| Department | Laboratory | Document no | LAB-095 | ||
| Title | HPLC Method Development & Validation Procedure | ||||
| Prepared by: | Date: | Supersedes: | |||
| Checked by: | Date: | Date Issued: | |||
| Approved by: | Date: | Review Date: | |||
Document Owner
Laboratory Manager
Affected Parties
All Laboratory staffs.
Purpose
To explain how to:
(i) Develop and optimise an HPLC method, and
(ii) How to validate the method.
Scope
It is the responsibility of all Laboratory Technicians who are trained in HPLC procedures.
EHS Statement
Safety glasses should be worn when handling chemicals.
MSDS (Material Safety Data Sheet) should be read before handling toxic chemicals.
Procedure
General
The validation of an analytical method is the process by which it is established that the performance characteristics of the method, such as Precision, Accuracy, Specificity, Linearity, Limit of Detection (LOD), Limit of Quantitation (LOQ) and Robustness meet the requirements for the intended applications.
This SOP refers specifically to HPLC. However, the same principles may be applied to validations of other types of analytical procedures.
Well-characterised reference materials with documented purity should be used to perform the validation.
The optimum wavelength for a method can be found by acquiring the chromatographic data on a PDA detector over a large wavelength range, (e.g. 200-400nm). The optimum wavelength is the wavelength, which maximises the response for all the components of interest, but is outside the absorbance for the mobile phase. Before validating an HPLC method, its Specificity must be determined. If the method does not comply with the Specificity requirements, the method must be modified until the acceptance criteria are met. Hence it is essential that the Specificity be adequate, before Precision, Linearity and Accuracy, etc. are performed.
1. Definitions used in determining Specificity
1.1. Capacity Factor (k’) is defined by the equation
k’ = VR ‑ VO
————
VO
Where,
VR = the distance along the baseline between the point of injection and a perpendicular dropped from the maximum of the peak of interest.
VO the distance along the baseline between the point of injection and a perpendicular dropped from the maximum of an unretained peak.
1.2. Resolution, R
Determine the resolution between adjacent peaks, A and B, using the following equation:
R = VB ‑ VA
——————
1/2 (WA + WB)
where
V = retention time of the peaks (expressed in mm)
W = peak widths at the baseline of tangents drawn on the peak (mm).
1.3. USP Tailing
1.3.1. The Tailing factor, T is an indicator of peak skewness and is calculated using the equation:
T = W
2F
where
W = Peak width at 5% of peak height
F = Distance between the perpendicular dropped from the peak maximum and the leading edge of the peak at 5% of the peak height.
2. Specificity (or Selectivity)
2.1. The Specificity of an analytical method is its ability to accurately measure the analyte in the presence of components that may be present in the sample matrix, (e.g. excipients, solvents, breakdown products (BDP’s) and impurities).
2.2. The method often also needs to be able to measure the level of any detectable BDP’s or impurities present. Stress Testing (refer 4.) enables the BDP’s to be characterised by their retention times.
2.3. The analyte peak must be well separated from any peaks due to excipients, breakdown products, impurities and void volume. Coelution between any of the peaks of interest should not occur.
2.4. For stability indicating chromatographic methods, peak purity (homogeneity) should be demonstrated. This can be done by peak slicing using diode-array UV detection (PDA) and comparing the results obtained with reference standard and drug degraded under stress conditions.
2.5. The specificity of a test method is determined by comparing test results from the analysis of samples containing impurities, degradation products, or placebo ingredients with those obtained from the analysis of samples without impurities, degradation products, or placebo ingredients. The bias of the assay is a measure of the specificity.
3. System Suitability Requirements
3.1. During method development, the following system suitability conditions need to be met for the analyte and breakdown product/impurity peaks:
3.1.1 k’ > 1:
Ensures that the compound is retained on the column and does not elute in the void volume.
3.1.2 k’ < 5 (preferably):
Avoids very long analysis times.
3.1.3 R’ > 1 (between adjacent peaks):
Ensures peaks are adequately resolved from each other.
3.1.4 USP Tailing = 1-2 (preferably):
The integration of peaks becomes less reliable as peak tailing increases, causing peak areas to become less accurate.
3.1.5 Peak purity (homogeneity):
No coelution to occur on any of the peaks of interest.
3.1.6 Peak area and/or height:
To be unaffected by the presence of the other components, (e.g. excipients, BDP’s).
3.2. If the conditions above could not be met by the existing method, the method must be changed. The following are suggested possible steps which could be used to modify the existing method*, so that it meets the required conditions.
*(If it is a REVERSE PHASE HPLC method)
3.2.1. k’ < 1 for the analyte, BDP or impurity.
The analyte, BDP or impurity needs to be retained on the column for longer. This could be achieved by decreasing the proportion of organic in the mobile phase. For example a mobile phase containing:
a. 60% Acetonitrile
b. 40% Phosphate Buffer pH 6.8
could be changed to:
a. 53% Acetonitrile
b. 47% Phosphate Buffer pH 6.8
to increase retention times. This is most easily done using a dual pump system and by a process of trial and error.
Alternatively, a mobile phase gradient may be set up.
3.2.2. k’ > 5 for the analyte, BDP or impurity.
When retention times are too long, this can often be rectified by increasing the proportion of organic in the mobile phase. This can be done either isocratically by trial and error, or by setting up a gradient.
3.2.3. R < 1 between adjacent peaks of interest.
The following procedures could improve resolution:
* Increase retention times as in (a) above, or
* Increase column length, or
* Decrease flow rate.
3.2.4. USP Tailing > 2
The following measures could help to reduce tailing:
a. Dilute the sample, or
b. Lower the injection volume, or
c. Change the pH of the mobile phase (between pH 2 ‑ 8), or
d. Add an ion-pairing reagent (e.g. PIC, hexanesulphonic acid) to the mobile phase.
If conditions 3.2.1 – 3.2.4 still cannot be met, a totally new HPLC method may need to be developed.
3.2.5. A certain number of System Suitability tests are chosen for evaluation when an analytical method is used. HPLC methods often specify a range of k’ values for a particular compound and the minimum resolution between specific peaks, for example.
4. Stress Testing (forced degradation)
Stress testing is conducted to provide data on forced decomposition products and decomposition mechanisms for the drug substance.
4.1. Stress testing involves subjecting the sample solution (including active and excipients) to extreme conditions so as to promote degradation.
Typically, sample solutions are treated with acid, base, hydrogen peroxide, heat, (and light for photosensitive products). They are then placed at 40°C for between 5-20 days.
4.2. The stressed samples are subsequently analysed via HPLC, using a photodiode array detector (PDA).
The baselines of the chromatograms are magnified and scrutinised for foreign peaks caused by degradation of the sample.
The peak purity (homogeneity) function on the PDA is also used, to detect any possible coelution of a degradate with the main peak.
The test method must be able to selectively and quantitatively distinguish between drug substance and degradant to be stability indicating.
5.1. The linearity of an analytical method may be defined as the ability of the method to produce test results that are proportional to the concentration of analyte in samples within a given range. The range of a method is the interval between upper and lower concentrations that have been demonstrated to be accurate, precise and linear by method validation studies.
5.2. At least 5 samples of differing concentrations must be tested in triplicate, (i.e. injected three times) to establish the linearity of the response and the concentration range over which this linear response applies.
The five concentrations are made up by:
a. Accurately weighing sample then transferring it into volumetric glassware, OR
b. Making appropriate dilutions from an accurately prepared stock solution.
5.3. Linear regression analysis is then applied to the results to demonstrate the linearity of the method over the range of concentrations studied. The range of concentrations studied is usually 50% ‑ 125% of the target concentration of the active in the sample. Degradant concentrations are evaluated for each particular method.
5.4. The correlation coefficient, r, should be > 0.99 for the range selected. This is the range where the slope is linear.
5.5. The method should have a linear response over at least 75-125% of the target concentration of the analyte.
6.1. The Precision of an analytical method refers to the degree of agreement among individual test results obtained from multiple sampling of the same homogeneous sample. Precision may be considered at 3 levels: Repeatability, Intermediate Precision and Reproducibility.
The precision of a method is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements.
6.2. Repeatability may be obtained by:
1) Repeatedly applying the analytical method to multiple samplings (at least 6) of a homogeneous sample at 100% of test concentration,
or;
2) at least 9 determinations covering the specified range for the procedure,
(e.g. 3 concentrations, 3 replicates each). The standard deviation of the results must be within < 2% of the mean for the method to meet precision requirements.
6.3. Intermediate Precision. This establishes the extent to which random events influence the precision of the method, within the one laboratory. Typical variations to be studied are: different days, different analysts, or different pieces of equipment.
6.4. Reproducibility refers to inter-laboratory trials. As a general rule the reproducibility should be within +/- 2% between laboratories for active drugs and 10% for degradation products.
7.1. Accuracy may be defined as the closeness of an individual test result to the true test result value. Thus, accuracy is a measure of the exactness of the analytical method. The results of a given method may be high in accuracy but low in precision, and vice versa. Accuracy may often be expressed as percent recovery by the assay of known, added amounts of analyte to the inert matrix.
7.2. Accuracy can be determined by preparing a matrix of the ingredients of the product with the exception of the active component. The active component is then added or ‘spiked’ in known amounts usually ranging from 75% to 125% of the dosage strength on at least
5 levels (25% – 125% for dissolution studies). The recovery of the known amount is then calculated.
7.3. A minimum of 3 concentrations should be studied, 3 injections per concentration. The accuracy of an analytical method is the closeness of test results obtained by that method to the true or theoretical value.
% Accuracy = Experimental Assay – Theoretical Assay x 100
Theoretical Assay
7.4. Typical accuracy acceptance criteria are > 98% and < 102%.
Typical % RSD acceptance criteria (over all concentration levels) is < 2%.
7.5. To validate the accuracy of a method, the analyst must have a standard material of characterised purity in order to know what response to expect in the test method.
8. Limit of Detection / Limit of Quantitation
8.1. For the quantitation of impurities and degradation products, linearity studies should be carried out in the presence of the drug substance. Such studies should be extended to low concentrations to experimentally define actual Limits of Detection (LOD) and Limits of Quantitation (LOQ).
8.2. The LOD is the lowest amount of an analyte in a sample that can be detected but not quantitated as an exact value. This can be defined as that concentration giving a peak height response three times greater than the baseline noise level.
8.3. The LOQ is the lowest amount of analyte in a sample that can be quantitatively determined with precision and accuracy. The limit of quantitation is used particularly for the determination of impurities and/or degradation products.
It can be defined as that concentration giving:
a. A signal: Noise ratio of at least 10:1 and < 10% precision, OR
b. A signal: Noise ratio of at least 20:1 and < 5% precision, (i.e. % RSD over 6 injections is < 5%).
9.1. The robustness of a method is a measure of its capacity to remain unaffected by small but deliberate variations in the method parameters and provides an indication of its reliability during normal usage within a laboratory. The evaluation of robustness enables a series of system suitability parameters to be established for the analytical method, to ensure the validity of the procedure is maintained whenever used.
9.2. Examples of typical variations are:
a. Stability of analytical solutions
b. Influence of the variation of mobile phase pH
c. Different columns
d. Temperature
e. Flow rate.
GLP for Pharmaceutical Laboratories, Pharma Systems 1995.
ICH guideline on “Validation of Analytical Procedures – Methodology Q2B dated 6/11/96.
BP
| Version # | Revision History |
| Lab-095 | New |