Evaluation of 384-well formatted sample preparation technologies for regulated bioanalysis

Area of Science:

• Pharmacokinetics and Drug Metabolism
• Analytical Chemistry
• Bioanalytical Method Development

Background:

• Regulated bioanalysis requires robust and efficient sample preparation techniques.
• Traditional 96-well formats present limitations in throughput for high-volume studies.
• The 384-well format offers potential for increased efficiency but requires careful evaluation for bioanalytical applications.

Purpose of the Study:

• To assess the capabilities and limitations of 384-well sample preparation technologies in regulated bioanalysis.
• To develop and validate assays for simultaneous quantitation of lopinavir and ritonavir using 384-well liquid-liquid extraction (LLE) and solid-phase extraction (SPE).
• To critically evaluate cross-contamination, sensitivity, assay throughput, and ruggedness of the 384-well format.

Main Methods:

• Two assays were developed: one using LLE and another using SPE, adapted from validated 96-well assays to a 384-well format.
• Simultaneous quantitation of lopinavir and ritonavir in plasma samples was performed using column-switching high-performance liquid chromatography with tandem mass spectrometric detection (HPLC/MS/MS).
• Cross-contamination was assessed by including potential contaminants (saquinavir or amprenavir) in adjacent wells.

Main Results:

• Both LLE and SPE methods in the 384-well format achieved the same lower limit of quantitation and assay range as the original 96-well assays.
• Critical evaluation showed minimal cross-contamination between adjacent wells in the 384-well plate.
• Sample preparation in the 384-well format, when combined with automation, demonstrated up to five times greater efficiency compared to the 96-well format.

Conclusions:

• The 384-well format is suitable for regulated bioanalysis, offering significant throughput advantages over the 96-well format.
• LLE and SPE sample preparation techniques are effectively transferable to the 384-well format with minor modifications.
• The 384-well format maintains assay sensitivity and ruggedness while minimizing cross-contamination risks, making it a viable option for high-throughput bioanalytical studies.