The 8th Mass Spectrometry CVG Annual Symposium - Full Day Event!!!
Presentation No. 11 - Systematic and Expanded Investigation of High and Unexpected Positive Deviation for QC Samples during GLP Incurred Samples Analysis by LC-MS/MS
Georges El-Kadissi; Mireille Nohra; Natasha Savoie; Ericka Franco; Véronik Gill; Milton Furtado; Chantal Menard; Mary Carbone; Fabio Garofolo
Algorithme Pharma Inc., Laval (Montreal), Quebec , CANADA
Novel Aspect: Importance of structured investigation by using of multiple reference standards to explain the salient high deviation for quality control samples.
Introduction: Montelukast is used in the treatment of prophylaxis, chronic asthma and seasonal allergic rhinitis. A bioanalytical method, able to quantify Montelukast, was successfully validated in human plasma. During incurred samples analysis, some high positive deviations for quality control samples were noticed. Consequently, an investigation was started. A preliminary investigation was first conducted to eliminate all general possible causes to this phenomenon due to error or oversight during sample preparation. However, as no assignable cause could be identified following this preliminary investigation, an expanded investigation was started. Hypotheses were listed: 1) Matrix effect causing response enhancement, 2) contamination of stock solutions, 3) solubility problems and impurities/alteration in reference standards. Then, a series of confirmatory investigations were conducted.
Methods: Montelukast-d6 was used as internal standard (IS). Montelukast method employed protein precipitation extraction using MeOH/Acetone 50/50%v/v. The extracted was injected on a Agilent Technologies 1100 pumps and autosampler coupled to a AB/Sciex-API3000 MS/MS equipped with heated nebulizer source in positive mode. The gradient mobile phase consisted of 0.1% HCOOH and Acetone on a Zorbax, SB-C18, 5µ, (50X2.1)mm. Montelukast and Montelukast-d6 (IS) eluted at approximately 0.83 and 0.82 minute, respectively. During validation, the method showed an intraday precision range of 2.9% to 4.3% and an accuracy range of 96.1% to 112.5% and an interday precision range of 3.8% to 7.0% and accuracy range of 95.1% to 113.9%.
Results: Verification of the first hypothesis (Matrix effect) yielded no significant enhancement from the matrix. Prior to sample analysis, matrix effect testing was conducted during method validation with different donors and no matrix effect was observed. Results from several tests suggested a problem with a particular stock solution (not due to contamination) used to prepare the quality control samples and/or the reference standard lot-B used to prepare this particular stock solution. Several tests were rigorously designed to investigate solubility problems and impurities/alteration in the reference standard. A new lot-C of reference standard was ordered and compared with reference standard lot-A used during method development and validation and with reference standard lot-B used during sample analysis. Results demonstrated a solubility problem of reference standards lots B and C in MeOH:H2O 50/50 % v/v for a period of time exceeding 2 days versus reference standard lot-A which was perfectly soluble in this solvent. During method development, reference standard lot-A showed acceptable solubility in MeOH:H2O 50/50 % v/v versus other solvents. Reference standards lots B and C were more soluble in pure MeOH. After further investigations on the synthetic procedures used to prepare the different lots, it was discovered that the reference standard lot-A went through an extra step of purification which explains the differences in solubility with lots B and C. As a remedial action, stock solutions prepared in MeOH:H2O 50/50 % v/v were only used between 0 and 2 days. However, as a long term corrective action, the solvent was changed to MeOH and the method went through a partial validation. In conclusion, rigorous tests, meticulous trend analysis and mainly investigating different lots of reference standard were able to reveal a severe and unusual analytical phenomenon that was not definitively apparent during method development and validation.
