Method Development and Validation of Hplc

8/8/2019 Method Development and Validation of Hplc

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HPLC Method Development and Validation forPharmaceutical AnalysisMar 1, 2004By: Ghulam A. Shabir

PHARMACEUTICAL TECHNOLOGY EUROPE

The wide variety of equipment, columns, eluent and operational parameters involved makes highperformance liquid chromatography (HPLC) method development seem complex. The process isinfluenced by the nature of the analytes and generally follows the following steps:

y step 1 - selection of the HPLC method and initial system

y step 2 - selection of initial conditions

y step 3 - selectivity optimization

y step 4 - system optimization

y step 5 - method validation.


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Depending on the overallrequirements and nature of thesample and analytes, some ofthese steps will not benecessary during HPLCanalysis. For example, a

satisfactory separation may befound during step 2, thus steps3 and 4 may not be required.The extent to which methodvalidation (step 5) isinvestigated will depend on theuse of the end analysis; forexample, a method required forquality control will require morevalidation than one developedfor a one-off analysis. Thefollowing must be consideredwhen developing an HPLCmethod:

y keep it simple

y try the most common columns and stationary phases first

y thoroughly investigate binary mobile phases before going on to ternary

y 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 whereastemperature has a minor effect and would only achieve small selectivity changes. pH will onlysignificantly affect the retention of weak acids and bases. A flow diagram of an HPLC system isillustrated in Figure 1.

HPLC method

development Step1 - selection of theHPLC method andinitial system.When developingan HPLC method,the first step isalways to consultthe literature toascertain whetherthe separation has

been previouslyperformed and if so, under what conditions - this will save time doing unnecessary experimentalwork. When selecting an HPLC system, it must have a high probability of actually being able toanalyse the sample; for example, if the sample includes polar analytes then reverse phase HPLCwould offer both adequate retention and resolution, whereas normal phase HPLC would be muchless feasible. Consideration must be given to the following:

Sample preparation.Does the sample require dissolution, filtration, extraction, preconcentration or

Figure 1: A flow diagram of an HPLC system.

Table I: HPLC detector comparison.


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clean up? Is chemical derivatization required to assist detection sensitivity or selectivity?

Types of chromatography. Reverse phase is the choice for the majority of samples, but if acidic orbasic analytes are present then reverse phase ion suppression (for weak acids or bases) or reversephase ion pairing (for strong acids or bases) should be used. The stationary phase should beC18 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 withthan plain silica for normal phase separations. For inorganic anion/cation analysis, ion exchangechromatography is best. Size exclusion chromatography would normally be considered for analysing

high molecular weightcompounds (.2000).

Gradient HPLC. This is only arequirement for complexsamples with a large number ofcomponents (.2030) becausethe maximum number of peaksthat can be resolved with a

given resolution is much higherthan in isocratic HPLC. This isa result of the constant peakwidth that is observed in

gradient HPLC (in isocratic HPLC peak width increases in proportion to retention time). The methodcan also be used for samples containing analytes with a wide range of retentivities that would, underisocratic conditions, provide chromatograms with capacity factors outside of the normally acceptablerange of 0.515.

Gradient HPLC will also give greater sensitivity, particularly for analytes with longer retention times,because of the more constant peak width (for a given peak area, peak height is inverselyproportional to peak width). Reverse phase gradient HPLC is commonly used in peptide and smallprotein analysis using an acetonitrilewater mobile phase containing 1% trifluoroethanoic acid.

Gradient HPLC is an excellent method for initial sample analysis.

Column dimensions. For most samples (unless they are very complex), short columns (1015 cm)are recommended to reduce method development time. Such columns afford shorter retention andequilibration times. A flow rate of 1-1.5 mL/min should be used initially. Packing particle size shouldbe 3 or 5 m.

Detectors. Consideration must be given to the following:

y Do the analytes have chromophores to enable UV detection?

y Is more selective/sensitive detection required (Table I)?

y What detection limits are necessary?

y Will the sample require chemical derivatization to enhance detectability and/or improve thechromatography?

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 thedetector.

UVwavelength. For the greatest sensitivity max should be used, which detects all sample

Table II: The basic types of analytes used in HPLC.


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components that contain chromophores. UV wavelengths below 200 nm should be avoided becausedetector noise increases in this region. Higher wavelengths give greater selectivity.

Fluorescence wavelength. The excitation wavelength locates the excitation maximum; that is, thewavelength that gives the maximum emission intensity. The excitation is set to the maximum valuethen 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 literaturedata, experience, expert system software and empirical approaches.

Step 2 - selectionof initialconditions. Thisstep determinesthe optimumconditions toadequately retain

all analytes; thatis, ensures noanalyte has acapacity factor ofless than 0.5 (poorretention could result in peak overlapping) and no analyte has a capacity factor greater than 1015(excessive retention leads to long analysis time and broad peaks with poor detectability). Selectionof the following is then required.

Mobile phase solvent strength. The solvent strength is a measure of its ability to pull analytes fromthe column. It is generally controlled by the concentration of the solvent with the highest strength; forexample, in reverse phase HPLC with aqueous mobile phases, the strong solvent would be theorganic modifier; in normal phase HPLC, it would be the most polar one. The aim is to find thecorrect concentration of the strong solvent. With many samples, there will be a range of solventstrengths that can be used within the aforementioned capacity limits. Other factors (such as pH andthe presence of ion pairing reagents) may also affect the overall retention of analytes.Gradient HPLC. With samples containing a large number of analytes (.2030) or with a wide rangeof analyte retentivities, gradient elution will be necessary to avoid excessive retention.

Determination of initial conditions. The recommended method involves performing two gradient runsdiffering 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.

Table III: HPLC optimization parameters.


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Step 3 - selectivity optimization. The aim of this stepis to achieve adequate selectivity (peak spacing).The mobile phase and stationary phasecompositions need to be taken into account. Tominimize the number of trial chromatogramsinvolved, only the parameters that are likely to have

a significant effect on selectivity in the optimizationmust be examined. To select these, the nature of theanalytes must be considered. For this, it is useful tocategorize analytes into a few basic types (Table II).

Once the analyte types are identified, the relevantoptimization parameters may be selected (Table III).Note that the optimization of mobile phaseparameters is always considered first as this is mucheasier and convenient than stationary phaseoptimization.Selectivityoptimization in gradient HPLC. Initially,gradient conditions should be optimized using abinary system based on either acetonitrile/water (oraqueous buffer) or methanol/water (or aqueousbuffer). If there is a serious lack of selectivity, adifferent organic modifier should be considered.

Step 4 - system parameter optimization. This is usedto find the desired balance between resolution andanalysis time after satisfactory selectivity has beenachieved. The parameters involved include columndimensions, column-packing particle size and flowrate. These parameters may be changed without

affecting capacityfactors orselectivity.Step 5 - methodvalidation. Propervalidation ofanalytical methodsis important forpharmaceuticalanalysis when

ensurance of thecontinuing efficacyand safety of each batch manufactured relies solely on the determination of quality. The ability tocontrol this quality is dependent upon the ability of the analytical methods, as applied under well-defined conditions and at an established level of sensitivity, to give a reliable demonstration of alldeviation from target criteria.

Analytical method validation is now required by regulatory authorities for marketing authorizationsand guidelines have been published. It is important to isolate analytical method validation from the

Figure 2: The chemical structure of progesterone and Figure 3:

Amount injected versus peak area of progesterone standard todemonstrate linearity.


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selection and development of the method. Method selection is the first step in establishing ananalytical method and consideration must be given to what is to be measured, and with whataccuracy and precision.Method development and validation can be simultaneous, but they are two different processes, bothdownstream 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, thecriteria to be used must be determined.

Analytical methods should be used within good manufacturing practice (GMP) and good laboratorypractice (GLP) environments, and must be developed using the protocols set out in the InternationalConference on Harmonization (ICH) guidelines (Q2A and Q2B).

1,2The US Food and Drug

Administration (FDA)3,4

and US Pharmacopoeia (USP)5

both refer to ICH guidelines. The mostwidely applied validation characteristics are accuracy, precision (repeatability and intermediateprecision), specificity, detection limit, quantitation limit, linearity, range, robustness and stability ofanalytical solutions. Method validation must have a written and approved protocol prior to use.

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This article reviews and demonstratespractical approaches to analytical methodvalidation with reference to an HPLC assayof progesterone (Figure 2) in a gelformulation. Progesterone is widely used fordysfunctional uterine bleeding oramenorrhoea,7,8 for contraception (eitheralone or with, for example, oestradiol ormestranol in oral contraceptives) and incombination with oestrogens for hormonereplacement therapy in postmenopausalwomen.9,10

Experimental Chemicals and reagents Allchemicals and reagents were of the highestpurity. HPLC-grade methanol was obtainedfrom Merck (Darmstadt, Germany).Progesterone reference standard waspurchased from Sigma Chemicals (St Louis,Missouri, USA). Deionized distilled waterwas used throughout the experiments.HPLC instrumentation The HPLC systemsused for the validation studies consistedofSeries 200UV/Visible Detector, Series

200 LC Pump, Series 200 Autosampler andSeries 200 Peltier LC Column Oven (all

Perkin Elmer, Boston, Massachusetts, USA). The data were acquired viaTotalChrom Workstation(Version 6.2.0) data acquisition software (Perkin Elmer), using Nelson Series 600 LINKinterfaces(Perkin Elmer).

All chromatographic experiments were performed in the isocratic mode. The mobile phase was amethanol/water solution (75:25 v/v). The flow rate was 1.5 mL/min and the oven temperature was 40C. The injection volume was 20 L and the detection wavelength was set at 254 nm. The

Equation 1 and Figure 4: HPLC chromatograms of (a) progesterone

reference standard; (b) separation of progesterone gel sample; (c) placeboformulation.


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chromatographic separation was on a 25034.6 mm ID, 10 m C18 -Bondapak column (Waters,Milford, Massachusetts, USA).

Results and discussionLinearity and

range The linearity of a testprocedure is its ability (within a

given range) to produce resultsthat are directly proportional to theconcentration of analyte in thesample. The range is the intervalbetween the upper and lowerlevels of the analyte that havebeen determined with precision,accuracy and linearity using themethod as written. ICH guidelinesspecify a minimum of fiveconcentration levels, along withcertain minimum specified ranges.For assay, the minimum specified

range is 80120% of thetheoretical content of active.

Acceptability of linearity data isoften judged by examining thecorrelation coefficient and y-intercept of the linear regressionline for the response versusconcentration plot. The regressioncoefficient (r

2) is .0.998 and is generally considered as evidence of acceptable fit of the data (Figure

3) to the regression line. The per cent relative standard deviation (RSD), intercept and slope shouldbe calculated.

In the present study, linearity was studied in the concentration range 0.0250.15 mg/mL (25150%of the theoretical concentration in the test preparation, n=3) and the following regression equationwas found by plotting the peak area (y) versus the progesterone concentration (x) expressed inmg/mL: y53007.2x14250.1 (r

251.000). The demonstration coefficient (r

2) obtained for the regression

line demonstrates the excellent relationship between peak area and concentration of progesterone.The analyte response is linear across 80-120% of the target progesterone concentration.

Table V: Demonstration of the repeatability of the HPLC assay for progesterone.


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AccuracyA methodis said to beaccurate if it givesthe correctnumerical answerfor the analyte.

The methodshould be able todetermine whetherthe material inquestion conformsto its specification(for example, itshould be able tosupply the exactamount ofsubstance

present). However, the exact amount present is unknown, which is why a test method is used toestimate the accuracy. Furthermore, it is rare that the results of several replicate tests all give the

same answer, so the mean or average value is taken as the estimate of the accurate answer.

Some analysts adopt a more practical attitude to accuracy, which is expressed in terms of error. Theabsolute error is the difference between the observed and the expected concentrations of theanalyte. Percentage accuracy can be defined in terms of the percentage difference between theexpected and the observed concentrations (Equation 1).Percentage accuracy tends to be lower at the lower end of the calibration curve. The term accuracyis 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 varietyof conditions but preferably in the formulation, bulk material or intermediate product to ensure thatother 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. Theper cent recovery should then be calculated. The accuracy criterion for an assay method is that themean recovery will be 100 2% at each concentration across the range of 80-120% of the targetconcentration. To document accuracy, ICH guidelines regarding methodology recommend collectingdata from a minimum of nine determinations across a minimum of three concentration levelscovering 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 knownamounts of progesterone reference standard to a known amount of gel formulation, to obtain threedifferent 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 spikedsamples met the evaluation criterion for accuracy (100 2.0% across 80 120% of targetconcentrations).Specificity Developing a separation method for HPLC involves demonstrating specificity, which is theability of the method to accurately measure the analyte response in the presence of all potentialsample components. The response of the analyte in test mixtures containing the analyte and allpotential sample components (placebo formulation, synthesis intermediates, excipients, degradationproducts and process impurities) is compared with the response of a solution containing only theanalyte. Other potential sample components are generated by exposing the analyte to stressconditions sufficient to degrade it to 8090% purity. For bulk pharmaceuticals, stress conditions such


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as heat (5060 C), 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. Theresulting mixtures are then analysed, and the analyte peak is evaluated for peak purity andresolution from the nearest eluting peak.

Once acceptable resolution is obtained for the analyte and potential sample components, thechromatographic parameters, such as column type, mobile phase composition, flow rate anddetection mode, are considered set. An example of specificity criterion for an assay method is thatthe analyte peak will have baseline chromatographic resolution of at least 2.0 from all other samplecomponents. In this study, a weight of sample placebo equivalent to the amount present in a samplesolution preparation was injected to demonstrate the absence of interference with progesteroneelution (Figure 4).

Precision Precision means that all measurements of an analyte should be very close together. Allquantitative results should be of high precision - there should be no more than a 2% variation in theassay 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 andintermediate precision. Repeatability is an indication of how easy it is for an operator in a laboratoryto obtain the same result for the same batch of material using the same method at different timesusing the same equipment and reagents. It should be determined from a minimum of ninedeterminations covering the specified range of the procedure (for example, three levels, threerepetitions each) or from a minimum of six determinations at 100% of the test or targetconcentration.

Intermediate precision results from variations such as different days, analysts and equipment. Indetermining intermediate precision, experimental design should be employed so that the effects (ifany) of the individual variables can be monitored. Precision criteria for an assay method are that theinstrument precision and the intra-assay precision (RSD) will be 2%.

In this study, the precision of the method (repeatability) was investigated by performing sixdeterminations of the same batch of product. The resulting data are provided in Table V, which showthat the repeatability precision obtained by one operator in one laboratory was 0.28% RSD forprogesterone peak area and, therefore, meets the evaluation criterion.


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The intermediate precision wasdemonstrated by two analysts,using two HPLC systems and whoevaluated the relative per centpurity data across the two HPLCsystems at three concentration

levels (50%, 100%, 150%) thatcovered the assay method range(0.0250.15 mg/mL). The meanand RSD across the systems andanalysts were calculated from the

individual relative per cent purity mean values at 50%, 100% and 150% of the test concentration.The data are presented in Table VI, and show 2.0% RSD, therefore, meeting the evaluationcriterion.

Limits of detection and quantitation The limit of detection (LOD) is defined as the lowest concentrationof an analyte in a sample that can be detected, not quantified. It is expressed as a concentration at aspecified signal:noise ratio,

2usually 3:1. The limit of quantitation (LOQ) is defined as the lowest

concentration of an analyte in a sample that can be determined with acceptable precision andaccuracy under the stated operational conditions of the method. The ICH has recommended asignal:noise ratio 10:1. LOD and LOQ may also be calculated based on the standard deviation of theresponse (SD) and the slope of the calibration curve(s) at levels approximating the LOD according tothe formulae: LOD53.3(SD/S) and LOQ510(SD/S).The standard deviation of the response can be determined based on the standard deviation of theblank, 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 documentedand supported, and an appropriate number of samples should be analysed at the limit to validate thelevel. In this study, the LOD was determined to be 10 ng/mL with a signal:noise ratio of 2.9. TheLOQ was 20 ng/mL with a signal:noise ratio of 10.2. The RSD for six injections of the LOQ solutionwas 2%.

Analytical solution stability Validation of sample and standard solution preparation may be divided intosections, each of which can be validated. These include extraction; recovery efficiency; dilutionprocess when appropriate; and addition of internal standards when appropriate. Although extractionprocesses do not actually affect the measuring stage they are of critical importance to the analyticaltest method as a whole. The extraction process must be able to recover the analyte from theproduct; it must not lose (for example, by oxidation or hydrolysis) any of the analyte in subsequentstages, and must produce extraction replicates with high precision. For example, during analysis ofan ester prodrug the extraction process involves the use of strongly alkaline or acid solutions, it maycause 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 beendeactivated 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.


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Samples and standards should be tested during a period of at least 24 h (depending on intendeduse), and component quantitation should be determined by comparison with freshly preparedstandards. For the assay method, the sample solutions, standard solutions and HPLC mobile phaseshould be stable for 24 h under defined storage conditions. Acceptable stability is 2% change instandard or sample response, relative to freshly prepared standards. The mobile phase isconsidered 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 valueobtained with fresh mobile phase.

In the present study, the stabilities of progesterone sample and standard solutions wereinvestigated. Test solutions of progesterone were prepared and chromatographed initially and after24 h. The stability of progesterone and the mobile phase were calculated by comparing arearesponse and area per cent of two standards with time. Standard and sample solutions stored in acapped volumetric flask on a lab bench under normal lighting conditions for 24 h were shown to bestable with no significant change in progesterone concentration during this period (Table VII).

Robustness Robustness measures the capacity of an analytical method to remain unaffected bysmall 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 areper cent organic content in the mobile phase or gradient ramp; pH of the mobile phase; bufferconcentration; temperature; and injection volume. These parameters may be evaluated one factor ata time or simultaneously as part of a factorial experiment. The chromatography obtained for asample containing representative impurities when using modified parameter(s) should be comparedwith the chromatography obtained using the target parameters.

Conclusion Method development involves a series of sample steps; based on what is known aboutthe sample, a column and detector are chosen; the sample is dissolved, extracted, purified andfiltered 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 thefinal separation; retention efficiency and selectivity are optimized as required for the purpose of theseparation (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 AnalyticalProcedures," Federal Register60(40), 1126011262 (1995).

2. International Conference on Harmonization, "Q2B: Validation of Analytical Procedures:Methodology; Availability," Federal Register62(96), 2746327467 (1997).

3. FDA, "Analytical Procedures and Methods Validation: Chemistry, Manufacturing and ControlsDocumentation; Availability,"Federal Register (Notices)65(169), 5277652777 (2000).

4. www.fda.gov/cder/guidance/cmc3.pdf

5. USP 25NF 20, Validation of Compendial Methods Section (1225) (United States PharmacopealConvention, Rockville, Maryland, USA, 2002) p 2256.

6. G.A. Shabir, "Validation of HPLC Chromatography Methods for Pharmaceutical Analysis.Understanding the Differences and Similarities Between Validation Requirements of FDA, the USPharmacopeia and the ICH," J. Chromatogr. A.987(1-2), 57-66 (2003).


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7. C.E. Wood, "Medicare Program; Changes to the Hospital Outpatient Prospective," Med. J.Aust.165, 510514 (1996).

8. A. Prentice, "Medical Management of Menorrhagia," Br. Med. J.319, 13431345 (1999).

9. D.T. Baired and A.F. Glasier, "Hormonal Contraception," New Engl. J. Med.328, 15431549

(1993).

10. P.E. Belchetz, "Hormonal Treatment of Postmenopausal Women," New Engl. J. Med.330,10621071(1994).

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Method Development and Validation of Hplc