Good Laboratory Practices (GLP) Part-3 : Pharmaceutical Guidelines

Good Laboratory Practices (GLP) Part-3

GLP about TOC analyzer, Friabilator, Statistical tools for evaluation of data, Microbiology testing, Records & Reports

·    TOC Analyzer

Ø  Ensure that the instrument is in calibrated condition before use.
Ø  Ensure all the connections of the instrument are proper before use.
Ø  Open carrier gas used for TOC (Zero air cylinder) with cylinder pressure and gas line pressure maintained at 4-6 Kg/cm2.
Ø  The pressure of the carrier gas i.e. Zero air should be 2 Kg/cm2 (200 kpa) and flow should be 150 ml/min. Adjust if necessary by opening the front door and adjusting gas pressure controller and mass flow controller.
Ø  Ensure level of the water in the humidifier should be kept between two marks shown on the bottle i.e. between (High and Low mark).
Ø  Get the raw data (printout) of the analysis checked.

Related: Calibration of TOC (Total Organic Carbon) Analyzer

·    Disintegration test Apparatus

Ø  Ensure that the instruments in calibrated condition before use.
Ø  Ensure all the connections of the instrument are proper before use.
Ø  Ensure, the bath is filled with water up to the level marked (without beakers)
Ø  Set the temperature to 37oC unless otherwise specified.
Ø  Clean the basket rack assembly, beakers, and discs after use.

Related: Calibration of Disintegration Test Apparatus

·    Friabilator

Ø  Ensure that the instrument s in calibrated condition before use.
Ø  Ensure all the connections of the instrument are proper before use.
Ø  For tablets with a unit weight equal to or less than 650 mg, take a sample of whole tablets corresponding as near as possible to 6.5 g. For tablets with a unit weight of more than 650 mg, take a sample of 10 whole tablets.
Ø  The tablets should be carefully dedusted prior to testing.
Ø  Accurately weigh the tablet sample, and place the tablets in the drum. After testing remove the tablets. Remove any loose dust from the tablets as before, and accurately weigh.
Ø  After conducting friability test, weighing of the tablets shall be done within 15 minutes.

Related: Calibration of Friability Test Apparatus

·    Statistical tools for evaluation of data

Ø  Mean and Standard deviation:

Mean (arithmetic mean) is given by the formula
            X1 + x2 + x3 + ……. xn-1 + xn
X   =     ---------------------------------------
Standard deviation (S), is given by the formula
                                ∑ (x - x)2
                      S =  -----------------
Relative standard deviation (RSD), is given by formula
               RSD = -------

Ø  Comparison of results

The comparison of the values obtained from a set of results with either the true value or with other sets of data makes it possible to determine whether the analytical procedure has been accurate and / or precise, or if it is superior to another method. There are two common method for comparing results, Student’s t-test and the variance ratio test (F-test).
Student’s t-test
      This is the test used for small samples; its purpose is to compare the mean from a sample with some standard value and to express some level of confidence in the significance of the comparison. It is also used to test the difference between the means of two sets of data x1 and x2.
The value of t is obtained from the equation
                     (x- µ)
              T  =  ----------
Where µ is the true value.
This is used to compare the precisions of two sets of data, e.g. the results of two different analytical methods or the results from two different laboratories. It is calculated from the equation
F   =     ------------------
The larger value of S is always used as the numerator, so the value of F is always greater than unity.
Ø  Comparing the means of two samples
When a new analytical method is being developed it is usual practice to compare the values of the mean and precision of the new (test) method with those of an established (reference) procedure.
The value of t when comparing two sample means x1 and x2 is given by the expression
                                         x1- x2
                               t  =  ------------------------------
                                  Sp  1/n1 + 1/n2
Where Sp, the pooled standard deviation, is calculated from the two sample standard deviations S1 and S2, as follows:
               (n1-1)s21 + (n2-1)s22
   Sp  =     --------------------------------
                     n+ n- 2
Note that there must not be a significant difference between the precisions of the methods. Hence the F-test is applied before using the t-test in equation.
When using instrumental methods it is often necessary to carry out a calibration procedure by using a series of samples (standards) each having a known concentration of the analyte to be determined. A calibration curve is constructed by measuring the instrumental signal for each standard and plotting this response against concentration. Provided the same experimental conditions are used for the measurement of the standards and for the test (unknown) sample,  the concentration of the test sample may be determined from the calibration curve by graphical interpolation.
Correlation coefficient
To establish whether there is a linear relationship between two variables x1 and y1, use Pearson’s correlation coefficient r :
                                         n∑ x1y1 - ∑x1∑y1
                         r   =   ---------------------------------------------------------
                                  {[n∑x21 – (∑x1)2][n∑y21 –(∑y1)2]}1/2
Where n is the number of data points.
The value of r must lie between +1 and -1; the nearer it is to ± 1, the greater the probability that a definite linear relationship exists between the variables x and y; values close to +1 indicate positive correlation and values close to -1 indicate negative correlation. Values of r that tend towards zero indicate that x and y are not linearly related (they may be related in a non-linear fashion).

·    Microbiology testing

Ø  Use correct incubation period and temperature specified in the method of analysis.
Ø  Ensure that the growth promotion test is performed for the media to be used for analysis.
Ø  Take all required precautions while handling the Microbiological cultures.
Ø  Ensure that the Microscope is calibrated and working properly.
Ø  Ensure that all the glassware's used for microbial analysis are decontaminated properly.
Ø  Culture / suspending / sub culturing should be traceable.

Related: SOP for Calibration of Microscope

·    General precaution


Ø  Crosscheck the exactness of instrument method / program & sequence with respect to the method of analysis.
Ø  Crosscheck the exactness of sequence with respect to the samples loaded in auto sampler.
Ø  Always compare response obtained with previous data as a tool of crosscheck.
Ø  Use correct excel sheet for calculation.
Ø  Avoid glassware breakage. Upon breakage of any glassware intimate to your respective supervisor so that the requirement for the same can be raised to avoid shortage of glassware.
Ø  Keep the allocated samples in the respective designated area during and after analysis duly labeled.
Ø  Record the temperature and humidity of the areas daily once and intimate to immediate supervisor if any excursion is observed for more than half an hour.

·    Interpretation

Ø  Interpretation should be aligned with the requirements in the method of analysis.
Ø  Interpretation should be done by considering the category of the method e.g. Related substances, assay etc.
Ø  Do not use manual integration events for quantification method unless otherwise specified in the method.
Ø  Compare the chromatographic data with specimen chromatogram.
Ø  If the results are not as per trend then inform immediately to immediate supervisor.

·    Records & Reports

Ø  Record the values & results online.
Ø  Check the entered values & figures for correctness online.
Ø  Calculate the results using correct formula (as per method of analysis) & recheck once online.
Ø  Do not overwrite the wrong entry. Cross it out with a line permitting the reading of original entry. Clearly write the correct entry near the cross out & sign the data along with the date on which correction is made.
Ø  Do not leave the blank spaces. Draw a line across the page from left to right.
Ø  All the document entries shall be made with indelible black ink in clear & legible hand writing.
Ø  Columns or rows not required shall be marked as ‘N.A.’ or ‘—’.
Ø  Validate excel sheet whenever required for calculation of results of CU/BU & dissolution (Profile/ Single point).
Ø  Use correct path of validated excel sheet for calculation of results.
Ø  Ensure that the usage and consumption entry is made in the respective instrument and column log book and working standard log book respectively.
Ø  Ensure that the respective index is updated when any SOP/ Document is created / revised.

Related: Validation of excel calculation sheets

Also see:

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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