The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample, which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/or degradation products.
The quantitation limit can be determine based on signal to noise ratio in which signal to noise ratio between 10:1 is acceptable and based on standard deviation method, in standard deviation method it can be expressed as,
LOQ = 10 ? / S
? = the standard deviation of the response
S = the slope of the calibration curve
The linearity of an analytical procedure is its ability (within a given range) to obtain test results.
The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. Which are directly proportional to the concentration (amount) of analyte in the sample.
1.6 BIOANALYTICAL METHODS 25
A Bioanalytical method is a set of all the procedures involved in the collection, processing Storing and analysis of a biological matrix for an analyte. Bioanalytical methods used for quantitative determination of drugs and their metabolites in biological fluids. Helpful in generating reproducible and reliable data which is used in the evaluation and interpretation of bioavailability, bioequivalence and pharmacokinetics. Method development involves evaluation and optimization of the various stages of sample preparation, chromatographic separation, detection and quantification.
To start these work there are main starting points which is of primary importance. Based on the information from this survey, the following can be done.
1 The choice of instrument: This is suitable for the analysis of your
analyte of interest includes the choice of the column associated with your
2 instrument of choice detector, the mobile phase in the high performance
liquid chromatography (HPLC) and the choice of carrier in gas
3 Choice of internal standard: which is suitable for your study, it must
have similar chromatographic properties to your analyte.
4 Choice of extraction procedure: which is time economical, gives the
highest possible recovery without interference at the elution time of the
analyte of interest and has acceptable accuracy and precision.
Method performance is determined primarily by the quality of the procedure itself. The two factors that are most important in determining the quality of the method are selective recovery and standardization. Analytical recovery of a method refers to whether the analytical method in question provides response for the entire amount of analyte that is contained in a sample. Recovery is usually defined as the percentage of the reference material that is measured, to that which is added to a blank. This should not be confused with the test of matrix effect in which recovery is defined as the response measured from the matrix (e.g. plasma) as a percentage of that from the pure solvent (e.g. water). Results of the experiment that compare matrix to pure solvent is referred to as relative recovery and true test of recovery is referred to as absolute recovery.
Another important issue in method development stage is the choice of internal versus external standardisation. Internal standardisation is common in bioanalytical methods especially with chromatographic procedures. The assumption for the use of internal standard is that the partition coefficient of the analyst and the internal standard are very similar. For internal standardization, a structural or isotopic analogue of the analyte is added to the sample prior to sample pre-treatment and the ratio of the response of the analyte to that of the internal standard is plotted against the concentration.
Another important point is that the tests performed at the stage of method development should be done with the same equipment that will actually be used for subsequent routine analysis. The differences found between individual instruments representing similar models from the same manufacturer is not surprising and should be accounted for.
The following two parameters must be determined at the method development stage as they are important for further work.
1.6.1 Lower limits of detection and quantification (LLOD and LLOQ)
The US pharmacopoeia (USP) defines the limit of detection (LLOD) as the lowest concentration of an analyte in a sample that can be detected but not necessarily quantitated. They also define the lowest limit of quantification (LLOQ), as the lowest amount of a sample that can be determined (quantitated) with acceptable precision and accuracy under the stated operational condition of the method.
The limits are commonly associated with the signal to noise ratio (S/N). In the case of LLOD, analysts often use S/N (signal to noise ratio) of 2:1 or 3:1, while a S/N of 10:1 is often considered to be necessary for the LLOQ. Typically the signal is measured from the base line to peak apex and divided by the peak-to-peak noise, which is determined from the blank plasma injection. 26
A calibration line is a curve showing the relation between the concentrations of the analyte in the sample and the detected response. The relationship between response and concentration must be demonstrated to be continuous and reproducible. In many cases, five to eight concentrations (excluding blank values) may define the standard curve 27.
1.6.2 Steps involved in method development and optimization
The method development and optimization in misanalysis is divided in to two steps.
1. Sample preparation