Increased Assay Robustness And Throughput Using Automated 96-well Solid Phase Extraction

S. Das, E. Fisher, T. Grever, and B. Burras
Bioanalytical Systems, Inc.West Lafayette IN, USA

ABSTRACT

Sample throughput and reproducibility for Olanzapine was increased using automated 96-well solid phase extraction. A manual solid phase extraction was transferred to the 96-well format using the Tomtec Quadra 96 . Sample volume was reduced from 1mL to 0.75mL. Prior to loading samples, the SPE plate is conditioned with methanol and phosphate buffer. After sample loading, the plate is rinsed with phosphate buffer and a wash solution before eluting with a basic elution solution. A successfully validated method transfer was obtained. The resulting assay was more sensitive, selective, accurate and precise than the manual method. Inter-assay precision and accuracy by the automated method ranged from 3.2% to 5.6%, and 1.4% to 1.9% respectively. Inter-assay precision and accuracy for the manual method ranged from 1.7% to 10.0%, and 1.6% to 3.7% respectively. The automated assay drastically reduces sample preparation time (at least a 3-4-fold decrease) decreases labor costs and increases throughput in clinical studies. Automation reduces the tedious nature of manual extraction and also decreases the incidence of lower IS recovery. This in turn leads to better batch to batch reproducibility.

PURPOSE OF AUTOMATION

  • The primary purpose of automation was to reduce the time involved with sample preparation
  • Automation will aid in reducing the cumbersome nature of solid phase extraction
  • In addition it will reduce labor costs and increase throughput in clinical studies

METHOD

Validation scheme for transferring the manual method to the automated method

  • Fresh calibration line
  • QCs at the limits of detection (n = 6)
  • 3 day inter-assay precision and accuracy

Comparison of General Assay Procedure

Manual Assay
Automated Assay
  • Sample volume: 1mL
  • Uses 130mg Bond Elut Certify
  • Erratic recovery for both drug and ISTD causing variability in the lower points of the calibration line
  • Line in singlet. Low, Mid and
  • High QCs
  • Sample volume: 0.75mL
  • Uses 10mg Oasis MCX
  • Recovery tracks well throughout the batch. Overall there was a better recovery for both drug and ISTD
  • Line in duplicate. QCs at the limits of detection

General Assay Procedure - Automation

  • Load Samples (calibrators, QCs, unknowns) onto 96-well plate
  • Add ISTD before loading it onto the automated platform
  • Methanol and buffer are used to condition the SPE plate. Samples diluted with buffer and subsequently loaded onto the SPE plate
  • Plate is rinsed with buffer and a wash solution before eluting with basic elution solution
  • Evaporate the organic and reconstitute before injecting onto HPLC system using a 96-well autosampler

Calibration Standard Statistics
(automated method)

 

Calibration Standard Statistics
(manual method)

 

Inter-Assay Quality Control
Sample Statistics
(automated method)

 

Inter-Assay Quality Control
Sample Statistics
(manual method)

 

Assay Specifics

  • Sample Volume: 750 ┬ÁL
  • Sample Preparation: Solid Phase Extraction
  • Validated Range: 0.250 - 100 ng/mL
  • Column: YMC Basic column
  • Mobile Phase: 75 mM PO buffer/MeOH/ACN 48/26/26 v/v/v
  • Quantitation: Linear regression with 1/concentration weighting.
    Quantitation by peak height ratio
  • Detection: BASi LC - 4C Electrochemical Detector

Representative 100ng/mL
Calibrator Chromatogram

 

Representative Blank Chromatogram

Assay throughput increased using automation
100 samples take about 6-7 hours to extract manually
400 samples take about 6-7 hours to extract using automation

Results

  • Automation improves precision and accuracy of the method
  • Changing to a polymer based cation exchanger SPE column eliminates the secondary silanol interactions and improves recovery for both drug and ISTD

Conclusion

  • Automation drastically reduces sample preparation time (3 - 4 fold decrease) and reduces the tedious nature of manual extractions
  • Changing from silica to polymer based SPE gives better batch to batch reproducibility
  • Better recoveries are obtained for both drug and ISTD improving low end robustness
  • Automating the assay helps with increasing throughput in clinical studies