One important advantage of FIA-MS analysis is the high throughput capabilities. Samples are analyzed routinely with less than a minute per injection with a single injector. Higher throughput is typically achieved by parallel injection from multiple injectors when injector washing becomes the rate-limiting step. Wang et al.  used a parallel eight-probe injector system to achieve an effective throughput of 7.5 s/sample. The method was used for the rate of information generation was obtained. Goetzinger et al.  investigated different packing materials, gradient methods, and sample solvents for the development of ultrafast gradient HPLC methods. Commercially available equipment and short columns (<30 mm) packed with small particles (<4 /m) were used to achieve a one minute total analysis time with a peak capacity of 49. These methods were used for quality control of spatially addressable combinatorial libraries. Pereira et al.  investigated different buffer types and column geometry effects in high-throughput LC-MS. All the tested buffers, phosphate, acetate, and acetic acid, exhibited good resolution, while the separation time varied from 4 minutes to 12 minutes. The use of acetic acid resulted in the shortest separation time. As the column length was shortened and the flow rate was increased, the separation time was reduced with no change in selectivity.
Fast LC-MS methods have been used to assess library quantity and purity, as well as to triage purification of compounds. Zeng et al.  developed one of the first fully automated analytical/preparative LC-MS systems for the characterization and purification of compound libraries derived by parallel synthesis. The system incorporated fast, reverse-phase LC/ESI-MS analysis (5-10 minutes). Post-data-acquisition purity assessment of compound libraries was performed automatically with software control. Compounds that were below a threshold level of purity were automatically purified with HPLC. The real-time purity assessment eliminated the need for postpurification analysis or pooling of fractions collected.
With the widespread application of combinatorial chemistry in drug discovery, there are an increased number of compounds that are tested for pharmaceutical profiling (solubility and ADME; see Chapter 15). The need for high throughput analysis of these compounds stimulated active research in the improvement of LC-MS-based quantitation techniques. Wu et al.  investigated monolithic columns for high throughput bioanalysis application. Due to the lower pressure-drop on a monolithic column than on a particulate column, a high flow rate (6 mL/min) was used for a 4.6 x 50-mm monolithic column. The separation efficiency and signal-to-noise ratios (S/N) for this separation remained almost constant at flow rates of 1, 3, and 6 mL/min. The chromatographic retention time, separation quality, peak response, and sensitivity were highly reproducible throughout a run of 600 plasma extracts. Romanyshyn et al.  examined the effects of column length and gradient time on ultrafast chromatographic resolution. By judicious adjustment of column length and gradient slope, the chromatographic integrity of chemically diverse analytes was maintained at a much faster elution speed. This optimized method development strategy enabled separations on 2 x 20-mm HPLC columns at flow rates of 1.5 mL/min to 2 mL/min with full linear gradients that could be achieved in one minute. Cheng et al.  described a simple and comprehensive LC/MS/MS strategy for the rapid analysis of a wide range of pharmaceutical compounds. The authors started with a column that provided a good peak capacity at short gradient run times; then employed high flow rates to achieve a good gradient peak capacity. This fast LC-MS/MS method was used to separate and identify a wide range of analytes with one-minute gradient analyses. Zhang et al.  described an isocratic LC-ESI/TOF-MS method for quantitation and accurate mass measurement of five tricyclic amine drugs fortified in human plasma with a per-sample run time of 18 seconds, with a short C-18 column (15 x 2.1-mm Id). The authors used a highly aqueous mobile phase at a flow rate of 1.4 mL/min. Samples were prepared by off-line liquid-liquid extraction. An acquisition speed of 0.2 s/spectrum accommodates these fast separation conditions. Accurate masses were determined by two-point internal mass calibration with postcolumn addition of standard. Results showed a mass error not greater than 9 ppm for all the target compounds. Zweigenbaum and Henion  demonstrated more than 2000 samples in 24 hours with LC-MS/MS separation and quantitation for compounds in control human plasma. The method includes sample preparation with liquid-liquid extraction in the 96-well format, an LC separation of the five compounds in less than 30 seconds. Hsieh et al.  developed a direct injection bioanalytical method based on a single column LC-MS/MS for pharmacokinetic analysis. Each plasma sample was mixed with a solution that contained an internal standard. The sample was directly injected into a polymer-coated mixed-function column for sample cleanup, enrichment, and chromatographic separation. The stationary phase incorporates both hydrophilic and hydrophobic groups that allow proteins and macromolecules to pass through the column due to restricted access to the surface of the packing, while the drug molecules are retained on the bonded hydrophobic phase. The analytes retained in the column were eluted with a strong organic mobile phase. The total analysis time was 5 min/sample. Yu et al.  developed a fast LC-MS method and compared the approach with an FIA-MS method for effective quantitation. With the application of fundamental concepts of fast LC such as the use of a small column (30-mm x 2.1-mm, 2.6-^m particle) at an elevated temperature (40oC) and the use of a high flow rate (1.0 mL/min), the authors were able to reduce the LC cycle time from more than 20 minutes to 2.7 minutes. The authors compared the limits of detection and quantitation, linearity, precision, and accuracy for each analyte. The results indicate that fast LC-MS is generally better than FIA-MS analysis. Romanyshyn et al.  developed an LC-MS/MS method for quantitation with rapid ("ballistic") gradients on narrow bore, short HPLC columns and compared the fast-gradient approach with the more traditional high-organic isocratic LC-MS/MS methods. Fast isocratic methods frequently elute the analytes of interest at the solvent front, the region of unretained salts.
The fast-gradient method, in contrast, retains analytes on-column until well after the solvent front has eluted. Overall sample throughput is increased with fast-gradient methods due to reduced analytical run time, decreased method development time, and fewer repeat analyses. Onorato et al.  used a multiprobe autosampler for parallel sample injection, short, small-bore columns, high flow rates, and elevated HPLC column temperatures to perform LC separations of idoxifene and its metabolite at 10 s/sample. Sample preparation employed liquid-liquid extraction in the 96-well format. An average run time of 23 s/sample was achieved for human clinical plasma samples.
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