Bioanalysis cycle time

The use of high flow and fast gradient HPLC has gained a lot of popularity because of the ability to reduce LC/MS/MS cycle times during bioanalysis. In the case of fast gradient HPLC, peak shapes were improved and method development times were minimized, especially when multiple analytes with diverse functionalities had to be separated. Flows as high as 1.5 to 2 mL/min were achieved on a 2.1 x 30 mm Xterra C18 column.7 Details are discussed in a recent review.8... 

Because the instability of the N-oxide metabolite, which was subjected to decomposition during sample preparation (solvent evaporation during offline SPE), online SPE LC/MS became the method of choice for the application. Hsieh et al. (2004) built a system with two TFC cartridges and one analytical column, and another system with two TFC cartridges and two analytical columns for GLP quantitative bioanalysis of drug candidates. A Turbo C18 (50 x 1.0 mm, 5 /.mi, Cohesive Technologies), an Xterra MS C18 (30 x 2.0 mm, 2.5 /mi), and a guard column were used. Protein precipitation preceded injection. The cycle times for the two systems were 0.8 and 0.4 min. 

The use of higher chromatographic flow rates has become an increasingly accepted technique to decrease the HPLC-MS cycle time during assay validation and bioanalysis. 

At the same time, the bioanalysis of LOR and DCL in rat, rabbit, mouse, and dog plasma was reported by others [64]. In order to get more rehable toxicology data, the bioanalysis in these four preclinical species is done simultaneously instead of on separate days. The sample pretreatment was SPE in a 96-well plate format, using a Tomtec Quadra hquid handling system and an Empore Cig 96-well extraction disk plate. Fom-channel parallel LC was done with four 100x2-mm-lD Cg colunms (5 pm) and a mobile phase of 85% methanol in 25 mmol/1 aqueous AmOAc (adjusted to pH 3.5). The mobile phase was delivered at a flow-rate of 800 pl/min and split into 200 pl/min over each of the four colunms. A multi-injector system was apphed with four injection needles. A post-column spht was applied to deliver 60 pEmin per column to a four-channel multiplexed ESI source (Ch. 5.5.3). The interspray step time was 50 ms. Positive-ion ESI-MS was performed in SRM mode with a dwell time of 50 ms for each of the four transitions, i.e., LOR, DCL, and their [DJ-ILIS, with 20 ms interchannel delay. The total cycle time was thus 1.24 s. The LOQ was 1 ng/ml for both analytes. QC samples showed precision ranging from 1 to 16% and accuracy from -8.44 to 10.5%. The interspray crosstalk was less than 0.08% at concentrations as high as 1000 ng/ml. 

Because of the serial nature of LC-MS, much of the current discussion has centered on ways to reduce LC-MS/MS cycle time. Often the rate-limiting step for drug-discovery bioanalysis lies in the speed with which methods (sample preparation and chromatography) can be prepared for NCEs. One of the frequently overlooked steps is the need to tune and optimize MS/MS transitions for the various analytes studied. Fortunately, most MS vendors now offer semi- or fully automated procedures to perform this task. The origin for these procedures can be traced to the seminal work of Whalen et al., who published an automated procedure known as AUTOSCAN [106]. Itis possible to establish experimental conditions with this procedure in the flow-injection analysis (FIA) mode for 96 analytes in less than one hour. 

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