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5. The Limit of Detection and Limit of Quantitation for the DR CALUX®,
Much can be hidden in the LODs and LOQs that some companies state. Often these are the LOD and LOQ of the core method when analysing standards – NOT true matrices. Some HRGCMS laboratories, for example, give results based on recoveries of spikes of less than 10%. In principle, anyone can obtain lower and lower LOD/LOQs by processing more and more matrix. However, this can introduce error and can result in the use of large quantaties of reagents and the generation of much waste. BDS’ DR CALUX® bioassay is fundamentally extremely sensitive and to get the LOQs for almost all applications we only need to process a few grams of material. BDS states openly and transparently how much matrix must be processed to achieve an LOD/LOQ.
The DR CALUX® Bioassay (LOD and LOQ of 0.3 and 1.0 pM in each well respectivly)
>For determining LOD and LOQ you need to know that dimethylsulfoxide (DMSO) is normally used as the solvent vehicle in the CALUX® bioassay and all test samples including the different concentrations of reference materials and blanks are measured in triplicate on a 96-well microtiter plate. The standard deviation of the test sample triplicates in DMSO, are interpolated against the calibration curve of the reference material resulting in a concentration of the test sample versus a reference chemical. If the results of blank samples on many microtiter plates are combined, an overall standard deviation for the DMSO blanks (expressed as concentration of the reference material) can be determined. This is the “background noise” in the bioassay.
To statistically determine that a signal is a true signal above background, a safety factor is applied. This results in the Limit of Detection (LOD). The safety factor for the LOD that BDS uses is 3x the standard deviation of DMSO blanks interpolated to the calibration curve of the reference chemical resulting in LOD in terms of a concentration of the reference chemical. This is done for a large number of microtiterplates which then takes account of small differences between different plates.
The same procedure is followed to derive a Limit of Quantitation except that a factor of 10x the standard deviation is used. Samples values above this 10x level are very highly unlikely to have a value the same as the DMSO blank.
This results for example in the DR CALUX® in a LOD and LOQ of 0.3 and 1.0 pM in each well respectively.
The extract (LOD and LOQ of 0.3 and 1.0 pg per extract respectively)
Although the LOD and LOQ of a specific matrix depends on the amount of material extracted, the LOD and LOQ of the extract in pg 2,3,7,8 TCDD can be determined for all matrices . With the core CALUX® LOD and LOQ given above, combined with the knowledge that a sample is diluted 125 times in the bioassay , a sample is normally dissolved in 25 µl DMSO and the molar mass of 2,3,7,8 TCDD is 322, the LOD and LOQ can readily be expressed in pg 2,3,7,8 TCDD TEQ for each test sample.
If a lower LOD and LOQ are required, BDS has an enhancement to the DR CALUX® bioassay which results in a LOD and LOQ of almost a factor three lower – this is without processing more matrix.
The normal procedure results for example in the DR CALUX® in a LOD and LOQ of 0.3 and 1.0 pg per extract respectively.
The samples (see table)
If you wish to know the LOD and LOQ that can be achieved for a particular sample you need to know how much matrix is used for the analysis. Whilst this may not be a big issue for animal feed, it is certainly an issue for human tissues where only limited amounts of material can be obtained. Lower and lower LODs and LOQs can be obtained if you process a lot of matrix, however this can result in unwieldy and impractical work-up procedures. BDS normally uses small practical quantities of matrix – typically 1 – 10 grams. Given the information above about the LOD and LOQ, matrix dependent LODs and LOQs can be determined (see table below for some typical examples)
|
matrix |
unit |
Normal amount for work-up |
pg LOD/matrix |
pg LOQ/matrix |
|
milk |
gram fat |
1 |
0.30 |
1 |
|
egg |
gram fat |
1.5 |
0.20 |
0.67 |
|
vegetable oil |
gram material |
3 |
0.10 |
0.33 |
|
fish oil |
gram fat |
1.5 |
0.20 |
0.67 |
|
cattle feed |
gram fat |
9 |
0.03 |
0.11 |
From the
table it can be seen that the EU limit values for foodstuffs and feedstuffs can
easily be achieved (a technique should be able to
meaure half of the limit value).
In the next few years, the limit values for feed and food in the EU may
go down significantly. Constrained by
reporting upper-bound values for individual congeners, already the vast majority
of HRGCMS labs are over estimating the amount of dioxins in samples (actually
reporting upper-bound levels which as sensitivity
is not enough non-detects are being reported above the EU limit values). With lower limit values likely, HRGCMS may
just not be sensitive enough for dioxins analysis
in future.
If you
want to know the specific LOD and LOQ for your matrix of interest please
contact us. The LOD and LOQ achievable for samples delivered to BDS for small
quantities may be insufficient for your needs as the amount of material becomes
the limiting factor - not the analysis procedure. This happens for example with serum samples.
Serum samples are normally delivered in quantities of 5 gram serum but because
the TEQ is normally expressed as 2,3,7,8-TCDD TEQ per gram fat, the LOD and LOQ
becomes dependent on the amount of fat present in the serum.
Finally …........
Be sure
to ask your analytical service provider how LOD and LOQ are defined. If they cannot answer this question
clearly - ask BDS !
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