflect the true capability of the laboratory.
The method outlined above was reported by John Taylor in “Quality Assurance of Chemical Measurements” and is used by many analytical
chemists as a more robust method for determining LOD and LOQ.

The issue of inaccurate standards compounds the impact on the LOD and LOQ determination. Laboratories should purchase Certified
Reference Materials (CRMs) from an ISO Guide 34 accredited provider for critical validation and calibration efforts. CRMs will ensure low
uncertainty and homogeneity across an instrument’s working concentration range. This limits drift and variability, allowing laboratories to
accurately determine capabilities like LOD and LOQ.

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January 2012  IH-PDF