Effective HPLC method development - A Detailed view

Effective HPLC method development

1. Introduction

Optimization of HPLC method development has been discussed extensively in many standard
textbooks. However, most of the discussions have been focused on the optimization of HPLC
conditions. This article will look at this topic from other perspectives. All critical steps in
method development will be summarized and a sequence of events that required to develop the
method efficiently will be proposed. The steps will be discussed in the same order as they would
be investigated during the method development process. The rational will be illustrated by
focussing on developing a stability-indicating HPLC-UV method for related substances
(impurities). The principles, however, will be applicable to most other HPLC methods.

In order to have an efficient method development process, the following three questions must be
answered:

1.1. What are the critical components for a HPLC method?

The 3 critical components for a HPLC method are: sample preparation, HPLC analysis
and standardization (calculations). During the preliminary method development stage,
all individual components should be investigated before the final method optimization.
This gives the scientist a chance to critically evaluate the method performance in each
component and streamline the final method optimization.

1.2. What should be the percentage of time spent on different steps of the method development?

The rest of the article will discuss the recommended sequence of events, and the
percentage of time that should be spent on each step in order to meet the method
development timeline. One common mistake is that most scientists focus too much
on the HPLC chromatographic conditions and neglect the other 2 components of
the method (i.e., sample preparation, standardization). The recommended timeline
would help scientists investigate different aspects of the method development and
allocate appropriate time in all steps.

1.3. How should a method development experiment be designed?

A properly designed method development experiment should consider the following
important questions:
What sample should be used at each stage?
What should the scientists look for in these experiments?
What are the acceptance criteria?
We will see these questions in the following discussions.



2. Method Development Timeline

The following is a suggested method development timeline for a typical HPLC-UV
related substance method. The percentage of time spent on each stage is proposed to
ensure the scientist will allocate sufficient time to different steps. In this approach, the
three critical components for a HPLC method (sample preparation, HPLC analysis and
standardization) will first be investigated individually. Each of these steps will be
discussed in more detail in the following paragraphs.

Step 1: Define method objectives and understand the chemistry (10%)
Determine the goals for method development (e.g., what is the intended use of the method?), and
to understand the chemistry of the analytes and the drug product.

Step 2: Initial HPLC conditions (20%)
Develop preliminary HPLC conditions to achieve minimally acceptable separations. These
HPLC conditions will be used for all subsequent method development experiments.

Step 3: Sample preparation procedure (10%)
Develop a suitable sample preparation scheme for the drug product.

Step 4: Standardization (10%)
Determine the appropriate standardization method and the use of relative response factors in
calculations.

Step 5: Final method optimization/robustness (20%)
Identify the “weaknesses” of the method and optimize the method through experimental design.
Understand the method performance with different conditions, different instrument set ups and
different samples.

Step 6: Method validation (30%)
Complete method validation according to ICH guidelines

3. Define Method Objectives

There is no absolute end to the method development process. The question is what is the
“acceptable method performance”? The acceptable method performance is determined by the
objectives set in this step. This is one of the most important considerations often overlooked by
scientists. In this section, the different end points (i.e., expectations) will be discussed in
descending order of significance.

3.1 Analytes:
For a related substance method, determining the “significant and relevant” related substances is
very critical. With limited experience with the drug product, a good way to determine the
significant related substances is to look at the degradation products observed during stress
testing. Significant degradation products observed during stress testing should be investigated in
the method development.
Based on the current ICH guidelines on specifications, the related substances method for active
pharmaceutical ingredients (API) should focus on both the API degradation products and
synthetic impurities, while the same method for drug products should focus only on the
degradation products. In general practice, unless there are any special toxicology concerns,
related substances below the limit of quantitation (LOQ) should not be reported and therefore
should not be investigated.
In this stage, relevant related substances should be separated into 2 groups:

3.1.1. Significant related substances: Linearity, accuracy and response factors should be
established for the significant related substances during the method validation. To limit the
workload during method development, usually 3 or less significant related substances should
be selected in a method.

3.1.2 Other related substances: These are potential degradation products that are not
significant in amount. The developed HPLC conditions only need to provide good
resolution for these related substances to show that they do not exist in significant levels.

3.2 Resolution (Rs)
A stability indicating method must resolve all significant degradation products from each other.
Typically the minimum requirement for baseline resolution is 1.5. This limit is valid only for 2
Gaussian-shape peaks of equal size. In actual method development, Rs = 2.0 should be used as a
minimum to account for day to day variability, non-ideal peak shapes and differences in peak
sizes.

3.3 Limit of Quantitation (LOQ)
The desired method LOQ is related to the ICH reporting limits. If the corresponding ICH
reporting limit is 0.1%, the method LOQ should be 0.05% or less to ensure the results are
accurate up to one decimal place. However, it is of little value to develop a method with an LOQ
much below this level in standard practice because when the method is too sensitive, method
precision and accuracy are compromised.

3.4 Precision, Accuracy
Expectations for precision and accuracy should be determined on a case by case basis. For a
typical related substance method, the RSD of 6 replicates should be less than 10%. Accuracy
should be within 70 % to 130% of theory at the LOQ level.

3.5 Analysis time
A run time of about 5-10 minutes per injection is sufficient in most routine related substance
analyses. Unless the method is intended to support a high-volume assay, shortening the run time
further is not recommended as it may compromise the method performance in other aspects (e.g.,
specificity, precision and accuracy.)

3.6 Adaptability for Automation
For methods that are likely to be used in a high sample volume application, it is very important
for the method to be “automatable”. The manual sample preparation procedure should be easy to
perform. This will ensure the sample preparation can be automated in common sample
preparation workstations.

4. Understand the Chemistry

Similar to any other research project, a comprehensive literature search of the chemical and
physical properties of the analytes (and other structurally related compounds) is essential to
ensure the success of the project.

4.1 Chemical Properties
Most sample preparations involve the use of organic-aqueous and acid-base extraction
techniques. Therefore it is very helpful to understand the solubility and pKa of the analytes.
Solubility in different organic or aqueous solvents determines the best composition of the sample
solvent. pKa determines the pH in which the analyte will exist as a neutral or ionic species. This
information will facilitate an efficient sample extraction scheme and determine the optimum pH
in mobile phase to achieve good separations.

4.2 Potential Degradation Products
Subjecting the API or drug product to common stress conditions provides insight into the
stability of the analytes under different conditions. The common stress conditions include acidic
pH, basic pH, neutral pH, different temperature and humidity conditions, oxidation, reduction
and photo-degradation. These studies help to determine the significant related substances to
be used in method development, and to determine the sample solvent that gives the best sample
solution stability.

In addition, the structures of the analytes will indicate the potential active sites for degradation.
Knowledge from basic organic chemistry will help to predict the reactivity of the functional
groups. For example, some excipients are known to contain trace level of peroxide impurities.
If the analyte is susceptible to oxidation, these peroxide impurities could possibly produce
significant degradation products.

4.3 Sample Matrix
Physical (e.g., solubility) and chemical (e.g., UV activity, stability, pH effect) properties of the
sample matrix will help to design an appropriate sample preparation scheme. For example,
Hydroxypropyl Methylcellulose (HPMC) is known to absorb water to form a very viscous
solution, therefore it is essential to use mostly organic solvents in sample preparation.

5. Initial Method Conditions

The objective at this stage is to quickly develop HPLC conditions for subsequent method
development experiments. A common mistake is that scientists spend too much time at this
stage trying to get a perfect separation.

5.1 Preliminary HPLC Conditions
In order to develop preliminary HPLC conditions in a timely fashion, scientists should use
artificial mixtures of active pharmaceutical ingredients and related substances at relatively high
concentrations (e.g., 1-2% of related substance relative to API) to develop the preliminary HPLC
conditions. The concentration ratio between API and the related substances should be maintained
to ensure the chromatography represents that of a real sample. Alternatively, a highly stressed
sample (e.g., 5% degradation) can also be used at this stage. With the known composition and
high levels of degradation products in the sample, one can evaluate the chromatography to
determine whether there are adequate separations for all analytes. The high concentrations of
related substances are used to ensure all peaks will be detected.

Computer assisted method development can be very helpful in developing the preliminary HPLC
conditions quickly. Since the objective at this stage is to quickly develop HPLC conditions for
subsequent method development experiments, scientists should focus on the separation of the
significant related substances (section 3.1.1) instead of trying to achieve good resolution for all
related substances. These significant related substances should be baseline resolved from each
other with Rs > 2.0. After the preliminary method development, the HPLC conditions can be
further fine-tuned at a later stage to achieve the required specificity for the other related substances.

5.2 Aged HPLC Column

An aged HPLC column should be used to develop the initial HPLC conditions. Usually it is
more difficult to achieve the required resolution with an aged column (e.g., column with about
200 injections). This will reflect the worst case scenario likely to be encountered in actual
method uses, and help the long-term method robustness.

In general, develop all methods with HPLC columns from the same vendor. The preferred brand
of HPLC column should be selected primarily based on the long term stability and lot to lot
reproducibility.

6. Sample Preparation

6.1 Selection of Sample Solvent
This stage focuses on the selection of the sample solvent (for extraction) and the proper sample
preparation procedures. Investigate the effect of sample solvents of different % organic, pH,
extraction volume and extraction procedure on accuracy, precision, sensitivity (LOQ) and the
changes in the chromatography (e.g., peak shape, resolution). Whenever possible use the mobile
phase in the sample preparation. This will ensure that there will not be any compatibility issues
between the sample solution and the HPLC conditions.

6.1.1 Accuracy:
To investigate the accuracy in sample preparation (i.e., extraction efficiency),
prepare a spiked solution by adding known amounts of related substances into a sample
matrix. Compare responses of the spike solutions and the neat standard solutions to assess the
recovery from the sample preparation. In this stage, since only one particular step is being
investigated (i.e., sample preparation), close to theoretical recovery should be observed at this
point (e.g., 90-110%).

6.1.2 Precision:
Use the stressed sample to represent the worst case scenario and perform
replicate sample preparations from the same sample composite. Investigate the consistency of
the related substance profile (i.e., any missing peaks?) and the repeatability results from these
preparations.
6.2 Another objective is to determine the sample concentration that gives an acceptable LOQ
(Signal to Noise ratio, S/N) in low level spike concentrations. The sample concentration should
be low enough to maintain linearity and precision, but high enough to achieve the desired LOQ.
For example, if the ICH reporting limit for this drug product is 0.1%, the LOQ of the method
should be less than 0.05% (i.e., desired LOQ, in %). By using spike sample solutions of very
diluted concentrations for the significant related substances, estimate the concentrations that give
a S/N of about 10 for the significant related substances. This estimated concentration is the
approximate LOQ concentration (i.e., estimated LOQ concentration, in g/mL).
The following equation can be used to estimate the target sample concentration for the method:
Target sample concentration =
estimated LOQ concentration (g/mL) x 1/desired LOQ (%) x 100%

7. Standardization

7.1 Area % method
If the response of the active pharmaceutical ingredient is linear from LOQ to the nominal sample
concentration, use the % area approach where the related substance is reported as % area. This is
the most straightforward approach, and doesn’t require the preparation of standard solutions. It
also has the highest precision since preparation to preparation variation will not affect the results.
However, in order to ensure the concentration is linear within this range, the sample
concentration is usually limited and this will reduce the method sensitivity (i.e., increase LOQ).
In general, use this approach as long as the desired LOQ can be achieved.

7.2 External Standard method
Use the external standard method if the response of the active pharmaceutical ingredient is not
linear throughout the whole range, or the desired LOQ can not be achieved by the area %
method. The concentration of standard solution should be high enough to ensure the standard
solution can be prepared accurately and precisely on a routine basis, it should be low enough to
approximate the concentration of related substance in the sample solution. In general, the
standard concentration should correspond to about 5 % of related substances.

7.3 Wavelength Selection and Relative Response Factor
Generate the linearity plot of API and related substances at different wavelengths. At this point,
Photodiode Array Detector can be used to investigate the linearity of the active pharmaceutical
ingredient and related substances in the proposed concentration range. By comparing the
linearity slopes of the active pharmaceutical ingredient and the related substances, one can
estimate the relative response factors of the related substances at different wavelengths.

Disregard of whether Area % or External Standard approach is used, if the relative response
factors of some significant related substances are far from unity, a response factor correction
must be applied.

The optimum wavelength of detection is the wavelength that gives the highest sensitivity (max)
for the significant related substances and minimizes the difference in response factors between
those of the active pharmaceutical ingredient and the related substances.

After the optimum wavelength is determined, use a highly stressed sample (e.g., 5% degradation)
to verify that the selected wavelength will give the highest % related substance results.

7.4 Overall accuracy

A final check of the method performance is to determine the overall accuracy of the method.
Unlike the accuracy from sample preparation (section 6.1.1), which simply compares the
response of the analyte with and without spiking with matrix, the overall accuracy compares the
% related substances calculated from an accuracy solution with that of the theoretical value.
The accuracy solutions are the solutions spiked with known concentrations of related substances
and matrix. Since the extraction efficiency, choice of wavelength and the bias in standardization
influence the calculated related substance result, this is the best way to investigate the accuracy
of the method. Overall accuracy reflects the true accuracy of the method.

8. Method Optimization/ Robustness

After the individual components of the method are optimized, perform the final optimization of
the method to improve the accuracy, precision and LOQ. Use an experimental design approach
to determine the experimental factors that have significant impact on the method. This is very
important in determining what factors need to be investigated in the robustness testing during the
method validation (see section 9). To streamline the method optimization process, use Plackett
Burmann Design (or similar approach) to simultaneously determine the main effects of many
experimental factors.

Some of the typical experimental factors that need to be investigated are:
HPLC conditions: % organic, pH, flow rate, temperature, wavelength, column age.
Sample preparation: % organic, pH, shaking/sonication, sample size, sample age.
Calculation/standardization: integration, wavelength, standard concentration, response
factor correction.

Typical responses that need to be investigated are:
Results: precision (%RSD), % related substance of significant related substances, total related
substances.
Chromatography: resolution, tailing factor, separation of all related substances (section 3.1.1 and
3.1.2).

9. Method validation

9.1 Robustness
Method validation should be treated as a “final verification” of the method performance and
should not be used as part of the method development. Some of the typical method validation
parameters should be studied thoroughly in the previous steps. In some cases, robustness can be
completed in the final method optimization before method validation. At this point, the
robustness experiments should be limited only to the most significant factors (usually less than 4
factors). In addition, unlike the final method optimization (see section 8), the experimental
factors should be varied within a narrow range to reflect normal day to day variation. During the
method validation, the purpose is to demonstrate that the method performance will not be
significantly impacted by slight variations of the method conditions.

9.2 Linearity, Accuracy, Response Factor
Linearity, accuracy and response factors should be established for the significant related
substances (section 3.1.1) during the method validation. In order to limit the workload of method
development, usually less than 3 significant related substances should be selected in a method.
Therefore, the other related substances (section 3.1.2) should not be included in these
experiments.

9.3 System suitability criteria
It is advisable to run system suitability tests in these robustness experiments. During the
robustness testing of the method validation, critical method parameters such as mobile phase
composition and column temperature are varied to mimic the day-to-day variability. Therefore,
the system suitability results from these robustness experiments should reflect the expected
range. Consequently, the limits for system suitability tests can be estimated from these
experiments.

10. Conclusion

All of the critical steps in method development have been summarized and prioritized. The steps
for method development are discussed in the same order as they would be investigated in the
actual method development process. These steps will ensure all critical method parameters are
optimized before the method validation.

In order to develop a HPLC method effectively, most of the effort should be spent in method
development and optimization as this will improve the final method performance. The method
validation, however, should be treated as an exercise to summarize or document the overall
method performance for its intended purpose.
If you have any doubts, write me to prabhusankarshasun@gmail.com
If you have any suggestions, please tell me to the above address.
With Regards,
R.Prabhusankar

Protein Assay by Bradford Method

PURPOSE
To estimate the quantity of protein present in a sample by Bradford assay.

PROCEDURE

RAW MATERIALS
1. Coomassie brilliant blue g 250
2. Orthophosphoric acid
3. Bovine serum albumin
4 96-wellplate
5. Autoclaved MilliQ water
6. Absolute alcohol (99.9% ethanol)

Preparation Of Bradford’s Reagent
Dissolve 50mg of Coomassie brilliant blue G 250 slowly in 25 ml of absolute alcohol.

Take 50 ml of Orthophosphoric acid in a burette and add it drop by drop to the dye solution kept
stirring on a magnetic stirrer.

Make up the volume to 100ml with autoclaved MilliQ water.

Store the reagent in an amber bottle at room temperature protected from light (The bulk dye can
be stored at 4 C).

Prepare fresh Bradford reagent in every 10 days.

Preparation of Protein Standard
Prepare a 1 mg/ml BSA stock in autoclaved MilliQ water. This is done by weighing out 50mg BSA into 50mL water.

Prepare a working stock from the above by diluting 1 ml to 10 ml with autoclaved MilliQ water (the final concentration should be 100ug/ml).


Protein Estimation

Pipet between 5 and 25 g of protein sample in 800 l total volume into a microfuge tube. If the approximate protein concentration is unknown, assay a range of dilutions (1,1:10, 1:100, 1:1000). Prepare duplicates of each sample.

For the standard curve, Pipet duplicate volumes of 50,100,150, 200 and 250 l of working standard solution into microfuge tubes and make each up to 800 l with autoclaved MilliQ water. Pipet 800l of water into a separate tube to make the reagent blank.

Add 200l of Bradford reagent to the blank, standard and sample tubes and make up the volume
to 1000l. Mix well by gentle vortex-mixing. Avoid foaming, which will lead to poor
reproducibility.

Pipet 200 l of each tube and put into a 96 well plate and incubate the plate at 30 C for 10
minutes and measure A 595 of the samples and standards against the reagent blank using a
spectrophotometer. The reading of the sample must lie within the standard reading. If the
reading is below the lowest standard reading, then use a lower dilution. If the reading is higher
than the highest standard reading, then use a higher dilution.

The software using the calibration curve automatically calculates the protein concentration of
unknown sample.

Calculation

The protein concentration of a sample can be calculated using the formula below:
X=Y-A
B
Where X=Concentration,Y=A 595,B=Slope and A= Intercept.

Analytical Method Validation Protocol for HPLC and GC

Analytical Method Validation Protocol for HPLC and GC


1 INTRODUCTION

All analytical test procedures must be validated before being issued for general use (Quality Control (QC) or Stability). The objective of an analytical method validation is to demonstrate that it is suitable i.e. it is accurate, precise specific, rugged, reliable and where required, capable of demonstrating the stability of a product with time as per ICH Q3B guidelines.

2 SCOPE

This SP describes the validation procedure for HPLC Quality Control (QC) and Stability Indicating Method (SIM). In the case of QC release methods, all stages are required except the forced degradation study. Upon completion of all required stages described in this SP and when all acceptance criteria met, a method may be defined as suitable and approved for use.

3 SAFETY

All relevant MSDS and COSHH assessments should be read prior to commencing work.

4 VALIDATION PROCEDURE

The following range of tests must be performed to demonstrate that the method is suitable

4.1 Specificity

Specificity demonstrates that the method is capable of resolving the analyte(s) and if applicable, degradant(s) of interest from any placebo-related interference.

The method must ideally be capable of resolving all peaks of interest and placebo-related peaks from each other to the extent that co-elution is not deemed to be significant. However, in the event that blank or placebo-related peaks do co-elute with any peaks of interest, the area of the interfering peak must not exceed 1% of the area of the peak of interest in the active compound sample. Degradant peaks must be suitably resolved from the main-active peak(s), other degradant peaks, related-substance peaks and blank / placebo-related peaks. Resolution between peaks of interest and nearest-neighbour peaks must be >1.5 at a given wavelength to facilitate satisfactory peak integration.
Peak-of-interest purity must also be ascertained when there is a potential for interference by co-eluting or neighbouring peaks. This can be carried out using any of the diode-array HPLC systems and the acceptance threshold is 0.990.


Linearity

4.2.1 The linearity test demonstrates that the mode of detection (UV, FID etc) has a linear response to concentration over the range of concentrations that can be realistically be expected for a given product.

4.2.2 Linearity is to be performed separately on all of the components of interest in a sample, using separate experiments for the analyte(s) and the degradant(s). The minimum number of points (or ‘levels’) in the line must be 5 over a range of 20 – 200% of the theoretical sample solution concentration. More points may be used if the experiment does not demonstrate linearity to 200% and more points are needed to determine the upper limit of linearity. When the concentrations over which the response is linear are demonstrated, the working linear range of the analytical method can be inferred. The following acceptance criteria for linearity must be met and quoted:

Active compound: Correlation coefficient (R2) ³ 0.999 (1 injection per level to be used).


For degradants and impurities, linearity is assessed over the range 10% – 200% of the products’ degradant specification limit using a minimum of 5 concentrations for each substance.

Degradants and Impurities: Correlation coefficient ³ 0.99 (1 injection per level to be used).


4.2.3 When performing the linearity test, an amount of placebo matrix equivalent to that found in an assay sample must be added to the sample solution at each concentration level. This is so the test demonstrates recovery of actives or degradants realistically, as if from a sample rather than from a simple solution.

4.2.4 The peak area at the y-axis intercept on the graph must be £ ±2.0% of the peak area at 100% (< +10% in the case of degradants). The y-intercept and slope are to be determined from the graph line equation of the form;

y = mx + c where m = gradient (slope) and c = y-intercept.

Report the correlation coefficients, bias, slope, y-intercept and linear range.

4.2.5 When the linearity graph has been plotted (using Excel®) and correlation determined, residuals will be obtained and the bias calculated. Bias must not exceed +2.0% in order to demonstrate that the results are not significantly affected by the analytical method itself.


Accuracy
4.3.1 Accuracy demonstrates the capability of the method to recover a known quantity of active or degradant etc from the placebo matrix. This indicates the efficiency of the method.

4.3.2 To demonstrate accuracy for active compounds, recovery is performed using solutions containing 80%, 100% and 120% of the theoretical active concentration in the finished product. Each level is performed in triplicate and the mean value for each level is calculated and reported.

4.3.3 The acceptance criteria for active compounds and main analytes in this test are that recovery for each of the concentration levels is within the limits 98.0 – 102.0%.


4.3.4 To demonstrate suitable accuracy for degradants and impurities, recovery is performed on solutions containing 2 x LOQ concentration, 50%, 100% and 200% of the degradants and impurities limit in the finished product. Each level is performed in triplicate and the mean value for each level is calculated and reported.


4.3.5 The acceptance criteria for degradants and impurities in this test are that for 2 x LOQ concentration, all recoveries must be within the limits 80% - 120% and all higher concentrations are within the limits of 90% - 110% of target for each concentration.



Precision
4.4.1 Precision demonstrates the capability of the method to generate similar results when carried out at different times.

4.4.2 There are three types of precision test required;

Instrument Precision: The capability of instruments to repeatedly generate similar results for the same set of samples.
Repeatability: The capability of the method to generate similar sets of results for a set of samples made from the same batch of sample.
Intermediate Precision: The capability of the method to generate similar results when carried out using the same set of samples, but by different analysts, using different instruments over a different time period (typically on different days).

4.4.3 For active compounds:

a) Instrument Precision – a working standard solution is prepared and analysed 10 times in succession.
(Limits RSD £ 1.0%)

b) Repeatability – 6 assay samples are prepared, and analysed Single injections are made of each sample. A total of six results will be achieved.
(Limits RSD £ 1.5%).

c) Intermediate Precision – 2 analysts prepare a set of six sample solutions each, on separate days and analyse their sample sets on separate instruments. Each analyst generates a set of six results.
(Limits: Each analyst set of six results has RSD <2.0%.
Both analysts results combined into a set of 12 results has RSD <2.0%)

4.4.4 For degradants and impurities:

a) Instrument Precision – a working standard solution is prepared and analysed 10 times in succession.
(Limits RSD £ 5.0%)

b) Repeatability – 6 assay samples are prepared, and analysed Single injections are made of each sample. A total of six results will be achieved.
(Limits RSD £ 5.0%).

c) Intermediate Precision – 2 analysts prepare a set of six sample solutions each, on separate days and analyse their sample sets on separate instruments. Each analyst generates a set of six results.
(Limits: Each analyst set of six results has RSD <5.0%.
Both analysts results combined into a set of 12 results has RSD <10.0%)



Limit of Detection (LOD) and Limit of Quantification (LOQ).
4.5.1 The Limit of Detection (LOD) determines the lowest concentration of active compound or degradant that can be determined but not reliably quantified.

4.5.2 Limit of detection is defined as 3 x S/N ratio (S/N = signal to noise). This must be determined for both the active, degradant and any impurities.

4.5.3 The Limit of Quantification (LOQ) is the lowest concentration of a active compound or degradant that can be quantified routinely with acceptable accuracy.

4.5.4 Limit of quantification is defined as 10 x S/N ratio. This must be determined for both the active, degradant and any impurities.

4.5.5 There are several ways to determine the LOQ and LOD. One is to determine the lowest level of concentration that will be considered significant for the test e.g. 0.05%. If a solution at this strength can be accurately quantified or detected, then that is the level set and no further work is required to determine the absolute limit.
If the response factor (extinction coefficient) of the entity being analysed is not particularly strong and the LOQ / LOD cannot be readily be determined in this way, then other methods may be employed providing they are scientifically sound (for example, ICH Q2(R1)). The source of any alternative determination must be written alongside any alternative determination to aid traceability.

4.5.6 Once these levels have been determined, the LOQ solution is prepared. The LOQ solution is analysed 10 times and the RSD of the peak area calculated. The LOD solution is prepared by diluting the LOQ solution further and is analysed once for demonstration purposes.

4.5.7 The acceptance criteria for the 10 analytical results is an RSD <20%.
If RSD of £20% cannot be readily obtained, the concentration of the solution is to be increased incrementally until this limit is achieved.



Robustness
4.6.1 Robustness demonstrates the capability of the method to reproduce results in the event of changes to test parameters or subtle instrument to instrument / analyst to analyst differences.
The ICH defines it as “ a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters”.

4.6.2 Given the functionality of the instrumentation, the following parameter variations are to be applied as appropriate to the method being validated;


HPLC METHODS
PARAMETER VARIATION

Column
Use different columns of the same specification but of different supplier lot #’s or batches.
Column Temperature
Set the column 5oC above and below the temperature Specified in the method.
Mobile Phase pH
Adjust the pH to 0.1 pH unit above and below the pH specified in the method.
Mobile Phase Aqueous Component
(Isocratic)
Adjust the aqueous component to 10% (Relative) above and below the proportion specified in the method if the channel being changed is <29%>30%.
Mobile Phase Organic Component
(Isocratic)
Adjust the organic component to 10% (Relative) above and below the proportion specified in the method if the channel being changed is <29%>30%.
Mobile Phase Components
(Gradient)
Adjust the organic mobile phase component at the gradient start-point as outlined above.
For three- or four-component systems, adjust the organic component with the largest proportion at the start of the gradient.
Gradient Slope
Extend the time of any gradient portion of the run-time by +10% relative. Extend the run-time accordingly.
E.g. a gradient slope over 20 mins will be extended to 22 mins and therefore the injection run-time will be extended by 2 mins also.
Ion-Pair Concentration
Adjust the ion-pair content to 10% relative above and below the concentration specified in the method.
Detection Wavelength (UV)
Set the UV detector wavelength to 4nm above and below the wavelength specified in the method.
Flow Rate
Set the flow rate to 0.5ml/min above and below the rate specified in the method.
If the flow is specified at 0.99ml/min or below, adjust the flow by 10% relative above and below that specified in the method.
Sample Extraction
Vary the sample extraction times by 50% above and below that specified in the method.
Injection Volume
Adjust the injection volume by 10% above and below that specified in the method.




GC METHODS
PARAMETER VARIATION
Column
Use different columns of the same specification but of different supplier lot #’s or batches.
Oven Temperature
Set the starting oven temperature 5oC above and below the temperature specified in the method.
Flow Rate
Set the column flow rate to 10% relative above and below that specified in the method.
Injection (Inlet) Temperature
Set the inlet temperature to 10oC above and below that specified in the method.
Hydrogen Component
Set the hydrogen component of the gas mix to 5ml/min above and below that specified in the method.
Split Ratio
Adjust the split ratio by 10% relative above and below that specified in the method.
Detector Temperature.
Set the Detector temperature to 5oC above and below that specified in the method.


4.6.3 With each method alteration, a system suitability sequence is to be run consisting of;
1 x Blank
4 x Calibration Stds
1 x QA Standard
Any resolution standards (as necessary)
Sample preparations (2 minimum)
Final Calibration Standard.

For each method alteration, the same sequence is to be run and the sample preparations must be from the same batch so that results can be compared.

4.6.4 Acceptance Criteria:

When the robustness study is performed, the following acceptance criteria should be applied to the resultant chromatography:

1) All analyte peaks must be resolved as expected for the specified method.
2) Sample peak RT must be within +1.5% of the 1o standard peak RT.
3) Sample assay results must be within ±2.0% relative of those obtained when the method is used as specified.

When the acceptance criteria are not met, the consequence is that the method is not wholly robust to the desired extent. The experiment must then move on to find the maximum parameter deviation that can be made before acceptance criteria are exceeded. This can often be done by extrapolation. The relevant limits are reported in the validation document.


Solution Stability

4.7.1 A timeframe is required to be determined through which samples and standards can be prepared and analysed with the confidence that they have not been compromised by degradation. To this end, the stability of both standard and sample solutions needs to be determined.

4.7.2 Duplicate calibration standards and assay sample solutions will be prepared and analysed (Day 0, T=0hrs). The remaining bulk of these solutions in their respective flasks will then be stored in a fridge at 5oC (+3oC), a dark cupboard, and on an ordinary laboratory bench for a period of 7 days.

4.7.3 Each day at approximately the same time, a fresh primary and secondary standard will be prepared and the initial stored solutions assayed against them. The results will be calculated as % w/w values for Day 1, T=24hrs >>> Day 2, T=48hrs >>> Day 4, T=96hrs >>> Day 7, T=168hrs.

Standard calculations will provide results in % anyway and assay results should be converted to % results for comparison.

4.4.4 At the end of the solution stability study (Day 7), the results will be tabulated, reviewed and the solution stability determined.

4.7.5 For sample and standard, the recovery must lie between the limits of 98.0 – 102.0% of day 0 (initial results).
For degradants and impurities, the recovery should lie within 90.0 – 110.0% (ie. standard and sample solutions within 10% relative to day 0 (initial) results).

4.7.6 When the data is reviewed, the solution stability is deemed to be the period within which the assay values were within 2.0% absolute of the Day 0 result.

4.7.7 It is acceptable if necessary, to extend the solution stability to a maximum of 4 weeks if supported by adequate analyte stability data and if there is an experimental / business need for it.


Forced Degradation Studies

4.8.1 It is useful for formulators and for analysts to know the degradation pathways for the active pharmaceutical ingredients (API’s) in new products or new formulations of existing products. It may be possible to obtain this information from the supplier or from a literature source but if this is not the case, then a ‘forced degradation study’ (sometimes called a ‘stress study’) is required.

4.8.2 To ensure that the major API degradants are observed, it is desirable to achieve a 5-20% degradation of the API’s. The methods used to achieve this are based upon the types of stress the product or API is most likely to encounter after manufacture, but at a much more powerful level than at ambient conditions. In this way the degradation can be thought of as accelerated.

4.8.3 Namely;

· Peroxide (to mimic the action of increased O2 concentration or exposure to oxidising chemicals or conditions).
· Heat (the product may be stored and marketed in hot climates or warehousing temperature may not be controlled).
· Light (some chemicals are photosensitive, especially those with extensive Pi-systems like many pharmaceuticals).
· Water (Water may react directly with some API’s or may cause some substances to ionise and react)
· Acid (‘Wet’ products are usually buffered or formulations are designed to become active upon application or after ingestion like tablets or capsules. Changes in pH may cause degradation.
· Alkali (see above).

4.8.4 The degree of stress must be considered since the aim is to cause degradation and not destruction e.g. it would be common to use 0.1M or 0.01M concentration of acid and not concentrated acid.

4.8.5 As appropriate, a forced degradation study is to be carried out on the placebo, the finished product and the raw API. API data may already be available from the manufacturer, Drug Master File or Certificate of Suitability (a piece of European Union documentation that demonstrates that the manufacturing process has been validated, the material is processed by cGMP and that it meets the specifications of the EP monograph).



4.9 Procedure for Forced Degradation Study

4.9.1 To avoid repetition of work, always check for previous API studies. Only new API’s and novel formulations should have this study performed.

4.9.2 Aliquots of the test material equivalent to assay sample quantities (API if new to site), placebo, finished product), suitable for analysis are to be accurately weighed into the appropriate number of labelled volumetric flasks as if for assay. These flasks are then stored under the following conditions:

a) In the dark and refrigerated (Control) & in a darkened cupboard at RT.
b) In an air oven at 70oC
c) As a solution or suspension in water at 70oC
d) As a solution or suspension in aqueous alkali (typically 1M NaOH) at 70oC
e) As a solution or suspension in aqueous acid (typically 1M HCl) at 70oC
f) As a solution or suspension in hydrogen peroxide (1% solution) at 70oC
g) Under constant light in a clear borosilicate glass container (this should be in the form of a light cabinet and the test should be done not less than 200whm2) (whm2 = Watt hours per square metre).
h) In a clear, open glass container subjected to high humidity (75%)

Containers in a) to g) should be securely sealed to prevent loss by sublimation or evaporation.

4.9.3 One flask per condition is recommended in order to increase confidence in any results obtained.

4.9.4 There will already be analysis of fresh API & fresh product (solution stability) and fresh placebo (specificity). These results along with those from the control samples can be used to compare results of the forced degradation study and determine the extent of degradation.

4.9.5 At the start of the study, samples should be stored under all conditions and examined at suitable time periods after going into solution. The first analysis is to be performed as soon after 2 hours storage as possible. If there has been none or an insignificant (less than LOD) amount of degradation, then further testing after 24 hours, one-week, two-weeks and four weeks may be appropriate.
If there has been none or an insignificant (less than LOD) amount of degradation after any of these storage-times conditions, then the time and / or severity of the conditions should be increased (e.g. 10x concentrations of alkai / acid / peroxide and higher temperatures taking the physical properties of the materials involved into account).
If the sample has been destroyed or severely degraded to the extent that chromatography becomes problematic, then less severe conditions are to be used (e.g. 10x reduction in concentrations of alkali / acid / peroxide and higher temperatures taking the physical properties of the materials involved into account).

4.9.6 Degradation products are usually the result of salt-breaks, hydrolysis and oxidation / reduction reactions. Even though the resulting compounds will have different properties to the parent molecules, HPLC / GC is still the best way to monitor degradation. For HPLC methods, full-spectrum diode array analysis is preferable since it will still detect degradants with chromophores that have a different max to the parent molecule

4.9.7 If the data requires, carry out a linearity calculation using data from the forced degradation study assays.
Plot assay results for the API and major degradants vs time to establish the relationship.
NOTE: Do not expect all relationships to be linear. It cannot be assumed that degradation is always 1st order.

4.9.8 If degradation products are observed then this degraded solution can assist in producing a stability indicating method for the product under test. However, as identification of the degradation products is usually required, further analytical work will be required .

4.9.9 Liquid Chromatography / Gas Chromatography – Mass Spectrometry (LC-MS / GC-MS) are powerful tools ideally suited for confirming the identity and structure of degradation compounds.
If the analysis is by GC then GC-MS can be used to examine degradation products. Likewise, if the analysis is by HPLC, then LC-MS can be employed to investigate.

4.9.10 It will be necessary to obtain samples of the degradation compounds for use as reference materials once their structure is known. However, relative retention times (RTT’s) may be used to determine the identity of degradant peaks if they are known.






5 DOCUMENTATION

All experiments must be well documented and the raw data is to be readily traceable for inspection by registration authorities if required.



Prepared by
R.Prabhusankar