- step 1 - selection of the HPLC method and initial system
- step 2 - selection of initial conditions
- step 3 - selectivity optimization
- step 4 - system optimization
- step 5 - method validation.
- keep it simple
- try the most common columns and stationary phases first
- thoroughly investigate binary mobile phases before going on to ternary
- think of the factors that are likely to be significant in achieving the desired resolution.
Mobile phase composition, for example, is the most powerful way of optimizing selectivity whereas temperature has a minor effect and would only achieve small selectivity changes. pH will only significantly affect the retention of weak acids and bases. A flow diagram of an HPLC system is illustrated in Figure 1.
Types of chromatography. Reverse phase is the choice for the majority of samples, but if acidic or basic analytes are present then reverse phase ion suppression (for weak acids or bases) or reverse phase ion pairing (for strong acids or bases) should be used. The stationary phase should be C18 bonded. For low/medium polarity analytes, normal phase HPLC is a potential candidate, particularly if the separation of isomers is required. Cyano-bonded phases are easier to work with than plain silica for normal phase separations. For inorganic anion/cation analysis, ion exchange chromatography is best. Size exclusion chromatography would normally be considered for analysing high molecular weight compounds (.2000).
Column dimensions. For most samples (unless they are very complex), short columns (10–15 cm) are recommended to reduce method development time. Such columns afford shorter retention and equilibration times. A flow rate of 1-1.5 mL/min should be used initially. Packing particle size should be 3 or 5 μm.
Detectors. Consideration must be given to the following:
- Do the analytes have chromophores to enable UV detection?
- Is more selective/sensitive detection required (Table I)?
- What detection limits are necessary?
- Will the sample require chemical derivatization to enhance detectability and/or improve the chromatography?
Fluorescence or electrochemical detectors should be used for trace analysis. For preparative HPLC, refractive index is preferred because it can handle high concentrations without overloading the detector.
UV wavelength. For the greatest sensitivity λmax should be used, which detects all sample components that contain chromophores. UV wavelengths below 200 nm should be avoided because detector noise increases in this region. Higher wavelengths give greater selectivity.
Fluorescence wavelength. The excitation wavelength locates the excitation maximum; that is, the wavelength that gives the maximum emission intensity. The excitation is set to the maximum value then the emission is scanned to locate the emission intensity. Selection of the initial system could, therefore, be based on assessment of the nature of sample and analytes together with literature data, experience, expert system software and empirical approaches.
Gradient HPLC. With samples containing a large number of analytes (.20–30) or with a wide range of analyte retentivities, gradient elution will be necessary to avoid excessive retention.
Determination of initial conditions. The recommended method involves performing two gradient runs differing only in the run time. A binary system based on either acetonitrile/water (or aqueous buffer) or methanol/water (or aqueous buffer) should be used.
Selectivity optimization in gradient HPLC. Initially, gradient conditions should be optimized using a binary system based on either acetonitrile/water (or aqueous buffer) or methanol/water (or aqueous buffer). If there is a serious lack of selectivity, a different organic modifier should be considered.
Step 4 - system parameter optimization. This is used to find the desired balance between resolution and analysis time after satisfactory selectivity has been achieved. The parameters involved include column dimensions, column-packing particle size and flow rate. These parameters may be changed without affecting capacity factors or selectivity.
Method development and validation can be simultaneous, but they are two different processes, both downstream of method selection. Analytical methods used in quality control should ensure an acceptable degree of confidence that results of the analyses of raw materials, excipients, intermediates, bulk products or finished products are viable. Before a test procedure is validated, the criteria to be used must be determined.
Analytical methods should be used within good manufacturing practice (GMP) and good laboratory practice (GLP) environments, and must be developed using the protocols set out in the International Conference on Harmonization (ICH) guidelines (Q2A and Q2B).1,2 The US Food and Drug Administration (FDA)3,4 and US Pharmacopoeia (USP)5 both refer to ICH guidelines. The most widely applied validation characteristics are accuracy, precision (repeatability and intermediate precision), specificity, detection limit, quantitation limit, linearity, range, robustness and stability of analytical solutions. Method validation must have a written and approved protocol prior to use.6
HPLC instrumentation The HPLC systems used for the validation studies consisted of Series 200 UV/Visible Detector, Series 200 LC Pump, Series 200 Autosampler and Series 200 Peltier LC Column Oven (all Perkin Elmer, Boston, Massachusetts, USA). The data were acquired via TotalChrom Workstation (Version 6.2.0) data acquisition software (Perkin Elmer), using Nelson Series 600 LINK interfaces (Perkin Elmer).
All chromatographic experiments were performed in the isocratic mode. The mobile phase was a methanol/water solution (75:25 v/v). The flow rate was 1.5 mL/min and the oven temperature was 40 ºC. The injection volume was 20 μL and the detection wavelength was set at 254 nm. The chromatographic separation was on a 25034.6 mm ID, 10 μm C18 μ-Bondapak column (Waters, Milford, Massachusetts, USA).
Percentage accuracy tends to be lower at the lower end of the calibration curve. The term accuracy is usually applied to quantitative methods but it may also be applied to methods such as limit tests. Accuracy is usually determined by measuring a known amount of standard material under a variety of conditions but preferably in the formulation, bulk material or intermediate product to ensure that other components do not interfere with the analytical method. For assay methods, spiked samples are prepared in triplicate at three levels across a range of 50-150% of the target concentration. The per cent recovery should then be calculated. The accuracy criterion for an assay method is that the mean recovery will be 100±2% at each concentration across the range of 80-120% of the target concentration. To document accuracy, ICH guidelines regarding methodology recommend collecting data from a minimum of nine determinations across a minimum of three concentration levels covering the specified range (for example, three concentrations, three replicates each).
In the present study, the accuracy of the method was evaluated by recovery assay, adding known amounts of progesterone reference standard to a known amount of gel formulation, to obtain three different levels (50, 100 and 150%) of addition. The samples were analysed, and mean recovery and %RSDs calculated. The data presented in Table IV show that the recovery of progesterone in spiked samples met the evaluation criterion for accuracy (100±2.0% across 80–120% of target concentrations).
Specificity Developing a separation method for HPLC involves demonstrating specificity, which is the ability of the method to accurately measure the analyte response in the presence of all potential sample components. The response of the analyte in test mixtures containing the analyte and all potential sample components (placebo formulation, synthesis intermediates, excipients, degradation products and process impurities) is compared with the response of a solution containing only the analyte. Other potential sample components are generated by exposing the analyte to stress conditions sufficient to degrade it to 80–90% purity. For bulk pharmaceuticals, stress conditions such as heat (50–60 ºC), light (600 FC of UV), acid (0.1 M HCl), base (0.1 M NaOH) and oxidant (3% H2O2) are typical. For formulated products, heat, light and humidity (70-80% RH) are often used. The resulting mixtures are then analysed, and the analyte peak is evaluated for peak purity and resolution from the nearest eluting peak.
Once acceptable resolution is obtained for the analyte and potential sample components, the chromatographic parameters, such as column type, mobile phase composition, flow rate and detection mode, are considered set. An example of specificity criterion for an assay method is that the analyte peak will have baseline chromatographic resolution of at least 2.0 from all other sample components. In this study, a weight of sample placebo equivalent to the amount present in a sample solution preparation was injected to demonstrate the absence of interference with progesterone elution (Figure 4).
Precision Precision means that all measurements of an analyte should be very close together. All quantitative results should be of high precision - there should be no more than a ±2% variation in the assay system. A useful criterion is the relative standard deviation (RSD) or coefficient of variation (CV), which is an indication of the imprecision of the system (Equation 2).
According to the ICH,2 precision should be performed at two different levels - repeatability and intermediate precision. Repeatability is an indication of how easy it is for an operator in a laboratory to obtain the same result for the same batch of material using the same method at different times using the same equipment and reagents. It should be determined from a minimum of nine determinations covering the specified range of the procedure (for example, three levels, three repetitions each) or from a minimum of six determinations at 100% of the test or target concentration.
Intermediate precision results from variations such as different days, analysts and equipment. In determining intermediate precision, experimental design should be employed so that the effects (if any) of the individual variables can be monitored. Precision criteria for an assay method are that the instrument precision and the intra-assay precision (RSD) will be ≤2%.
In this study, the precision of the method (repeatability) was investigated by performing six determinations of the same batch of product. The resulting data are provided in Table V, which show that the repeatability precision obtained by one operator in one laboratory was 0.28% RSD for progesterone peak area and, therefore, meets the evaluation criterion.
The standard deviation of the response can be determined based on the standard deviation of the blank, on the residual standard deviation of the regression line, or the standard deviation of y-intercepts of regression lines. The method used to determine LOD and LOQ should be documented and supported, and an appropriate number of samples should be analysed at the limit to validate the level. In this study, the LOD was determined to be 10 ng/mL with a signal:noise ratio of 2.9. The LOQ was 20 ng/mL with a signal:noise ratio of 10.2. The RSD for six injections of the LOQ solution was ≤2%.
Analytical solution stability Validation of sample and standard solution preparation may be divided into sections, each of which can be validated. These include extraction; recovery efficiency; dilution process when appropriate; and addition of internal standards when appropriate. Although extraction processes do not actually affect the measuring stage they are of critical importance to the analytical test method as a whole. The extraction process must be able to recover the analyte from the product; it must not lose (for example, by oxidation or hydrolysis) any of the analyte in subsequent stages, and must produce extraction replicates with high precision. For example, during analysis of an ester prodrug the extraction process involves the use of strongly alkaline or acid solutions, it may cause some of the prodrug to be hydrolysed and, therefore, give false results.
Reference substances should be prepared so that they do not lose any of their potency. Thus it is necessary to validate that the method will give reliable reference solutions that have not been deactivated by weighing so little that an error is produced; adsorption onto containers; decomposition by light; and decomposition by the solvent. If the reference is to be made up from a stock solution then it must be validated that the stock solution does not degrade during storage. Reagent preparation should be validated to ensure that the method is reliable and will not give rise to incorrect solutions, concentrations and pH values.
Samples and standards should be tested during a period of at least 24 h (depending on intended use), and component quantitation should be determined by comparison with freshly prepared standards. For the assay method, the sample solutions, standard solutions and HPLC mobile phase should be stable for 24 h under defined storage conditions. Acceptable stability is ≤2% change in standard or sample response, relative to freshly prepared standards. The mobile phase is considered to have acceptable stability if aged mobile phase produces equivalent chromatography (capacity factors, resolution or tailing factor) and the assay results are within 2% of the value obtained with fresh mobile phase.
In the present study, the stabilities of progesterone sample and standard solutions were investigated. Test solutions of progesterone were prepared and chromatographed initially and after 24 h. The stability of progesterone and the mobile phase were calculated by comparing area response and area per cent of two standards with time. Standard and sample solutions stored in a capped volumetric flask on a lab bench under normal lighting conditions for 24 h were shown to be stable with no significant change in progesterone concentration during this period (Table VII).
Robustness Robustness measures the capacity of an analytical method to remain unaffected by small but deliberate variations in method parameters. It also provides some indication of the reliability of an analytical method during normal usage. Parameters that should be investigated are per cent organic content in the mobile phase or gradient ramp; pH of the mobile phase; buffer concentration; temperature; and injection volume. These parameters may be evaluated one factor at a time or simultaneously as part of a factorial experiment. The chromatography obtained for a sample containing representative impurities when using modified parameter(s) should be compared with the chromatography obtained using the target parameters.
Conclusion Method development involves a series of sample steps; based on what is known about the sample, a column and detector are chosen; the sample is dissolved, extracted, purified and filtered as required; an eluent survey (isocratic or gradient) is run; the type of final separation (isocratic or gradient) is determined from the survey; preliminary conditions are determined for the final separation; retention efficiency and selectivity are optimized as required for the purpose of the separation (quantitative, qualitative or preparation); the method is validated using ICH guidelines. The validated method and data can then be documented.
References 1. International Conference on Harmonization, "Q2A: Text on Validation of Analytical Procedures," Federal Register 60(40), 11260–11262 (1995).
2. International Conference on Harmonization, "Q2B: Validation of Analytical Procedures: Methodology; Availability," Federal Register 62(96), 27463–27467 (1997).
3. FDA, "Analytical Procedures and Methods Validation: Chemistry, Manufacturing and Controls Documentation; Availability," Federal Register (Notices) 65(169), 52776–52777 (2000).
5. USP 25–NF 20, Validation of Compendial Methods Section (1225) (United States Pharmacopeal Convention, Rockville, Maryland, USA, 2002) p 2256.
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