Hi experts,
I am doing an external standard method to determine concentration of an analyte using AAS instrument. But two of my samples showed signals beyond the signals produced by my standards, so I have difficulty to determine the concentration using calibration curve obtained. Appreciate your kind suggestions and recommendations.
Hi,
"Samples for which the absorbance value exceeds the highest point of the standard range should be re-analyzed at a higher dilution." this step is very often used within different methods and should be a part of method validation.
https://www.mabtech.com/knowledge-center/assay-principles/elisa-assay-principle/determination-sample-concentration
This specific test in validation is called dilution integrity
"Dilution of samples should not affect the accuracy and precision. If applicable, dilution integrity should be demonstrated by spiking the matrix with an analyte concentration above the ULOQ and diluting this sample with blank matrix (at least five determinations per dilution factor). Accuracy and precision should be within the set criteria, i.e. within ±15%. Dilution integrity should cover the dilution applied to the study samples. Evaluation of dilution integrity may be covered by partial validation. Use of another matrix may be acceptable, as long as it has been demonstrated that this does not affect precision and accuracy."
https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-bioanalytical-method-validation_en.pdf
"If the method measures diluted samples, the integrity of the dilution should be monitored during validation by diluting QC samples above the ULOQ with like matrix to bring to within quantitation range, and the accuracy and precision of these diluted QCs should be demonstrated. Dilutions used during the validation should mimic the expected dilutions in the study. The prozone effect should be demonstrated in LBAs."
https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf
Those criteria and general philosophy could be used in any method and analytical technique,
Best regards
Tomasz
two approaches:
a) dilute the sample
b) add additional calibration levels and adapt the calibration curve.
Both steps need to be included in the method validation, as stated by Tomasz.
Good luck with your research
Detlef.
Dear Colleague,
As a supplement to Prof. Grabowski's and Dr. Jensen's answers, which are actually a combination of the two previous ones:
(c) the appropriate rotation of the burner head, up to a position perpendicular to the optical path if necessary, in combination with Dr. Jensen's (b) and Prof. Grabowski's instructions;
(d) The use of an appropriate (less „sensitive“ i.e. „secondary“) wavelength for some elements. Topical where there is not significantly higher signal noise compared to the „basic“ wavelength and especially out of the influence of the matrix. For example, for Mn, Cu, Ni, Fe. Verification as instructed by Prof. Grabowski and point b) of Dr. Jensen. From the above, the impression is that the simplest procedure is to dilute the original sample. If, however, larger batches of samples with a matrix of similar composition are used, the universal procedure (c) is probably preferable, especially if the device allows automatic rotation of the burner head, or (d), respective. However, always after prior verification in accordance with Prof. Grabowski's notes and point (b) of Dr. Jensen.
Good Luck!
Karel.
Dear Profs Tomasz Grabowski , Detlef Jensen , Petr Karel Mrak-Fiala
Thank you very much for all the helpful answers.
Use a high dilution factor to bring the sample reading signal within the calibration curve.
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