Analytical Test Method Validation for API Raw Material, In Process Control, and Early Intermediate Material Tests
Quantitation and Detection Limit
Introduction
This procedure provides guidance for the validation of analytical test methods. These analytical test methods include those tests which evaluate API Raw Materials, In Process samples (e.g. reaction monitoring) and early intermediate materials (prior to the introduction of the first critical intermediate).
Per ICH Q7A, the degree of analytical validation performed should reflect the purpose of the analysis and the stage of the API production process.
Guidance for quantitation and detection limit testing is provided, including approaches, recommended data and acceptance criteria.
Quantitation Limit
The quantitation limit is an important parameter when dealing with quantitative assays for low levels of compounds in sample matrices. The quantitation limit is normally established and confirmed for methods used to determine impurities or degradation products.
In many cases it may be appropriate to prescribe a quantitation limit, which is at an analytically significant level, rather than necessarily attempting to establish the absolute level, at which quantitation becomes unacceptable. Impurities methods normally fall into this category. The ICH standards Q3A and Q3B define thresholds for reporting of impurities and degradation products below which impurities need not be quantitated or reported. For these cases, it should be determined that the quantitation limit is at or below the reporting level.
Several approaches for determining the quantitation limit are possible, depending on whether the procedure is a non-instrumental or instrumental method. Approaches other than those listed below may be acceptable. Determination of quantitation limit is not normally required for assay or identification tests.
Based on Visual Evaluation
Visual examination 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 samples
with known concentrations of analyte and by establishing the minimum level at which the analyte can be quantified with acceptable accuracy and precision. The quantitation limit must not be
greater than 50% of the specification, where technically feasible.
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 comparing measured signals from samples with known low concentrations of analyte with those of blank samples, 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 Capability of the Instrument
In some cases the instrument itself is the limiting factor for the analysis regardless of the sample. An example of this is an LOD test using an analytical balance. In this case a discussion of the quantitation limit may be constructed in the validation documentation based on the calibration tolerance of the equipment rather than analysis of actual samples. The actual limit of quantitation would still be presented in numerical terms relevant to the assay method based on the discussion.
Another example of this may be for KF titration assays where the ability of the instrument to deliver a minimum amount of titrant would be the limiting factor. It is recommended that experiments to determine this minimum amount of sample should be conducted for the specific instrument model if this approach is taken. The experiment(s) could then be referred to in any validation that utilizes the same model of equipment.
Based on the Standard Deviation of the Response and the Slope.
The quantitation limit (QL) may be expressed as: QL = 10 σ/ S where, σ= the 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 σ is carried out in a variety of ways including:
Based on the Standard Deviation of the Blank:
Analyzing an appropriate number of blank samples and calculating the standard deviation of these responses and perform measurement of the magnitude of analytical background response.
Based on the Calibration Curve:
A specific calibration curve should be studied using samples containing an analyte in the range of the 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. In all cases, the quantitation limit can be subsequently validated by the analysis of a suitable number of samples known to be near or prepared at the quantitation limit or reporting level.
Two possible approaches include:
A) Three replicate preparations of a spiked sample are prepared at the quantitation level or reporting level and analyzed. Calculate the % recovery. Calculate the average of the replicates and % RSD.
B) Alternatively, accuracy or repeatability experiments at or near the quantitation limit or reporting level can be used for this determination.
Recommended QL Data:
The quantitation limit and the method used for determining the quantitation limit should be presented. For validation of the actual quantitation limit or reporting level:
For case (A) it is suggested to report the average of replicates, % recovery and RSD. For case (B) it is suggested to report the accuracy or repeatability of replicate experiments conducted at or near the reporting level.
The quantitation limit should be expressed as the amount actually measured, as well as the corresponding percentage of the target analyte concentration. If applicable, representative chromatograms can be presented at an expansion that allows visual inspection of the signal vs. noise and integrations that impact quantitation.
Recommended QL Criteria:
It is suggested that accuracy and precision criteria are met.
Detection Limit
The detection limit may be an important parameter for non-quantitative tests. In some cases it may be desirable to establish the detection limit for quantitative tests but this is generally not required.
Several approaches for determining the detection limit are possible depending on whether the procedure is non-instrumental or instrumental. Approaches other than those listed below may also be acceptable.
The limit of detection may be estimated by any one of the following.
- A solution of the impurity at a concentration corresponding to the lowest level demonstrated to be within the established linear range of the method is injected six times. The detection limit is estimated as the concentration that corresponds to 3 times the standard deviation of the responses for the six injections.
- 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 samples 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 comparing measured signals from samples with known low concentrations of analyte with those of blank samples 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 σ/ S where, σ= 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 σ maybe carried out in a variety of ways, for example:
- Based on the Standard Deviation of the blank: Analyzing an appropriate number of blank samples and calculating the standard deviation of these responses and perform measurement of the magnitude of analytical background response.
- Based on the Calibration Curve: A specific calibration curve can be studied using samples 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.
The assay need not give results that are directly proportional to the concentration (amount) of analyte in the sample for the test method to be valid. However, the desire to have a linear relationship reflects a practical consideration, since a linear relationship should be accurately described with fewer standards.
A validated method may be sufficiently linear to meet accuracy requirements in the concentration range in which it is intended to be used. When inferring accuracy from a linearity study, linearity could be considered acceptable if results, as compared to a standard, meet the accuracy criteria. A plot of the data should visually appear to be linear. Suggested acceptance criteria (for API Raw Material, In Process Control, and early intermediate material tests) for an acceptable linear relationship may be a test method having a minimum correlation coefficient (r) of > 0.95.
Range:
The range is the interval between the upper and lower levels of analyte concentration for which acceptable linearity, accuracy (recovery), and precision are obtained. It is recommended that the range be established to include all specification limits for a method and the expected results. The range should include at least five points to establish linearity. Values outside of the validated range can be reported as estimates. Range should be established by summarizing the accuracy (where appropriate), the linearity, and the precision data.
The following minimum specified ranges are taken from ICH and may be considered as minimum start points for test methods within the scope of this document.
- For the assay, the ICH range is normally from 80% to 120% of the test concentration. If assay and purity are performed together as one test and only a 100% standard is used, linearity should cover the range from the reporting level of the impurities to 120% of the assay specification.
- For determination of an impurity; the range of concentrations used to evaluate the linearity should consist of the quantitation limit and at least 120% greater than the concentration that would be the impurity specification limit.
- For example, if the concentration at the specification limit was 0.2% w/w, and the limit of quantitation was 0.08% w/w then the range should span 0.08% (w/w) to 0.24% w/w. For example, the concentrations for the linearity experiment might be 0.08%, 0.12%, 0.16% 0.20% and 0.24%. More solutions may be evaluated if the linearity range must be extended.
- In cases where specified impurities/degradation products are not available a surrogate material such as a compound with similar structure or API may be used to demonstrate linearity. In these cases, a rationale for the use of a surrogate should be given.
- For impurities known to be unusually potent or to produce toxic or unexpected pharmacological effects, the detection/quantitation limit should be commensurate with the level at which the impurities must be controlled.
- For validation of impurity test procedures carried out during development, it may be necessary to consider the range around a suggested (probable) limit.
In cases where an estimated value for the detection limit is obtained by calculation or extrapolation, this estimate may subsequently be validated by the independent analysis of a suitable number of samples known to be near or prepared at the detection limit.
Determination of detection limit is not normally required for assay or identification tests.
Recommended DL Data:
It is recommended that the detection limit and the method used for determining the detection limit should be presented. If DL is determined based on visual evaluation or based on signal-to-noise ratio, the presentation of the relevant chromatograms is considered acceptable for justification.
The detection limit can be expressed as the amount actually measured. If applicable, representative chromatograms, spectra or other instrumental outputs can be presented.
Recommended DL Acceptance Criteria:
It is suggested that for limit tests, the detection limit should be less than one-half the specification limit where technically feasible. The detection limit should be less than the required reporting level.
For Example, if the method is a limits test, the detection limit must be below the lowest residue limit.
Sensitivity: For limit of quantitation of analyte the article suggests that an analyst determines the smallest quantity of analyte detectable at the most sensitive instrument settings. This limit is suggested to be twice the signal to noise ratio. The detection limit may prove to be outside the range examined in the linearity test. For the limit of quantitation of minor components it is accepted that the LOQ be 10 times the signal to noise ratio.