2.6: Post-selection IVT and journal club - Biology

2.6: Post-selection IVT and journal club - Biology

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You'll begin today by setting up your post-column IVT reactions. Then we will move to a nearby classroom for the first round of journal club presentations.


Part 1: IVT

Refer to the Day 3 protocol to prepare your IVT Master Mix, except this time shoot for a total volume of 40 μL rather than 80 μL per reaction. (Thus, you will add 6.6 μL of RT-PCR mixture to each reaction instead of 13.1 μL as you did before.) Note the time that you put your reactions on the 37 °C heat block.

Part 2: Journal Club

Half of the students have signed up give their presentations today; the other half will present on Day 8. See the Module 1 Journal Club assignment page for more information.

For Next Time

  • For everyone...
    • No additional lab report homework will be due. However, it is strongly suggested that you continue to work on your report, particularly the RT-PCR figure and experimental schematic. As always, I am happy to provide feedback even when I am not providing a grade.
  • If you presented for journal club today...
    • You should work on Part III of the computational assignment, and hand in a picture of the full-length 8-12 sequence with the requested highlighting on Day 7.
    • An awareness of your own strengths and weaknesses can often help you improve your future work. After you give your presentation today, write a brief self-evaluation. Specifically, describe (in a short phrase or sentence each) two things that you thought you did well, and two that could use improvement. Feel free to include both big-picture and detail-oriented comments. This assignment is due by email within 48 hours after your presentation.

Reagent List

Same list as for Day 3.

Validation of Analytical Methods and Procedures Author: Dr. Ludwig Huber(Source:labcompliance)

Method validation is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Results from method validation can be used to judge the quality, reliability and consistency of analytical results it is an integral part of any good analytical practice.

Analytical methods need to be validated or revalidated

Method validation has received considerable attention in the literature and from industrial committees and regulatory agencies.

The USP has published specific guidelines for method validation for compound evaluation (7). USP defines eight steps for validation:

The FDA has also published a guidance for the validation of bioanalytical methods (8). The most comprehensive document is the conference report of the 1990 Washington conference: Analytical Methods Validation: Bioavailability, Bioequivalence and Pharmacokinetic Studies, which was sponsored by, among others, the American Association of Pharmaceutical Scientists (AAPS), the AOAC and the U.S. FDA (70). The report presents guiding principles for validating studies of both human and animal subjects. The report has also been used as a basis for the FDA industry guidance document (8).

Representatives of the pharmaceutical and chemical industry have published papers on the validation of analytical methods. Hokanson (9,10) applied the life cycle approach, developed for computerized systems, to the validation and revalidation of methods. Green (11) gave a practical guide for analytical method validation, with a description of a set of minimum requirements for a method. Renger and his colleagues (12) described the validation of a specific analytical procedure for the analysis of theophylline in a tablet using high-performance thin layer chromatography (HPTLC). The validation procedure in this particular article is based on requirements for EU multistate registration.

Wegscheider (13) has published procedures for method validation with a special focus on calibration, recovery experiments, method comparison and investigation of ruggedness. Seno et al. (14) have described how analytical methods are validated in a Japanese QC laboratory. The AOAC (15) has developed a Peer-Verified Methods validation program with detailed guidelines on exactly which parameters should be validated. Winslow and Meyer (16) recommend the definition and application of a master plan for validating analytical methods. J.Breaux and colleagues have published a study on analytical methods development and validation (17). The key point is to develop methods for easy validation and revalidation. O. Krause published a guide for analytical method transfer, comparability, maintenance and acceptance criteria for the testing of biopharmaceuticals (18).

This primer gives a review and a strategy for the validation of analytical methods for both methods developed in-house as well as standard methods, and a recommendation on the documentation that should be produced during, and on completion of, method validation. It also describes what is important when transferring a method.

Strategy for the Validation of Methods

The validity of a specific method should be demonstrated in laboratory experiments using samples or standards that are similar to unknown samples analyzed routinely. The preparation and execution should follow a validation protocol, preferably written in a step-by-step instruction format. Possible steps for a complete method validation are listed in Table 1. This proposed procedure assumes that the instrument has been selected and the method has been developed. It meets criteria such as ease of use ability to be automated and to be controlled by computer systems costs per analysis sample throughput turnaround time and environmental, health and safety requirements.

Table 1. Steps in Method Validation

Successful acceptance of the validation parameters and performance criteria, by all parties involved, requires the cooperative efforts of several departments, including analytical development, QC, regulatory affairs and the individuals requiring the analytical data. The operating procedure or the Validation Master Plan (VMP) should clearly define the roles and responsibilities of each department involved in the validation of analytical methods.

The scope of the method and its validation criteria should be defined early in the process. These include the following questions:

The method’s performance characteristics should be based on the intended use of the method. It is not always necessary to validate all analytical parameters that are available for a specific technique. For example, if the method is to be used for qualitative trace level analysis, there is no need to test and validate the method’s limit of quantitation, or the linearity, over the full dynamic range of the equipment. Initial parameters should be chosen according to the analyst’s experience and best judgment. Final parameters should be agreed between the lab or analytical chemist performing the validation and the lab or individual applying the method and users of the data to be generated by the method. Table 2 gives examples of which parameters might be tested for a particular analysis task.

The scope of the method should also include the different types of equipment and the locations where the method will be run. For example, if the method is to be run on a specific instrument in a specific laboratory, there is no need to use instruments from other vendors or to include other laboratories in the validation experiments. In this way, the experiments can be limited to what is really necessary.

Major compounds and traces

Table 2. Validation parameters for specific tasks

The validation experiments should be carried out by an experienced analyst to avoid errors due to inexperience. The analyst should be very well versed in the technique and operation of the instrument. Before an instrument is used to validate a method, its performance specifications should be verified using generic chemical standards. Satisfactory results for a method can be obtained only with equipment that is performing well. Special attention should be paid to those equipment characteristics that are critical for the method. For example, if detection limit is critical for a specific method, the instrument’s specification for baseline noise and, for certain detectors, the response to specified compounds should be verified.

Any chemicals used to determine critical validation parameters, such as reagents and reference standards, should be

Any other materials and consumables, for example, chromatographic columns, should be new and be qualified to meet the column’s performance criteria . This ensures that one set of consumables can be used for most experiments and avoids unpleasant surprises during method validation.

Operators should be sufficiently familiar with the technique and equipment. This will allow them to identify and diagnose unforeseen problems more easily and to run the entire process more efficiently.

If there is little or no information on the method’s performance characteristics, it is recommended to prove the suitability of the method for its intended use in initial experiments. These studies should include the approximate precision, working range and detection limits. If the preliminary validation data appear to be inappropriate, the method itself, the equipment, the analysis technique or the acceptance limits should be changed. Method development and validation are, therefore, an iterative process. For example, in liquid chromatography, selectivity is achieved through the selection of mobile phase composition. For quantitative measurements, the resolution factor between two peaks should be 2.5 or higher. If this value is not achieved, the mobile phase composition needs further optimization. The influence of operating parameters on the performance of the method should be assessed at this stage if this was not done during development and optimization of the method.

There are no official guidelines on the correct sequence of validation experiments, and the optimal sequence may depend on the method itself. Based on the author’s experience, for a liquid chromatographic method, the following sequence has proven to be useful:

The more time-consuming experiments, such as accuracy and ruggedness, are included toward the end. Some of the parameters, as listed under (2) to (6), can be measured in combined experiments. For example, when the precision of peak areas is measured over the full concentration range, the data can be used to validate the linearity.

During method validation, the parameters, acceptance limits and frequency of ongoing system suitability tests or QC checks should be defined. Criteria should be defined to indicate when the method and system are beyond statistical control. The aim is to optimize these experiments so that, with a minimum number of control analyses, the method and the complete analytical system will provide long-term results to meet the objectives defined in the scope of the method.

Once the method has been developed and validated, a validation report should be prepared that includes the following:

Verification of Standard Methods

A laboratory applying a specific method should have documented evidence that the method has been appropriately validated. This holds for methods developed in-house, as well as for standard methods, for example, those developed by organizations such as the EPA, American Society for Testing and Materials (ASTM), ISO or the USP.

A number of questions usually arises about the validation of standard methods: Firstly, should these methods be revalidated in the user’s laboratory and, if so, should method revalidation cover all experiments, as performed during initial validation? Secondly, which documentation should be available or developed in-house for standard methods? Official guidelines and regulations are not explicit about validating standard methods. Only CITAC/EURACHEM guide (19) includes a short paragraph that reads as follows:

The validation of standard or collaboratively tested methods should not be taken for granted, no matter how impeccable the method’s pedigree - the laboratory should satisfy itself that the degree of validation of a particular method is adequate for the required purpose, and that the laboratory is itself able to match any stated performance data.

There are two important requirements in this excerpt:

Further advice comes from FDA’s 21 CFR 194 section(a)2: “If the method employed is in the current revision of the United States Pharmacopeia, National Formulary, Association of Official Analytical Chemists, or in other recognized standard references, or is detailed in an approved new drug application and the referenced method is not modified, a statement indicating the method and reference will suffice. The suitability of all testing methods used shall be verified under actual conditions of use.” The spirit of this text is in line with the two requirements listed above. 

This section elaborates on what these statements mean in practice, and it gives a strategy for validating standard methods. Like the validation of methods developed in-house, the evaluation and verification of standard methods should also follow a documented process that is usually the validation plan. Results should be documented in the validation protocol. Both documents will be the major source for the validation report.

Figure 1. Workflow for evaluation and validation of standard methods

An example of a step-by-step plan for the evaluation and validation of standard methods is shown as a flow diagram in Figure 1. As a first step, the scope of the method, as applied in the user’s laboratory, should be defined. This should be done independently of what is written in the standard method and should include information such as

As a second step, the method’s performance requirements should be defined in considerable detail, again irrespective of what has been validated in the standard method. General guidelines on validation criteria for different measurement objectives and procedures for their evaluation are discussed later in this chapter.
The results of these steps lead to the experiments that are required for adequate method validation and to the minimal acceptance criteria necessary to prove that the method is suitable for its intended use. Third, required experiments and expected results should be compared with what is written in the standard method.
In particular, the standard method should be checked for the following items:

Figure 2. Steps for validating complete analytical procedures. Standard methods should be checked if all steps are included in the validation data. 

If either the scope, the validation parameters or the validation results do not meet the user’s requirements, adequate validation experiments should be defined, developed and carried out. The extent of these experiments depends on the overlap of the user requirements with the scope and results, as described in the standard method. If there is no overlap, a complete validation should be carried out. In the case of a complete overlap, validation experiments may not be necessary.

If method validation experiments are unnecessary, the user should prove the suitability of the method in his or her laboratory. This evidence should confirm that the user’s equipment, the people, the reagents and the environment are qualified to perform the analysis. The experiments may be an extract of the full method validation and should focus on the critical items of the method. Guidelines for these tests should have been developed during method development. If not, they should be developed and carried out at this stage. Typical experiments may include precision of amounts and limits of quantitation. The validation report should include a reference to the standard method.

Validation of Non-routine Methods

Frequently, a specific method is used for only a few sample analyses. The question should be raised as to whether this method also needs to be validated using the same criteria as recommended for routine analysis. In this case, the validation may take much more time than the sample analysis and may be considered inefficient, because the cost per sample will increase significantly. The answer is quite simple: Any analysis is worthwhile only if the data are sufficiently accurate otherwise, sample analysis is pointless. The suitability of an analysis method for its intended use is a prerequisite to obtaining accurate data therefore, only validated methods should be used to acquire meaningful data. However, depending on the situation, the validation efforts can be reduced for non-routine methods. The CITAG/ EURACHEM guide (19) includes a chapter on how to treat non-routine methods. The recommendation is to reduce the validation cost by using generic methods, for example, methods that are broadly applicable. A generic method could, for example, be based on capillary gas chromatography or on reversed phase gradient HPLC. With little or no modification, the method can be applied to a large number of samples. The performance parameters should have been validated on typical samples characterized by sample matrix, compound types and concentration range.

If, for example, a new compound with a similar structure in the same matrix is to be analyzed, the validation will require only a few key experiments. The documentation of such generic methods should be designed to easily accommodate small changes relating to individual steps, such as sample preparation, sample analysis or data evaluation.

The method’s operating procedure should define the checks that need to be carried out for a novel analyte in order to establish that the analysis is valid. Detailed documentation of all experimental parameters is important to ensure that the work can be repeated in precisely the same manner at any later date. 

Quality Control Plan and Implementation for Routine

For any method that will be used for routine analysis, a QC plan should be developed. This plan should ensure that the method, together with the equipment, delivers consistently accurate results. The plan may include recommendations for the following:

In many cases, methods are developed and validated in service laboratories that are specialized in this task. When the method is transferred to the routine analytical laboratory, care should be taken that the method and its critical parameters are well understood by the workers in the departments who apply the method. A detailed validation protocol, a documented procedure for method implementation and good communication between the development and operation departments are equally important. If the method is used by a number of departments, it is recommended to verify method validation parameters and to test the applicability and usability of the method in a couple of these departments before it is distributed to other departments. In this way, problems can be identified and corrected before the method is distributed to a larger audience. If the method is intended to be used by just one or two departments, an analyst from the development department should assist the users of the method during initial operation. Users of the method should be encouraged to give constant feedback on the applicability and usability of the method to the development department. The latter should correct problems if any arise.

Transferring Validated Routine Methods

Validated routine methods are transferred between laboratories at the same or different sites when contract laboratories offer services for routine analysis in different areas or when products are manufactured in different areas. When validated routine methods are transferred between laboratories and sites, their validated state should be maintained to ensure the same reliable results in the receiving laboratory. This means the competence of the receiving laboratory to use the method should be demonstrated through tests, for example, repeat critical method validation experiments and run samples in parallel in the transferring and receiving laboratories. The transfer should be controlled by a procedure, The recommended steps are:

Most likely some method parameters have to be changed or adjusted during the life of the method if the method performance criteria fall outside their acceptance criteria. The question is whether such change requires revalidation. In order to clarify this question upfront, operating ranges should be defined for each method, either based on experience with similar methods or else investigated during method development. These ranges should be verified during method validation in robustness studies and should be part of the method characteristics. Availability of such operating ranges makes it easier to decide when a method should be revalidated. A revalidation is necessary whenever a method is changed, and the new parameter lies outside the operating range. If, for example, the operating range of the column temperature has been specified to be between 30 and 40°C, the method should be revalidated if, for whatever reason, the new operating parameter is 41°C.

Revalidation is also required if the scope of the method has been changed or extended, for example, if the sample matrix changes or if operating conditions change. Furthermore, revalidation is necessary if the intention is to use instruments with different characteristics, and these new characteristics have not been covered by the initial validation. For example, an HPLC method may have been developed and validated on a pump with a delay volume of 5 mL, but the new pump has a delay volume of only 0.5 mL.

Figure 3. Flow diagram for revalidation

Part or full revalidation may also be considered if system suitability tests, or the results of QC sample analysis, lie outside preset acceptance criteria and where the source of the error cannot be traced back to the instruments or any other cause.

Whenever there is a change that may require part or full revalidation, the change should follow a documented change control system. A flow diagram of such a process is documented in Figure 3. The change should be defined, authorized for implementation and documented. Possible changes may include

An evaluation should determine whether the change is within the scope of the method. If so, no revalidation is required. If the change lies outside the scope, the parameters for revalidation should be defined. After the validation experiments, the system suitability test parameters should be investigated and redefined, if necessary.

Parameters for Method Validation

The parameters for method validation have been defined in different working groups of national and international committees and are described in the literature. Unfortunately, some of the definitions vary between the different organizations. An attempt at harmonization was made for pharmaceutical applications through the ICH (4,5), where representatives from the industry and regulatory agencies from the United States, Europe and Japan defined parameters, requirements and, to some extent, methodology for analytical methods validation. The parameters, as defined by the ICH and by other organizations and authors, are summarized in Table 3 and are described in brief in the following paragraphs.

Table 3. Possible analytical parameters for method validation

(1) Included in ICH publications, (2) Included in USP

(3) Terminology included in ICH publication but not part of required parameters


The terms selectivity and specificity are often used interchangeably. A detailed discussion of this term, as defined by different organizations, has been presented by Vessmann (20). He particularly pointed out the difference between the definitions of specificity given by IUPAC/WELAC and the ICH.

Although it is not consistent with the ICH, the term specific generally refers to a method that produces a response for a single analyte only, while the term selective refers to a method that provides responses for a number of chemical entities that may or may not be distinguished from each other. If the response is distinguished from all other responses, the method is said to be selective. Since there are very few methods that respond to only one analyte, the term selectivity is usually more appropriate. The USP monograph (7) defines the selectivity of an analytical method as its ability to measure accurately an analyte in the presence of interference, such as synthetic precursors, excipients, enantiomers and known (or likely) degradation products that may be expected to be present in the sample matrix. Selectivity in liquid chromatography is obtained by choosing optimal columns and setting chromatographic conditions, such as mobile phase composition, column temperature and detector wavelength. Besides chromatographic separation, the sample preparation step can also be optimized for best selectivity.

It is a difficult task in chromatography to ascertain whether the peaks within a sample chromatogram are pure or consist of more than one compound. Therefore, the analyst should know how many compounds are in the sample or whether procedures for detecting impure peaks should be used.

While in the past chromatographic parameters such as mobile phase composition or the column were modified, now the application of spectroscopic detectors coupled on-line to the chromatograph is being used. UV/visible diode-array detectors and mass spectrometers acquire spectra on-line throughout the entire chromatogram. The spectra acquired during the elution of a peak are normalized and overlaid for graphical presentation. If the normalized spectra are different, the peak consists of at least two compounds.

The principles of diode-array detection in HPLC and their application and limitations with regard to peak purity are described in the literature (21). Examples of pure and impure HPLC peaks are shown in Figure 4. While the chromatographic signal indicates no impurities in either peak, the spectral evaluation identifies the peak on the left as impure. The level of impurities that can be detected with this method depends on the spectral difference, on the detector’s performance and on the software algorithm. Under ideal conditions, peak impurities of 0.05 to 0.1 percent can be detected.

Selectivity studies should also assess interferences that may be caused by the matrix, e.g., urine, blood, soil, water or food. Optimized sample preparation can eliminate most of the matrix components. The absence of matrix interferences for a quantitative method should be demonstrated by the analysis of at least five independent sources of control matrix.

Figure 4. Examples of pure and impure HPLC peaks. The chromatographic signal does not indicate any impurity in either peak. Spectral evaluation, however, identifies the peak on the left as impure.

Precision and Reproducibility

The precision of a method (Table 4) is the extent to which the individual test results of multiple injections of a series of standards agree. The measured standard deviation can be subdivided into 3 categories: repeatability, intermediate precision and reproducibility (4, 5). Repeatability is obtained when the analysis is carried out in a laboratory by an operator using a piece of equipment over a relatively short time span. At least 6 determinations of 3 different matrices at 2 or 3 different concentrations should be performed, and the RSD calculated.

The ICH (4) requires precision from at least 6 replications to be measured at 100 percent of the test target concentration or from at least 9 replications covering the complete specified range. For example, the results can be obtained at 3 concentrations with 3 injections at each concentration.

The acceptance criteria for precision depend very much on the type of analysis. Pharmaceutical QC precision of greater than 1 percent RSD is easily achieved for compound analysis, but the precision for biological samples is more like 15 percent at the concentration limits and 10 percent at other concentration levels. For environmental and food samples, precision is largely dependent on the sample matrix, the concentration of the analyte, the performance of the equipment and the analysis technique. It can vary between 2 percent and more than 20 percent.

The AOAC manual for the Peer-Verified Methods program (15) includes a table with estimated precision data as a function of analyte concentration (Table 4).

Intermediate precision is a term that has been defined by ICH (4) as the long-term variability of the measurement process. It is determined by comparing the results of a method run within a single laboratory over a number of weeks. A method’s intermediate precision may reflect discrepancies in results obtained

Table 4. Analyte concentration versus precision (Ref. 15)

The objective of intermediate precision validation is to verify that in the same laboratory the method will provide the same results once the development phase is over.

Reproducibility (Table 5), as defined by the ICH (4), represents the precision obtained between different laboratories. The objective is to verify that the method will provide the same results in different laboratories. The reproducibility of an analytical method is determined by analyzing aliquots from homogeneous lots in different laboratories with different analysts, and by using operational and environmental conditions that may differ from, but are still within, the specified parameters of the method (interlaboratory tests). Validation of reproducibility is important if the method is to be used in different laboratories.

Table 5. Typical variations affecting a method’s reproducibility

Table 6 summarizes factors that should be the same, or different, for precision, intermediate precision and reproducibility.

batches of accessories e.g. chrom. columns

Batches of material, e.g., reagents

Environmental conditions, e.g., temperature

Table 6. Variables for measurements of precision, intermediate precision and reproducibility

Accuracy and Recovery

The accuracy of an analytical method is the extent to which test results generated by the method and the true value agree. Accuracy can also be described as the closeness of agreement between the value that is adopted, either as a conventional, true or accepted reference value, and the value found.

The true value for accuracy assessment can be obtained in several ways. One alternative is to compare the results of the method with results from an established reference method. This approach assumes that the uncertainty of the reference method is known. Secondly, accuracy can be assessed by analyzing a sample with known concentrations (e.g., a control sample or certified reference material) and comparing the measured value with the true value as supplied with the material. If certified reference materials or control samples are not available, a blank sample matrix of interest can be spiked with a known concentration by weight or volume. After extraction of the analyte from the matrix and injection into the analytical instrument, its recovery can be determined by comparing the response of the extract with the response of the reference material dissolved in a pure solvent. Because this accuracy assessment measures the effectiveness of sample preparation, care should be taken to mimic the actual sample preparation as closely as possible. If validated correctly, the recovery factor determined for different concentrations can be used to correct the final results.

The concentration should cover the range of concern and should include concentrations close to the quantitation limit, one in the middle of the range and one at the high end of the calibration curve. Another approach is to use the critical decision value as the concentration point that must be the point of greatest accuracy.

Table 7. Analyte recovery at different concentrations (Ref 9)

The expected recovery (Table 7) depends on the sample matrix, the sample processing procedure and the analyte concentration. The AOAC manual for the Peer-Verified Methods program (15) includes a table with estimated recovery data as a function analyte concentration.

The ICH document on validation methodology recommends accuracy to be assessed using a minimum of nine determinations over a minimum of three concentration levels covering the specified range (e.g., three concentrations/three replicates each). Accuracy should be reported as percent recovery by the assay of known added amount of analyte in the sample or as the difference between the mean and the accepted true value, together with the confidence intervals.

Linearity and Calibration Curve

The linearity of an analytical method is its ability to elicit test results that are directly proportional to the concentration of analytes in samples within a given range or proportional by means of well-defined mathematical transformations. Linearity may be demonstrated directly on the test substance (by dilution of a standard stock solution) and/or by using separate weighings of synthetic mixtures of the test product components, using the proposed procedure.

Linearity is determined by a series of 3 to 6 injections of 5 or more standards whose concentrations span 80� percent of the expected concentration range. The response should be directly proportional to the concentrations of the analytes or proportional by means of a well-defined mathematical calculation. A linear regression equation applied to the results should have an intercept not significantly different from 0. If a significant nonzero intercept is obtained, it should be demonstrated that this has no effect on the accuracy of the method.

Frequently, the linearity is evaluated graphically, in addition to or as an alternative to mathematical evaluation. The evaluation is made by visually inspecting a plot of signal height or peak area as a function of analyte concentration. Because deviations from linearity are sometimes difficult to detect, two additional graphical procedures can be used. The first is to plot the deviations from the regression line versus the concentration or versus the logarithm of the concentration, if the concentration range covers several decades. For linear ranges, the deviations should be equally distributed between positive and negative values.

Another approach is to divide signal data by their respective concentrations, yielding the relative responses. A graph is plotted with the relative responses on the y-axis and the corresponding concentrations on the x-axis, on a log scale. The obtained line should be horizontal over the full linear range. At higher concentrations, there will typically be a negative deviation from linearity. Parallel horizontal lines are drawn on the graph corresponding to, for example, 95 percent and 105 percent of the horizontal line. The method is linear up to the point where the plotted relative response line intersects the 95 percent line. Figure 5 shows a comparison of the two graphical evaluations on a sample of caffeine using HPLC.

The ICH recommends, for accuracy reporting, the linearity curve’s correlation coefficient, y-intercept, slope of the regression line and residual sum of squares. A plot of the data should be included in the report. In addition, an analysis of the deviation of the actual data points from the regression line may also be helpful for evaluating linearity. Some analytical procedures, such as immunoassays, do not demonstrate linearity after any transformation. In this case, the analytical response should be described by an appropriate function of the concentration (amount) of an analyte in a sample. In order to establish linearity, a minimum of five concentrations is recommended. Other approaches should be justified.

Figure 5. Graphical presentations of linearity plot of a caffeine sample using HPLC.

Plotting the sensitivity (response/amount) gives clear indication of the linear range. Plotting the amount on a logarithmic scale has a significant advantage for wide linear ranges. Rc = Line of constant response.

The range of an analytical method is the interval between the upper and lower levels (including these levels) that have been demonstrated to be determined with precision, accuracy and linearity using the method as written. The range is normally expressed in the same units as the test results (e.g., percentage, parts per million) obtained by the analytical method.

For assay tests, the ICH (5) requires the minimum specified range to be 80 to 120 percent of the test concentration, and for the determination of an impurity, the range to extend from the limit of quantitation, or from 50 percent of the specification of each impurity, whichever is greater, to 120 percent of the specification.

Figure 6. Definitions for linearity, range, LOQ, LOD

The limit of detection is the point at which a measured value is larger than the uncertainty associated with it. It is the lowest concentration of analyte in a sample that can be detected but not necessarily quantified. The limit of detection is frequently confused with the sensitivity of the method. The sensitivity of an analytical method is the capability of the method to discriminate small differences in concentration or mass of the test analyte. In practical terms, sensitivity is the slope of the calibration curve that is obtained by plotting the response against the analyte concentration or mass.

In chromatography, the detection limit is the injected amount that results in a peak with a height at least two or three times as high as the baseline noise level. Besides this signal/noise method, the ICH (4) describes three more methods:

Figure 7. Limit of detection and limit of quantitation via signal to noise

Limit of Quantitation

The limit of quantitation is the minimum injected amount that produces quantitative measurements in the target matrix with acceptable precision in chromatography, typically requiring peak heights 10 to 20 times higher than the baseline noise.

If the required precision of the method at the limit of quantitation has been specified, the EURACHEM (22) (Figure 8) approach can be used. A number of samples with decreasing amounts of the analyte are injected six times. The calculated RSD percent of the precision is plotted against the analyte amount. The amount that corresponds to the previously defined required precision is equal to the limit of quantitation. It is important to use not only pure standards for this test but also spiked matrices that closely represent the unknown samples.

For the limit of detection, the ICH (5) recommends, in addition to the procedures as described above, the visual inspection and the standard deviation of the response and the slope of the calibration curve.

Figure 11. Limit of quantitation with the EURACHEM (80) method.

Any results of limits of detection and quantitation measurements must be verified by experimental tests with samples containing the analytes at levels across the two regions. It is equally important to assess other method validation parameters, such as precision, reproducibility and accuracy, close to the limits of detection and quantitation. Figure 6 illustrates the limit of quantitation (along with the limit of detection, range and linearity). Figure 7 illustrates both the limit of detection and the limit of quantitation.

Ruggedness is not addressed in the ICH documents (4,5) Its definition has been replaced by reproducibility, which has the same meaning as ruggedness, defined by the USP as the degree of reproducibility of results obtained under a variety of conditions, such as different laboratories, analysts, instruments, environmental conditions, operators and materials. Ruggedness is a measure of reproducibility of test results under normal, expected operational conditions from laboratory to laboratory and from analyst to analyst. Ruggedness is determined by the analysis of aliquots from homogeneous lots in different laboratories.

Robustness tests examine the effect that operational parameters have on the analysis results. For the determination of a method’s robustness, a number of method parameters, for example, pH, flow rate, column temperature, injection volume, detection wavelength or mobile phase composition, are varied within a realistic range, and the quantitative influence of the variables is determined. If the influence of the parameter is within a previously specified tolerance, the parameter is said to be within the method’s robustness range.

Obtaining data on these effects helps to assess whether a method needs to be revalidated when one or more parameters are changed, for example, to compensate for column performance over time. In the ICH document (5), it is recommended to consider the evaluation of a method’s robustness during the development phase, and any results that are critical for the method should be documented. This is not, however, required as part of a registration.

Many solutes readily decompose prior to chromatographic investigations, for example, during the preparation of the sample solutions, extraction, cleanup, phase transfer or storage of prepared vials (in refrigerators or in an automatic sampler). Under these circumstances, method development should investigate the stability of the analytes and standards.

The term system stability has been defined as the stability of the samples being analyzed in a sample solution. It is a measure of the bias in assay results generated during a preselected time interval, for example, every hour up to 46 hours, using a single solution (Figure 9). System stability should be determined by replicate analysis of the sample solution. System stability is considered appropriate when the RSD, calculated on the assay results obtained at different time intervals, does not exceed more than 20 percent of the corresponding value of the system precision. If, on plotting the assay results as a function of time, the value is higher, the maximum duration of the usability of the sample solution can be calculated.

Figure 9. Schematics of stability testing

The effect of long-term storage and freeze-thaw cycles can be investigated by analyzing a spiked sample immediately after preparation and on subsequent days of the anticipated storage period. A minimum of two cycles at two concentrations should be studied in duplicate. If the integrity of the drug is affected by freezing and thawing, spiked samples should be stored in individual containers, and appropriate caution should be employed for the study of samples.

Which Parameters Should Be Included in Method Validation?

For an efficient validation process, it is of utmost importance to specify the right validation parameters and acceptance criteria. The more parameters, the more time it will take to validate. The more stringent the specifications or acceptance limits, the more often the equipment has to be recalibrated, and probably also requalified, to meet the higher specifications at any one time. It is not always essential to validate every analytical performance parameter, but it is necessary to define which ones are required. This decision should be based on business, regulatory and/or accreditation requirements:

Guidance from different agencies(Source:(cGALP))

Guidance document on part 11-Electronic signature & Electronic record & Computer System Validation(CSV)

Stability study

The purpose of stability testing is to provide evidence on how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors such as temperature, humidity, and light, and to establish a re-test period for the drug substance or a shelf life for the drug product and recommended storage conditions. Stability testing is a routine procedure performed on drug substances and products. It is involved at various stages of product development.

In early stages, accelerated stability testing (at relatively high temperatures and/or humidities) can be used as a “worst case” evaluation to determine what types of degradation products may be found after long-term storage.

Testing under more gentle conditions (those recommended for long-term shelf storage), and slightly elevated temperatures, can be used to determine a product’s shelf life and expiration dates.

In these types of studies, the product is analyzed at regular intervals for various parameters, which may include assay of the active ingredient, measurement of known degradation products, dissolution time, appearance, etc.

Quality Guidelines : (Stability)

Guidelines for stability testing of pharmaceutical products containing well established drug substances in conventional dosage forms Annex 5, WHO Technical Report Series 863, 1996

Item 10.1 TRS 937:
Item 10.1, WHO Technical Report Series 937, 2006

Update, Item 11.1 TRS 908
Item 11.1, WHO Technical Report Series 908, 2003

Stability testing of active pharmaceuticals ingredients and finished pharmaceuticals products - Annex 2

WHO Technical Report Series 953, 2009

Consultation of Stability studies in a global environment

Stability testing of new drug substances and products

Australian Regulatory Guidelines for Complementary Medicines(ARGCM)

TGA : Questions & answers on the stability testing of Listed complementary medicines

Stability study of drug product

Stability testing of drug substances and pharmaceutical products

Decision Tree for Data Evaluation for Retest Period or Shelf Life Estimation for Drug Substances or Products (excluding Frozen Products)

Case 1 : No significant change in accelerated

a) No or little change in long term and accelerated

Extrapolation (Y) = up to 2X, but not exceeding X + 12 months

or if refrigerated, Y = up to 1.5X, but not exceeding X + 6 months

b) Change in in long term & acclerated

i)If backed by statistical analysis and relevant supporting data

Extrapolation (Y) = up to 2X, but not exceeding X + 12 months

or if refrigerated, Y = up to 1.5X, but not exceeding X + 6 months

ii)If backed by relevant supporting data

Extrapolation (Y) = up to 1.5X, but not exceeding X + 6 months

or if refrigerated, Y = up to X + 3 months

Case 2 : Significant change within 6 month accelerated

a) if refrigerated, and significant change within 3 months - No extrapolation

b) if refrigerated, and no significant change within 3 months- No extrapolation

c) if not refrigerated and significant change at intermediate - No extrapolation

d) if not refrigerated and no significant change at intermediate condition

i) If backed by relevant supporting data:Y = up to X + 3 months

ii) If backed by statistical analysis and relevant supporting data

Y = up to 1.5X, but not exceeding X + 6 months

"Significant change" for a drug substance is defined as failure to meet its specification

In general, "significant change" for a drug product is defined as:

1. A 5% change in assay from its initial value or failure to meet the acceptance criteria for potency when using biological or immunological procedures

2. Any degradation product’s exceeding its acceptance criterion

3. Failure to meet the acceptance criteria for appearance, physical attributes, and functionality test (e.g., color, phase separation, resuspendibility, caking, hardness, dose delivery per actuation) however, some changes in physical attributes (e.g., softening of suppositories, melting of creams) may be expected under accelerated conditions and, as appropriate for the dosage form:

4. Failure to meet the acceptance criterion for pH or

5. Failure to meet the acceptance criteria for dissolution for 12 dosage units.

FIP Position Paper on Qualification of Paddle and Basket Dissolution Apparatus -To read click the link FIP Position paper dissolution

FDA publishes guidance document on capillary Electrophoresis

The ICH Steering Committee recommends that the analytical procedures described in the official pharmacopoeial texts, Ph. Eur. 2.2.47. Capillary Electrophoresis, JP General Information. Capillary Electrophoresis, and USP General Information Chapter <1053> Biotechnology-derived Articles – Capillary Electrophoresis, can be used as interchangeable in the ICH regions. To view the guidance, CE FDA Guidance

To get the copy of Analytical instrument qualification from Agilent technologies click the link - Analytical Instrument Qualification

Guidance for UK Manufacturer’s Licence and Manufacturer’s Authorisation (for Investigational Medicinal Products) Holders on the use of UK Stand Alone Contract Laboratories

• Defines a stand alone contract laboratory in relation to quality control testing of medicinal products.

• Provides guidance as to when a contract laboratory must be named on a manufacturer’s licence for relevant medicinal products for human and veterinary use and/or a manufacturer’s authorisation for investigational medicinal products.

• Is applicable to all manufacturing licence holders, i.e. import, export, herbals and specials.

• Provides guidance as to when a contract laboratory is not required to be named on a manufacturer’s licence or authorisation.

• Outlines the MHRA’s criteria for inspection of contract laboratories.

This guidance can be downloaded by clicking Guidance .

Analytical Balance

HPLC Column comparison

To find out alternative column for your column of interest.

HPLC Troubleshooting guide

Instrument calibration

1. Disolution Apparatus 1 & 2 - Mechanical calibration

Calibration of Dissolution Apparatus 1&2

Recently FDA has released a new guidance on Calibration of disolution apparatus 1 & 2. This guidance document more emphasis on enhanced mechanical calibration than chemical performance verification test. This guidance also recommends to manufacturer take appropriate control to handle recognised source of significant variablity during dissolution testing like dissolved gases, vibration and vessel dimensions. For more information go through the link .Guidance to industry -Dissolution

Dissolution procedure toolkit

USP released the version 2 of dissolution tool kit procedure for mechanical calibration and performance verification test apparatus 1 & 2. The dissolution toolkit provides a description of best practices associated with the mechanical calibration and performance verification test for the USP basket and paddle dissolution apparatuses and test assemblies.This second version of the dissolution toolkit represents a continuing effort to provide detailed information describing the procedures that if used will assure a properly qualified dissolution test assembly. For more information click Disolution Toolkit Procedure Version-2.

Further USP established a new acceptance criteria for current lot of Prednisone tablet. For more information click the link Performance of Equipment to Test Dissolution of Medications Further Assured

2. Prednisone tablet for PVT - New lot - P1I303

Prednisone Tablet for PVT - A new lot is released on March 01, 2010 Lot No.P1I300 valid through Feb 29, 2012 with test procedure an optional two stage test and calculation example. On April 30, 2010, Lot P0E203 will no longer be official.

Note: There was an error in the initial USP certificate dated Feb.22, 2010. It is now corrected. For corrected certificate click Prednisone Tablet for PVT - New lot released on March 2010.

Instrument Qualification

Result & Reports

This paper is concerned with challenges related to the maintenance of appropriate transit conditions during transport as products move through all stages of the manufacturing and wholesale distribution system from active substance through the wholesale distribution system to ensure that patients and animals receive safe and efficacious medicinal products.

From a product’s perspective, transport is a mobile form of storage but where there are weaker controls than storage in fixed sites – therefore similar levels of controls should exist. Compliance at all stages of manufacture and distribution becomes more important as the number of transport stages increase, including transport (i.e. import) into the EU. Several examples have been seen where sea-freight significantly exceeds 30 days. Any increases in the length of time and/or the climactic challenge at each stage will also impact on compliance. It is also increasingly difficult to be aware of and to assess the cumulative effects of adverse incidents at different stages.

Many sites of manufacture are now located in tropical zones and/or where transport infrastructure may be difficult. Therefore the challenges arising from transport between such sites and from these sites to the EU may take finished products, or their earlier stages of manufacture, outside of the conditions defined in the EU Marketing Authorisation for storage of the product. Also, the risk of freezing during transport and the effects on the products should be considered.

It is important to understand the susceptibility of different products at the different stages and whilst the principles of quality risk management would be applied on a product by product basis at each stage of manufacture and transport would be expected, the reality is that different products and different product stages are frequently co-shipped. It is therefore evident that a simple set of readily understood general rules should apply to transit conditions to reduce this complexity and risk of error.

Although widely acknowledged, there is no explicit requirement for the need to conduct transport studies under worst case conditions and no requirement for routine monitoring during transport.

Primer for GLP & GMP for Analytical Lab - Agilent

Top ten deficiencies found during first assessment of new applications from October to December 2009 - EDQM

This document is a summary of the main questions resulting from the first assessment of new applications for Certificates of Suitability (CEP) for chemical purity. It is based on the content of 108 deficiency letters sent to the applicants on applications treated from October to December 2009.

The Top 10 questions are listed below with additional recommendations regarding EDQM requirements added. By including these recommendations - together with the requirements described in the EDQM Guideline "Content of the dossier for chemical purity" PA/PH/CEP (04) 1 (current version) which is available on our website - applicants can improve the quality of their dossiers with a view to facilitating and speeding up the granting of their CEP.

TOP 1 (3.2.S.2.2) / (3.2.S.2.3): Redefinition of starting material

TOP 2 (3.2.S.2.3): Absence of discussion on the carry-over of impurities/by-products from key materials

TOP 3 (3.2.S.2.3): Absence of discussion for Class 1 solvent as contaminant of another solvent

TOP 4 (3.2.S.3.2): Genotoxic impurities

TOP 5 (3.2.S.4.4): Absence of comparison of the quality of the final substance obtained with starting materials from different suppliers

TOP 6 (3.2.S.2.3): Incomplete specifications for the declared starting materials

TOP 7 (3.2.S.4.3): Suitability of the monograph to control the impurity profile of the final substance

TOP 8 (3.2.S.6): Specification for container closure system

TOP 9 (3.2.S.3.2): Compliance with the requirements of the Ph. Eur General Monograph 2034: limit for unspecified impurities

TOP 10 (3.2.S.2.3): Solvent recovery

For more information click the weblink

How to do document - New revised APIC guide - GMP for API

It describes the intrepretation of ICH Q7 with revision in quality management, personnel & agents,brokers,traders,distributors,repackers and relabellers - Version 6.

Example of Quality Risk Management (QRM) Implementation by PIC/S

An informal working group within PIC/S has developed an example of methodology for the implementation of Quality Risk Management (QRM) in industry. For download example

EU GMP Guide chapter-7 revised - Outsourced activities -contract manufacturer and analysis

Chapter 7 of the EU GMP Guide "Contract Manufacture and Analysis" has been revised and was published on the GMP-information site of the European Commission on 9 November 2010.

Reasons for changes: in view of the ICH Q10 guideline on the Pharmaceutical Quality System, Chapter 7 of the GMP Guide has been revised in order to provide updated guidance on outsourced GMP regulated activities beyond the current scope of contract manufacture and analysis operations. The title of the Chapter has been changed to reflect this.

This guideline will align with the general framework described within other current international papers on this subject.

Principles of quality risk management- Four primary principles of QRM are:

•the evaluation of the risk to quality should be based on scientific knowledge and
ultimately link to the protection of the patient
• QRM should be dynamic, iterative and responsive to change
• the level of effort, formality and documentation of the QRM process should be
commensurate with the level of risk and
• the capability for continual improvement and enhancement should be embedded in the QRM process.

Change in ICH classification of residual solvent - Cumene(Isopropyl Benzene) - From class 3 to class 2

Cumene is listed in the ICH Q3C(R4) guideline in class 3. A revision to the ICH Q3C(R4) guideline is proposed in which it is recommended that cumene be placed into class 2 to take account of new toxicity data.

To go through draft guideline and send comment click Draft guideline

Out-Of-Specification (OOS)

Investigation is must for any product failures to find out the root cause and Corrective And Preventive Action(CAPA). USFDA come up with definite guidance on this subject. This guideline helps to handle OOS data and procedure for investigation. For more information refer the below link. FDA Guidance - OOS

Out-Of-Trend(OOT) Analytical Results

OOT means an analytical result which fall with in the specification limit but does not follow with in the trend or unexpected result. Normally any analytical result which fall in Out-Of-Specification requires a detailed investigation to find a root cause failure and folowed by a currettive And Preventive Action(CAPA).

Though regulation demands investigation to be completed with in thirty days but most of the cases industries fails to complete the investigation with in stipulated period to find root cause. Meanwhile its end up with few more failures. To avoid such things happen the current practice starts investigation when results appear to be out-of-trend.

The following links help to know more about OOT

Revised Annex 13 on Investigational Medicinal Products (IMP) coming into operation
The European Commission has published on the EudraLex - Volume 4 webpage the new Annex 13 (Investigational Medicinal Products) of the EU Guidelines to Good Manufacturing Practice. The new Annex is coming into force in July 2010.

Part 2 EU GMP Guide on APIs Will No Longer Be Identical to ICH Q7
The European Commission published a revised Part 2 text on GMP for APIs which will enter into force by 31 July 2010.

A template for API quality agreement

This Quality Agreement template was developed by the Bulk Pharmaceutical Task Force (BPTF), an affiliate organization of the Society of Chemical Manufacturers and Affiliates (SOCMA), as a guide for drafting a Quality Agreement relating to the manufacture and release of substances regulated by the Food and Drug Administration. The template is based on the collective experience of industry members. This can be downloaded by clicking Quality agreement template

WHO released draft guidance for production and control of specified starting material

Specified starting material means any substance which is primarily or mainly used as a starting material for the production of an API, but which could be used directly as an API.

This guideline is intended to assist applicants or MA holders in assessing the required level of quality of “specified starting materials” that will be used for the manufacture of an API. It is also intended to help API master file holders (APIMF) in the compilation of their APIMFs

The control of the “specified starting materials” should be designed to detect isomeric or other impurities which are potentially reactive and could be carried through to the final product of the synthesis.

Verification of Compendial Procedures - Changes Planned in the USP General Chapter <1226> -USP 35

In the Pharmacopeial Forum from November/December 2010, the USP proposed to revise the General Chapter <1226> Verification of Compendial Methods.

Changes planned are as below. USP planning to implement in USP 35.

The word "method" should be replaced by "procedure"

Aims to clarify the purpose and the scope of the verification process. "The verification process for compendial test procedures is the assessment of whether the procedure can be used for its intended purpose, under the actual conditions of use for a specified drug substance and/or drug product matrix."

"The process of assessing the suitability of a compendial analytical test procedure under the conditions of actual use may or may not require actual laboratory performance of each analytical performance characteristic."

The following will be added to the points which must be taken into consideration for the Verification of Compendial Procedures:

drug substance's synthetic route

method of manufacture for the drug product

effect of the matrix on the recovery of impurities

suitability of chromatographic conditions and column

appropriateness of detector signal response

The revision of the General Chapter <1226> Verification of Compendial Procedures should be published in the USP 35.

Clarification not provided by USP is "Whether the laboratory needs to prove the performance of each validation parameter for the assessment is left open".

Analytical Method Development

Analytical Method Validation

Validated analytical method only can give accurate results. Validation of analytical method is must for pharmaceutical analytical laboratory.

There is guideline for analytical method validation from ICH & US FDA.

ICH-Q2(R1)- Validation of Analytical Procedures - Text and Methodology

US FDA draft guidance on analytical procedures & method validation

Analytical Method Transfer

There is no definite guidance from USFDA on this subject. Recently USP has come up with a stimuli article on this subject. For more information refer the link below.

Transfer of analytical procedures-A new general information chapter

Auditing pharmaceuticals quality control Laboratory

Auditing quality control laboratory is must for continuous improvement and regualotory requirements. It helps to keep QC laboratory in high compliance level and maintain best practices through continuos improvement by training and gap analysis.

There is a guideline from US FDA for inspection pharmaceuticals QC laboratory it includes chemical lab and Micro lab.

Pharmaceutical Quality Control Labs

Audit check list for Pharmaceutical Quality Control Labs by PICScheme

Microbiological Pharmaceutical Quality Control Labs

World Health Organization Public Inspection Reports (WHOPIR)

The World Health Organization Public Inspection Reports (WHOPIR) is a summary of the inspection report of

a manufacturing site for Active Pharmaceutical Ingredients (APIs)

a manufacturing site for Finished Products (FPs)

an organization such as a Contract Research Organization where a bioequivalence study or other clinical study had been performed (CROs)

a quality control laboratory

The WHOPIR reflects the inspection report and gives a summary of the observations and findings made during the inspection, but excludes confidential proprietary information. It indicates also the date and duration of the inspection as well as the scope of the inspection.

The reported inspection reports are from India & china companies. Few examples are Ranbaxy, Cipla, Matrix, Lupin, IPCA, Aurobindo, Sitec lab,Vimta lab etc

Watch the video: ERN Biomedical Journal Club Session 2 - Lead by Dr. Hossam Elgabarty (June 2022).


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