HPLC Method Development and Validation

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J Pharm Educ Res Vol. 4, Issue No. 1, June 2013

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can detect a wide array of compounds

Analytic methods are intended to establish theidentity, purity, physical characteristics and potency ofthe drugs that we use. Methods are developed to supportdrug testing against specications during manufacturing

and quality release operations, as well as during long-term stability studies. Methods may also support safetyand characterization studies or evaluations of drug

performance. Once a stability-indicating method is in place, the formulated drug product can then be subjectedto heat and light in order to evaluate potential degradationof the API in the presence of formulation excipients 3,4.

The validation of an analytic method demonstrates thescientic soundness of the measurement or characterization.It is required to varying extents throughout the regulatory

submission process. The validation practice demonstratesthat an analytic method measures the correct substance,in the correct amount, and in the appropriate range for theintended samples. It allows the analyst to understand the

behavior of the method and to establish the performancelimits of the method 5,6. The goal is to identify the critical

parameters and to establish acceptance criteria for methodsystem suitability 7. The HPLC system with its differentcomponents is shown in Figure 2.

Fig 2: HPLC system with its different components

METHOD DEVELOPMENT

The three critical components for a HPLC method are:sample preparation (% organic, pH, shaking/sonication,sample size, sample age), HPLC analysis conditions (%

organic, pH, ow rate, temperature, wavelength, andcolumn age) and standardization (integration, wavelength,standard concentration, and response factor correction).During the preliminary method development stage, allindividual components should be investigated before

the nal method optimization. This gives the scientista chance to critically evaluate the method performancein each component and streamline the final methodoptimization 8. The percentage of time spent on eachstage is proposed to ensure the scientist will allocatesufcient time to different steps. In this approach, thethree critical components for a HPLC method (sample

preparation, HPLC analysis and standardization) will rst be investigated individually 9-11 .

The degraded drug samples obtained are subjectedto preliminary chromatographic separation to study thenumber and types of degradation products formed undervarious conditions 12 . Scouting experiments are runand then conditions are chosen for further optimization13 . Resolving power, specificity, and speed are keychromatographic method attributes to keep in mind duringmethod development 14. Selectivity can be manipulated bycombination of different factors like solvent composition,type of stationary phase, mobile phase, buffers and pH.Changing solvents and stationary phases are the mostcomfortable approaches to achieve the separation. The

proper range of pH is an important tool for separation ofionizable compounds. Acidic compounds are retainedat low pH while basic compounds are more retained athigher pH. The neutral compounds remain unaffected. The

pH range 4-8 is not generally employed because slightchange in pH in this range would result in a dramatic shiftin retention (up-slope or down-slope of curve) (Figure 3).However, by operating at pH extremes (2-4 or 8-10), notonly is there a 10-30 fold difference in retention that can

Figure 3: Reversed-phase retention behavior as pH is varied


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be exploited in method development but also the methodcan be made more robust which is a desirable outcomewith validation in min 15,16 .

Method development within the different pH rangefrom 1 to 12 for better chromatographic resolution between

two or more peak of an analyte depends upon three mainfactors that are -Column efciency, Selectivity, Retentiontime. The ionizable analytes are either bases or acids andit effects above three factors dramatically with change in

pH. Retention time can be improved by changing the pHthat will lead to easy separation of ionizable analytes fromnon-ionized form. By changing the mobile phase pH canalso improve column efciency because it altered both theionization of the analyte and the residual silanols and italso minimizes secondary interactions between analytesand the silica surface that will lead to poor peak shape. Toachieve optimum resolution, it require change in the pHof mobile phase. Method development can proceeds byinvestigating parameters of chromatographic separationsrst at low pH and then at higher pH until optimum resultsare achieved 17.

Mobile phase composition (or solvent strength) playsan important role in RP-HPLC separation. Acetonitrile(ACN), methanol (MeOH) and tetrahydrofuran (THF)are commonly used solvents in RP-HPLC having lowUV cut-off of 190, 205 and 212nm respectively. These

solvents are miscible with water. Mixture of acetonitrileand water is the best initial choice for the mobile phaseduring method development. An HPLC column packedwith stationary phase of C 18-bonded silica (C 18 Column)and C 8-bonded silica (C 8 Column) is used in RP-HPLCseparation of a wide range of organic compounds. Theseparation selectivity for certain components vary betweenthe columns of different manufacturer as well as betweencolumn production batches from the same manufacturer.Column dimensions, silica substrate properties and bondedstationary phase characteristics are the main ones. The use

of silica-based packing is favoured in most of the presentHPLC columns due to several physical characteristics.Silica substrates are available in spherical or irregularshapes and can be prepared with different surface areas,

pore sizes and particle sizes, which make them suitablefor most HPLC applications. Totally porous silica particleswith 5 m diameter provide the desired characteristics formost HPLC separations 14,16,18 . Zirconia-based columnsare revolutionary HPLC phases. Zirconia particles are

mechanically stable, and have a porous structure similarto that of silica. However, zirconias main advantage oversilica is that it is very stable in a wide range of eluent pH;indeed the ZirChrom-EZ and ZirChrom-MS phasesare stable over the pH range of 1-10.

Separation of many samples can be enhanced byselecting the right column temperature. Higher columntemperature reduces system backpressure by decreasingmobile phase viscosity, which in turn allows use of longercolumns with higher separation efciency. However, anoverall loss of resolution between mixture components inmany samples occurs by increasing column temperature.The optimum temperature is dependent upon nature ofthe mixture components. The overall separation can beimproved by simultaneous changes in column temperatureand mobile phase composition 19-21. Recently, normal phaseHPLC is back popular with the birth of HILIC technologythat proved to improve reproducibility in separating

polar and hydrophilic compounds such as peptides,carbohydrate, vitamins, polar drugs and metabolites.In order to develop a HPLC method effectively, mostof the effort should be spent in method developmentand optimization as that will improve the nal method

performance 22,23 . Method validation is important tocomplete method development.

METHOD VALIDATION

Validation is dened by the International Organizationfor Standardization (ISO) as verication, where thespecied requirements are adequate for an intended use,where the term verication is dened as provision ofobjective evidence that a given item fullls speciedrequirements 24.

The applicability and scope of an analytical methodshould be dened before starting the validation process.It includes dening the analytes, concentration range,description of equipment and procedures, validation level

and criteria required. The validated range is dened byIUPAC as the interval of analyte concentration withinwhich the method can be regarded as validated 25,26 . Thisrange does not have to be the highest and lowest possiblelevels of the analyte that can be determined by the method.Instead, it is dened on the basis of the intended purposeof the method 27,28 . The method can be validated for useas a screening (qualitative), semi-quantitative (e.g. 5-10


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ppm) or quantitative method. It can also be validatedfor use on single equipment, different equipments in thelaboratory, different laboratories or even for internationaluse at different climatic and environmental conditions.The criteria of each type of validation will of course

be different with the validation level required24,29

. Thevarious validation parameters include linearity, accuracy, precision, ruggedness, robustness, LOD, LOQ andselectivity or specicity.

Linearity

The linearity of an analytical procedure is its ability(within a given range) to obtain test results that are directly

proportional to the concentration of analyte in the sample30,31 . It is essential to determine the useful range at whichthe instrumental response is proportional to the analyteconcentration. Generally, a value of co-relation coefcient(r 2) > 0.998 is considered as evidence of an acceptablet of the data to the regression line 32. Signicance ofdeviation of intercept of calibration line from the originvalue of zero can be evaluated statistically by determiningcondence limits for the intercept, generally at the 95 %level 33,34 .

Linearity is determined by a series of three to sixinjections of five or more standards. Peak areas (orheights) of the calibration standards are usually plottedin the Y-axis against the nominal standard concentration,

and the linearity of the plotted curve is evaluated throughthe value of the co-relation coefcient (r 2). Becausedeviations from linearity are sometimes difcult to detect,two additional graphical procedures can be used. Therst one is to plot deviations from regression line versusconcentration or versus logarithm of concentration. Forlinear ranges, the deviations should be equally distributed

between positive and negative values. Another approachis to divide signal data by their respective concentrationsyielding the relative responses 35.

A graph is plotted with the relative responses onY-axis and the corresponding concentrations on X-axis ona log scale. The obtained line should be horizontal overthe full linear range. At higher concentrations, there willtypically be a negative deviation from linearity. Parallelhorizontal lines are drawn in the graph corresponding to,for example, 95 % and 105 % of the horizontal line. Themethod is linear up to the point where the plotted relativeresponse line intersects the 95 % line. A comparison of

the two graphical evaluations using HPLC is shown inFigure 4 where Rc = Line of constant response 36,37 .

Figure 4: Graphical presentations of linearity plot of caffeinesample using HPLC

Accuracy

Accuracy is defined by ISO as closeness ofagreement between a measured quantity value and atrue quantity value of a measurand. It is a qualitativecharacteristic that cannot be expressed as a numericalvalue. It has an inverse relation to both random and

systematic errors, where higher accuracy means lowererrors 33,38. Accuracy is evaluated by analyzing test drug atdifferent concentration levels. Typically, known amountsof related substances and the drug substance in placeboare spiked to prepare an accuracy sample of knownconcentration of related substance. Samples are preparedin triplicate. ICH recommends accuracy evaluation usinga minimum of nine determinations over a minimum ofthree concentration levels covering the range specied.It is determined by comparing the found concentrationwith the added concentration. The methods of determining

accuracy include analysis of analysis of an analyte ofknown purity (i.e., reference material), comparisons ofresults of the proposed analytical procedure with thoseof a second well-characterized procedure and standardaddition method. The accuracy may also be inferred once

precision, linearity and specicity have been established39,40 .


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Precision

It expresses closeness of agreement (degree of scatter) between a series of measurements obtained from multiplesampling of the same homogeneous sample under the

prescribed conditions. Precision may be considered at

three levels: repeatability, intermediate precision andreproducibility 29,41 . Repeatability is also referred to asintra-assay precision. It is a measure of precision ofanalysis in one laboratory by one operator using one

piece of equipment over a relatively short time-span.It is degree of agreement of results when experimentalconditions are maintained as constant as possible, andexpressed as RSD of replicate. ICH recommends aminimum of nine determinations covering the speciedrange for the procedure (e.g., three concentrations/threereplicates as in the accuracy experiment), or a minimum

of six determinations at 100% of the test concentrationfor evaluation of repeatability which should be reportedas standard deviation, relative standard deviation(coefcient of variation) or condence interval. ICHdenes intermediate precision as long-term variability ofthe measurement process and is determined by comparingthe results of a method run within a single laboratoryover a number of weeks. It is also called as interday

precision 40. It re ects discrepancies in results obtained by different operators, from different instruments, withstandards and reagents from different suppliers, with

columns from different batches or a combination of these but in the same laboratory 29. The objective of intermediate precision validation is to verify that the method will provide same results in the same laboratory once thedevelopment phase is over. Reproducibility expresses

precision of analysis of the same sample by differentanalysts in different laboratories using operational andenvironmental conditions that may differ but are stillwithin the specied parameters of the method 24,42 . Theobjective is to verify that the method will provide thesame results despite differences in room temperatureand humidity, variedly experienced operators, differentcharacteristics of equipments (e.g., delay volume of anHPLC system), variations in material and instrumentconditions (e.g. in HPLC, mobile phase composition, pH,ow rate of mobile phase), equipments and consumablesof different ages, columns from different suppliers ordifferent batches and solvents, reagents and other materialwith different quality 29,43 .

Selectivity and Specifcity

Selectivity and specificity are sometimes usedinterchangeably to describe the same concept in methodvalidation. Selectivity of an analytical method is dened

by the ISO as property of a measuring system, used with

a specied measurement procedure, whereby it providesmeasured quantity values for one or more measurandssuch that the values of each measurand are independent ofother measurands or other quantities in the phenomenon,

body, or substance being investigated 24. Specicity is theability to assess unequivocally the analyte in the presenceof components that may be expected to be present. Thespecicity of a test method is determined by comparing testresults from an analysis of samples containing impurities,degradation products, or placebo ingredients with thoseobtained from an analysis of samples without impurities,

degradation products, or placebo ingredients. Specicitycan best be demonstrated by resolution between the analyte

peak and the other closely eluting peak(s) 29,44 .

Detection limit (LOD) and Quantitation limit (LOQ)

LOD of an analytical procedure is the lowestconcentration of an analyte in a sample which can bedetected but not necessarily quantitated as an exact valuewhere as LOQ is the lowest amount of analyte in a samplewhich can be quantitatively determined with suitable

precision and accuracy. ICH guidelines describe three

methods for determining LOD and LOQ that include:Visual evaluation

It may be used for both non instrumental andinstrumental methods. The LOD and LOQ is determined

by analysis of samples with known concentrations ofanalyte and by establishing the minimum level at whichthe analyte can be reliably detected or quantied withacceptable accuracy and precision respectively.

Figure 5: Limit of detection and limit of quantitation via signal tonoise ratio (S/N)


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S/N Ratio approach

This method can only be applied to analytical procedures which exhibit baseline noise. It is determined by comparing measured signals from samples with knownlow concentrations of analyte with those of blank samples

and establishing minimum concentration at which theanalyte can be reliably detected. A S/N ratio of 3:1 isconsidered acceptable for estimating LOD (with RelativeStandard Deviation (RSD) 10%) whereas for whereasfor LOQ S/N ratio of 10:1 is considered appropriate (withRelative Standard Deviation (RSD) 3%) described inFigure 5.

Standard deviation of the response and slope

The LOD and LOQ may be expressed as:LOD = 3.3 x /S and LOD = 10 x /S

where = the standard deviation of the response, S = theslope of the calibration curve of analyte. The slope S may

be estimated from the calibration curve of the analyte.The value of may be taken from as standard deviationof analytical background responses of an appropriatenumber of blank samples.

Alternatively, it can be taken as residual standarddeviation of a regression line or standard deviation ofy-intercepts if regression lines obtained after samplescontaining an analyte in the range of LOD and LOQ 40.

RangeThe range of an analytical procedure is the interval

between the upper and lower concentration of analyte inthe sample (including these concentrations) for whichit has been demonstrated that the analytical procedurehas a suitable level of precision, accuracy and linearity(Figure 6) 45 . It is established by conrming that theanalytical procedure provides an acceptable degree oflinearity, accuracy and precision when applied to samplescontaining amounts of analyte within or at the extremes ofthe specied range of the analytical procedure. The rangeof an analytical method varies with its intended purpose.It is generally 80-120% of the test concentration for assayof a drug substance or a nished (drug) product, 70-130%of the test concentration for content uniformity, 20%over the specied range for dissolution testing, reportinglevel of an impurity

to 120% of the specication for

determination of an impurity and it should commensuratewith LOD or LOQ (the control level of impurities), for

impurities known to be unusually potent or to producetoxic or unexpected pharmacological effects 40.

Fig. 6: Range

Robustness

The robustness of an analytical procedure is a measureof its capacity to remain unaffected by small, but deliberatevariations in method parameters and provides an indicationof its reliability during normal usage. For determinationof method robustness, a number of chromatographic

parameters, for example, ow rate, column temperature,injection volume, detection wavelength or mobile phasecomposition are varied within a realistic range andquantitative in uence of the variables is determined. Ifin uence of the parameter is within a previously speciedtolerance, the parameter is said to be within the methodsrobustness range. Obtaining data on these effects willallow to judge whether a method needs to be revalidatedwhen one or more of parameters are changed, for exampleto compensate for column performance over time 46,47 .Variation in method conditions for robustness should besmall and re ect typical day-to-day variation. Critical

parameters are identied during the method development process. Only these critical method parameters should be investigated for robustness. Common critical method parameters can be divided into two categories. The HPLCconditions include HPLC column (lot, age, and brand),

Mobile-phase composition (pH 0.05 unit, organic content 2%) and HPLC instrument (dwell volume, detectionwavelength 2 nm, column temperature 5C, owrate). The sample preparation variations include samplesolvent (pH 0.05 unit, organic content 2%), sample

preparation procedure (shaking time, different membranelters) and HPLC solution stability. The variations inchromatographic parameters for robustness study aregiven in the Table 29, 40 .


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Table 1: Variations in chromatographic parameters for robustnessstudy

S.No Robustness parameter Change

Detection wavelength 5 nm

Flow rate 0.05 ml/min

Buffer pH 0.1 unitMobile phase 2 ml

Column Different brand or batch number

CONCLUSION

The method development and validation are continuousand interrelated processes that are conducted throughoutthe drug development process. The analytical validationveries that a given method measures a parameter asintended and establishes the performance limits of the

measurement. Reproducible quality HPLC results canonly be obtained if proper attention has been paid to themethod development, validation and systems suitabilityto carry out the analysis.

The validated methods produce results withinknown uncertainties that are helpful to continuing drugdevelopment and provide emerging knowledge supportingthe product. The time and effort that is devoted intodeveloping scientically sound and robust analyticmethods should be aligned with the drug developmentstage. The resources that are constantly used during

method development and validation must be balanced withregulatory requirements and the probability for productcommercialization.

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HPLC Method Development and Validation

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