Increasing high‐throughput Discovery bioanalysis using automated selected reaction monitoring compound optimization, ultra‐high‐pressure liquid chromatography, and single‐step sample preparation workflows

Abstract

QuickQuan is an integrated software package for Thermo Scientific triple quadrupole mass spectrometers that allows users to automate routine operations ranging from method development to data processing. QuickQuan automated optimization of compound-selected reaction monitoring (SRM) transitions by evaluating both positive and negative polarities during an infusion. Whichever mode produces the most intense Q1 scan is then carried to product ion spectra. QuickQuan then writes these SRM methods to a shared network database. The total volume of compound needed is 100 microL infused over approximately 1.6 min. The auto-optimization is carried out in 96-well plates and does not require an operator present. The SRM database was shared between two identical TSQ Quantum mass spectrometers. For data acquisition, QuickQuan automatically created a sequence file complete with a data processing method pre-populated with compound IDs and corresponding SRM transitions. To increase throughput we coupled each Finnigan Quantum with ultra-high-pressure liquid chromatography (uHPLC) accomplished using 4x Ultra Flux quaternary pumps that were designed to handle pressures up to 15 000 psi. The total run time for all analyses was 1.2 min using BEH 1.7 microm particle C18 columns. Further time reductions were realized with sample preparation accomplished using Strata Impact protein precipitation plates which provided an in-well protein crash and 0.20 micron filtering in a one-step process. Pharmacokinetic data turnaround time was significantly improved by combining these three techniques of automated method development with the speed efficiency of uHPLC and a single step in well sample preparation.

Ultrafast mass spectrometry based bioanalytical method for digoxin supporting an in vitro P‐glycoprotein (P‐gp) inhibition screen

“…[28] Smalley et al suggested the requirement of a minimum of 10 data points across a chromatographic peak for adequate bioanalytical quantitation with UPLC-MS/MS in a discovery setting. [29] In our current method, the RF-MS/MS peaks for both digoxin and the internal standard were approximately 2 s wide at base. By using an SRM dwell time of 70 ms each for digoxin and internal standard, we ensured that more than 13 data points were obtained across the peak to achieve reliable quantitation, as demonstrated by the accurate and precise data presented in Tables 1-4 and Fig.…”

Development of a Highly Sensitive Methodology for Quantitative Determination of Fexofenadine in a Microdose Study by Multiple Injection Method Using Ultra-High Performance Liquid Chromatography with Tandem Mass Spectrometry

“…21) The flow injection analysis was conducted using a standard solution of fexofenadine and the Automaton ® software for automated optimization of SRM to obtain the precursor and product ions of fexofenadine. 26,27) The precursor ion of fexofenadine was m/z 502 of protonated ions [M+H] + . The product ion at m/z 466 represented the highest ion intensity in the product ions from m/z 502; moreover, the product ion at m/z 171 was detected (Fig.…”

Analytical procedures for the determination of the selective serotonin reuptake inhibitor antidepressant citalopram and its metabolites

“…The market entry of the techniques of ultra‐high‐pressure liquid chromatography (UHPLC or UPLC) has managed to reduce the time analysis and the effectiveness of the separations. Thus, methods have been developed for the determination of CIT and other compounds by UPLC‐MS‐MS with a total run time for all analyses of 1.2 min using BEH 1.7 micron particle C 18 columns (Smalley et al ., 2009). Similarly, using Q‐TOF mass analyzers, Nielsen et al .…”