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Development and Validation of a RP-HPLC method
- 1. DEVELOPMENT AND VALIDATION OFA RP- HPLC METHOD SUBMITTED BY, Usha Khanal M.Pharm 1st year Roll no.-18HMPA06 Pharmaceutical Analysis&QualityAssurance
- 2. Method: Amethod is a set of experimental conditions designed to create a good analysis of a particular sample. Developing a method: Method development encompasses many stages and can take months to complete, depending on the complexity and goals of the method. The process usually includes the following steps:
- 3. STEPS IN HPLCMETHOD DEVELOPMENT Information on sample, define separation goal Need for special HPLC procedure, sample, pretreatment, etc. Choose detector and detector settings Choose LC method; preliminary run; estimate best separation conditions
- 4. Optimize separation conditions Checkfor problems or requirements for special procedure Recover purified material Quantitative calibration Qualitative method Validate method for release to routine laboratory
- 5. NATURE OF THESAMPLE To know the composition and properties of sample; particularly the properties likely to be affected by the analysis. The sample properties we need to know before developing a method are: Sample property Details Matrix To know the compounds other than the analyte are in the sample. Concentration The amount of the compound is present in the sample. The concentration range (low or high).
- 6. Sample property Details QuantityThe number compounds are present in the sample. Chemical/physical properties •pKa values • molecular size and weight •electrical charge • sample solubility •sample volatility • stability and toxicity • hydrophobicity/polarity • chemical/biological reactivity •UV spectra
- 7. METHOD GOALS Method goalDetails Detect The compound is present or not in the sample. Quantitate The amount of the compound is present in the sample Identify Compound identification Characterize Properties of the compound Purify/ isolate To collect the compound for further use, if required
- 8. DETERMINE THE ANALYSIS REQUIREMENT These are variables associated with the method goals. Method goal Analysis requirement Detection Detection technique that can be used to analyze the sample Quantitation Method for quantify (e.g., internal standard, external standard) The concentration range or sample amount (low or high) Levels of accuracy and precision are required Identification Process required for identifying the compound To determine purity
- 9. Method goal Analysisrequirement Characterization To determine the properties or property levels Purification/isolation To isolate purified material To recover 100% of your sample Sample matrix If there is more than one sample matrix before analysis and either the sample matrix interfere with analysis Properties The analysis technique allow you to determine sample properties
- 10. CONDUCT RESEARCH Conductresearch to determine if the analysis has been performed before. Previously developed methods with quantitation and sample matrices that are close to the requirements can form a starting point for the method. Resources to consult include: • Internet • United States Pharmocopeia (USP) • FDA requirements • EPA requirements • USDA methods • Colleagues • Professional/technical journals and meetings
- 11. SELECT THE ANALYSISTECHNIQUE Analysis technique Capabilities Liquid chromatography (LC) Separates samples in solution based on physical properties such as polarity, ionic strength, and molecular size. Liquid chromatography-mass spectrometry (LC-MS) Combines the physical separation capabilities of LC with the mass analysis capabilities of MS
- 12. SAMPLE PRETREATMENT AND DETECTION Samples come in various forms: Solutions ready for injection Solutions that require dilution buffering, addition of an internal standard or other volumetric manipulation Solids that must first be dissolved or extracted Samples that require sample pretreatment to remove interferences and/or protect the column or equipment from damage
- 13. Most HPLCanalysis require weighing and/or volumetric dilution before injection. Best results are often obtained when the composition of the sample solvent is close to that of the mobile phase, since this minimizes baseline upset and other problems. This means that it is important to know the nature of the sample matrix and the probable concentrations of various analytes. Before the first sample is injected during HPLC method development, we must be reasonably sure that the detector selected will sense all sample components of interest. variable-wavelength ultraviolet (UV) detectors normally are the first choice, because of their convenience and applicability for most samples.
- 14. For thisreason, information on the UV spectra can be an important aid tor method development. UV spectra can be found in the literature, estimated from the chemical structures of sample components of interest, measured directly (if the pure compounds are available), or obtained during HPLC separation by means of a photodiode-array (PDA) detector. When the UV response of the sample is inadequate, other detectors are available (fluorescence, electrochemical, etc.), or the sample can be derivatized for enhanced detection.
- 15. DEVELOPING THE SEPARATION(METHOD) Selecting an HPLC Method and Initial Conditions Based on a knowledge of sample composition and the goals of separation. If HPLC is chosen for the separation, the next step is to classify the sample as regular or special. SAMPLE HPLC Regular Special
- 16. Using information aboutthe sample to select conditions for the initial experimental separation. Regular Special Neutral Ionic exploratory run (reversed-phase) •Inorganic ions •Isomers •Enantiomers •Biological samples Peptides Carbohydrates Neuclotides Macromolecules Proteins Carbohydrates Nucleic acids Synthetic polymers
- 17. When thegoal of separation is the isolation of purified material, an optimized final HPLC method will differ from one developed for routine quantitative analysis. However, the beginning of the method development proceeds in exactly the same way for both cases. Thus method can be developed using one of the following approaches: a. stepwise incremental (one-factor-at-a-time) approach based on results from previous experiment b. systematic screening protocol, in which we evaluate factors such as stationary phases, solvents, pH and retention and thereby enhance resolution.
- 18. Preferred ExperimentalConditions for the Initial HPLC Separation Separation Variable Preferred Initial Choice Column Dimensions 15 x 0.46 cm Particle size 5µm Stationary phase C8 or C18 Mobile phase Solvent A &B Buffer and acetonitrile % B 80-100% Buffer (compound, pH, concentration) 25 mM potassium phosphate, 2.0<Ph<3.0 Additives (e.g., amine modifiers, ion- pair reagents) Do not use initially Flow rate 1.5-2.0 mL/min Temperature 35-45°C Sample Size Volume Weight < 25µL < 100µg
- 19. GETTING STARTED ONMETHOD DEVELOPMENT we assume that the sample is regular unless noted otherwise. Although the initial and final conditions required for special samples will differ from those listed for regular samples, the general strategy and approach to method development is similar for both regular and special samples. Thus the separation of regular samples will therefore prove applicable in many respects to method development for special samples. Start with an isocratic mobile phase composition use water & Methanol or ACN according to solubility in 50:50 ratio. Feed other parameter like wavelength, ambient temperature, 1ml/min Flow rate.
- 20. If thereare more than 10 components in particular sample it is usually not recommended to begin method development with an intermediate strength mobile phase. A better alternative is to use a very strong mobile phase ( e.g., 100-80% B), then reduce the %B as necessary. Example: For the separation of mixture of triazine herbicides (6 components) as a function of mobile phase conditions. Conditions: 25 x 0.46-cm C18 column; water- methanol mobile phase; ambient temperature; 1.7 mL/min. (a) 50% B; (b) 100% B; (c) 80% B; (d) 60% B; (e)5-100%B in 20 min (gradient) (f) 70%B (isocratic)
- 25. IMPROVING THE SEPARATION GoalComment Resolution Precise and rugged quantitative analysis requires that R, be sgreater than 1.5. Separation time <5-10 min is desirable for routine procedures. Quantitation ≤2% for assays; ≤5% for less-demanding analyses; ≤15% for trace analyses Pressure <150 bar is desirable, <200 bar is usually essential. Peak height Narrow peaks are desirable for large signal/noise ratios. Solvent consumption Minimum mobile-phase use per run is desirable.
- 26. METHOD OPTIMIZATION Theexperimental conditions should be optimized to get desired separations and sensitivity after getting appropriate separations. Stability indicating assay experimental conditions will be achieved through planned/systemic examination on parameters including pH (if ionic), mobile phase components and ratio, gradient, flow rate, sample amounts, injection volume and diluents solvent type.
- 27. VALIDATION OF METHOD Validation of an analytical procedure is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. The methods validation process for analytical procedures begins with the planned and systematic collection by the applicant of the validation data to support analytical procedures. The validation of analytical methods is done as per ICH guidelines Q2(R1).
- 28. COMPONENTS OF METHODVALIDATION The following are typical analytical performance characteristics which may be tested during methods validation: 1. System Suitability 2. Accuracy 3. Precision 4. Repeatability 5. Intermediate precision 6. Linearity 7. Detection limit 8. Quantitation limit 9. Specificity 10. Range 11. Robustness 12. Forced degradation studies
- 29. 1. System Suitability System suitability testing originally believed by the industry of pharmaceuticals to decide whether a chromatographic system is being utilized day to day in a routine manner in pharmaceutical laboratories where quality of results is most important which is suitable for a definite analysis. The parameters used in the system suitability tests (SST) report are as follows: 1. Number of theoretical plates or Efficiency (N). 2. Capacity factor (K). 3. Separation or Relative retention (α). 4. Resolution (Rs). 5. Tailing factor (T).
- 30. 1. Number oftheoretical plates/Efficiency (N) In a specified column, efficiency is defined as the measurement of the degree of peak dispersion. The efficiency is conveyed in terms of number of theoretical plates. The formula of calculation of N is illustrated below in the following
- 31. 2. Capacity ratioor Capacity factor (k) K’=tR-tM/tM The above said capacity factor sometimes is called as a retention factor which has no dimension and independent from flow rate of mobile phase as well as column dimensions which is the measure of extent of retention relating to an analyte relative to an un-retained peak. Where tR implies retention time of the sample peak and retention time of an un- retained peak is tM. k' = 0 means no compound is left in the column. Generally the value of k' is > 2.
- 32. 3. Relative retentionor separation factor (α) α = t2-ta/t1-ta α = Relative retention. t2= Retention time calculated from point of injection. ta= Unretained peak time (Retention time (tR) of an inert component not retained by the column). t1= the retention time from the point of injection of reference peak defined. (Suppose no reference peak is found, value would be zero).
- 33. 4. Resolution (Rs) Resolution is the capability of the column to separate 2 drugs in 2 individual peaks or chromatographic zones and it is improved by enhancing column length, reduction of particle size and rising temperature, altering the eluent or stationary phase. By using the following formula resolution is calculated.
- 34. Determination of resolutionbetween two peaks. tR1 and tR2 are the retention times for the two peaks of components. tw1 and tw2 = At the baseline lies between tangents drawn to the sides of the peaks.
- 35. 5.Tailing factor orAsymmetry factor Chromatographic peak assumed to have a Gaussian shape under ideal conditions. However in practical conditions, there is always a deviation from normal distribution which indicates non- uniform migration and non-uniform distribution process. The asymmetry factor and tailing factor are roughly same and rarely accurate and equal in most cases. Values should normally between 1.0-1.5 and values greater than 2 are unacceptable. The peak asymmetry is computed by utilizing the following formula. As = B/A Where: As = peak asymmetry factor. B = distance from the point at peak midpoint to the trailing edge. (measured at 10 % of peak height). A = distance from the leading edge of peak to the midpoint. (measured at 10 % of peak height).
- 36. Ideally, peaksshould be Gaussian in shape or totally symmetrical. Determination of tailing and asymmetric factor.
- 38. 2. Specificity Specificityis the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradants, matrix, etc. Lack of specificity of an individual analytical procedure may be compensated by other supporting analytical procedure(s). This definition has the following implications: Identification: To ensure the identity of an analyte. Purity Tests: Ensure that all the analytical procedures performed allow an accurate statement of the content of impurities of an analyte, i.e. related substances test, heavy metals, residual solvents content, etc. Assay (content or potency): To provide an exact result which allows an accurate statement on the content or potency of the analyte in a sample.
- 39. 3. Accuracy Theaccuracy 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. This is sometimes termed trueness.
- 40. 4. Precision Theprecision 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 under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility. Precision should be investigated using homogeneous, authentic samples. However, if it is not possible to obtain a homogeneous sample it may be investigated using artificially prepared samples or a sample solution.
- 41. Repeatability Repeatability expressesthe precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision . Intermediate precision Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc. Reproducibility Reproducibility expresses the precision between laboratories (collaborative studies, usually applied to standardization of methodology).
- 42. 5. Detection limit The detection limit of 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.
- 43. 6. Quantitation limit 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 quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/or degradation products.
- 44. 7. Linearity Thelinearity 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.
- 45. 8. Range Therange of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity
- 46. 9. Robustness Therobustness 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.
- 47. Forced Degradation Studies Forced degradation or stress studies are undertaken to deliberately degrade the sample. These studies are used to evaluate an analytical method’s ability to measure an active ingredient and its degradation products, without interference, by generating potential degradation products. During validation of the method, drug substance are exposed to acid, base, heat, light and oxidizing agent to produce approximately 10% to 30% degradation of active substance. The studies can also provide information about the degradation pathways and degradation products that could form during storage. These studies may also help in the formulation development, manufacturing, and packaging to improve a drug product.
- 48. REFERENCES Lloyd R.Snyder, Joseph J. Kirkland, Joseph L. Glajch. Practical HPLC method development. 2nd edition,1988,1-15 Vibha Gupta et al,. Development and validation of HPLC method. International Research Journal of Pharmaceutical and Applied Sciences. 2(4),2012,17-25. Pallavi Nemgonda Patil. HPLC method development. Journal of Pharmaceutical Research & Education. 1(2),2017,243-260. ICH, Validation of Analytical Procedures: Text and Methodology. International Conference on Harmonisation, EMEA.2006,2-15.