Calibration & VAlidation - SYED TABRAIZULLAH HUSSAINIComputer system validation. IMPORTANCE OF...

UNIT V - Calibration & Validation � .1

NTRODUCTION TO CALIBRATION:

DEFINTION AND GENERAL PRINCIPLES:

alibration: Calibration is a process by which we ensure that an instrument readings are accurate with reference to established standards. Calibration is performed using primary reference standards. Instruments need to be calibrated before using. For example- weighing balance, pH meter. etc

Need for calibration. Calibration can be called for: • a new instrument. • a specified time period is elapsed. • a specified usage (operating hours) has elapsed. • when an instrument has had a shock or vibration which potentially may have put it out of calibration. • sudden changes in weather. • whenever observations appear questionable.

Calibration is a process that demonstrates a particular instrument or device produces results within specified limits, as compared to those produced by a traceable standard over an appropriate range of measurements. Calibration activities must be performed with qualified instruments by an accredited laboratory. Calibration is of utmost importance in building a solid Quality System Management with experts and well-trained specialists. Additional external support is also requested when refurbishing, upgrading and building departments or plants, when buying new equipment.

The word calibration is defined as “a test during which known values of measurement are applied to the transducer and corresponding output readings are recorded under specified conditions.”

Calibration is a comparison of measuring equipment against a standard instrument of higher accuracy to detect, correlate, adjust, rectify and document the accuracy of the instrument being compared. Typically, calibration of an instrument is checked at several points throughout the calibration range of the instrument.

The calibration range is defined as “the region between the limits within which a quantity is measured, received or transmitted, expressed by stating the lower and upper range values.


GENERAL PRINCIPLES OF CALIBRATION:

Calibration Tolerance: Every calibration should be performed to a specified tolerance. The terms tolerance and accuracy are often used incorrectly. In ISA’s The Automation, Systems, and Instrumentation Dictionary, the definitions for each are as follows: Accuracy: The ratio of the error to the full scale output or the ratio of the error to the output, expressed in percent span or percent reading, respectively.

Tolerance: Permissible deviation from a specified value; may be expressed in measurement units, percent of span, or percent of reading.

Calibration tolerances should be determined from a combination of factors. These factors include: • Requirements of the process • Capability of available test equipment • Consistency with similar instruments at your facility • Manufacturer’s specified tolerance.

Accuracy Ratio: This term was used in the past to describe the relationship between the accuracy of the test standard and the accuracy of the instrument under test. Traceability: All calibrations should be performed traceable to a nationally or internationally recognized standard. The standards from the calibration lab are periodically checked for calibration by “higher level” standards, and so on until eventually the standards are tested against Primary Standards maintained by NIST or other internationally recognized standard. The calibration technician’s role in maintaining traceability is to ensure the test standard is within its calibration interval and the unique identifier is recorded on the applicable calibration data sheet when the instrument calibration is performed. Additionally, when test standards are calibrated, the calibration documentation must be reviewed for accuracy and to ensure it was performed using NIST traceable equipment.

Definition and general principles of validation: Validation is the process of establishing documentary evidence demonstrating that a procedure, process, or activity carried out in testing and then production maintains the desired level of compliance at all stages. In the pharmaceutical industry, it is very important that in addition to final testing and compliance of products, it is also assured that the process will consistently produce the expected results.


Since a wide variety of procedures, processes, and activities need to be validated, the field of validation is divided into a number of subsections including the following: Equipment validation Facilities validation HVAC system validation Cleaning validation Process Validation Analytical method validation Computer system validation.

IMPORTANCE OF VALIDATION: The pharmaceutical industry is one of highly regulated industries, with many rules and regulations enforced by the government to protect the health and well-being of the public. Facilities and processes involved in the pharmaceutical production impact significantly on the quality of the products. At the same time, the objective of the pharmaceutical industry is to get high quality at low cost.

OBJECTIVE: High quality and low cost.

Therefore, efficient use of resources is the key to success. Thus, validation becomes an integral part of the quality assurance. It is requirements of current Good Manufacturing Practices (cGMPs) for finished pharmaceutical (21CFR211) and medical devices (21 CFR 820).

*****According to the Food and Drug Administration (FDA) Validation is to establish documented evidence which provides high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality characteristics.*****

Validation is thus the action of proving that any procedure, process, equipment, material activity will actually lead to expected results and produce a quality product. It includes activities starting from analytical methods used for quality control of drug substance to drug product validation. There are four components of validation - man, machine, material, method.

Validation has many benefits as described below:

• Fulfillment of regulatory requirement. • Increased output. • Reduction in rejections reworks.


• Safety • Avoidance of capital expenditures. • Fewer complaints about process related failures. • Reducing testing in the process and finished goods. • More rapid and accurate investigations into process deviations. • Easier and reliable startup of new equipment. • Easier scale-up from development work. • Easier maintenance of the development. • Improved employee awareness of process. • More rapid automation.

SCOPE OF VALIDATION: The scope of validation involves providing the specific elements to verify that all the components of the system operate as predetermined specified to achieve the desired results. For example, operational qualification of dryer report assures that the dryer works according to the acceptance of criteria involving the highest and lowest working limits. Following requirements are come under scope of validation.

• Validation should be done in structured way according to documentation including procedures and protocols.

• Validation requires an appropriate and sufficient infrastructure including: organization documentation, personnel and finances.

• Personnel with appropriate qualifications and experience. • Extensive preparations and planning before validation is performed. • A specific program for validation activities. • Validation should be performed: for new premises, equipment utilities and systems, and

procedures and processes; at periodic intervals; and when major changes have been made.

• Manufacturers or top officials to identify type of validation needed to work. • Involvement of management and quality assurance personnel. • Significant changes in facilities, equipment, processes should be validated. • Risk assessment approach should be used to determine in specific scope and extent of

validation needed case-by- case basis.

TYPES OF VALIDATION: 1. Process Validation 2. Equipment Validation and 3. Analytical Validation.


I. PROCESS VALIDATION: According to FDA, assurance of product quality is derived from careful and systemic attention number of important factors including, selection of components and materials adequate product and process design and control of process through "in process and end product testing". The latest guidance document for validation is published by USFDA is process validation:

The USFDA guidance describe process validation activities in three states Stage 1 process design Stage 2 process qualification Stage 3 continuous process verification

Definition: the collection and evaluation of data from process design stage throughout production, which establishes scientific evidence that a processes is capable of consistently delivering quality products.

There are four types of process validation 1. Prospective validation( pre-market validation):to prove that the process will work in accordance to the process with validation protocol. 2. Concurrent validation: in some exceptional cases it may be acceptable not to complete a validation process before routine production starts this approach involves monitoring of critical processing and end product testing of current production to show that the manufacturing process is in a state of control. 3. Retrospective validation: only acceptable for when established processes whose manufacturing processes are considered stable on the basis of economic consideration only. 4. Revalidation: revalidation of equipment and process in pharmaceutical revalidation in pharmaceutical industry is very important as it helps to maintain the validated status of equipments, plant, manufacturing process and computer systems. Its objective is to make sure that systems are working to a good standard.

II. EQUIPMENT VALIDATION: It is predominantly a documentation exercise in which details of the physical components of the system are recorded as definition of the equipment.

Qualification is broken down into three phases:

1. Design qualification (DQ): it provides documented evidence that all key as that of the design improvement at here to the approved design intention and that all the manufacturers recommendations have been suitably consider it defines the functional


and operational specification of the instrument it helps in selection of the supplier for example setting wrong functional specification substantially increases the workload for OQ testing adding missing function at a later stage will be much more expensive than including them in the initial specifications and selecting a vendor with insufficient support probability. Demonstrate design compliance to GMP.

Equipment validation at a glance.

2 . Installation qualification (IQ): it provides documented evidence that all three aspects of the insulation added to the to design intention and that all the manufacturers recommendations have been switched be considered it should be performed on you on modified facilities, systems and equipment. It provides following benefits:

A. Assure proper installation B. Application of materials of construction. C. Assure all operating manual are available D. Determine calibration requirements.


This is the first step in validation test and shows that system recruitment and its components are installed correctly and operating according to manufacturer specifications. for eg. it is important to demonstrate that during solid dosage equipment application that calibrations of components on all moveable equipment is unaffected by movement to change location and the figures of cleaning using organic solvents and or water. Water this includes the strain gauges that control weight on automated tablet presses.

3. Operational qualification (OQ): it provides documented certification that the system and subsystem of rate as Alarm and interlocks are tested to verify the proper operation of the system throughout all anticipated operating range according to sop. Demonstrate that system works acceptable. it challenges the system to operating condition. Test to include a condition or a set of conditions and compass in upper and lower operating limits sometimes referred to as of “worst case”conditions. For eg: power failure and recovery test a form to document defect of this event on the control of the system.

OQ involve development of sop and training of personnel full stocks all test data and Measurement must be documented the completion of a successful operational qualification should allow the. 1) Finalization of calibration operating and cleaning procedures, or operator 2) training and preventive maintenance requirements. 3) It should format of formal “release” of facilities, systems and equipment. It is possible to run oppressive batch during this phase to minimize the financial loss in case of an equipment failure. 3. Performance qualification (PQ): this is the final phase of requirement validation it provides document verification that the system performs acceptability and for what it is made for. PQ is performed on the manufacturing process as the whole. Individual components of the system not tested individually. The tests are done using production materials qualified substitutes for stimulator product that have been developed from knowledge of the process and the facilities, systems or equipment. Tests include condition or set of conditions encompassing upper and lower operating limits.

III. ANALYTICAL VALIDATIONS

Analytical method validation is the process used to prove that the analytical procedure employed for a specific test is fit for its intentional use. Outcome from method validation can be used to Judge the quality, reliability and consistency of analytical results. It is an integral part of any good analytical practice. There must be assurance that "the accuracy,


sensitivity, specificity, and reproducibility of test methods employed by the firm are established and documented."(CFR Title 21- part 211)".

According to ICH Guidelines, the following four types of methods require validation

Identification tests- Identification tests are intended to ensure the identity of an Analyte in a sample. This is normally achieved by comparison of a property of the sample (e.g., spectrum, chromatographic behavior, chemical reactivity, etc.) to that of a reference standard. Quantitative tests for impurities content -Testing for impurities can be either a quantitative test or a limit test for the Impurity in a sample. Either test is intended to accurately reflect the purity characteristics of the sample. Limit tests for the control of impurities- Limit test is defined as quantitative or semi quantitative test designed to identify and control small quantities of impurity which is likely to be present in the substance Assay- Quantitative tests of the active in samples of drug substance or drug product or the selected components in the drug product. An analytical method should be validated when It Is necessary to verify that its performance parameters are adequate for use for a particular analytical problem. For example

• Method just developed • Revised method or established method adapted to new problems • When a review of quality control Indicates an established method is changing with time • when an established method Is used In a different laboratory, with different analysts or with different equipment. • Demonstration of the equivalence between two methods, e.g a new method and a standard.

Types of analytical method validation 1. specificity (Selectivity) 2. Linearity 3. Range 4. accuracy 5. precision 6. Detection Limit 7. Quantitation Limit 8. Robustness 9. System Suitability Tenting


1. Specificity (Selectivity): It is the ability of the method to assess unequivocally the analyte in presence of components which may be expected to be present. ((typically impurities, degradants, matrix)). This should include samples stored under relevant stress conditions: light, heat, humidity, acid/base hydrolysis and oxidation. This definition has the following implications: • Identification: to ensure the identity of an analyte. Suitable identification tests should be able to discriminate between compounds of closely related structures which are likely to be present. The discrimination of a procedure may be confirmed by obtaining positive results from samples containing the analyte, coupled with negative results from samples which do not contain the analyte. The identification test may be applied to materials structurally similar to or closely related to the analyte to confirm that a positive response is not obtained. • Purity Tests: to ensure that all the analytical procedures performed allow an accurate statement of the content of impurities of an analyte. the discrimination may be established by spiking drug substance or drug product with appropriate levels of impurities and demonstrating the separation of these impurities individually and/or from other components in the sample matrix. • 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. When impurities are available, this should involve demonstration of the discrimination of the analyte in the presence of impurities and/or excipients. This can be done by spiking pure substances with appropriate levels of impurities and/or excipients and demonstrating that the assay result is unaffected by the presence of these materials . if impurity or degradation product standards are unavailable, specificity may be demonstrated by comparing the test results of samples containing impurities or degradation products to a second well characterized procedure e.g. phamacopeial method or other validated procedure.

2. Linearity: Ability (Within a given range) to obtain test s results Which are directly proportional to the concentration amount of analyte in the sample. Linearity should be evaluated by visual inspection of a plot of signals as a function of analyte concentration or content. For the establishment Of linearity, a minimum of five concentrations is recommended. Linearity results should be established by appropriate statistical methods. Transformations are also acceptable and may include log, square root, or reciprocal (other transformations are acceptable). If linearity is not attainable, a nonlinear model may be used. The goal is to have a model (Whether linear or non-linear) that describes closely the concentration-response relationship.


The following parameters should be determined: • correlation coefficient- indicates the relationship chosen is coned • y-intercept indicates response for no analyte (interference) • slope of the regression line-indicates sensitivity of the method • Residual sum of-squares- indicates uncertainty of intercept(in blank response) Acceptance criteria: correlation coefficient should not be less than 0.999 for assay, dissolution and 0.99 for impurities test method. The range of the procedure is validated by verifying that the analytical procedure provides acceptable precision, accuracy and linearity when applied to samples containing analyte at the extremes of the range as well as within the range. 3. Range : Range of an analytical procedure is the interval between the upper and lower concentration 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. The specified range is normally derived from linearity studies and depend on the intended application of the procedure The following minimum specified ranges should be considered: • for the assay of an active substance or a finished product normally from 80 to 120

Percent of test concentration. • for content uniformity, covering a minimum of 70 to 130 percent of the test

concentration. • for dissolution testing +/- over the specified range eg. if the specification for a

controlled released product cover a region from 20%, after 1 hour, up to 90%. after 24 hours, the validated range would be 0-110% of the label claim.

4. Accuracy: Accuracy means closeness of test results obtained by that method to the true value. The accuracy of an analytical procedure expresses the closeness of agreement between the value, which is accepted which is accepted either as a conventional true value or an accepted reference value and the value found. Sometimes it is termed trueness.

Procedure: 1. Assay/Dissolution: known amount of drug substance is spiked with synthetic mixtures of drug product components (excepients) - minimum of 3 levels (80%, 100%, and 120% of test concentration) each level is triplicate. 2. Impurities- drug substance/drug product spiked with known amounts of impurities- minimum of 3 levels and triplicates.


Acceptance criteria: 1. Assay: recovery should be between 98%-102% 2. Dissolution: 95%-105% 3. Impurities- if, specifications is <0.2%: 85%-115%, if specifications is >0.2%:90%- 110%.

Concept of Accuracy and Precision

5. Precision: Degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of a homogenous sample. A sufficient number of aliquots of a homogeneous sample are assayed to be able to calculate statistically valid estimates of standard deviation or relative standard deviation. Minimum 9 determinations over a minimum of 3 concentration levels (e.g, 3 concentrations/3 replicates each of the total analytical procedure). Precision may be considered at three levels: repeatability, intermediate precision and reproducibility.

a. Repeatability: repeatability expresses the perception under the same operating conditions of a short interval of time. (Within a laboratory of a short period of time using the same analyst with the same equipment). Repeatability is also termed as intra assay precision. b. Intermediate precision-intermediate precision expresses within laboratory variations. (Within Laboratory variation, as on different days or different analysts for different equipments.) c. Reproducibility: precision between laboratories. Can be considered during the standardization of a procedure before it is submitted to the pharmacopoeia. As per CDER guidelines, it is not normally expected of intermediate position is accomplished.

6. Detection Limit/ limit of detection(LOD): detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated, under the stated experimental conditions. Several approaches for determining the detection limit are possible, depending on whether the procedure is a non-instrumental or instrumental. • Based on visual examination • Based on signal to noise ratio (baseline noise) 2:l or 3:1

• Based on the Standard Deviation of the Response and the Slope.


7 . Q u a n t i t a t i o n l i m i t / l i m i t o f Quantification: limit of quantification of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. Several approaches for determining the detection limit are possible depending on whether the procedure is non instrumental instrumental. a. Based on visual examination b. Based on signal noise ratio(10:1) c. Based on the standard deviation of the response and the slope. Procedure: SD of response and Slope (S): prepare linearity curve with the series of related substances solutions are different concentrations (3 concentrations below 50% of specification level and three more concentrations above 50% specification level). RSD criteria: prepare a series of related substance solutions of concentrations below specification level (generally about 10%,20%, 30%, 40% and inject 6 replicate into HPLC) Precision should be established (if predicted from other than RSD criteria) at LOQ and LOD levels as per ICH, USP and EP guidelines.

8. Robustness: robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations (changes like pH, mobile phase composition, temperature) in method parameters and provide an indication of its reliability during normal usage. The evaluation of robustness should be considered during the development phase and depends on the type of procedure under study. Consequence development of system suitability parameters. If the measurements are susceptible to variations and analytical conditions, the analytical condition should be suitably controlled or a precautionary statement should be included in the procedure, such as: use solutions within 24 hours, maintain temperature below 25 degrees. In the case of liquid chromatography, examples of typical variations are: influence of variations of pH in mobile phase, influence of variations in mobile phase composition different columns (different lot/and suppliers) temperature flow rate. In case of gas chromatography, examples of typical variations are: different columns ( different lot/and suppliers) temperature flow rate.


9. System suitability testing: System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analysed constitute an integral system that can be evaluated as such. Hidden sure that the system is working properly at the time of analysis. determination made our repeatability, tailing factor (T), capacity factor (k'), resolution (R), and theoretical plates( N).


alidation Master plan

It consists of following elements

1. Introduction: firm validation policy and general description. 2. Organizational structure: description of personal responsibility for all validation activities. 3. Plant process product description: description of the plant layout, process and product for completing the documents in all aspect. 4. Specific process requirements- mention of the important characteristics of the plant. 5. List of products, process, system to be validate matrix system comprising the total list of validations required 6. Key acceptance criteria: explanation and listening to acceptance criteria for all above mentioned validations 7. documentation format: the format used for profession should be save and referred 8. SOP- A list of relevant SOP's 9. Planning and scheduling description of overall planning including Human resource equipment, time plan. 10. Change control description of the methods to control changes in in critical components including materials, facilities, equipments or process.

Some other elements are - - Facility validation - Analytical method validation - Equipment qualification - Qualification of input material - Product quality specification and test methods - Method validation - Process validation - Equipment Cleaning method validation - Train manpower and competency of personal - Stability studies data evaluation - Revalidation criteria - Validation report at each step


ALIBRATION OF PH METER

The calibration of a pH meter is important to ensure that the readings returned from the meter is accurate. Digital & analog PH meters offer calibration buttons or dials that are used to adjust the sensitivity of the meter.

STEPS FOR CALIBRATION:-

1. Turn on pH meter: Allow adequate time for the meter to warm up. This generally takes around 30 minutes, operating manual for exact times should be checked.

2. Clean the electrode: Take the electrode out of its storage solution distilled water under an empty waste beaker. Once rinsed, blot dry. Be sure to rinse your electrode in a waste beaker that is different from the beaker you will be calibrating in. Avoid rubbing the electrode as it has a sensitive membrane around it.

3. Prepare buffers: Generally more than one buffer is needed for calibrating a pH meter. The first is a “neutral" buffer with a pH of 7, and the second should be near the expected sample pH, either a pH of 4 or 9.21. Buffers with a higher pH (9.21) are best for measuring bases, whereas buffers with a low pH (4) are best for measuring acidic samples. Once the buffers the buffers are selected allow them to reach the same temperature as the pi meter because pH readings are temperature dependent. Buffers should be kept in a beaker for no longer than two hours discard the buffer when you are finished. Do not return it to its original container.

4. Place electrodes in the buffer with a pH value of 7 and begin reading: Press the "measure" or calibrate button to begin reading the pH once the electrode is placed in the buffer. Allow the pH reading to stabilize before letting it sit for approximately 12 minutes.

5. Set the pH: Once a stable reading appears, set the pH meter to the value of the buffer's pH by pressing the measure button a second time. Setting the pH meter once the reading has stabilized allows for more accurate and tuned readings

6. Rinse the electrode with distilled water: Rinse and pat dry with a lint-free tissue in between buffers.

7. Place the electrode in the appropriate buffer for sample reading: Press the button to begin reading the pH once the electrode is placed in the buffer.

8. Set the pH a second time: Once the reading has stabilized, set the pH meter to the value of the buffer's pH by pressing the measure button.

9. Rinse the electrode: Distilled water can be used to rinse. A lint-free tissue should be used to dry the electrode.


UALIFICATION OF UV VISIBLE SPECTROPHOTOMETER

The suitability of a specific instrument for a given procedure is ensured by a stepwise life cycle evaluation for the desired application from selection to instrument retirement: Design Qualification (DQ), Installation Qualification (IQ), an initial performance-to-specification qualification, also known as Operational Qualification (OQ), and an ongoing Performance Qualification.

As with any spectrometric device, a UV-Vis spectrophotometer must be qualified for both wavelength (x-axis) and photometric (y-axis) accuracy and precision, and fundamental parameters of stray light and resolution must be established. OQ is carried out across the operational ranges required within laboratory for both the absorbance and wavelength scales.

• Installation Qualification: The IQ requirements provide evidence that the hardware and software are properly installed in the desired location.

• Operational Qualification: Specifications for particular instruments and applications can vary depending upon the analytical procedure used and the desired accuracy of the final result. Instrument vendors often have samples and test parameters available as part of the IQ/OQ package. Wherever possible in the procedures detailed as follows, certified reference materials (CRMs) are to be used in preference to laboratory-prepared solutions. These CRM should be obtained from a recognized accredited source and include independently verified traceable value assignments with associated calculated uncertainty. The CRMs must be kept clean and free from dust.

• Control Of Wavelengths: Ensure that accuracy of the wavelength axis over the intended operational range is correct within acceptable limits. For non-diode array instruments, wavelength accuracy and precision are determined over the operational range using at least six replicate measurements. For wavelength accuracy, the difference of the mean ensure that accuracy of the wavelength axis over the intended operational range is correct within acceptable limits. For non-diode array instruments, wavelength accuracy and precision are determined over the operational range using at least six replicate measurements. For wavelength accuracy, the difference of the mean measured value to the certified value of the CRM must be within + or – 1nm in the uv region and in the visible region it must be


+ or - 2nm. For wavelength precision, the standard deviation of the mean must not exceed 0.5nm. For diode array instruments, only one wavelength accuracy is required, and no precision determination needs to be performed.

• Control Of Absorbance: To establish the transmittance accuracy, precision, and linearity of the given system, it is necessary to verify the absorbance accuracy of a system over its intended operational range by using acidic potassium chromate solutions in 0.001M perchloric acid as appropriate for the wavelength and absorbance range is required.

• Limit Of Stray Light (stray radiant energy): Although the measurement of the absorbance or transmittance is a ratio measurement of intensities and therefore theoretically is independent of monochromatic source intensity, particle measurements are affected by the presence of unwanted radiation called “stray radiant energy”.

• Resolution: If accurate absorbance measurements must be made on benzoin compounds or other compounds with sharp absorption bands, the spectral bandwidth of the spectrophotometer used should not be greater than 1L8th of the natural bandwidth of the compounds absorption.

• Performance Qualification: The purpose PQ is to determine that the instrument is capable of meeting the users requirements for all the parameters that may affect the quality of the measurement and to ensure that it will function properly over extended period of time.

PROCEDURE: With few exceptions, compendia spectrophotometric tests and assays call for comparison against a USP reference standard. This help ensure measurements under identical conditions for the test specimens and the reference substance. These condition could include wavelength setting, spectral bandwidth selection, cell placement and correction, and transmittance levels. Cells that exhibit identical transmittance at a given wavelength may differ considerably in transmittance at other wavelengths. Appropriate cell corrections should be established and used where required. Comparisons of a test specimen with a reference standard are best made at a peak of spectral absorption for the compound concerned. Assays that prescribe spectrophotometry give the commonly accepted wavelength for peak spectral absorption of the substance in question. Different spectrophotometer may show minor variation in the apparent wavelength of this peak. Good practice demands that comparison be made at the wavelength at which peak


comparison occurs. The expression “similar preparation” and “similar solution” as used in tests and assays involving spectrophotometry indicate that the reference comparator, generally a UP Reference standard, should be prepared and observed in an identical manner for all practical purposes to that used for the test specimen. Usually when analysts make up the solution of the specified reference standard, they prepare solution of about the desired concentration, and they calculate the absorptivity on the basis of the exact amount weighed out. If a previously dried specimen of the reference has not been used, the absorptivity is calculated on the anhydrous basis. The expressions “concomitantly determine” and “concomitantly measure” as used in tests and assays involving spectrophotometry indicate that the absorbance of the both the solutions containing the test specimen and the solution containing the reference specimen, relatives to the specified test blanks, must be measured in immediate succession.

• Sample Solution Preparation: For determination using UV or visible spectrophotometry, the specimen is generally is dissolved in a solvent. Unless otherwise directed in the monograph, analysts make determinations at room temperature using a path length of 1cm. many solvents are suitable for ranges, including water, alcohols, lower hydrocarbons, ethers and dilute solutions of strong acids and alkalis. Precautions should be taken. For the solvent, analysts typically should use water – free methanol or alcohol or alcohol denatured by the addition of methanol but without benzene or other interfering impurities. Solvents of special spectrophotometric quality, guaranteed to be free from contaminants, are available commercially from several sources. Some other analytical reagent-grade organic solvents may contain traces of impurities that absorb strongly in the UV region. New lots of these solvents should be checked for their transparency, and analysts should take care to use the same lot of solvent for preparation of the test solution, the standard solution, and the blank. The best practice is to use solvents that have NLT 40% transmittance at the wavelength of interest. Assays in the visible region usually call for concomitantly comparing the absorbance produced by the assay preparation with that produced by a standard preparation containing approximately an equal quantity of a USP reference standard. Under certain circumstances, the absorbance found in the assay result can be calculated. Such standard curves should be confirmed frequently and always when a new spectrophotometer or new lots of reagents are put in use.


eneral principles of analytical method validation

Principle Guideline presents information on the characteristics to be considered. Manufacturer to demonstrate – analytical procedure is suitable for its intended purpose. Validate analytical methods – whether they indicate stability or not. Validate by R and D before being transferred to the quality control unit when appropriate.

Analytical procedures to be validated • Identification test. • Quantitative test for impurities content. • Limit test for control of impurities. • Quantitative test of the active mighty of the drug substance a drug product or the other selected components in the drug product. • Dissolution testing and determination of particle size.

What is the purpose for validation? When should verification or revalidation be done?

1. Changes in the purposeful synthesis of the drug substance. 2. Changes in the composition of the finished product. 3. Changes in the analytical procedure. 4. Transfer of messages from one laboratory to another. 5. Changes in major pieces of equipment instrument.

Documentations of validation

Protocol: includes procedure and acceptance criteria Report: documented results. Justification needed when non-pharmacopoeia methods are used. Justification to include: Data example comparisons with the pharmacopoeia or other methods. Detail standard test methods.

Validation characteristics: Characteristic that should be considered during validation of analytical methods include:

➢ Specificity ➢ Linearity ➢ Range


➢ Accuracy ➢ Precision ➢ detection limit ➢ quantitation limit ➢ Robustness

Accuracy: The degree of agreement of test results with the true value or the closeness of the results opened by the procedure to the true value. It is normally established on samples of the material to be examined that have been prepared to quantitative accuracy. Accuracy should be established across the specified range of the analytical procedure.

Precision: The precision of the analytical procedure expresses the closeness of agreement between a series of measurement obtained from multiple sampling of the same homogeneous sample under the prescribed condition.

Repeatability: a minimum of nine determination covering the specified range for the procedure. Example 3 concentrations or three replicates each, or a minimum of six determination at hundred percent of the test concentration.

Intermediate precision: within laboratory variation usually on different days, different analyst and different equipment.

Reproductive ability: precision between laboratories.

Robustness: The ability of the procedure to provide analytical results of acceptable accuracy and precision during a variety of conditions. The results from separate samples are influenced by changes in the operational or environmental conditions. It should be considered during the development phase, and should show the reliability of the analysis when deliberate variations are made in method parameters. Factors that can have effects on robustness when performing chromatographic analyses include:

i. Stability of present standard samples and solutions. ii. Reagents iii. Different columns iv. Extraction time v. variation of pH of mobile phase vi. variation in mobile phase composition vii. temperature viii. Flow rate


Linearity: It indicates the ability to produce results that are directly proportional to the concentration of analyte examples. Series of sample should be prepared in which the analyte concentration span the claim range of the procedure. If there is a linear relationship, test results should be evaluated by appropriate statistical methods. A minimum of five concentrations should be used.

Range: It is an expression of the lowest and highest level of analyte that have been demonstrated to be determinable for the product.

The specified range is normally derived from linearity studies.

Specificity: It is the ability to measure unequivocally the desired analyte in the presence of components such as excipients and impurities that may also be expected to be present. An investigation of specificity should be conducted during the validation of identification tests, the determination of impurities and assay.

LOD DETECTION LIMIT (LIMIT OF DETECTION): It is the smallest quantity of an analyte that can be detected, and not necessarily determined, in a qualitative fashion. Approaches (instrumental or non-instrumental): • Visual evaluation. • Signal to noise ratio. • Standard derivation of the response and the slope. • Standard derivation of the blank. • Calibration cure.

LOQ QUANTINATION LIMIT (limit of quantination): It is the lowest concentration of an analyte in a sample that may be determined with acceptable accuracy and precision.

• Visual evaluation. • Signal to noise ratio. • Standard derivation of the response and the slope. • Standard derivation of the blank. • Calibration cure.


LOQ, LOD AND SNR:

Limit of Quantification Limit of Detection Signal To Noise Ratio

Calibration & VAlidation - SYED TABRAIZULLAH HUSSAINIComputer system validation. IMPORTANCE OF...

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Transcript of Calibration & VAlidation - SYED TABRAIZULLAH HUSSAINIComputer system validation. IMPORTANCE OF...