LAB-085 Laboratory Analytical Determinations

5.1 Preparation of Solutions

5.1.1 All analysts conducting the laboratory analytical analyses must prepare mobile phase, diluent / media as directed in the relevant testing document.  The mobile phase must be filtered through 0.45µm PTFE/HV membrane filter (or equivalent) and degassed unless otherwise specified in the test method.

5.1.2 Reference standard and sample solutions should be prepared by using “A” grade pipettes and volumetric glassware.

5.1.3 The actual amount of standards and samples weighed must not deviate more than ± 10% from the value stated in the test method.

5.1.4 Labelling of Glassware for Samples and Standards

Write batch details (e.g. product, batch number) on a laminated A4 sheet.

Place the sheet on top of the bench clearly visible for other people.

For each piece of glassware enter the last 2 numbers of the batch number, sample number, initial and date.

5.1.5 If conducting a Composite Assay, Degradation, Content Uniformity (CU) or Dissolution analysis, prepare 2 reference standard solutions (Working Standard and Check Standard).

Exceptions to this are:

5.1.5.1 Methods that require a calibration curve (e.g. three different standard concentrations) to calculate the samples.

5.1.5.2 Degradation methods that require the reference to be prepared as a dilution from the sample solution (e.g. pharmacopoeia degradation methods were peak area comparison is used as calculation).

5.1.6 If conducting a Composite Assay or Degradation analysis, prepare the sample solutions as directed in the following:

5.1.6.1 For In-Process and Finished Product Testing (except for Blend testing refer to Section 5.1.6.2):  2 sample solutions must be prepared for each batch / lot unless otherwise directed in the relevant testing document.

5.1.6.2 Product Blend Sample Testing:  Only one sample solution should be prepared for each sample to be analysed unless otherwise specified in a Laboratory Analytical Testing Report for Non Standard Testing Form (Form-715) or as required for laboratory investigation requirements.

5.1.6.3 For Raw Material Testing:  2 sample solutions must be prepared if there is one sample or one pooled sample to be analysed.  Only one sample solution should be prepared if there are 2 or more than 2 samples or pooled samples to be analysed.

5.1.6.4 For Stability Testing:  Sample preparation as described in the relevant test method.  If not outlined in test method 1 sample preparation is carried out.

5.2 System Operation

Note: The analyst must complete the relevant HPLC, UV-VIS or other instrument logbook as well as the associated HPLC column logbook, if applicable, as per SOP.

5.2.1 The analyst must set up and operate the instrument:

5.2.1.1 Setup and operation of the HPLC instrument must follow appropriate SOP

5.2.1.2 Setup and operation of the UV-VIS Spectrophotometer must follow appropriate SOP

5.2.1.3 Setup and operation of any other instruments must follow the relevant SOP(s).

5.3 Guidelines for SST, Test Sequence and Standard Bracketing

In order to show that the system is operating in a stable condition, the analyst conducting the HPLC and UV-VIS Spectrophotometer Analyses must follow the guidelines for SST, test sequence and standard bracketing as detailed in the following sections.

The system suitability criteria outlined in this document and in the relevant test method must be met before sample injections are allowed to be run.  This is required to avoid LIR’s (Laboratory Investigation Reports) because of failed system suitability results.

The analyst needs to consult the relevant Manager in cases where the system suitability injections cannot be finished and checked prior to leaving for the day.

5.3.1 HPLC SST Guidelines

5.3.1.1 HPLC system must be setup under the conditions specified in the analytical method.

Minor adjustments may be required in order for a system to pass system suitability requirements.  However, allowable adjustments are only those that have been documented in the test method validation (i.e. robustness parameters).

For methods that don’t have robustness included, the following can be followed.

Adjustment of the composition of the mobile phase:

The amount of the minor solvent component may be adjusted by ± 30% relative or 2% absolute, whichever is the larger.  No component is altered by more than 10% absolute (BP).

Example:  20% Acetonitrile / 80% Buffer Solution ± 30% relative adjustment of 20% Acetonitrile is ±6%

This is larger than ±2% absolute adjustment and lower than 10% absolute adjustment. Therefore the maximum allowed change for Acetonitrile is ±6%.

5.3.1.2 Test injection(s) of blanks, resolution solutions and working standards can be performed prior to the start of the SST analysis to ensure proper retention time and column equilibration are achieved.

5.3.1.3 HPLC SST must be run prior to injecting samples, or when a significant change occurs.  A significant change includes:

a. Fresh mobile phase or a new column.

b. Turning off any component of the HPLC system.

c. Breaking any plumbing connection.

Changing the detector wavelength(s) established in system suitability. Note: The maximum gap between the completion of the SST and the start of analysis is defined as 8 hours if the instrument keeps running.

5.3.2 HPLC Test Sequence and Standard Bracketing Guidelines

5.3.2.1 The Laboratory Analyst must perform the following test sequence unless otherwise specified in the relevant test document.

Refer to Table 1

Continue injecting samples to completion ensuring all samples are bracketed by 4 working standard injections.  Ensure there are no more than 12 sample injections between bracketing standards.

5.3.2.2 Samples are injected in singular.

5.3.2.3 Sample injections must be bracketed by 4 working standard injections.

5.3.2.4 To ensure that the system is operating in a stable manner, a standard solution must be injected after no more than 12 injections of samples, or every 8 hours (whichever occurs first) and again at the end of the sequence.

5.3.2.5 For external standard analysis, the average peak response (peak area or peak height as stated in the test method, if not specified in test method peak area should be used) of the 4 injections of bracketing standard should be used to calculate the sample results.

5.3.2.6 For internal standard analysis, the average ratios of peak response (peak area or peak height as stated in the test method) of 4 injections of bracketing standard will be used to calculate the sample results.

5.3.3 HPLC SST Requirements

5.3.3.1 Using the chromatographic software, calculate the SST results.

5.3.3.2 Blank Injection

a. One blank injection is carried out to show that the system is clean and no carry over peaks are present from a previous run.

b. No peak should elute with the same retention time as the peaks of interest (main active peak, degradation products).

c. If a peak elutes in the blank chromatogram it will be compared to the peak with the same retention time of the standard chromatogram.

d. Check for acceptance criteria.

5.3.3.3 Unless otherwise specified in the relevant testing document, the criteria in Table 2 must be met for the %RSD of the peak response (area or height) and retention time for the 6 injections of the Working Standard.  The test samples should not be run unless all the acceptance criteria have been met.  For internal standard the peak ratios of the peak response (area or height) will be used.

5.3.3.4 All other system suitability parameters specified in the relevant test document, e.g. plate count, tailing factor and resolution, must also be met.  If any of the above SST requirement(s) is/are not met, the Team Leader or designee must be informed.

Refer to – Table 3

5.3.3.5 Unless otherwise specified in the relevant testing document, the criteria in the Table 3 must be met for the % Ratio between the working standard and the check standard using the calculation below:

% Ratio =

Response Check Std (1 injection) x Weight Working Std x 100% / Response Working Std (average 6 injections) x Weight of Check Std

5.3.3.6 The four bracketing standards must be assessed and must meet the acceptance criteria outlined in Table 2 for the peak response (area or height).

5.3.4 UV-VIS Analyses Guidelines

5.3.4.1 Unless otherwise specified in the test method the following procedure needs to be followed

5.3.4.2 Test sequence, refer to Table 4.

Note: The instrument must be blanked by the diluent or media prior to the UV-VIS analysis.

Continue reading samples to completion ensuring all samples are bracketed by 4 working standard readings.  Ensure there are no more than 12 sample readings between bracketing standards.

5.3.4.3 The analyst conducting UV-VIS spectrophotometer analyses must conduct SST prior to an UV–VIS analysis.

5.3.4.4 Samples must be read singly and must be bracketed by 4 working standard readings.

5.3.4.5 The average absorbance response of the 4 bracketing standards will be used to calculate the sample results.

5.3.4.6 The criteria in Table 5 must be met for the %RSD of the absorbance response for the 6 replicates of the Working Standard.

5.3.4.6 The criteria in Table 6 must be met for the %RSD of the absorbance response for the 6 replicates of the Working Standard.

5.3.4.9 All other system suitability parameters specified in the relevant test document must also be met.  If any of the above SST requirement(s) is/are not met, the Team Leader or designee must be informed.

5.4 Other Analyses

5.4.1 Proceed as per the relevant testing document in conjunction with relevant instrument operation procedures.

5.4.2 Labelling of TLC Plates

The spots are numbered on top of the TLC plate to make sure the TLC plate coating is not damaged before the plate is developed.  The key explaining the numbers is written in the workbook / worksheet at the time the plate is spotted.

5.4.3 Examination of TLC Plates Using a Spray Reagent

As spots may fade between spraying and taking the picture the following procedure needs to be followed.

As soon as the TLC plate is sprayed a second analyst needs to verify the TLC plate for any results obtained.

Results must be written into workbook / worksheet and co-signed by second analyst.

A photograph will then be taken.

5.5 Calculation

The analyst must perform the following calculations to establish the SST requirements:

5.5.1 The statistical calculation

a. Average / Mean (Ave) =   X / n

b. Standard Deviation (SD) = Root sign  å (X – Ave)2 / n – 1

c. Relative Standard Deviation (%RSD) = SD / Ave x 100 6.0