Chapter-2. Analytical Method Development and Validation. Overview:

Transcription

1 Chapter-2 Analytical Method Development and Validation Overview: The primary focus of this chapter is on general approaches and considerations toward development of chromatographic (HPLC/UPLC) methods for separation, identification, and quantification of pharmaceutical compounds, which may be applied within the various functions in the drug development continuum. The chapter also discusses the issues and parameters that must be considered in the validation of analytical methods. At the end of the chapter, a scope of the present research study is covered. 50

2 Analytical Method Development and Validation 2.1 GENERAL INTRODUCTION The number of drugs introduced into the market is increasing every year. These drugs may be either new entities or partial structural modification of the existing one. Very often there is a time lag from the date of introduction of a drug into the market to the date of its inclusion in pharmacopoeias. This happens because of the possible uncertainties in the continuous and wider usage of these drugs, reports of new toxicities (resulting in their withdrawal from the market), development of patient resistance and introduction of better drugs by competitors. Under these conditions, standards and analytical procedures for these drugs may not be available in the pharmacopoeias. There is a scope, therefore to develop newer analytical methods for such drugs. Analytical methods development and validation play important roles in the discovery, development, and manufacture of pharmaceuticals [1-4]. Pharmaceutical products formulated with more than one drug, typically referred to as combination products, are intended to meet previously unmet patients need by combining the therapeutic effects of two or more drugs in one product. These combination products can present daunting challenges to the analytical chemist responsible for the development and validation of analytical methods. The official test methods that result from these processes are used by quality control laboratories to ensure the identity, purity, potency, and performance of drug products. Identification and quantification of impurities is a crucial task in pharmaceutical process development for quality and safety [5-7]. Related components are the impurities in pharmaceuticals which are unwanted chemicals that remain with the active pharmaceutical ingredients (APIs), or develop during stability testing, or develop during formulation or upon aging of both API and formulated APIs to medicines. The presence of these unwanted chemicals even in small amounts may influence the efficacy and safety of the pharmaceutical products. Various analytical methodologies are employed for the determination of related components in 51

3 Chapter-2 pharmaceuticals. There is a great need for development of new analytical methods for quality evaluation of new emerging drugs. Basic criteria for new method development of drug analysis: [8] The drug or drug combination may not be official in any pharmacopoeias. A proper analytical procedure for the drug may not be available in the literature due to patent regulations. Analytical methods may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients. Analytical methods for the quantitation of the drug in biological fluids may not be available. Analytical methods for a drug in combination with other drugs may not be available. The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable. 2.2 DEVELOPMENT PROCESS There are three phases in the Analytical Method Development Phase-1 (Planning) Determine the purpose and regulatory requirements of the method to be developed. Gather existing information (technical and safety) of the drug substance, known related substances, and formulation ingredients. Understand the QC expectations (e.g. analysis time, ease of use, analysis costs, available instrumentation, etc ). Estimate the requirements and availability of resources (reference standards, impurity standards, required sensitivity, selectivity, chirality s, chemicals, etc ). 52

4 Analytical method development and validation In case of troubleshooting of earlier method, review all the data generated by using the earlier method and plan the study. The samples analyzed by earlier method needs to be tested by new revised method. In case compendial method is available, preferably use this method Phase-2 (Method development) Once the planning phase is completed and the parameters are selected for the initial development, perform the trials and optimize the final method. The method should be capable of following; Method should be practical and efficient. Method is capable to be validated (perform few method validation experiments during development like Specificity, Accuracy, Solution stability and LOQ). Method should be specific and stability indicating. Meaningful system suitability criteria needs to be incorporated for daily usage of the method like % RSD of area of standard, theoretical plates, tailing factor and resolution between the closely eluting peaks. If the method is a troubleshooting of the earlier developed method the samples and analyzed by earlier method to be analyzed by the new revised method and results needs to be discussed with advantages of new revised method. In case of pharmacopoeia method minimum study of excipient interference, Specificity and solution stability has to be performed in order to check the suitability of the method for the in-house formulation. In case of residual solvents it is preferred to adopt the drug substance manufacturer test method for establishing the workability at our laboratory. If the method is not working or if there is any specific constraint in such cases alternate test method shall be adopted which should be validated completely. 53

5 Chapter-2 In case of Non-compendial product, as far as possible use the Drug substances method. This will reduce the time of method development and discrepancies related to Relative response factors, elution pattern, and resolution of degradants from the main analyte. Use the available columns in the laboratory in order to develop the method. In case the separation is inadequate use the readily available columns in the markets. During development evaluate the critical factors which has impact on the Repeatability, reproducibility, Accuracy and Resolution between the critical closely eluting peaks. Add a precautionary note in the final test method Phase-3 Issuance of analytical method for routine analysis and method validation Method issued for routine analysis and Method validation should include at least following parameters Name of the method and name of drug substance or drug product for which the method is issued. A list of reagents (with grades), standards, instrumentation and equipments. A list of solution preparations (mobile phase, standards, system suitability solution, system sensitivity solution and samples). A list of method conditions (flow rate, wavelength, gradient profile, equilibration procedure, column temperature, etc ). System suitability requirements for chromatography and how to calculate. Sample sequence scheme with diluents, placebo, system suitability, system sensitivity, standards, samples and bracketing standards. Calculation formulas. 54

6 Analytical method development and validation A table of analyte relative retention times (RRTs) and relative response factors (RRFs) or response factor (RF). A representative labeled chromatogram of diluents, placebo, system suitability, system sensitivity solution, standards and sample solution Review of technical package A comprehensive review should be made on the technical package after the receipt of the same from the drug substance manufacturer. Following aspects needs to be taken into consideration: Adequacy of analytical methods described. Discussion on the impurities. Polymorphic characteristics wherever applicable. Accelerated stability data available. Forced degradation studies. Method validation reports Evaluation of drug substances Drug substance lots received from the drug substance manufacturer (procured by purchase department) should be evaluated for critical parameters using the methods chosen for testing. The results of the same should be compared against that reported in the COA (certificate of analysis) of manufacturer. The consistency in quality within the lots should be evaluated. Any discrepancies observed in the test result should be intimated to purchase and the manufacturer. 2.3 PRELIMINARY ANALYTICAL METHOD Analytical method development involves continuous process of method improvement in terms of various experiments and experience being gathered on the project. At the beginning of the 55

7 Chapter-2 product development a comprehensively developed methods may not be available in absence of the availability of compendial monograph for the drug product. In such cases the initial analysis shall be carried out using the preliminary analytical methods. Following aspects should be looked into while adopting the preliminary analytical method: Dissolution by UV method No interference from placebo. Wavelength selection (for interested compound). Working concentration having optimum absorbance. Standard and sample solution stability. Compatibility with inline filter and syringe filter Dissolution by HPLC/UPLC/RRLC method Column selection. Mobile phase/ solvent selection. Mobile phase/ solvent ph selection. No interference from placebo. No interference from major degradation product. No interference from dissolution media. Working concentration having optimum peak response. Optimum injection volume. Wavelength selection Assay by HPLC/UPLC/RRLC method Column selection. Mobile phase/ solvent selection. Mobile phase/ solvent ph selection. 56

8 Analytical method development and validation No interference from placebo. No interference from major degradation product. Working concentration having optimum peak response. Diluents selection based on solubility of drug substance. Dilution selection based on sample matrix. Primary extraction condition (essential for extended release product). Optimum injection volume. Wavelength selection. Compatibility with inline filter and syringe filter. Solution stability Related substances by HPLC/UPLC/RRLC method Column selection. Mobile phase/ solvent selection. Mobile phase/ solvent ph selection. No interference from placebo at the RT (retention time) of interested peak. Separation of major degradation product. Working concentration having optimum peak response. Diluents selection based on solubility of drug substance and impurities. Dilution selection based on sample matrix. Primary extraction condition (essential for extended release product). Optimum injection volume. Wavelength selection. Compatibility with inline filter and syringe filter. Solution stability. Tentative response factor or related response factor. Method sensitivity (target LOQ, 0.05 %). 57

9 Chapter Blend analysis and content uniformity/ dose content uniformity/ spray content uniformity by HPLC/UPLC/RRLC method In absence of the placebo interference, an UV method will be preferred for the content uniformity analysis. The same method shall be adopted for the blend uniformity of the in-process blend and composite assay of blend with suitable dilutions. Wherever significant placebo interference (i.e. more than 2.0 %) is observed in the UV methodology, HPLC/UPLC/RRLC method adopted for assay shall be employed for the content uniformity with suitable dilutions. The workability of the content uniformity method should be verified by preparing sample preparation from ten individual dosage units of the final formulation. Following aspects should be looked into while adopting the preliminary analytical method Column selection. Mobile phase/ solvent selection. Mobile phase/ solvent ph selection. No interference from placebo. No interference from major degradation product. Working concentration having optimum peak response. Diluents selection based on solubility of drug substance. Dilution selection based on sample matrix. Primary extraction condition (essential for extended release product). Optimum injection volume. Wavelength selection. Compatibility with syringe filter. Solution stability. 58

10 Analytical method development and validation 2.4 AVAILABLE INFORMATION FOR HPLC / UPLC / RRLC ANALYTICAL METHOD DEVELOPMENT Buffer selection A partial list of common buffers and their corresponding useful ph range is supplied. Perhaps the most common buffer in HPLC is the phosphate ion. Although, with the growth of LC-MS, volatile buffers such as TFA, acetate, formate, and ammonia are becoming more frequently used. Remember, the purpose of a buffer in the mobile phase is to inhibit a ph change in the mobile phase after the introduction of a sample. When developing a method, it is important to select a mobile phase with a final ph at least one ph unit away from any analytes pk value. As a rule of thumb, one should work within a ±1 ph unit of the buffer pka. Typical buffer concentrations for HPLC tend to be mill molar level. Buffer ph range, pka and its UV cutoff are shown in Table 2.1 and Table 2.2. Physical properties of acids are also summarized in Table 2.3. Table 2.1 Buffers for reversed phase UPLC/HPLC [9] Buffer pka Buffer Range UV Cutoff (nm) Phosphate pk pk pk Citrate pk pk pk Formate Acetate Tris (hydroxymethyl) aminomethane Borate Triethylamine

11 Chapter-2 Table 2.2 Buffers information for UPLC/HPLC, MS and ELSD Buffer ph range UV range, nm MS, ELSD TFA >210 + Acetic acid > Formic acid > H 2 SO >195 + HClO >195 - H 3 PO > TEA phosphate > Sodium phosphate > Sodium phosphate > Ammonium phosphate > TEA formate >220 + TEA Acetate >220 + Ammonium formate > Ammonium acetate > Table 2.3 Physical properties of acids (for HPLC/ UPLC/ RRLC application) Name Formula Density, g/ml Boiling point, o C UV range, nm Ion strength of 0.1% solution, mm ph of 0.1% solution Sulfuric acid H 2 SO high > Perchloric HClO high > Phosphoric acid H 3 PO high > Trifluoroacetic acid (TFA) Methanesulfonic acid CF 3 COOH > CH 3 SO 3 H high > Formic acid HCOOH > Acetic acid CH 3 COOH >

12 Analytical method development and validation Mobile phase preparation It should be understood that slight variations in ph and buffer concentration could have a dramatic affect on the chromatographic process, consistent and specific techniques should be a regular practice in the preparation of mobile phases. A common practice is to place a sufficient amount of pure water into a beaker and add an accurate amount of buffer. Dissolve and then dilute to final volume of water. The ph of the solution should be adjusted, if necessary, Then, add a volumetrically measured amount of organic solvent to obtain the final mobile phase. Degassing and filtering is required prior to use. 2.5 DETECTOR SELECTION Chemical nature of analyte and interferences. Limit of detection and quantification are required. Availability and cost of the detector Ultraviolet/ visible absorbance (UV/VIS) Analyte must absorb more light than the sample matrix at some wavelengths. Photo-Diode Array (PDA) detector can monitor multiple wavelengths. Choose the wavelength that maximizes the sensitivity and specificity. Solvents used may cause slight shift in UV max from published values (2-5nm) check absorbance in mobile phase. Mobile phase solvents have UV cut-offs, operating below the cutoff wavelength will reduce the sensitivity and increase baseline noise Mass spectrometer (MS) Analyte must be ionizable. Can discriminate between the co-eluting peaks in selective ion mode. For best sensitivity, work at ph where analytes are ionized. 61

13 Chapter-2 For acids work in ph range of 7-9. For bases work in ph range of Refractive index (RI); Monitors difference in refractive index sample cell and the reference cell. RI detector is non-selective and performance is concentration dependant. Sensitivity is typically 100X-1000X times less than the UV/VIS detector. Cannot be used with Gradient elution directly, requires few precautions. Different solvent refractive indexes and its UV cutoff are summarized in Table 2.4. Table 2.4 Solvent refractive index and its UV cutoff Solvent Refractive Index UV cutoff i-octane n-hexane Cyclohexane n-decane Carbon tetrachloride i-propyl ether Toluene Benzene Chloroform Dichloromethane Methylene chloride Tetrahydrofuran Ethylene dichloride Methyl ethyl ketone Acetone Acetonitrile Ethyl acetate Methyl acetate

14 Analytical method development and validation Pyridine Methoxyethanol Propanol Ethanol Methanol Acetic acid Water Evaporative light scattering (ELSD) Detector is mass dependant and non-selective. Ideal for high molecular weight compounds, sugars and less volatile acids. Amount of light scattering is related to the molecular mass of analyte. Can be used with gradient elutions. Solvents should be volatile for the best results Fluorescence detector (FLD) Analyte must fluoresce. Excite at one wavelength, measure the emission at longer wavelength. Up to 1000X more sensitive than UV/VIS. High specificity. Concentration dependant. Operation similar to UV/VIS detector Electrochemical (EC) Analytes should be oxidizable or reducible by Electric current. More sensitive than the fluorescence detector. Not as selective as fluorescence detector. Not compatible with Gradient elution. 63

15 Chapter HPLC MODE SELECTION Part A (If molecular mass is less than 2000) Part B (If molecular mass is greater than 2000) 64

16 Analytical method development and validation Basic column selection chemistry Bonded group Polarity Retention mechanisms Comments C 18, C 8, C 4 Non-polar Vander Waals C 8 does not retain hydrophobic compounds as strongly as C 18 Phenyl Non-polar Hydrophobic and pi-pi NA Cyano Amino Polar (-NH 2 ) or ionic (-NH 3 + ) Intermediate Hydrophobic, dipoledipole and pi-pi Dipole-dipole and H- bonding Resolve polar organic compounds by reversed-phase or normal-phase chromatography Normal-phase or ion-exchange separation; separates carbohydrates, polar organic compounds, and inorganic ions; reacts with aldehydes and ketones Bare silica Very polar H-bonding Normal-phase separations 2.7 DISSOLUTION METHOD DEVELOPMENT Precision: Dissolution results may be considered highly variable if the relative standard deviation (RSD) is greater than 20% at time points of 10 minutes or less and greater than 10% RSD at later time points. Discrimination: The procedure should be appropriately discriminating, capable of distinguishing significant changes in a composition or manufacturing process that might be expected to affect in vivo performance. Also it should reflect changes over period of stability. For compounds with high solubility and high permeability (as defined by the Biopharmaceutics Classification System), the choice of medium and apparatus may be influenced by the referenced FDA Guidance. Centrifugation: Centrifugation of samples is not preferred, because dissolution can continue to occur and because there may be a concentration gradient in the supernatant. A possible exception might be for compounds that adsorb onto all common filters. Media selection: In case of media selection the solubility and solution state stability of the drug as a function of the ph value needs to study. When selecting the composition of the medium, the 65

17 Chapter-2 influence of buffers, ph value, and surfactants on the solubility and stability of the drug need to be evaluated. One goal is to have sink conditions, defined as the volume of medium at least three times that required in order to form a saturated solution of drug substance. When sink conditions are present, it is more likely that dissolution results will reflect the properties of the dosage form. Using an aqueous organic solvent mixture as a dissolution medium is discouraged. The dissolution characteristics of an oral formulation should be evaluated in the physiologic ph range of 1.2 to 6.8 (1.2 to 7.5 for modified-release formulations). If the compound dissolves quickly in the stomach and is highly permeable, the dissolution test should demonstrate that the drug is released quickly under typical gastric (acidic) conditions. On the other hand, if dissolution occurs primarily in the intestinal tract (e.g., for a poorly soluble, weak acid), a higher ph range (e.g., simulated intestinal fluid with a ph of 6.8) may be more appropriate. Typical media for dissolution may include the following (not listed in order of preference): dilute hydrochloric acid, buffers in the physiologic ph range of 1.2 to 7.5, simulated gastric or intestinal fluid (with or without enzymes), water, and surfactants (with or without acids or buffers) such as polysorbate 80, sodium lauryl sulfate, and bile salts. Media Volume: Normally, for basket and paddle apparatus, the volume of the dissolution medium is 500 ml to 1000 ml, with 900 ml as the most common volume. The volume can be raised to between 2 and 4 L, using larger vessels and depending on the concentration and sink conditions of the drug; justification for this procedure is expected. Deaeration: The significance of deaeration of the medium should be determined, because air bubbles can interfere with the test results, acting as a barrier to dissolution if present on the dosage unit or basket mesh. Further, bubbles can cause particles to cling to the apparatus and vessel walls. On the other hand, bubbles on the dosage unit may increase buoyancy, leading to an increase in the dissolution rate, or may decrease the available surface area, leading to a decrease in the dissolution rate. A dearation method includes heating the medium, filtering, and drawing a 66

18 Analytical method development and validation vacuum for a short period of time. Media containing surfactants are not usually deaeration because the process results in excessive foaming. Sinkers: When sinkers are used, a detailed description of the sinker must be stated in the written procedure. It may be useful to evaluate different sinkers, recognizing that sinkers can significantly influence the dissolution profile of a dosage unit. Apparatus selection: For solid oral dosage forms, Apparatus 1 and Apparatus 2 are used most frequently. Apparatus 3 (Reciprocating Cylinder) has been found to be especially useful for bead-type modified-release dosage forms. Apparatus 4 (Flow-Through Cell) may offer advantages for modified-release dosage forms that contain active ingredients with limited solubility. In addition, Apparatus 3 or Apparatus 4 may have utility for soft gelatin capsules, bead products, suppositories, or poorly soluble drugs. Apparatus 5 (Paddle over Disk) and Apparatus 6 (Rotating Cylinder) have been shown to be useful for evaluating and testing transdermal dosage forms. Apparatus 7 (Reciprocating Holder) has been shown to have application to nondisintegrating oral modified-release dosage forms, as well as to transdermal dosage forms. Some changes can be made to the apparatus; for example, a basket mesh size other than the typical 40-mesh basket (e.g., 10, 20, 80 mesh) may be used when the need is clearly documented by supporting data. Agitation: For immediate-release capsule or tablet formulations, Apparatus 1 (baskets) at 100 rpm or Apparatus 2 (paddles) at 50 or 75 rpm are most commonly used. Time points: Dissolution profiles of immediate-release products typically show a gradual increase reaching 85% to 100% at about 30 to 45 minutes. Thus, dissolution time points in the range of 15, 20, 30, 45, and 60 minutes are usual for most immediate-release products. For rapidly dissolving products, including suspensions, useful information may be obtained from earlier points, e.g., 5 to 10 minutes and 85% or more of the active drug substance dissolved within 15 minutes. For an extended-release dosage form, at least three test time points are 67

19 Chapter-2 chosen to characterize the in vitro drug release profile for Pharmacopeial purposes. Additional sampling times may be required for drug approval purposes. An early time point, usually 1 to 2 hours, is chosen to show that there is little probability of dose dumping. An intermediate time point is chosen to define the in vitro release profile of the dosage form, and a final time point is chosen to show the essentially complete release of the drug. Infinity point: The paddle or basket speed is increased at the end of the run for a sustained period (typically 15 to 60 minutes), after which time an additional sample is taken. Although there is no requirement for 100% dissolution in the profile, the infinity point can provide data that may supplement content uniformity data and may provide useful information about formulation characteristics during initial development or about method bias. Filters: Filtration removes insoluble excipients that may otherwise cause high background or turbidity. Filters can be in-line or at the end of the sampling probe or both. The pore size can range from 0.45 to 70 µm. 2.8 METHOD DEVELOPMENT FOR RESIDUAL SOLVENT BY GC Classification of residual solvents by risk assessment: Class-1, residual solvents: Solvents to be avoided, known human carcinogens strongly suspected human carcinogens environmental hazards. Solvent Concentration limit (ppm) Concern Benzene 2 Carcinogen Carbon tetrachloride 4 Toxic and environmental hazard 1,2-Dichloroethane 5 Toxic 1,1-Dichloroethane 8 Toxic 1,1,1-Trichloroethane 1500 Environmental hazard 68

20 Analytical method development and validation Class-2, residual solvents: Solvents to be limited nongenotoxic, animal carcinogens or possible causative agents of other irreversible toxicity, such as neurotoxicity or teratogenicity. Solvents suspected of other significant but reversible toxicities. Solvent PDE (mg/day) Concentration limit (ppm) Acetonitrile Chlorobenzene Chloroform Cyclohexane ,2-Dichloroethene ,2-Dimethoxyethane N,N-Dimethylacetamide N,N-Dimethylformamide ,4-Dioxane Ethoxyethanol Ethylene glycol Formamide Hexane Methanol Methoxyethanol Methylbutylketone Methylcyclohexane Methylene chloride N-Methylpyrrolidone Nitromethane Pyridine Sulfolane Tetrahydrofuran Tetralin Toluene Trichloroethylene Xylene (Usually 60 % m-xylene, 14 % p-xylene, 9 % o-xylene with 17 % ethyl benzene)

21 OPTIONS FOR DETERMINING LEVELS OF CLASS 2 RESIDUAL SOLVENTS Chapter-2 Option-1: The concentration limits in ppm stated in above table (Class-2 section) are used. They were calculated using equation (1) below by assuming a product weight of 10 g administered daily x PDE Concentration (ppm) = equation (1) Dose Where, PDE is given in terms of mg per day and dose is given in terms of g per day These limits are considered acceptable for all drug substances, excipients, and drug products. Therefore, this option may be applied if the daily dose is not known or fixed. If all drug substances and excipients in a formulation meet the limits given in Option-1, these components may be used in any proportion. No further calculation is necessary provided the daily dose not exceed 10 g. Products that are administered in doses greater than 10 g per day are to be considered under Option-2. Option-2: The PDE in terms of mg per day as stated in above table (Class-2 section) can be used with the known maximum daily dose and equation (1) above to determine the concentration of residual solvent allowed in a drug product. Option-2 may be applied by adding the amounts of a residual solvent present in each of the components of the drug product. The sum of the amounts of solvent per day should be less than that given by the PDE. Class-3, residual solvents: Solvents with low toxic potential solvents with low toxic potential to humans; no health-based exposure limit is needed. 70

22 Analytical method development and validation Unless otherwise stated in the individual monograph, Class-3 residual solvents are limited to not more than 50 mg per day (corresponding to 5000 ppm or 0.5% under Option-1). If a Class-3 solvent limit in an individual monograph is greater than 50 mg per day, that residual solvent should be identified and quantified. Acetic acid Heptane Ethyl acetate Acetone Isobutyl acetate Ethyl ether Anisole Isopropyl acetate Ethyl formate 1-Butanol Methyl acetate Formic acid 2-Butanol 3-Methyl-1-butanol 1-Propanol Butyl acetate Methylethylketone 2-Propanol Tert-butylmethyl ether Methylisobutylketone Propyl acetate Cumene 2-Methyl-1-propanol Ethanol Dimethyl sulfoxide Pentane 1-Pentanol Other Residual Solvents (For which no adequate toxicological data was found) 1,1-Diethoxypropane 1,1-Dimethoxymethane 2,2-Dimethoxypropane Isooctane Isopropyl ether Methyl isopropyl ketone Methyltetrahydrofuran Hexane Trichloroacetic acid Trifluoroacetic acid Method development: In case of Class-1 and Class-2, Residual Solvent method development is based on the solubility of the material, there are different procedures for the water soluble and water insoluble materials mentioned in the USP, BP and EP. If only class-3 solvents are present, the level of residual solvents is to be determined as directed under loss on drying. If the loss on drying value is greater than 0.5 %, a water determination should be performed on the test sample as directed under water determination. 71

23 Chapter METHOD WORKABILITY STUDY (HPLC/UPLC and GC METHOD) Once the final formulation for scaling-up clinical batch is available method workability should be studied to assess the following: Whether the proposed method fulfils specificity, precision and accuracy criteria. To optimize a suitable sample preparation according to the final formula. To optimize the stress study conditions that is to be followed for method validation. For method workability checking below mentioned parameters can be performed during analytical method development. Sr. No. Parameter Assay Related Substances Dissolution 1 System precision Yes Yes Yes 2 System suitability parameters Yes Yes Yes 3 Specificity a) Interference from placebo Yes Yes Yes b) Interference from degradants Yes Yes (major degradants) 4 Extraction time Yes Yes Filter paper compatibility Yes Yes Yes 6 Accuracy (Recovery) Yes Yes Method precision Yes Yes Yes 8 Stability of analytical solution Yes Yes Yes 9 Selectivity (Impurity elution) Yes Yes Robustness ---- Yes System precision, system suitability and selectivity (Impurity elution) The purpose of this study is to establish the precision of the HPLC/ UPLC/ RRLC system being used for the analysis. Following sequence can be followed. 72

24 Analytical method development and validation Sr. No. Injection sequence No. of Injections 1. Mobile phase 1 2. Diluents 1 3. Resolution solution (If applicable) 1 4. Standard solution (specification level) 6 5. Standard preparation in duplicate 1 6. Impurity standard solution 1 7. Composite impurity solution with drug substance 1 Prefer acceptance criteria: In case of pharmacoepial methods, % RSD of standard should be less than that mentioned in the individual test method. In case of in-house assay methods, % RSD of standard should be below 2.0 %. Similarity between two standard preparations should be within 0.98 to 1.02 or 0.95 to Theoretical plates for analyte peak in Assay should not be less than 3000 (if applicable). Tailing factor for analyte peak should not be more than 2.0. Resolution of the analyte with closest eluting impurity should be at least 1.5. % RSD of six replicate standard injections in Related Substances should be below % RSD of six replicate standard injections in GC should be below Extraction time This experimental study is conducted to establish the minimum time required for the complete extraction of active ingredient from the formulation matrix. Prepare three sample solutions by weighing a suitable quantity of sample and sonicate the above three preparations for different time period (e.g. 15 minutes, 30 minutes and 45 minutes) after adding a suitable quantity of diluents. After completion of sonication suitably dilute and 73

25 Chapter-2 determine the assay of these samples. Compare the assay values obtained using different extraction time. Following sequence can be followed. Sr. No. Injection sequence No. of Injections 1. Mobile phase 1 2. Diluents 1 3. Resolution solution (If applicable) 1 4. Standard solution-1 (specification level) 6 5. Standard solution Sample Sample Sample-3 1 Prefer acceptance criteria: The time which gives the complete extraction (between 98.0% - 102%) should be incorporated in the final test procedure. Similarity between two standard preparations should be within 0.98 to 1.02 or 0.95 to Method precision This experimental study is conducted to establish study the repeatability of test results obtained by this method. Prepare three samples as per the test method representing a single batch. Determine the assay (related substances) of these samples and evaluate the precision of the method by computing the percentage-relative standard deviation of the assay results. In case of dissolution perform the dissolution as the test procedure on six dosage forms and evaluate the precision. Following sequence can be followed. 74

26 Analytical method development and validation Sr. No. Injection sequence No. of Injections 1. Mobile phase 1 2. Diluents 1 3. Resolution solution (If applicable) 1 4. Standard preparation-1 (system suitability) 6 5. Standard preparation-2 (for similarity) 1 6. Precision sample-1 to Bracketing standard (from standard preparation-1) 1 Prefer acceptance criteria: In case of assay % RSD for assay of three preparations is NMT 2.0. In case of content uniformity % RSD for assay of six/ten preparations is NMT 6.0. In case of preservatives/antioxidants content % RSD for assay of three preparations is NMT In case of residual solvents % RSD for three preparations is NMT In case of related substances below mentioned limit can be follow. Impurity level % RSD 0.05 % to 0.10 % 25.0 % 0.11 % to 0.50 % 15.0 % 0.51 % to 1.0 % 10.0 % More than 1.0 % 5.0 % Accuracy (recovery) This experimental study is conducted to establish the recovery of test results obtained by the method. Assay: Prepare recovery samples (i.e. spiking placebo {containing every colors of all strengths} with known quantities of API) at the level of 50 %, and 150 % of target concentration. Prepare the recovery samples in triplicate. 75

27 Chapter-2 Related Substances: Prepare recovery samples (i.e. spiking placebo {containing every colors of all strengths} with known quantities of all known impurities of API) at the level of LOQ (i.e %), 50 %, and 150 % of target concentration. Prepare the recovery samples in triplicate. Dissolution: Perform from 25% of lowest (lower) strength to 150% of highest (higher) strength. Sr. No. Injection sequence No. of Injections 1. Mobile phase 1 2. Diluents 1 3. Resolution solution (If applicable) 1 4. Standard preparation-1 (system suitability) 6 5. Standard preparation-2 (for similarity) 1 6. Recovery (LOQ) preparation Recovery (LOQ) preparation Recovery (LOQ) preparation Recovery (at 50 %) preparation Recovery (at 50 %) preparation Recovery (at 50 %) preparation Recovery (at 150 %) preparation Recovery (at 150 %) preparation Recovery (at 150 %) preparation Bracketing standard (from standard preparation-1) 1 Prefer acceptance criteria: Test % Recovery % RSD Assay 98.0 to % Dissolution/ content uniformity 95.0 to % Preservative content/ anti oxidants 90.0 to % Related Substances Impurity levels Acceptable % recovery Between 0.05 and 0.10 % Between 50.0% to 150.0% Between 0.11 and 0.50 % Between 70.0% to 130.0% Between 0.51 and 1.0 % Between 80.0% to 120.0% More than 1.0 % Between 90.0% to 110.0% 76

28 Analytical method development and validation Recovery from plate for swab analysis: The recovery at LOQ level shall not be less than 50 %. The recovery at 50 %, 100 % and 150 % level shall not be less than 70 % Filter compatibility This experimental study is designed to verify the compatibility of filter with sample solution. Prepare standard and sample solution as per test method. Filter a portion of standard solution and sample solution through a selected syringe filter (0.45 or 0.22 m HVLP/PVDF/HDPE/Nylon). Inject unfiltered and filtered standard solution, unfiltered sample solution (centrifuged sample) and filtered sample solution. Calculate the difference in response between unfiltered and filtered solutions of standard and sample. Prefer acceptance criteria: In case of assay, dissolution, content uniformity, preservative content and anti oxidant content. The difference between the responses of unfiltered and filtered solution should not be more than 2 %. In case of related substances the difference between the individual impurity content of unfiltered and filtered sample should not be more than 0.05% and in case of total impurity contents the content of unfiltered and filtered sample should not be more than 0.1 % Stability of analytical solution This experimental study is designed to demonstrate the stability of analytical solutions (i.e., standard and sample solution) at room temperature (i.e., about 25 C) and Refrigerator (2-8 C). In case of dissolution conduct it at 37.5 C. Prepare standard and sample solution as per the test method and chromatograph these solutions into HPLC/UPLC/RRLC system at regular intervals 77

29 Chapter-2 for minimum 12 hours. Monitor the area of the analyte and impurity peaks for both standard and sample solutions. Calculate the % deviation of analyte and impurity peaks area from initial for both standard and sample solutions. For assay, dissolution, preservative and antioxidant content: The response of standard and sample solution should not differ by more than 2.0 % from the initial response. For related substances: The individual impurity should not differ by more than 0.05 and total impurities by more than 0.1 from the initial value. The response obtained for each component shall be above the corresponding LOQ level response. The System suitability solution shall be used as long as the elution pattern matches with that of chromatogram injected during system suitability experiment. For swab analysis by HPLC/UPLC/RRLC: The response of standard solution should not differ by more than 10.0 % from the initial response. For dissolution/assay by UV: The deviation in test result should not be more than 2 % of the initial value Specificity The purpose of this study is to examine stability indicating nature of the test methodology. This will be studied in two parts. 78

30 Part-A: Interference from placebo and known impurities Analytical method development and validation In case of look-alike formulations placebo of lowest (lower) strength shall be prepared. In case of dose proportional formulations placebo of highest strength shall be prepared. In case of multiple colored formulations placebo with all colors mixed shall be prepared. Prefer acceptance criteria: There should be no interference of placebo at the retention time of Analyte as well as known impurities of Drug substances. The known impurities should not co-elute with the main active peak, at the same time the process related impurities should not co-elute with the degradants. Peak purity of analyte peak should be not less than 990/0.995 Swab absorbance should not be more than 10.0 % of the standard absorbance. In case of UV method: Placebo absorbance should not be more than 2.0 % of the standard absorbance. Interference from capsule shell should not be more than 2% of the labeled content. Part-B: Interference from degradants Before planning this study, evaluate the stability of the drug substance in different environmental conditions based on the available stability data and forced degradation data provided in the technical package. To evaluate the interference from degradation product, carry out the forced degradation study. API, placebo (with all colors, wherever applicable) and formulation (with all colors, wherever applicable), can be analyzed by the following conditions. 79

31 Chapter-2 Stress Type Acid hydrolysis Alkali hydrolysis Peroxide oxidation Thermal degradation Humidity degradation Photolytic degradation Condition Treat with suitable concentration of HCl for specific period Treat with suitable concentration of NaOH for specific period Treat with suitable concentration of hydrogen peroxide solution for specific period Expose the sample at 100 C for suitable period. Expose the sample at 40 C / 75 % RH for suitable period. Expose under UV light for 24 hours in a photo stability chamber ( under degradation mode) For highly unstable products, apply mild conditions to achieve about 5 to 10 % degradation. For highly stable products, apply strong conditions to achieve maximum possible degradation (e.g. stress period up to 12 hours). Chromatograph the above stress samples into HPLC/ UPLC/ RRLC system equipped with a diode array detector. Evaluate the peak purity of analyte peak and the assay of formulation under each stressed condition. Prefer acceptance criteria: Peak purity of analyte should be above 990. All degradants should be well separated from the analyte peak, impurity peaks and placebo peaks. ICH guidance: As per ICH guideline the unknown impurities obtained during the Stability studies needs Identified, characterized as per the thresholds proposed in the guideline. Degradation study will help in predicting these unknown degradants well before the submission batch stability. Drug substances impurity threshold are presented in Table

32 Analytical method development and validation Table 2.5 Drug substances impurities thresholds Maximum daily dose a Reporting threshold b c Identification threshold c Quantification threshold c < 2 g/day 0.05 % 0.01 % or 1.0 mg/day intake (whichever is less) 0.15 % or 1.0 mg/day intake (whichever is less) > 2 g/day 0.03 % 0.05 % 0.05 % a The amount of drug substance administered per day. b Higher reporting threshold should be scientifically justified. c Lower threshold can be appropriate if the impurities are unusually toxic. Drug product impurity threshold are presented in the Figure 2.1. Figure 2.1 ICH Q3B(R) Drug products impurity thresholds 81

33 Chapter FLOW OF METHOD DEVELOPMENT IN HPLC 82

34 Analytical method development and validation 2.11 METHOD SELECTION OF DRUG SUBSTANCES ASSAY AND RELATED SUBSTANCES 83

35 Chapter METHOD SELECTION FOR DRUG PRODUCT ASSAY 2.13 METHOD SELECTION FOR DRUG PRODUCT RS 84

36 Analytical method development and validation 2.14 METHOD SELECTION FOR DISSOLUTION AND CU 2.15 METHOD VALIDATION Once an analytical method is developed for its intended use, it must be validated. The extent of validation evolves with the drug development phase. Usually, a limited validation is carried out to support an Investigational New Drug (IND) application and a more extensive validation for New Drug Application (NDA) and Marketing Authorization Application (MAA). Typical parameters recommended by FDA, USP, and ICH are as follow [10]: 85

37 Chapter-2 1. Specificity 2. Linearity & Range 3. Precision (A) Method precision (Repeatability) (B) Intermediate precision (Ruggedness) 4. Accuracy (Recovery) 5. Solution stability 6. Limit of Detection (LOD) 7. Limit of Quantification (LOQ) 8. Robustness Method validation is vast area which includes many validation parameters with different approaches for different level of requirement based on intended use of analytical method, criticality and regulatory requirements. Validated method also can give the unpredicted or unknown problem during the course of routine usage, because validated method has also limited level of confidence, as method was validated for known or predicted variable parameters or every method can fail sooner or later [11]. But still after method development it needs to be validated as per requirement which gives certain level of confidence for its intended use. A common method validation protocol is followed for all the method developed during the research project (FDA, ICH Q2A & Q2B, 2005) Specificity Specificity is the ability of the method to measure the analyte in the presence of other relevant components those are expected to be present in a sample. The relevant components might include impurities, degradants, matrix, etc. Lack of specificity of an individual procedure may be compensated by other supporting analytical procedure(s). Specificity can also be demonstrated by verification of the result with an independent analytical procedure. In the case of chromatographic separation, resolution factors should be obtained for 86

38 Analytical method development and validation critical separation. Tests for peak homogeneity, for example, by diode array detection (DAD) or mass spectrometry (MS) are recommended. The evaluation of the specificity of the method was determined against placebo. The interference of the excipients of the claimed placebo present in pharmaceutical dosage form is derived from placebo solution. Further the specificity of the method toward the drug is established by means of checking the interference of the degradation products in the drug quantification for assay during the forced degradation study. The peak purity of analyte peak was evaluated in each degraded sample with respect to total peak purity and three point peak purity. The peak purity value must be more than (for Agilent system) or purity angle is less than threshold (for Waters system) in every case. Force degradation studies: [12] These studies are undertaken to elucidate inherent stability characteristics. Such testing is part of the development strategy and is normally carried out under more severe condition than those used for accelerated stability studies. Force degradation of the drug substance can help identify the likely degradation products, which can in turn help establish the degradation pathways and the intrinsic stability of the molecule and validate the stability indicating power of the analytical procedures used. The nature of the stress testing will depend on the individual drug substance and the type of drug product involved. Examining degradation products under stress conditions is useful in establishing degradation pathways and developing and validating suitable analytical procedures. So, as per the guidelines the stress studies for all the drug under investigation are done in the same conditions, the only difference is in temperature and the time required for each drug to degrade up to 5-20% level. Usually, the drugs are kept at solution and solid state stability in the following stability studies: Solution state stability: Acidic hydrolysis Alkaline hydrolysis Hydrolytic 87

39 Chapter-2 Oxidative degradation Solid state stability: Thermal degradation Photolytic degradation Linearity and Range The linearity of an analytical procedure is its ability (within a given range) to obtain test results, which are directly proportional to the concentration (amount) of analyte in the sample. A linear relationship should be evaluated across the range of the analytical procedure. It is demonstrated directly on the drug substance by dilution of a standard stock solution of the drug product components, using the proposed procedure. For the establishment of linearity, minimum of five concentrations are recommended by ICH guideline. The value of correlation co-efficient (r 2 ) should fall around Precision The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample. Precision may be considered at two levels: repeatability and intermediate precision. The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements. Repeatability: Repeatability study is performed by preparing a minimum of 6 determinations at 100% of the test concentration and analyzed as per the respective methodology. Intermediate Precision: The extent to which intermediate precision should be established depends on the circumstances under which the procedure is intended to be used. The analyst should establish the effects of random events on the precision of the analytical procedure. Typical variations to be studied include days, analysts, equipment, etc. It is not considered necessary to study these effects individually. Here, intermediate precision of the method is 88

40 Analytical method development and validation checked by carrying out six independent assays of test sample preparation on the different day by another person under the same experimental condition and calculated the % RSD of assays Accuracy The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. The evaluation of accuracy has got very prime importance as it deliberately force the method to extract the drug and impurities at higher and lower level Solution stability Drug stability in pharmaceutical formulations/active pharmaceutical ingredients is a function of storage conditions and chemical properties of the drug, preservative and its impurities. Condition used in stability experiments should reflect situations likely to be encountered during actual sample handling and analysis. Stability data is required to show that the concentration and purity of analyte in the sample at the time of analysis corresponds to the concentration and purity of analyte at the time of sampling. Stability of sample solution was established by storage of sample solution at ambient temperature (25 C) for 24h Limit of detection The limit of detection (LOD) for an individual analytical procedure is the lowest amount of analyte in a sample, which can be detected but not necessarily quantitated as an exact value. Determination of the signal-to-noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected. A signal-to-noise ratio between 3 or 2:1 is generally considered acceptable for estimating the detection limit. The limit of detection is evaluated by serial dilutions of analyte stock solution in order to obtain signal to noise ratios of 3:1. 89

41 Chapter Limit of quantitation The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample, which can be quantitatively determined with suitable precision and accuracy. The limit of quantitation (LOQ) is a parameter of quantitative assays for low levels of compounds in sample matrices. Determination of the signal-to-noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and by establishing the minimum concentration at which the analyte can be reliably quantified. A typical signal-to-noise ratio is 10:1. The limit of quantification was evaluated by serial dilutions of analyte stock solution in order to obtain signal to noise ratios of 10: Robustness The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. In the case of liquid chromatography, examples of typical variations are: Influence of variations of ph in a mobile phase Influence of variations in mobile phase composition Different columns (different lots and/or suppliers) Temperature Flow rate The factors chosen for all the drugs under investigation were the flow rate, mobile phase composition, ph of a mobile phase and using different lot of LC column. The observation shall be summarized and critical parameters shall be listed out in the validation report. System suitability parameter must be within the limit of acceptance criteria as mentioned in the method. 90

42 Validation characteristics of the tests Analytical method development and validation Validation characteristics of the various types of tests are listed in Table 2.6. Table 2.6 Validation characteristics of the tests* Advantages of analytical method validation The advantages of the analytical method validation are as follow: [8] The biggest advantage of method validation is that it builds a degree of confidence, not only for the developer but also to the user. Although the validation exercise may appear costly and time consuming, it results inexpensive, eliminates frustrating repetitions and leads to better time management in the end. Minor changes in the conditions such as reagent supplier or grade, analytical setup are unavoidable due to obvious reasons but the method validation absorbs the shock of such conditions and pays for more than invested on the process. 91