Druganalysis means identification, characterization and determination of drugs in mixtures such aspharmaceutical formulations and biologicalfluids. There is largenumber of cardiovascular and anti-diabetic drugs are introduced into themarket at an alarming rate.
These drugs may be either newentity in the market or partial structural modifications of theexisting drugs. Newer analytical methods are developed for these drugs in theircombined dosage form because of the followingreasons:· The drug combinations may not beofficial in any official compendia· Literature survey reveal no analyticalmethods are reported· Analytical methods ishard in drug combinations due to theinterference from excipient.Onthe other hand, the existing procedure may, · Needed expensive instruments, thereagents, solvents for analysis· Involve time consuming or tedious extraction or separation steps· Not berapid, accurate, reliable and sensitive Thenewly developed analytical method finds their importance in various fields like · Government and private Research institutions· Quality control department in industries· Approved drug testing laboratories· Bio-pharmaceutics and Bio-equivalence studies· Clinical pharmacokinetic studies Estimation of Drugs in combinedDosage Forms: Administration of twoor more drugs at a time becomes crucial for several therapeutic considerations.The combined dosage form have gained lot of importance now days due to greater patient acceptability, increasedpotency, multiple action, fewer side effectsand quicker relief. The quantitative determining of combined dosage forms(CDF)are complex in nature as onecomponent does not interfere with the estimation of the other. There is a need ofanalysis of such formulations without prior separation. For theestimation of multi-component formulation, the instrumental techniques commonlyemployed, are spectrophotometry, GLC, HPTLC, HPLC etc.
these methods are based upon the measurement ofspecific and nonspecific physicalproperties of the substances. Of all the above techniques HPLCis most widely considered quantitative analysis of drugs in CDF.HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Amongall the chromatographic techniques HPLC8 is one of the most widely usedanalytical techniques providing superior qualitative and quantitative results, reproducibility and higher detectionsensitivity and high reliability of analyticaldata. In high performance liquid chromatography(HPLC) the liquid mobile phase is passed through the stationary phase underpressure. Chromatographic separation in HPLCis the result of specificinteractions between sample molecules with both stationary and mobile phases.HPLC offers a great variety of stationary phases, which allows a greater variety of these selective interactions and more possibilities for separation.
Asimple HPLC system consisting of a solvent reservoir to hold the mobile phase,a pumping system to pressurize the mobile phase, and injector to introduce asmall volume of the sample mixture under high pressure, a column containingstationary phase, a detector to detect the presence of components as they exit the column, and a recorder to record the detector signal. HPLCused for analysingü Nonvolatile substancesü Substanceswith high polarity or ionic samplesü Substanceswith high molecular weightü Thermallyunstable and decomposable substances In HPLC there are variousmodes of separations. normal phase modestationary phase polar & mobilephase nonpolar, for this nonpolar compoundseluted first reason lowaffinityof nonpolar compounds & polar stationaryphase. take more time toseparate . The stationary phases used are SiO2, Al2O3,-NH2, -CN, -Diol, -NO2.The mobile phases used areheptanes, hexane, cyclohexane, chloroform, dichloromethane.Reversed phase mode in analytical field ofchemicalbiological pharmaceuticalbiomedical sciences, stationary phase nonpolargroups attached silicagel & mobilephase polarsolvent.
Coloumns are ODS viz:C8 & C4 C18 The mobile phases used are water, acetonitrile, methanol and buffers. 3 Ion exchange chromatography, thestationary phase contains cation exchanger (SO -, COO-)and anion exchanger (NR +,NHR +), used separating ofinorganic ions, organic acids, organic bases, proteins and nucleic acids. Ion pair chromatographyFor acidic substances tetraalkylammonium salts and for basic substances alkyl sulfonates are used as ion pairreagents.
Affinity chromatography10 involves the covalent attachment of immobilized biochemical to solidsupport highly specific biochemical interactions for separation. When sample is passed through thecolumn only solutes that selectively bind to complementary ligand is retained while other sample components elutewithout retaining in column. Thestationary phase contains specific groups of moleculescalled ligands, This technique was usedto seperate biological samples like proteinsenzymes&antibodies in High mixtures.Sizeexclusionchromatographyused separation in molecular mass. LSM first and the SSM elute later, itlater defined into GPC and GFC. Instrumentation in HPLC 11, 12 Mobilephase reservoir system with pumps Sampleinjection system Column Detectors Mobile phase reservoir system with pumps TheHPLC pump is very important componentof the system. Pumps are required todeliver a constant flow of mobilephase at pressure ranging from 14.
6 to 8000 psi. Pumps capable of pressures up to 8000 psi provide a wide range of mobile phase flowrate typically from 0.01- 10 ml / min.Displacementviscosity backpressure nondepending withlimitedcapacity.Reciprocatingpump:35 -400 ?l ,outputpressure 10,000 psi &constantflowrate & pulsed flow.
Pneumaticpump:. less than 2000 psi.There are twotype of elution process, i.e. isocratic and gradient, Isocratic:In this system, the things are keptconstant throughout the run. In the case of pumping of mobile phase, the mobilephase composition is kept constant throughout the run. The nominal flow rateaccuracy required is ±1% of the set flow.
Gradient:There issome change purposely incorporated during the particular sample run to achieve a better or/and fasterseparation. In case of pumping mobile phase, the composition ofmobile phase is continuously varied during the particular run. The gradient accuracy of ±1% ofthe step gradient composition is typical.
Column TheColumns in HPLC are fabricated withstand high pressure. The internal diameter of analytical column are 4-5 mm and of length10-30 cm. The shorter column with 3-6 cm in lengthis also available. The particle sizes in column packing are generally 3 or 5 µm.
For analysis of single componentshorter columns are sufficient but forseparation two or more componentslonger column are preferable.Column packing: There are threemain types of column packing in HPLC. a. polymericporusbeds b. Porous bedlayered c. Totally Porous silica particles Detectors .
The ideal detector should respond to a particular property of thesubstance being separated, it should be sensitive and give a linear responserange over a wide concentration range.Detectors arebasically two typesBulkProperty Detectors &Solute Property Detectors:.The commonlyused in HPLC analysis of pharmaceutical substances are i. Photometric detectors: Thesenormally operate in ultraviolet region of spectrum and most widely used. Thesedetectors consist of light source, dispersing element, flow cell,photomultiplier cell or diode. These are of five typesSinglewavelength detectors Multi wavelength detectors Variable wavelength detectorProgrammable detector Diode array detectorsii.
Fluorescencedetector it is more sensitive when compared photometric detectors. ii.Refractive index detectors these detectorsrespond to change in bulk property of refractive index of solution of thecomponent in mobile phase. Its sensitivity is less when compared to bulkproperty detectors.iv.Electrochemical detectors are detector based onelectrochemical principles involving amperometry, voltammetry, andpolarography.Thereare several detectors available in the market. However UV Visible detector,photo diode array detector, fluorescence detector, conductometric andcoulometric detector are more commonly used.
The new ELSD detector is provingto be important detector, while the MS detector is outstanding. Fig.1.1. Instrumentation of HPLC Quantitativeanalysis in HPLC 8 Thequantitative analysis in HPLC can performed three methods External Standardcalibration:The external Standard is the samesubstance as that being analysed in the sample.
This method is most commonly used for analysis. The external Standard must be pure and its composition must beknown through prior analysis. By injecting Standardsolution in different concentration peak response is plotted vs.concentration. Unknown samples are analysed in similar manner and theirconcentration determined from the calibrationcurve.
Internal Standardmethod:The effect of minor variables in separation parameters, peak size andinjection errors. In this method ofanalysis, a fixed concentration of knowncompound addconcentration separate peaks. InternalStandard shouldhave following properties ü Musthave completely resolved peaks without interference from other compound presentin sample.ü Shouldhave similar chemical properties as the compound of interest.ü Shouldmimic the analyte in any sample preparationmethod.ü Mustbe stable, compatible with column packing, mobile phase and chemically inerttowards components of sample.
ü Mustbe easily available and with reasonably highpurity. Acalibration is produced by analyzing various concentrations of pure drug with afixed concentration of internal Standard. From the chromatogram response factoris calculated. RF= Ap/AISTD Similarly the analyte is to added to the fixedconcentration of internal Standard and response factor is calculatedRF = Ax/AISTD Where Axis thepeak areaof analyteand AISTD isthe peakarea ofinternal Standard. From the interpolation of calibration curve theconcentration of analyte calculated.Useof internal Standard addition method is not always advantageous as precision isless when compared to external Standard method. Standard additionmethod: Inthese method different concentrations of.The response from Standard additionto extrapolated to zero addition concentration.
The negative intercept on theX-axis will be the concentration of analyte. The important aspect in this method is detector response should be adequateto produce S/N ratio >10 otherwise results are not precise. Method Development and design ofseparation method 13 Plate number (N): Itis an important property of the column. It reflectsits quality of separation and itsability to produce sharp, narrow peaks and goodresolution of peaks. Column with N ranging from5,000 to 1,00,000 plates/meter are ideal for a good system. The factorsinfluencing plate number areØ Wellpacked columnØ Larger columnsØ Lessflow rateØ Lowviscosity of mobile phaseØ Smallsample molecules Resolution factor (RS):It measuresthe quality of separation of adjacent bands. It is defined as distance centers of band peaks divided by average band width. RS =Rt2- Rt1/ 0.
5(W1 + W2) Rtension1&Rtension2 retention time and W1 & W2peak width The fundamentalwhich influences the resolution factor are Capacityfactor Selectivity factorColumn efficiency Columnefficiency: It representsthe number of theoretical plates per meter (N) and HETP. HETP height equivalentto theoretical plates is a section in a column in whichmobile phase and stationary phase areinequilibrium. Lower the HETP value more efficiency is the columnHETP = L/N L=length of column N= no. of theoretical plates Its value depends upon particle size, flow rate,viscosity of mobile phase and quality of packing Peak Assymetry (As): Itis a practical measurement of peak shape. Peak asymmetry is measured at 10% offull peak height. Good columns produce peaks with As values of 0.95-1.1.
VALIDATION OF ANALYTICAL METHOD 14-17 Validationis the assessment of the defined test method. The result of any successfulvalidation is a comprehensive set of datathat will support the suitability of test method for its intended use.The validationparameters are:Specificity Precision LinearityAccuracyLimit of detectionLimit of quantitationRuggednessRobustness Stability solutionsSpecificitya. inject sample as wellas other related compounds decomposition/ degradation products, intermediates,solvent, chemicals used in analysis.All compounds should separate from mainanalyte peak.b. placeboto demonstrate the lack of interferencefrom excipients. c.
stressed placebo toshow that degradation products from excipients will not interfere withdegradation products of drug substance.d. sample subjected todegradation studies to achieve 10-20 % of degradation of analyte in conditions of 0.1 N HCl, 0.1 N NaOH, 3% H2O2 ,50 °C , UV radiation. All the degradation products if any should beseparated from analyte. Precision a.
Repeatability of method can be determined by multiplereplicate preparations of sample.These can be done either by multiplesample preparations (n=6) in same experiment or by preparing three replicatesat three different concentrations. % RSD should be less than 2 %.
b. Intermediate %RSD should be less than 2 %.c. Reproducibilityexpressesthe precision between laboratories. Linearityof analytical procedure is its abilityto obtain test results that is directly proportional to the concentration ofanalyte in the sample. The following results should be reported slope,correlation coefficient, y intercept and residual sum of squares.
Thisis to verify that the method performance is not affected by typical changes in normalexperiments. The method parameters that is investigated for robustness are¨ HPLCcolumn (lot, age, brand)¨ Mobilephase composition (pH ± 0.05 unit, percent organic ± 2 % )¨ HPLC instrument(detection wavelength ± 2 nm , column temperature ± 5 °C, flow rate ± 0.2 ml/min)Ø Differentoperators in same laboratoryØ Differentequipment in same laboratory Ø Differentsource of reagents and solutionsØ Differentsource of columnØ Different laboratory Stabilitysolutions used for the analysis is required to bestable for a period more than 24 hr toproduce precise and reliable results. Advantages of analytical method validation ü Reliabilityof analytical results and assurance of qualityproductü Performancecapability of the method can be confirmed by analysts using themethodü Awarenessabout importance of protocols forvalidation workü Motivationfor improvement in quality of workü Providesopportunity for training to QC staffü Helpsin scientific communication ontechnical matters FORCED DEGRADATIONSTUDIES AND STABILITY INDICATING ANALYTICAL METHOD 18Thepharmaceutical molecules chemical stability is a matter of great worth as it make impact on safety and efficacy of the drug product. To study the FDA andICH laid guidelines for the requirement of analytical stability testing data.
Knowledge about stability of molecule beneficialin choosing proper formulation and packaging requirements as well as storageconditions maintenance and shelflife, which is essential in filling documentation for regulatory. In Forced degradation studies the drug products and drugsubstances undergo degradation at conditions more intense than accelerated conditionsand thus produce degradation productswhich can be used to assess thestability of the molecule. According to theICH guideline the intention in stresstesting is further helps investigation of theintrinsic stability of the molecule and elucidatingpossible degradation pathways, and for validation of the stability indicatingmethod used. For filling the new drug moiety in registration dossier it ismandatory to perform stability studies. The duration for stability studies in long term studies is 12 monthsand for accelerated stability studies it is 6months. The intermediate studies are performed for 6 months can be at mildconditions when compare to accelerated studies.
So it will take longtime for separation, identification and quantification of degradation products.When compared to stability studies inforced degradation studies the degradationproducts are generating in shorter time. The samples generated from forceddegradation studies was further useful for the development of the stabilityindicating methods which can be applied for the analysis of samples generated from accelerated and long term stability studies. Objectiveof FDS Forceddegradationstudies are carried out achieve following purposes: v Toinvestigate degradation pathways of drug substances and drug products.v Todifferentiate degradation products of drug products from those of non-drug product in a formulation.v Forstructural elucidation of degradation products.v Toassess the intrinsic stability of adrug substance in formulation.v Toreveal the drug degradation mechanisms in pure and formulationsv Toexamine the stability indicating nature of adeveloped method.
v Tounderstand the chemical properties of drug molecules.v Toproduce more stable formulations.v Topredict stability-related problems Astability indicating method (SIM) isan analytical procedure which is used to estimate the decrease in the concentration of the active pharmaceutical ingredient(API) in drug product when it exposed to various degradation conditions. As per FDA guidance document astability-indicating method is a validated quantitative analytical procedure that can be used for the study in variation in the stability of the drug substances and drug products with time. Astability-indicating method accurately measures the changes in activeingredients concentration without interference from other generated degradationproducts, related impurities and formulation excipients. The suitable platformin pre-formulation studies, stability studies and proper storage conditions fordrug substance and drug product. Degradation conditions Hydrolytic conditions Hydrolysisis one of the two most common mechanisms of drugdegradation over a wide range of pH. Hydrolysis is a chemicalprocess that involves degradation of a chemical compound by reaction withwater.
Hydrolytic reactions are mainly acid or base catalyzed. For hydrolyticstudy acidic, neutral and basic conditions should be employed which susceptiblyattacks at ionizable functional groups present in the molecule. The type and concentrationsof acid or base used for hydrolysis isbased on the stability of the drugsubstance. Generally HCl or H2SO4 in the concentration range of 0.1–1 M areused foracid hydrolysis and NaOH orKOH in the concentration range of 0.1–1Mfor base hydrolysis. If thecompounds have low solubility in water, then inert water miscible co- solventscan be used.
Hydrolytic stress studies is normally performed at room temperature and if there isno sign of degradation then elevated temperature (50–70 °C) are recommended. The duration of stress testing should not exceed more than one week. Oxidation conditions Oxidativereactions are one of the two most common mechanisms of drug degradation.Hydrogen peroxide is most widely used reagent for oxidation of drug substances in forced degradationstudies but other oxidizing agents such as metal ions, oxygen, and radicalinitiators (e.g., azobisisobutyronitrile, AIBN) can also be used.
Selection ofan oxidizing agent, its concentration, and conditions depends on the drugsubstance. It is recommended toexpose sample with 0.3–3 % hydrogen peroxide at neutral pH and room temperaturein dark for seven days or 5- 20 % degradation could potentially producerelevant degradation products. Photolytic conditions Photolyticdegradation is a degradation that results fromexposure of drug substance ordrug product to UV or visible light in the wavelength range of 300-800 nm.Exposure to radiation at wavelengths < 300 nm is not needed because pharmaceuticalwon't expose to such light during its life cycle. The drug substance or drugproduct should be exposed to a minimum of 1.
2 million lux-hrs and 200 Wh/m2light. The maximum allowable