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Requirements for Analytical Method Development and Validation for HPLC

Analytical method development and validation are key elements of any pharmaceutical development program. HPLC analysis method is developed to identify, quantity or purifying compounds of interest. Effective method development ensures that laboratory resources are optimized, while methods meet the objectives required at each stage of drug development. Method validation, required by regulatory agencies at certain stages of the drug approval process, is defined as the “process of demonstrating that analytical procedures are suitable for their intended use”.

Analytical methods are intended to establish the identity, purity, physical characteristics and potency of the drugs that we use. Methods are developed to support drug testing against specifications during manufacturing and quality release operations, as well as during long term stability studies. Methods may also support safety and characterization studies or evaluations of drug performance.

The three critical components for a HPLC method are:

1. Sample preparation:
  • % organic, 
  • pH, 
  • Shaking/sonication, 
  • Sample size, 
  • Sample age,

2. Analysis conditions
  • % organic, 
  • pH, 
  • Flow rate, 
  • Temperature, 
  • Wavelength, 
  • Column age

3. Standardization
  • Integration, 
  • Wavelength, 
  • Standard concentration, 
  • Response factor correction.

  1. Information on sample
  2. Defining separation goals
  3. Special procedure requirement, sample pretreatment, if any
  4. Detector selection and setting
  5. Separation conditions optimization
  6. Checking for problems or special procedure requirements
  7. Recovery of purified material
  8. Quantitative calibration/ Qualitative method
  9. Method validation for release to laboratories

Physicochemical properties of drugs play important part in method development. One has to study properties like solubility, polarity, pH and pKa of drug molecule. Polarity helps in deciding the solvent and composition of mobile phase. The solubility of molecules can be explained on the basis of polarity of molecules. 

Selection of diluents is based on the solubility of analyte. The analyte must be soluble in the diluent and must not react with any of the diluent components. The diluent should match to the starting eluent composition of the assay to ensure that no peak distortion will occur, especially for early eluting components. 

The buffer region extends over the approximate range pKa ± 2, though buffering is weak outside the range pKa ± 1, [A]/[HA] = 10 or 1/10. If the pH is known, the ratio may be calculated. This ratio is independent of the analytical concentration of the acid. When the pKa and analytical concentration of the acid are known, the extent of dissociation and pH of a solution of a monoprotic acid can be easily calculated.


Buffer Selection
Choice of buffer is typically governed by the desired pH. The typical pH range for reserved-phase on silica-based packing is pH 2 to 8. It is important that the buffer has a pKa close to the desired pH since buffer controls pH best at their pKa. A rule is to choose a buffer with pKa value < 2 units of the desired mobile phase pH.

Buffer Concentration
Generally, a buffer concentration of 10-50 mM is adequate for small molecules. Generally, no more than 50% organic should be used with a buffer. This will depend on the specific buffer as well as its concentration. Phosphoric acid and its sodium or potassium salts are the most common buffer systems for reversed-phase HPLC.

Selection of Detector
Selection of detector depends on the chemical nature of analytes, potential interference, limit of detection required availability and cost of detector. UV-Visible detector is versatile, dual wavelength absorbance detector for HPLC. 

UV-Visible detectors are typical in many laboratories as they can detect a wide array of compounds. Others detectors used in HPLC instrument include photodiode array detector, fluorescence detector, conductivity detector, refractive index detector, electrochemical detector, mass spectrometer detector and evaporative light scattering detector.

Column Selection
The heart of a HPLC system is the column. Changing a column will have the greatest effect on the resolution of analytes during method development. Generally, modern reverse phase HPLC columns are made by packing the column housing with spherical silica gel beads which are coated with the hydrophobic stationary phase. 

In general, the nature of stationary phase has greatest effect on capacity factor, selectivity, efficiency and elution. Silica matrices are robust, easily derivatized, manufactured to consistent sphere size, and does not tend to compress under pressure. C18 columns are the commonly used columns in HPLC method analysis. C8 or Octyl bonded phases are also used occasionally. Like C18, they are non-polar, but not as hydrophobic. 

Therefore, retention times for hydrophobic compounds are typically shorter. Also, they may show somewhat different selectivity than C18 due to increased base silica exposure unique selectivity results in proton interaction of the bonded phase with electron deficient functional groups of solute molecules.

Column Dimensions
This refers to the length and internal diameter of the packing media bed within the column tube. Short columns (30-50mm) offer short run times, fast equilibration, low back pressure and high sensitivity. Long columns (250-300mm) provide higher resolving power, but create more backpressure, lengthen analysis times and use more solvent. 

Narrow column (2.1mm and smaller) beds inhibit sample diffusion and produce narrower, taller peaks and a lower limit of detection. They may require instrument modification to minimize distortion of the chromatography. Wider columns (10-22mm) offer the ability to load more sample.

Mobile Phase Composition
Mobile phase composition (or solvent strength) plays an important role in RP-HPLC separation. Acetonitrile (ACN), methanol (MeOH) and tetrahydrofuran (THF) are commonly used solvents in RP -HPLC having low UV cut- off of 190, 205 and 212nm respectively. These solvents are miscible with water. Mixture of acetonitrile and water is the best initial choice for the mobile phase during method development.

Mobile Phase pH
Change in the mobile phase pH can also improve column efficiency because it alters both the ionization of the analyte and the residual silanols and it also minimizes secondary interactions between analytes and the silica surface that lead to poor peak shape. 

To achieve optimum resolution, it requires change in the pH of mobile phase. Method development can proceed by investigating parameters of chromatographic separations first at low pH and then at higher pH until optimum results are achieved.

Column Temperature
Separation of many samples can be enhanced by selecting the right column temperature. Higher column temperature reduces system backpressure by decreasing mobile phase viscosity, which in turn allows use of longer columns with higher separation efficiency. However, an overall loss of resolution between mixture components in many samples occurs by increasing column temperature. 

The optimum temperature is dependent upon the nature of the mixture components. The overall separation can be improved by the simultaneous changes in column temperature and mobile phase composition.

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.

According to ICH guideline Q2 Validation of analytical procedure is the process for proving that an analytical procedure is suitable for its intended purpose. Results obtained from method validation study can be used to judge the quality, reliability and consistency of analytical results.

Chemicals, such as reagents and standards, should be available in sufficient quantities, accurately identified, sufficiently stable and checked for purity. Other materials and consumables, for example, chromatographic columns, should be qualified to meet the column’s performance criteria. 

The validation experiments should also be carried out by an experienced analyst to avoid errors due to inexperience. Validation on the analytical procedure should be performed with homogeneous samples, and validation data should be obtained by repeat LOD analysing aliquots of a homogeneous sample, each of which has been independently prepared according to the analytical method procedure.

Revalidation is necessary whenever a method is changed and the new parameter is outside the operating range. The operating parameters need to be specified with ranges clearly defined. In case of methods for quantitation of impurities, if a new impurity is found that makes the method deficient in its specificity, it needs modification and revalidation. 

Changes in equipment or chemical quality may also have critical effects on method. So any such change needs revalidation. The various validation parameters include
  1. Linearity
  2. Accuracy
  3. Precision
  4. Ruggedness
  5. Robustness
  6. LOD
  7. LOQ
  8. Selectivity or specificity

1. Linearity
ICH definition: The linearity of an analytical procedure is its ability (within a given range) to obtain test results that are directly proportional to the concentration (amount) of analyte in the sample.

It is essential to determine the useful range at which the instrumental response is proportional to the analyte concentration. Generally, a value of correlation coefficient (r) > 0.998 is considered as the evidence of an acceptable ft of the data to the regression line. The significance of the deviation of the intercept of the calibration line from the origin can be evaluated statistically by determining confidence limits for the intercept, generally at a 95% level. 

Linearity is determined by a series of three to six injections of five or more standards. Peak areas (or heights) of the calibration standards are usually plotted on the Y-axis against the nominal standard concentration, and the linearity of the plotted curve is evaluated through the value of the correlation coefficient (r). Because deviations from linearity are sometimes difficult to detect, two additional graphical procedures can be used to evaluate the linearity of the plot. 

The first one is to plot deviations from the regression line versus concentration or versus the logarithm of concentration. For linear ranges, the deviations should be equally distributed between positive and negative values. Another approach is to divide signal data by their respective concentrations yielding the relative responses. A graph is plotted with the relative responses on Y-axis and the corresponding concentrations on X-axis on a log scale. 

The obtained line should be horizontal over the full linear range. At higher concentrations, there will typically be a negative deviation from linearity. Parallel horizontal lines are drawn in the graph corresponding to, for example, 95 % and 105 % of the horizontal line. The method is linear up to the point where the plotted relative response line intersects the 95 % line.

2. Accuracy
Definition: The accuracy of an analytical procedure expresses the closeness of agreement between the value that is accepted either as a conventional true value or as an accepted reference value and the value found.

It is a qualitative characteristic that cannot be expressed as a numerical value. It has an inverse relation to both random and systematic errors, where higher accuracy means lower errors. Accuracy is evaluated by analyzing test drug at different concentration levels. Typically, known amounts of related substances and the drug substance in placebo are spiked to prepare an accuracy sample of known concentration of related substance. Samples are prepared in triplicate. 

ICH recommends accuracy evaluation using a minimum of nine determinations over a minimum of three concentration levels covering the range specified. It is determined by comparing the found concentration with the added concentration. The methods of determining accuracy include analysis of analysis of known purity (reference material), comparison of results of the proposed analytical procedure with those of a second well characterized procedure and standard addition method. The accuracy may also be inferred once precision, linearity and specificity have been established.

3. Precision
The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple samples of the same homogeneous sample under prescribed conditions. 

Precision is usually investigated at three levels: repeatability, intermediate precision, and reproducibility. For simple formulation it is important that precision be deter- mined using authentic homogeneous samples. A justification will be required if a homogeneous sample is not possible and artificially prepared samples or sample solutions are used.

A. Repeatability (Precision)
Repeatability is a measure of the precision under the same operating conditions over a short interval of time. It is sometimes referred to as intra assay precision. Two assaying options are allowed by the ICH for investigating repeatability:
  • A minimum of nine determinations covering the specified range for the procedure (e.g., three concentrations/three replicates as in the accuracy experiment), or
  • A minimum of six determinations at 100% of the test concentration.

The standard deviation, relative standard deviation (coefficient of variation), and confidence interval should be reported as required by the ICH. The true value of the samples and the variation of the assay may be between 97.5 and 99.1%.

B. Intermediate (Precision)
Intermediate precision is defined as the variation within the same laboratory. The extent to which intermediate precision needs to be established depends on the circumstances under which the procedure is intended to be used. Typical parameters that are investigated include day-to-day variation, analyst variation, and equipment variation. Depending on the extent of the study, the use of experimental design is encouraged. 

Experimental design will minimize the number of experiments that need to be performed. It is important to note that the ICH allows exemption from doing intermediate precision when reproducibility is proven. It is expected that the intermediate precision should show variability that is in the same range or less than repeatability variation. The ICH recommended the reporting of standard deviation, relative standard deviation (coefficient of variation), and confidence interval of the data.

C. Reproducibility
Reproducibility measures the precision between laboratories as in collaborative studies. This parameter should be considered in the standardization of an analytical procedure (e.g., inclusion of procedures in pharmacopoeias and method transfer between different laboratories). 

To validate this characteristic, similar studies need to be performed at other laboratories using the same homogeneous sample lot and the same experimental design. In the case of method transfer between two laboratories, different approaches may be taken to achieve the successful transfer of the procedure. However, the most common approach is the direct method transfer from the originating laboratory to the receiving laboratory. 

The originating laboratory is defined as the laboratory that has developed and validated the analytical method or a laboratory that has previously been certified to perform the procedure and will participate in the method transfer studies. The receiving laboratory is defined as the laboratory to which the analytical procedure will be transferred and that will participate in the method transfer studies. 

In direct method transfer it is recommended that a protocol be initiated with details of the experiments to be performed and acceptance criteria (in terms of the difference between the means of the two laboratories) for passing the method transfer.

4. Robustness
Definition: The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in the analytical procedure parameters. The robustness of the analytical procedure provides an indication of its reliability during normal use. The evaluation of robustness should be considered during development of the analytical procedure.

The aim of the robustness study is to identify the critical operating parameters for the successful implementation of the method. These parameters should be adequately controlled and a precautionary statement included in the method documentation. In case of an HPLC method, robustness study involves method parameters like pH, flow rate, column temperature and mobile phase composition which are varied within a reasonable range. 

The system suitability parameters obtained for each condition are studied to check the parameter which significantly affects the method. Stability of the analytical solution and extraction time are other parameters which are also evaluated as additional parameters during robustness study. Stability of analytical solution is determined by assessing the results obtained by subjecting the analytical solution to the method parameters for longer period of time e.g. 4 hrs, 12 hrs, 24 hrs, 48 hrs, etc. The acceptance criteria are based on relative difference between initial value and the value at specified solution stability time. For drug substances and products difference should be ≤ 2.0 % and for impurity determination, it should be ≤ 10 %.

5. Specificity
Definition: Specificity is the ability to assess unequivocally an analyte in the presence of components that may be expected to be present. In many publications, selectivity and specificity are often used interchangeably. However, there are debates over the use of specificity over selectivity. For the purposes of this chapter, the definition of specificity will be consistent with that of the ICH.

The specificity of a test method is determined by comparing test results from an analysis of samples containing impurities, degradation products, or placebo ingredients with those obtained from an analysis of samples without impurities, degradation products, or placebo ingredients. For the purpose of a stability- indicating assay method, degradation peaks need to be resolved from the drug substance. However, they do not need to be resolved from each other.

Critical separations in chromatography should be investigated at the appropriate level. Specificity can best be demonstrated by the resolution of two chromatographic peaks that elute close to each other. In the potency assay, one of the peaks would be the analyte peak.

6. Limit of quantification and limit of detection
ICH definition: The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be determined quantitatively with suitable precision and accuracy. The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be detected but not necessarily quantitated as an exact value.

Two types of approaches can be used to determine the quantitation limit or detection limit, as described below.

Signal-to-Noise Approach. Quantitation limit is defined as the concentration of related substance in the sample that will give a signal-to-noise (S/N) ratio of 10: 1. Detection limit (LOD) corresponds to the concentration that will give a signal-to-noise ratio of 3: 1. The quantitation limit of a method is affected by both the detector sensitivity and the accuracy of sample preparation at such a low concentration. In practice, the quantitation limit should be lower than the corresponding ICH reporting limit.

To investigate the effect of both factors (i.e., sample preparation and detector sensitivity), solutions of different concentrations near the ICH reporting limits are prepared by spiking known amounts of related substances into excipients. Each solution is prepared according to the procedure and analyzed repeat LOD to determine the S/N ratio. The average S/N ratio from all analyses at each concentration level is used to calculate the LOQ or LOD. The following equation can be used to estimate the LOQ at each concentration level. Since different concentration levels give different LOQs, typically the worst-case LOQ will be reported as the LOQ of the method.

LOQ= 10 X concentration (in %related substance)/ (S/N)

Alternatively, the spike solution can be diluted serially to lower concentrations. The S/N ratio at each concentration level is determined. The concentration level (in percent related substance) that gives an S/N value of about 10 will be reported as the LOQ.

Standard Deviation Approach. The following equations can be used to determine quantitation limit and detection limit by standard deviation of the response at low concentrations:

LOQ= 10 X SD/ S LOD= 3.3 X SD/S

where SD is the standard deviation of the response near LOQ and S is the slope of the linearity curve near LOQ.

There are two ways to determine SD:
  • Using experiments similar to those given for the signal-to-noise approach, determine the standard deviation of the responses by repeat analysis of a solution near the targeted LOQ.
  • Construct a calibration curve near the targeted LOQ:
  1. Determine the residual standard deviation of the regression line of calibration, or
  2. Determine the standard deviation of the y-intercept.

7. System suitability
System suitability testing (SST) is an integral part of many analytical procedures. The tests are based on the concept that the equipment, analytical operations and samples are the integral part of the system that can be evaluated as such. System suitability test provide the added assurance that on a specific occasion the method is giving, accurate and precise results. System suitability test are run every time a method is used either before or during analysis. 

The results of each system suitability test are compared with defined acceptance criteria and if they pass, the method is deemed satisfactory on that occasion. In case of HPLC methods, system suitability tests ensure the adequacy for performing the intended application on daily basis. The primary SST parameters considered are resolution (Rs), repeatability (% RSD of peak response and retention time), column efficiency (N), and tailing factor (Tf). The other SST parameters include retention factor (k) and separation factor (α).

SST Limits:
  • Resolution (Rs) >2.0
  • Repeatability (RSD) <1.0% for five replicates
  • Plate count (N) >2000
  • Tailing factor (Tf) ≤2.0
  • Separation factor (α) >1.0

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