Qualification: B.Pharm / M.Pharm / M.Sc
Experience: 2 to 6 years
Work Location: Kothur (Near to Shadnagar)
Preferably from Formulation background.
Job Requirements
Quality Control
Experience in Method Validations, Method Verifications and Analytical Method Transfers with Regulatory exposure.
Experience in Finished Product, In-Process, Stability and GLP activities
Exposure in all types of Analysis (Instrumental HPLC / GC / Dissolution etc.)
Microbiology (Injectables)
Experience in Environmental Monitoring in Aseptic Area (Isolator technology)
Gowning Qualification, Personnel Monitoring.
Experience in Water Analysis include Purified water, WFI, Pure Steam, Media Preparation & LIMS activities.
Candidates who are interested and willing to relocate to Kothur location are only requested to attend the Interview.
Walk-in interview details
Date: 30th March 2024 i.e. Saturday
Time: 09:00 AM to 02:00 PM.
Venue: Natco Pharma Limited, Natco House, Road No.-2, Banjara Hills, Hyderabad – 34. Opp: A.P. Productivity Council of Hyderabad.
INTRODUCTION:
Doxazosin Mesylate is a quinazoline compound that is a selective inhibitor of the alpha1 subtype of alpha-adrenergic receptors. Doxazosin mesylate is freely soluble in dimethylsulfoxide, soluble in dimethylformamide, slightly soluble in methanol, ethanol, and water (0.8% at 25°C), and very slightly soluble in acetone and methylene chloride.
Doxacard-2 Tablets are quiniline yellow WS coloured, uncoated and round shaped tablets.
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This topic was modified 2 years, 6 months ago by
.
Overview
Analytical procedures used to measure the quality of pharmaceutical products span almost the entire range of currentlyavailable technologies and techniques. From immunoassay and electrophoretic techniques used to characterize protein moeities, and chromatographic and potentiometric methods used to evaluate the qualities of small molecules, the variety of procedures (and approaches necessary to prove these methods’ validity and usefulness) can be overwhelming. However, when evaluating available procedures to determine which are best for your intended use, it is important to keep in mind that the most important aspect of any analytical method is the quality of the data it ultimately produces.
Perhaps the most useful and widely-consulted guidance in the industry is the USP’s General Chapter 1225entitled, “Validation of Compendial Methods”. This Chapter opens by referencing the Federal Food, Drug and Cosmetics Act (and hence, stressing the legal status of USP test procedures), then continues with a formal definition of “validation” as it applies to analytical methods. Directly quoted, the Chapter states that “Validation of an analytical method is the process by which it is established, by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical applications.”
The most significant point raised by this definition is that the validity of a method can be demonstrated only through laboratory studies. It is not sufficient to simply review historical results; instead, laboratory studies must be conducted which are intended to validate the specific method, and those studies should be pre-planned and described in a suitable protocol. The protocol should clearly indicate the method’s intended use and principles of operation, as well as the validation parameters to be studied, and a rationale for why this method and these parameters were chosen. The protocol also must include pre-defined acceptance criteria and a description of the analytical procedure, written with sufficient detail to enable persons “skilled in the art” to replicate the procedure.
Validation Parameters – Assays
USP General Chapter 1225, as well as the ICH Guideline for Industry (Text on Analytical Procedures), provide cursory descriptions of typical validation parameters, how they are determined, and which subset of each parameter is required to demonstrate validity, based on the method’s intended use. For example, it would be inappropriate to determine limits of detection or quantitation for an active ingredient using an assay method intended for finished product release. However, if the method was intended to detect trace quantities of the active ingredient for purposes of a cleaning validation study, then knowledge of the detection and quantification limits are appropriate and necessary. For this reason, validation of each assay or test method should be performed on a case-by-case basis, to ensure that the parameters are appropriate for the method’s intended use. This is even more important when validating stability-indicating assay methods, because these validations are more complex – for example, they may require forced degradation, samples spiked with known degradates, literature searches, etc.
The following definitions, taken from the ICH Guideline for Industry (Text on Analytical Procedures), will provide a background for subsequent discussion:
Analytical Procedure.
The analytical procedure refers to the way of performing the analysis. It should describe in detail the steps necessary to perform each analytical test. This may include, but is not limited to, the sample, the reference standard and the reagents preparations, use of the apparatus, generation of the calibration curve, use of the formulae for the calculation, etc.
Specificity.
Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically, these might include impurities, degradants, matrix, etc. Lack of specificity of an individual analytical procedure may be compensated by other supporting analytical procedure(s).
This definition has the following implications:
• IDENTIFICATION: To ensure the identity of an analyte.
• PURITY TESTS: To ensure that all the analytical procedures performed allow an accurate statement of the content of impurities of an analyte, i.e., related substances test, heavy metals, residual solvents content, etc.
• ASSAY (Content or Potency): To provide an exact result which allows an accurate statement on the content or potency of the analyte in a sample.
Accuracy.
The closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value, and the value found.
Note: When measuring accuracy, it is important to spike placebo preparations with varying amounts of active ingredient(s). If a placebo cannot be obtained, then a sample should be spiked at varying levels. In both cases, acceptable recovery must be demonstrated.
Precision.
The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility.
Precision should be investigated using homogeneous, authentic (full scale) samples. However, if it is not possible to obtain a full-scale sample it may be investigated using a pilot-scale or bench-top scale sample or sample solution.
The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements. Refer to this month’s “The Regulatory Clinic” for a discussion of AccuReg’s consensual interpretations of the following terms that express precision:
a. Repeatability. Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision.
b. Intermediate Precision. Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc.
c. Reproducibility. Reproducibility expresses the precision between laboratories (collaborative studies usually applied to standardization of methodology).
Detection Limit.
The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.
Quantitation Limit.
The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/or degradation products.
Linearity.
The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.
Note: Measurements using clean standard preparations should be performed to demonstrate detector linearity, while method linearity should be determined concurrently during the accuracy study. Classical linearity acceptance criteria are 1) that the correlation coefficient of the linear regression line is not more than some number close to 1, and 2) that the y-intercept should not differ significantly from zero.
When linear regression analyses are performed, it is important not to force the origin as (0,0) in the calculation. This practice may significantly skew the actual best-fit slope through the physical range of use.
Range.
The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.
Robustness.
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate, variations in method parameters and provides an indication of its reliability during normal usage.
Note: Ideally, robustness should be explored during the development of the assay method. By far the most efficient way to do this is though the use of a designed experiment. Such experimental designs might include a Plackett-Burman matrix approach to investigate first order effects, or a 2k factorial design that will provide information regarding the first (main) and higher order (interaction) effects.
In carrying out such a design, one must first identify variables in the method that may be expected to influence the result. For instance, consider an HPLC assay which uses an ion-pairing reagent. One might investigate: sample sonication or mixing time; mobile phase organic solvent constituency; mobile phase pH; column temperature; injection volume; flow rate; modifier concentration; concentration of ion-pairing reagent; etc. It is through this sort of a development study that variables with the greatest effects on results may be determined in a minimal number of experiments.
The actual method validation will ensure that the final, chosen ranges are robust.
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Other points to consider include:
System Suitability
In addition, prior to the start of laboratory studies to demonstrate method validity, some type of system suitability must be done to demonstrate that the analytical system is performing properly. Examples include: replicate injections of a standard preparation for HPLC and GC methods; standardization of a volumetric solution followed by assays using the same buret for titrimetric methods; replicate scanning of the same standard preparation during UV-VIS assays, etc. When the method in question utilizes an automated system such as a chromatograph or an atomic absorption spectrophotometer, a suitable standard preparation should be intermittently measured during the sample analysis run. The responses generated by the standard should exhibit a reasonable relative standard deviation. This is done primarily to demonstrate the stability of the system during sample measurements. System suitability for dissolution studies should be performed using both USP non-disintegrating and disintegrating tablets prior to the validation of dissolution methods.
Validity Checks – General Tests
It is important to realize that assays are not the only tests important in evaluating the qualities of a drug product. The USP contains numerous identity tests of a chemical nature. In these types of tests, one should treat a placebo preparation with the reaction reagent to ensure a negative result is achieved. Otherwise, the test has no meaning. Dissolution tests, for instance, should be evaluated for adequate sink conditions (i.e., adequate solubility in an adequate volume of the dissolution media) prior to development.
Protocols
As mentioned earlier, prior to initiating a validation study, a well-planned validation protocol should be written and reviewed for scientific soundness and completeness by qualified individuals. The protocol should describe the procedure in detail, and should include pre-defined acceptance criteria and pre-defined statistical methods. Following approved by the appropriate corporate and Quality Control authorities, the protocol should be executed in a timely manner. A typical assay validation will require the preparation of product placebo(s), standards, and many samples.
How many times should an assay be repeated to ensure “validity”? Although 3 sequential replicates are often considered the “magic number,” a far more definitive number is one produced by a sound scientific rationale, usually with the assistance of statistical analyses.
Subsequent to the execution of the protocol, the data must be analyzed with results, conclusions and deviations presented in an official validation summary report. Provided the pre-defined acceptance criteria are met, and the deviations (if any) do not affect the scientific interpretation of the data, the method can be considered valid. A statement of the method’s validity should be placed at the beginning of the final summary report, along with the signatures and titles of all significant participants and reviewers.
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In the final analysis, the purpose of validating methods is to ensure the procurement of high quality data. After all, if the quality of data is questionable, no meaningful conclusions can be reached about the quality of the product – which will have seriously detrimental affects on stability study data reviews, process validation data reviews, and annual batch reviews, to name a few. Time invested in validating analytical methods in the beginning pays big dividends in the long run.
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This topic was modified 2 years, 6 months ago by
The principles of qualification and validation which are applicable to the manufacture of medicinal products.
It is a requirement of GMP that manufacturers identify what validation work is needed to prove control of the critical aspects of their particular operations. Significant changes to the facilities, the equipment and the processes, which may affect the quality of the product, be validated. A risk assessment approach should be used to determine the scope and extent of validation.
The key elements of a validation programme be clearly defined and documented in a validation master plan (VMP) or equivalent documents.
The VMP be a summary document which is brief, concise and clear.
The VMP contain data on the following:
(a) Validation policy;
(b) Organisational structure of validation activities;
(c) Summary of facilities, systems, equipment and processes to be validated;
(d) Documentation format: the format to be used for protocols and reports;
(e) Planning and scheduling;
(f) Change control;
(g) Reference to existing documents.
VALIDATION DOCUMENTATION
A written protocol should be established that specifies how qualification and validation will be conducted. The protocol be reviewed and approved. The protocol should specify critical steps and acceptance criteria.
A report that cross-references the qualification and/or validation protocol be prepared, summarising the results obtained, commenting on any deviations observed, and drawings including the necessary conclusions, including recommending changes necessary to correct deficiencies. Any changes to the plan as defined in the protocol should be documented with appropriate justification.
QUALIFICATION
DESIGN QUALIFICATION
The documented verification that the proposed design of the facilities, systems and
equipment is suitable for the intended purpose.
The first element of the validation for new facilities, systems or equipment is design qualification (DQ).
INSTALLATION QUALIFICATION
The documented verification that the facilities, systems and equipment, as installed or
modified, comply with the approved design and the manufacturer’s recommendations.
Installation qualification (IQ) has to be performed on new or modified facilities, systems and equipment.
IQ includes the following:
(a) Installation of equipment, piping, services and instrumentation has to be confirmed to the current engineering drawings and specifications;
(b) Collection and collation of supplier operating and working instructions and maintenance requirements;
(c) Calibration requirements;
(d) Verification of materials of construction.
OPERATIONAL QUALIFICATION
The documented verification that the facilities, systems and equipment, as installed or
modified, perform as intended throughout the anticipated operating ranges.
Operational qualification (OQ) has to be performed after the successful completion of Installation qualification.
OQ includes the following:
(a) Tests that have been developed from knowledge of processes, systems and equipment;
(b) Tests to include a condition or a set of conditions encompassing upper and lower operating limits, sometimes referred to as “worst case” conditions.
The completion of a successful Operational qualification allows the finalisation of calibration, operating and cleaning procedures, operator training and preventative maintenance requirements. It permits a formal “release” of the facilities, systems and equipment.
PERFORMANCE QUALIFICATION
The documented verification that the facilities, systems and equipment, as connected
together, can perform effectively and reproducibly, based on the approved process
method and product specification.
Successful completion of Installation qualification and Operational qualification follows Performance qualification (PQ).
PQ includes the following:
(a) tests, using production materials, qualified substitutes or simulated product,
those have been developed from knowledge of the process and the facilities,
systems or equipment;
(b) tests to include a condition or set of conditions encompassing upper and lower operating limits.
Qualification of established facilities, systems and equipment
Evidence should be available to support and verify the operating parameters and limits for the critical variables of the operating equipment. Additionally, the calibration, cleaning, preventative maintenance, operating procedures and operator training procedures and records should be documented for further use.
PROCESS VALIDATION
The documented evidence that the process, operated within established parameters, can
perform effectively and reproducibly to produce a medicinal product meeting its predetermined specifications and quality attributes.
Process validation applicable to the manufacture of pharmaceutical dosage forms. They cover the initial validation of new processes, subsequent validation of modified processes and
re-validation.
Process validation normally should be completed prior to the distribution and sale of the medicinal product (prospective validation). In exceptional circumstances, where this is not possible, it may be necessary to validate processes during routine production (concurrent validation). Processes in use for some time should also be validated (retrospective validation).
Facilities, systems and equipment to be used should have been qualified and analytical testing methods should be validated.
Facilities, systems, equipment and processes should be periodically evaluated to verify that they are still operating in a valid manner.
Prospective validation
Validation carried out before routine production of products intended for sale.
Prospective validation should include the following:
(a) Short description of the process;
(b) Summary of the critical processing steps to be investigated;
(c) List of the equipment/facilities to be used (including measuring/
monitoring/recording equipment) together with its calibration status
(d) finished product specifications for release;
(e) List of analytical methods, as appropriate;
(f) Proposed in-process controls with acceptance criteria;
(g) Additional testing to be carried out, with acceptance criteria and analytical
validation, as appropriate;
(h) sampling plan;
(i) methods for recording and evaluating results
(j) functions and responsibilities;
(k) proposed timetable.
Using this defined process (including specified components) a series of batches of the final product may be produced under routine conditions. In theory the number of process runs carried out and observations made should be sufficient to allow the normal extent of variation and trends to be established and to provide sufficient data for evaluation. It is generally considered acceptable that three consecutive batches/runs within the finally agreed parameters would constitute a validation of the process.
Batches made for process validation should be the same size as the intended industrial scale batches.
If it is intended that validation batches be sold or supplied, the conditions under which they are produced should comply fully with the requirements of Good Manufacturing Practice, including the satisfactory outcome of the validation exercise, and with the marketing authorisation.
Concurrent Validation
Validation carried out during routine production of products intended for sale.
In exceptional circumstances it may be acceptable not to complete a validation programme before routine production starts.
The decision to carry out concurrent validation must be justified, documented and approved by authorised personnel.
Documentation requirements for concurrent validation are the same as specified for prospective validation.
Retrospective validation
Validation of a process for a product which has been marketed based upon accumulated
manufacturing, testing and control batch data.
Retrospective validation is only acceptable for well-established processes and will be inappropriate where there have been recent changes in the composition of the product, operating procedures or equipment.
Validation of such processes should be based on historical data. The steps involved require the preparation of a specific protocol and the reporting of the results of the data review, leading to a conclusion and a recommendation.
The source of data for this validation should include, but not be limited to batch processing and packaging records, process control charts, maintenance log books, records of personnel changes, process capability studies, finished product data, including trend cards and storage stability results.
Batches selected for retrospective validation should be representative of all batches made during the review period, including any batches that failed to meet specifications, and should be sufficient in number to demonstrate process consistency. Additional testing of retained samples may be needed to obtain the necessary amount or type of data to retrospectively validate the process.
For retrospective validation, generally data from ten to thirty consecutive batches should be examined to assess process consistency, but fewer batches may be examined if justified.
CLEANING VALIDATION
Cleaning validation is documented evidence that an approved cleaning procedure will provide equipment which is suitable for processing medicinal products.
Cleaning validation should be performed in order to confirm the effectiveness of a cleaning procedure. The rationale for selecting limits of carryover of product residues, cleaning agents and microbial contamination should be logically based on the materials involved. The limits should be achievable and verifiable.
Validated analytical methods having sensitivity to detect residues or contaminants should be used. The detection limit for each analytical method should be sufficiently sensitive to detect the established acceptable level of the residue or contaminant.
Normally only cleaning procedures for product contact surfaces of the equipment need to be validated. Consideration should be given to noncontact parts. The intervals between use and cleaning as well as cleaning and reuse should be validated. Cleaning intervals and methods should be determined.
For cleaning procedures for products and processes which are similar, it is considered acceptable to select a representative range of similar products and processes. A single validation study utilising a “worst case” approach can be carried out which takes account of the critical issues.
Typically three consecutive applications of the cleaning procedure should be performed and shown to be successful in order to prove that the method is validated.
“Test until clean”. is not considered an appropriate alternative to cleaning validation
Products which simulate the physicochemical properties of the substances to be removed may exceptionally be used instead of the substances themselves, where such substances are either toxic or hazardous.
CHANGE CONTROL
Written procedures should be in place to describe the actions to be taken if a change is proposed to a starting material, product component, process equipment, process environment (or site), method of production or testing or any other change that may affect product quality or reproducibility of the process. Change control procedures should ensure that sufficient supporting data are generated to demonstrate that the revised process will result in a
product of the desired quality, consistent with the approved specifications.
All changes that may affect product quality or reproducibility of the process should be formally requested, documented and accepted. The likely impact of the change of facilities, systems and equipment on the product should be evaluated, including risk analysis. The need for, and the extent of, requalification and re-validation should be determined.
REVALIDATION
Facilities, systems, equipment and processes, including cleaning, should be periodically evaluated to confirm that they remain valid. Where no significant changes have been made to the validated status, a review with evidence that facilities, systems, equipment and processes meet the prescribed requirements fulfils the need for revalidation.
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This topic was modified 2 years, 6 months ago by
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?
The author will answer 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 (see section 8, method optimization/ robustness) 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 mg/mL).
The following equation can be used to estimate the target sample concentration for the method:
Target sample concentration =
estimated LOQ concentration (mg/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 (lmax)
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.
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Common Mistakes in Method Development:
Inadequate Formulation of Method Goals: Failure to define clear method objectives, such as resolution, selectivity, sensitivity, and throughput, can result in inefficient method development. Without well-defined goals, analysts may struggle to optimize parameters effectively, leading to suboptimal separations.
Little Knowledge of Chemistry of Analyte Mixture: Insufficient understanding of the chemical properties, interactions, and behavior of analytes within the sample matrix can impede column selection. Without a thorough grasp of analyte chemistry, analysts may choose columns unsuitable for achieving the desired separation.
Use of the First Reversed Phase C18 Column Available: Relying solely on a standard C18 column without considering alternative stationary phases or column chemistries limits the versatility and effectiveness of method development. Different analytes may require specific column characteristics to achieve optimal separation, and a one-size-fits-all approach may lead to suboptimal results.
Trial and Error with Different Columns and Mobile Phases: A common but inefficient practice involves a trial-and-error approach to column and mobile phase selection, which can prolong method development timelines and waste valuable resources. Without a systematic approach, analysts may overlook critical parameters and fail to identify optimal conditions.
These Mistakes Result In:
Laborious, Time-consuming Development Projects: Inefficient method development processes characterized by repeated experimentation and optimization consume valuable time and resources. Analysts may struggle to achieve satisfactory results, leading to frustration and delays in project timelines.
Methods that Fail to Meet the Needs of the Analyst: Suboptimal methods developed through haphazard approaches often fail to fulfill the requirements of the analytical task at hand. Inadequate separations, poor resolution, or low sensitivity can compromise the reliability and accuracy of analytical results, impacting downstream applications.
Conclusion:
To mitigate these challenges and streamline HPLC method development, analysts must adopt a systematic and knowledge-driven approach to column selection. This involves clearly defining method objectives, leveraging comprehensive understanding of analyte chemistry, exploring diverse column chemistries, and employing rational optimization strategies. By avoiding common mistakes and adopting efficient practices, analysts can accelerate method development, optimize chromatographic performance, and meet the diverse analytical needs of their applications.
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