HPLC CALIBRATION SOP

                                                         HPLC CALIBRATION SOP 

PURPOSE:

All instruments should be calibrated and the appropriate correction applied to the original measurements.  This calibration system should bring about trust in decisions or measures. 

1.1          To check the performance of the instrument. 

1.2          To avoid the variation/wrong result

1.3          To achieve the result according to the specification.

2.0        PROCEDURE:

VALIDATION:

Validation of an analytical method is a process by which it is established, by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical applications.  

ANALYTICAL PERFORMANCE CHARACTERISTICS

1.    Accuracy

2.    Precision

3.    Specificity

4.    Detection Limit

5.    Quantitation Limit

6.    Linearity

7.    Range

1- ACCURACY

Definition

The closeness of test results obtained by that method to the true value.  The accuracy of an analytical method should be established across its range.

Determination–In the case of the assay of a drug substance, accuracy may be determined by application of the analytical method to an analyte of known purity (e.g. a Reference Standard) or by comparison of the results of the method with those of a second, well-characterized method, the accuracy of which has been stated or defined.

In the case of the assay of a drug in a formulated product, accuracy may be determined by the application n of the analytical method to synthetic mixtures of the drug product components to which known amounts of analyte have been added within the range of the method.  If it is not possible to obtain samples of all drug product components, it may be acceptable either to add know quantities of the analyte to the drug product (i.e., “to spike”) or to compare results with those of a second, well-characterized method, the accuracy of which has been stated or defined.

Accuracy is calculated as the percentage of recovery by the assay of the known added amount of analyte in the sample, or as the difference between the mean and the accepted true value, together with confidence intervals.

It is recommended that accuracy should be assessed using a minimum of nine determinations over a minimum of three concentration levels, covering the specified range (i.e. three concentrations and three replicates of each concentration).

2-PRECISION

Definition–The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of a homogeneous sample.  The precision of an analytical method is usually expressed as the standard deviation or relative standard deviation (coefficient of variation) of a series of measurements.

 Definition–The precision of an analytical method is the deter determination mine assaying a sufficient member of aliquots of a homogeneous sample to be able to calculate statistically valid estimates of standard deviation or relative standard deviation (coefficient of variation).  Assays in this context are independent analyses of samples that have been carried through the complete analytical procedure from sample peparation to the final test of the result.

It is recommended that repeatability should be assessed using a minimum of nine determinations covering the specified range for the procedure (i.e. three concentrations and three replicates of each concentration or using a minimum of six determinations at 100% of the test concentration.

3-SPECIFICITY

Definition – the ICH documents define specificity as the ability to assess unequivocally the analyte in the presence of components that may be expected to the present, such as impurities, degradation products, and matrix components.  Lack, The lack of specificity of an individual analytical procedure may be compensated by other supporting analytical procedures.

 4-DETECTION LIMIT

Definition – the detection limit is a characteristic of limit tests.  It is the lowest amount of analyte in a sample that can be detected, but not necessarily, under the stated experimental conditions.  Thus, limit tests merely substantiate that the amount of analyte is above or below a certain level.  The detection limit is usually expressed as the concentration of analyte (e.g. percentage, parts per billion) in the sample.

DETERMINATION – for non-instrumental methods, the detection limit is generally determined by the analysis of samples with known concentrators of analyte and by establishing the minimum level at which the analyte can be reliably detected.

For instrumental procedures non instrumental, the same method may be used as for non-instrumental.   5-QUANTITATION LIMIT

Definition – the quantitation limit is a characteristic of quantitative assays for low levels of compounds in sample matrices, such as impurities in bulk drug substances and degradation products in finished pharmaceuticals.  It is the lowest amount of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions.  The quantitation limit is expressed as the concentration of analyte (e.g. percentage, parts per billion) in the sample.

DETERMINATION – for noninstrumentalnoninstrumental methods, the quantitation limit is generally determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be determined with acceptable accuracy and precision.

For instrumental procedures, the same method may be used as for noninstrumental.

noninstrumental LiNEARITY AND RANGE

Definition of Linearity – the linearity of an analytical method is its ability to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to the concentration in for analyte in samples within a given range.

Definition of range – range of an analytical is the interval between the upper and lower levels of analyte (including these levels) that have been demonstrated to be determined with a suitable level of precision, accuracy, and linearity using the method as written.  The ranges are normally expressed in the same units as test results (e.g. percent, parts per million) obtained by the analytical method.

RUGGEDNESS

Definition – the ruggedness of an analytical method is the degree of reproducibility of test results obtained by the analysis of the same samples under a variety of conditions, such as different laboratories, different analysts, different instruments, different lots of reagents, different elapsed assay times, different assay time, different assay temperatures, different days, etc.  Ruggedness is normally expressed as the lack of influence on test results of operational and environmental variables of the analytical method.  Ruggedness is a measure of reproducibility of test results under the variation in conditions normally expected from laboratory to laboratory and from analyst to analyst.

Determination – The ruggedness of an analytical method is determined by theanalyanalysisysis of aliquots from homogeneous lots in different laboratories, by different analysts, using operational and environmental conditions that may differ but are still within the specified parameters of the assay.  The degree of reproducibility of test results is then determined as a function of the assay variables.  This reproducibility may be compared to the precision of the assay under normal conditions to obtain a measure of the ruggedness of the analytical method.

ROBUSTNESS

Definition – The robustness of an analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and indicates its reliability during normal usage.

Data Elements Required for Assay Validation

 Analytical Performance Characteristics

CALIBRATION:

Our contractor performs calibration and service of HPLC after a regular period

To ascertain the effectiveness of the final operating system, it should be subjected to a suitability test before use  Specific data are collected from replicate injections of the assay preparation or standard preparation.  These are matches to specified maximum and minimum values, such as efficiency internal precision, tailing factor, response.

The calculation is expressed by the following equation:

                                 SR(%) =          100      [     1 = 1        (X1  - X 2)       ]      ½

                                                                           N – 1

In which SR is the standard deviation in percent X, is the mean of the set of N

 X1 is an individual measurement.  The term X1 refers to the measurement of the peak response ratio, Rs (Where an internal standard is employed).

 

            Xi         =          Rs.       =          rs

                                                RI

RI which rs is the peak response corresponding to the reference stan,r, d, and RI RI, the peak 

response corresponding to the internal standard or the peak response or an external standard method.

 

COLUMN EFFICIENCY

Theoretically,membermber of plate counts is calculated by the following formula:

 

                        n          =          16 ( t )2  

                                                      w  

Where t  =  retention time measured from fronhehehe time of injection to the time the o elution of peak maximum.  W  =  width of peak measured by extrapolating the relating straight side to the baseline.

 

COLUMN PERFORMANCE

 

Column performance may be evaluated from the number of theoretical plates per meter (n) calculated from the expression:

 

                        n          =          5.54  V2R   

                                                 Ll Wh

 

Where VR =  retention volume for the component of interest.

 

L          =          length of the column in meters

Wh       =          the width of the peak of interest at half peak and the retention volume is

the distance along the basebaselineween the point of injection and a

perpendicular dropped from the maximum of the peak of interest.