Increasing Throughput in Hplcmsms

Several reports have discussed the utility of increasing the speed of HPLC-MS/ MS by using various techniques [6, 28, 72, 86-96]. Typical strategies make use of advances in chromatographic columns. For example, Hsieh et al. [97] describe the use of small HPLC columns and a fast HPLC gradient to provide a method for assaying a compound in a discovery PK study in about one minute per sample. In a report by Tiller and Romanyshyn [98], the authors stated that fast gradients were generally better than isocratic HPLC systems at keeping the HPLC column clean. Recently, Dunn-Meynell et al. [99] reported on the development of a fast generic HPLC method that could be used for discovery PK studies; the method was tested specifically for its ability to be used for CARRS samples. The method used a generic 1-min ballistic gradient and an optimized autosam-pler method that results in an 85-s cycle time (time from injection to injection). Using this methodology, one could assay a set of 96 samples in less than 2.5 h.

Another area of some interest has been the development of monolithic HPLC columns. These columns are unusual in that they can be used under higher flow rate conditions [86, 95, 100-107]. In some applications, these columns have been used for HPLC-MS/MS assays where the flow rate was set to 5-6 mL min-1. This higher flow rate allows one to get higher sample throughput by reducing the gradient time. For example, Hsieh et al. [86] demonstrated that HPLC gradients could be completed in 30 s by using monolithic columns. One disadvantage of these columns is that the high flow rates translate into a much higher use of mobile phase solvents and the need to dispose of them as waste solvents after they have been used.

Another approach for speeding up sample throughput has been the use of parallel HPLC columns. This was first explored by Korfmacher et al. [108], who demonstrated that the effluent from two HPLC systems could be combined and assayed by using the SRM capabilities of the MS/MS system. Jemal et al. [109] also demonstrated that two parallel HPLC systems could be combined for analysis using one MS/MS system. This parallel HPLC technology was further enhanced by the development of the MUX system, in which four HPLC columns could be assayed by a single triple quadrupole MS/MS system [52, 57, 110]. Another variation that has been used more recently is the staggered parallel analysis strategy. Using the staggered strategy, multiple HPLC columns (typically four) are used to assay samples, but the injection time is staggered so that the ''analytical window'' (the portion of the chromatographic procedure) can be selected sequentially in order to maximize the use of the MS/MS system and increase sample throughput [111, 112]. For example, King et al. [112] described a four column staggered HPLC-MS/MS system that was able to be validated to meet GLP standards for a bioanalytical assay. In this example, the assay throughput was increased almost four-fold while still maintaining good chromatographic resolution [112].

In addition to increasing throughput, researchers are finding ways to utilize the increased sensitivity of the new HPLC-MS/MS systems. For example, Xu et al. [113] recently described the development of a low sample volume assay for preclinical studies. In this assay, only a 10-mL plasma sample volume is required for the analysis. The small volume is prepared by protein precipitation (1:6 = plasma:acetonitrile) using a special low volume 96-well plate. Only 5 mL of the precipitated sample is injected onto the HPLC-MS/MS system. In spite of these low volumes, the example assay is reported to have a limit of quantitation (LOQ) of 0.1 ng mL-1. It can be predicted that there will be more reports of improved LOQs and reduced sample volumes as new LC-MS/MS instrumentation is introduced to more laboratories.

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