Increased Sample Throughput For Ketoconazole Analysis By Automated 96-well Sample Preparation And Multiplexed Hplc

K. Szczap, C. Loop, D. Gray, H. Freiser, R. Shoup, A. Witkowski
BASi Analytics, 2701 Kent Avenue, West Lafayette, IN, 47906, USA

ABSTRACT

Purpose

In order to increase the sample throughput of a manual LC-FL assay for ketoconazole, the method was automated and a multiplexed HPLC system was employed.

Methods

The original ketoconazole method involved manual protein precipitation, followed by HPLC-FL analysis with a run time of approximately 15 minutes. The sample preparation was converted to the 96 well format and automated using the Tomtec Quadra96. In addition, a multiplexed HPLC system was configured to cut the HPLC run times in half. The LC configuration enabled alternate sample injections to travel down one of two HPLC columns. The injections were staggered such that the eluting peaks could be diverted to a single FL detector.

Results

The resulting assay was fully validated and readily met ±15/15/15% acceptance criteria. Based on the assay's performance statistics, the automated method was found to be robust, even though two separate analytical columns were used for the same batch. Additional data using high-flow HPLC on a monolithic rod column also looks very promising.

EXPERIMENTAL

Matrix: 250 µL heparinized human plasma

HPLC Column: Waters Symmetry C18 (150 x 3.9 mm) with Javelin BDS C18
precolumn (20 x 3 mm) or Chromolith Performance RP-18e column (100 x 4.6
mm)

Mobile Phase: 40% ACN : 60% 50 mM Phosphate Buffer, pH 7

Detector: Hitachi L-7480 fluorescence detector. λ_ex = 260 λ_em = 375

Validated Range: 0.2 to 50 µg/mL

Manual Sample Prep: Protein precipitation by combining 250 µL heparinized human plasma and 500 µL acetonitrile. Vortex mix, then centrifuge for 10 min. Transfer 500 µL of the supernatant to an autosampler vial, dilute with an equal volume of water, vortex mix, then inject 100 µL.

96-Well Sample Prep: Aliquot 250 µL of each sample into a 1.2 mL 96-well plate. The Tomtec Quadra96 adds 500 mL acetonitrile. Cap plate, vortex, and centrifuge for 5 min. The Tomtec then transfers 250 µL of the supernatant to a fresh plate, dilutes with an equal volume of water, and mixes. Centrifuge plate for 5 minutes, apply foil seal, then inject 100 µL.

Multiplexing Configuration: An autosampler alternately injects onto one of 2 columns via a switching valve. Another synchronized switching valve selects one column to the detector and one column to waste. (see schematic) Since the peak of interest elutes near the end of the chromatogram, the first half of each run is diverted to waste.

RESULTS

Throughput Comparison

Configuration	Sample Prep	LC Run	Total time
Manual - Sequential	1.5 hr	26.0 hr	27.5 hr
TomTec - Multiplexed	0.5 hr	13.0 hr	13.5 hr
TomTec - Chromolith	0.5 hr	6.0 hr	6.5 hr

Manual - Sequential

Nominal Concentration (ng/mL) Average Concentration (ng/mL) Standard Deviation Precision (%) Accuracy (%) N

40000 40279 763 1.9% 100.7% 18

20000 20356 561 2.8% 101.8% 18

600 623 36.0 5.8% 103.9% 18

TomTec - Multiplexed

Nominal Concentration (ng/mL) Average Concentration (ng/mL) Standard Deviation Precision (%) Accuracy (%) N

40000 40424 961 2.4% 101.1% 18

20000 20134 334 1.7% 100.7% 18

600 598 17.5 2.9% 99.6% 18

TomTec - Chromolith

Nominal Concentration (ng/mL) Average Concentration (ng/mL) Standard Deviation Precision (%) Accuracy (%) N

40000 39319 1151 2.9% 98.3% 18

20000 18997 341 1.8% 95.0% 18

600 627 27.5 4.4% 104.6% 18

CONCLUSIONS

• Use of the Tomtec Quadra96 and multiplexed HPLC allows significant time savings without a loss of quality.
• Although typically associated with LC/MS/MS assays, multiplexed HPLC is useful with other detectors as well.
• The Chromolith HPLC column appears to provide definite speed advantages without sacrificing chromatographic performance.
• The data demonstrate the ability to injection a single batch onto two HPLC columns without compromising the assay.