What are Compendial and non-Compendial analytical procedures ? What is analytical validation ?
Compendial is nothing but Pharmacopoeial and non-com pendial is nothing but non-Pharmacopoeial. The analytical procedure refers to the way of performing the analysis. It should describe in detail the steps necessary to perform each analytical test. This may include, but is not limited to, the sam ple, the reference standard and the reagents preparations, use of the apparatus, generation of the calibration curve, use of the formulae for the calculation, etc.
Standard parameters for inclusion in a study of analytical validation:
Specificity / Selectivity
Accuracy
Precision
Linearity and Range
LOD / LOQ
Ruggedness
Standard and Sample Solution Stability
Robustness (Optional)
Specificity / Selectivity : Specificity is the ability to assess unequivocally the analyte in the presence of com ponents which may be expected to be present. Typically these might include impurities, degradants, matrix, etc.
Lack of specificity of an individual analytical procedure may be com pensated by other supporting analytical procedure(s).
This definition has the following implications:
Identification: to ensure the identity of an analyte.
Purity Tests: to ensure that all the analytical procedures performed allow an accurate statement of the
content of impurities of an analyte, i.e. related substances test, heavy metals, residual solvents content, etc.
Assay (content or potency): to provide an exact result which allows an accurate statement on the content or potency of the analyte in a sam ple.
Accuracy : The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. This is sometimes termed trueness.
Precision :
The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sam pling of the sam e homogeneous sam ple under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility.
Precision should be investigated using homogeneous, authentic sam ples. However, if it is not possible to obtain a homogeneous sam ple it may be investigated using artificially prepared sam ples or a sam ple solution.
The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements.
Repeatability :Repeatability expresses the precision under the sam e operating conditions over a short interval of time. Repeatability is also termed intra-assay precision.
Intermediate Precision : Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc.
Reproducibility : Reproducibility expresses the precision between laboratories (collaborative studies, usually applied to standardization of methodology).
Detection Limit (LOD) : The detection limit of an individual analytical procedure is the lowest amount of analyte in a sam ple which can be detected but not necessarily quantitated as an exact value.
Several approaches for determining the detection limit are possible, depending on whether the procedure is a non- instrumental or instrumental. Approaches other than those listed below may be acceptable.
Based on Visual Evaluation : Visual evaluation may be used for non-instrumental methods but may also be used with instrumental methods. The detection limit is determined by the analysis of sam ples with known concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected.
Based on Signal-to-Noise : This approach can only be applied to analytical procedures which exhibit baseline noise. Determination of the signal-to-noise ratio is performed by com paring measured signals from sam ples with known low concentrations of analyte with those of blank sam ples and establishing the minimum concentration at which the analyte can be reliably detected. A signal-to-noise ratio between 3 or 2:1 is generally considered acceptable for estimating the detection limit.
Based on the Standard Deviation of the Response and the Slope : The detection limit (DL) may be expressed as : DL = 3.3 Stddev / S
where = the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibration curve of the analyte. The estimate of may be carried out in a variety of ways, for example:
Based on the Standard Deviation of the Blank : Measurement of the magnitude of analytical background response is performed by analyzing an appropriate number of blank sam ples and calculating the standard deviation of these responses.
Based on the Calibration Curve : A specific calibration curve should be studied using sam ples containing an analyte in the range of DL. The residual standard deviation of a regression line or the standard deviation of y-intercepts of regression lines may be used as the standard deviation.
Quantitation Limit (LOQ) : The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sam ple which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of com pounds in sam ple matrices, and is used particularly for the determination of impurities and/or degradation products.
Several approaches for determining the quantitation limit are possible, depending on whether the procedure is a non- instrumental or instrumental. Approaches other than those listed below may be acceptable.
Based on Visual Evaluation : Visual evaluation may be used for non-instrumental methods but may also be used with instrumental methods. The quantitation limit is generally determined by the analysis of sam ples with known concentrations of analyte and by establishing the minimum level at which the analyte can be quantified with acceptable accuracy and precision.
Based on Signal-to-Noise Approach : This approach can only be applied to analytical procedures that exhibit baseline noise. Determination of the signal-to-noise ratio is performed by com paring measured signals from sam ples with known low concentrations of analyte with those of blank sam ples and by establishing the minimum concentration at which the analyte can be reliably quantified. A typical signal-to-noise ratio is 10:1.
Based on the Standard Deviation of the Response and the Slope : The quantitation limit (QL) may be expressed as: QL = 10 Stddev / S
W here = the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibration curve of the analyte. The estimate of may be carried out in a variety of ways for example:
Based on Standard Deviation of the Blank : Measurement of the magnitude of analytical background response is performed by analyzing an appropriate number of blank sam ples and calculating the standard deviation of these responses.
Based on the Calibration Curve : A specific calibration curve should be studied using sam ples, containing an analyte in the range of QL. The residual standard deviation of a regression line or the standard deviation of y-intercepts of regression lines may be used as the standard deviation.
Linearity : The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sam ple.
Range : The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sam ple (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.
Robustness : 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.
Examples of typical variations are:
- stability of analytical solutions;
- extraction time.
In the case of liquid chromatography, examples of typical variations are:
- influence of variations of pH in a mobile phase;
- influence of variations in mobile phase com position;
- different columns (different lots and/or suppliers);
- temperature;
- flow rate.
In the case of gas-chromatography, examples of typical variations are:
- different columns (different lots and/or suppliers);
- temperature;
- flow rate.
Sample Solution Stability : This study determines the time period after sam ple preparation during which the com pound of interest remains stable in the HPLC apparatus under the described analytical conditions. Data to support the sam ple solution stability under normal laboratory conditions for the duration of the test procedures, e.g., 24 hours should be generated and provides an indication of its reliability during normal usage.
Other points to consider include:
Capacity Factor (k') : The capacity factor is a measure of where the peak of interest is located with respect to the
void volume, i.e., elution time of the non-retained com ponents. The peak should be well resolved from other peaks
and the void volume. Generally, the value of k' is >2.
Precision/Injection repeatability (RSD) Injection precision expressed as RSD (relative standard deviation) or coefficient of variation (C.V.) indicates the performance of the HPL chromatograph which includes the plumbing, column, and environmental conditions, at the time the sam ples are analysed. It should be noted that sam ple preparation and manufacturing variations are not considered. A C.V. of ≤ 1% for n ≥ 5 is desirable.
Relative retention ( α ) : Relative retention is a measure of the relative location of two peaks. This is not an essential parameter as long as the resolution (R s) is stated.
Resolution (Rs) : Rs is a measure of how well two peaks are separated. For reliable quantitation, well separated peaks are essential for quantitation. This is a very useful parameter if potential interference peak(s) may be of concern. The closest potential eluting peak to the analyte should be selected.
Rs of >2 between the peak of interest and the closest potential interfering peak (impurity, excipient, degradation product, internal standard, etc.) is desirable.
Tailing factor (T) : The tailing factor, a measure of peak sym metry, is unity for perfectly sym metrical peaks and its value increases as tailing becom es more pronounced. As peaks asym metry increases, integration and hence precision becom es less reliable. T of ≤ 2 is desirable.
Theoretical plate number (N) :Theoretical plate number is a measure of column efficiency, i.e., how many peaks can be located per unit run-time of the chromatogram.
N is fairly constant for each peak on a chromatogram with a fixed set of operating conditions. H or HETP, the height equivalent of a theoretical plate, measures the column efficiency per unit length (L) of the column.
Parameters which can affect N or H include peak position, particle size in column, flow rate of mobile phase, column temperature, viscosity of mobile phase and molecular weight of the analyte.
The theoretical plate number depends on elution time but in general should be >2000.
Methods have been developed and validated in the following technologies:
Confirmation of identity by NMR, IR, MS
Assay of com ponents by GC, HPLC, W et Chemistry
Organic impurities by GC, Ion chromatography Trace elements and ions by Ion Chromatography Chiral Purity by NMR and Chromatographies
A wide range of physical property measurements on products and formulations can also be carried, for example, viscosity (for liquid products), porosity (for marcroporous materials), and XRD (for crystalline materials).
