I know that there are several method to determine limit of detection and limit of quantitation such as calibration method, signal-to-noise method.
For example, the author of the article below used calibration method to determine LOD and LOQ:
" .. Seven ascending concentrations of 100, 500, 1,000, 2,500, 5,000, 7,500 and 10,000 ng mL-1 were selected as calibrators for piperacillin, while another seven concentrations of 30, 150, 300, 750, 1,500, 2,250 and 3,000 ng mL-1 were selected for tazobactam..."
How can I estimate the concentration of solution used for calibration curve like in that article and also the concentration at which is used to determine to S/N ratio?
The information you cited has nothing to do with LOD and LOQ. It just stated the serial dilution and concentration of two analytes they used to construct the calibration curves . Apparently the sensitivity of tazobactam is better than piperacillin in the method (more sensitive). I assume that they did by experimenting it. The LOQ calculation should not be done by by injecting standard alone but has to do with sample matrix due to matrix effect (suppression or enhancment). In case of suppression, the response of analyte in solvent will be higher than the response of analyte in sample matrix at the same concentration. Therefore, you will see lower concentration of the analyte in solvent than the concentration in matrix (at the same concentration). You can estimate the LOQ (10 times background noise) and LOD (3 times) by determining the signal to noise of analytel in matrix at know concentration. For instance, if you have matrix with 100 ng/g the analyte that gives you the peak with signal to noise of 100, your "estimated" LOQ is 10 ng/g and LOD is 3 ng/g
Hey Hong,
For the first part of your question: either you estimate the right concentrations required for obtaining standard calibration curve from valid articles which have developed/analyzed same analyte (in your case Piperacillin) with HPLC equipment similar to yours OR you determine them yourself by injecting a high-concentration standard and then start diluting it/preparing lower concentrations until your HPLC instrument is unable to detect it.
For the second part of your question, as you already know, they are a number of methods, namely:
1- Based on visual evaluation
2- Based on Signal-to-Noise Approach
3- Based on the Standard Deviation of the Response and the Slope
The second option has been explained by Dr. Chamkasem. However, in my opinion, the most easy one is their determination based on standard deviation of the response as well as the slope according to below equation:
DL= Xσ/S
where σ is the standard deviation of the response, S is the slope of the calibration curve, and x is a factor that is 3.3 for the LOD and 10 for the LOQ.
Please check the sections "Calibration Curve" and "Limit of Detection (LOD) and Limit of Quantification (LOQ)" in the my attached article as an example.
Good Luck
Thank you for your answer.
Actually, the author of that article uses the calibration curve to determine LOD and I also know it is one of methods to obtain LOD basing on the slope and standard deviation. My problem is that I don't know which concentration of solution used for calibration curve.
With signal-to-noise method, it is said that determination of signal-to-noise ratio is performed by comparing measured signals from samples with known low concentration of analyte with those of blank samples and by establishing the minimum concentration at which the analyte can be reliably detected. However, how low the concentration of analyte? 100ng/ml or another value?
As low as you can see, you want to know what is the concentration of analyte in sample matrix that will give you s/n of 10. Again, it is the "estimated" value, just to show how low you can quantify with good confident vs blank matrix.
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