Investigating Out of Specification Results Fiona McGuinness If you want to remove slide numbering follow these steps: View Master view - Slide master Master layout - Remove tick on Footer

Introduction What do we mean by OOS, OOT, atypical or unexpected results? Documented procedure Investigation What happens when you can’t find the cause? Examples Questions

What do we mean…? Out of Specification (OOS) Out of trend (OOT) Result that does not comply with stated accepted criteria Out of trend (OOT) A stability result that does not follow the trend of other stability results during a stability study A result that does not follow the trend based on data already collected with the same compound Atypical May be within specification but unexpected or questionable All unexpected results!

Documented Procedure Important that all laboratories have a fully documented OOS investigation procedure Clearly state what the investigation will be based on, what you will do in each case, actions and outcomes from the investigation Clients and regulatory agencies like to see a procedure and that it is being followed!

Initial Investigation First step is to QC Check the data generated to ensure that no identifiable calculation or preparation errors have been made Investigate the analysis…. Calibration standards, extraction volumes, dilution steps etc Instrument set-up, injection volumes, mobile phases, diluents, instrument working…

Re-dilution An investigative run Has a dilution error been made? What if dilution results are different? Don’t cherry pick results! Confirm results by a 3rd dilution!

Preparation of the Formulation Check the request, method, weights of test substance and vehicle as appropriate. Check calculations

Contingency Analysis No obvious analytical error (including dilutions), no obvious preparation error… Perform analysis of contingency samples

What happens when we can’t find the cause of an unexpected result? Meeting Root Cause Analysis investigation Corrective and Preventative Actions Analysis of next formulated occasion Discussion and agreement with senior members of department and SD

Fishbone

5 Whys

Analysed concentration (mg/ml) Example 1 Week of Formulation Group Nominal Analysed concentration (mg/ml)   RME % Difference from Mean inclusion Analysis 1 Analysis 2 Mean (%) (mg/ml) 13 1 ND - 2 0.75 2.6904 2.734 2.71 261.3 -0.80 0.80 3 1.5 1.4978 1.5041 1.50 0.0 -0.21 0.21 4 2.75 0.61669 0.60652 0.61 -77.7 0.83 -0.83 Group 2 samples are above the quantification range Group 4 samples are below the quantification range Obvious error?

Investigation Samples had been booked into the department in the wrong order Analyst followed the booking in rather than looking at the labels of the samples New samples were taken from the bulk sample provided by Pharmacy

Analysed concentration (mg/ml) Re-sampled Analysis Week of Formulation Group Nominal Analysed concentration (mg/ml)   RME % Difference from Mean inclusion Analysis 1 Analysis 2 Mean (%) (mg/ml) 13 1 ND - 2 0.75 0.72346 0.71577 0.720 -4.0 0.53 -0.53 3 1.5 1.4978 1.5041 1.50 0.0 -0.21 0.21 4 2.75 2.7186 2.5703 2.64 2.80 -2.80 All samples now within specification Original results for Groups 2 and 4 not reported due to an identified analyst error

Analysed concentration (mg/ml) Example 2 Week of Formulation Group Nominal Analysed concentration (mg/ml)   RME % Difference from Mean inclusion Analysis 1 Analysis 2 Mean (%) (mg/ml) 1 ND - 2 10 9.6428 8.1326 8.89 -11.1 8.50 -8.50 3 30 27.686 26.675 27.2 -9.3 1.86 -1.86 4 60 58.827 57.72 58.3 -2.8 0.95 -0.95 Investigation shows no calculation errors Re-dilution confirms the results No preparation errors found with formulation preparation

Analysed concentration (mg/ml) Contingency Analysis Week of Formulation Group Nominal Analysed concentration (mg/ml)   RME Precision % Difference from Mean inclusion Analysis 1 Analysis 2 Mean (%) from mean (mg/ml) 1 2 cont 10 9.9601 10.022 9.99 -0.1 0.44 -0.31 0.31 Contingency analysis showed that results were acceptable What should we do about the original analysis? Outlier test?

Outlier Test – Dixon’s Q Test   n 3 4 5 6 7 8 9 10 Q90% 0.941 0.765 0.642 0.560 0.507 0.468 0.437 0.412 Q95% 0.970 0.829 0.710 0.625 0.568 0.526 0.493 0.466 Q99% 0.994 0.926 0.821 0.740 0.680 0.634 0.598

Analysed concentration (mg/ml) Reportable Results 8.1326 mg/mL was rejected from the mean as an outlier with 90% confidence. Group Nominal Analysed concentration (mg/ml)   RME Precision % Difference from Mean inclusion 1 2 3 4 Mean (%) from mean (mg/ml) 10 9.6428 [8.1326] 9.9601 10.022 9.87 -1.3 2.06 -2.35 - 0.86 1.49

Example 3 UV Method – controls usually reportable as <LOQ One sample (from 3) showed a response >LOQ Full spectrum scan did not show a test item response No dilutions available to re-read Time sensitive as pre-dose analysis!

Contingency analysis The next 3 samples were read <LOQ Full scans were done of all controls = clear of test item response Rejected the initial sample result as anomalous as 5/6 controls were <LOQ Full investigation was not required as able to demonstrate the control group was clear by full spectrum scans (of all controls) and results of 5/6 controls

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