Analytical run times

Merely, the choice of column dimension demonstrates the awareness of cost reduction for organic solvents as well as analytical run time and thus of sample throughput and economic efficiency. Only 2 % of published reports make use of a column with an inner diameter (I.D.) as small as 1 mm [12] corresponding to a flow of 50 ul/min. Columns with an I.D. of 2.0-2.1 mm are used in 48 % of reports determining a flow between 200 and 500 ul/min and half of all applications is performed with a column I.D. between 3.0 and 4.6 mm and a flow ranging from 500 to 1,000 ml/min (Tables 5-8). 

Atmospheric pressure ionization LC/MS has several distinct advantages over other more established quantitative approaches for these biochemical markers. The HPLC/fluorescence methods that have been reported require involved sample preparation, analyte extraction, and long chromatographic run times and are prone to interferences. The reported enzyme assays are less selective in that they cannot differentiate between the two compounds in a single run. Pyridinoline must therefore be quantified by difference. The API LC/MS/MS approach has allowed for a decrease in chromatographic run time, a streamlining of the sample preparation approach (dilute/inject as opposed to extraction), and a decrease in the analytical run time, while allowing simultaneous determination of both components. 

Figure 2.31 Peak capacity as a function of analytical run time. The graph is valid for isocratic reversed-phase systems which are run at their van Deemter optimum. The maximum retention factor /ris 20, i.e. the maximum retention time is to 21, then the separation ends. The figure is only valid for small analytes with a diffusion coefficient ofapprox. 1-10 m s and not for macromolecules. Dotted lines represent the particle diameter, dashed lines the column length, and solid lines the pressure, respectively.

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. [90] 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. 

Sample preparation usually involves a protein precipitation cleanup step. Fast chromatography methods that provide run times of less than 2 min are generally preferred and attained. The LC-MS/MS method is optimized for each CYP substrate s metabolite SRM transition. The selectivity of MS/MS reduces the need for complete chromatographic resolution of individual components. Therefore, analytical run times are significantly reduced. Data analysis must be highly streamlined using automated data analysis and reporting. 

The use of short HPLC columns and ultrafast gradients has been explored in order to decrease HPLC-MS/MS analytical run times [27-30], Typical cycle times ranged from 85s to 2min and used 2x30mm, 4pm HPLC columns (Table 8.1). The lmin HPLC method is shown in Table 8.1. Mobile phases A and B are 0.01 M ammonium acetate in water/methanol (80 20) and methanol (100%), respectively. A ballistic gradient from 1% to 70% B was run over 0.01 min and held for 0.09 min. A linear gradient was then employed from 70% to 95% B for 0.6min and held for 0.1 min. The column was then reequilibrated to 1% B over 0.2 min at a constant flow rate of 0.8mL/min. 

The combination of LC with MS has created a powerful tool for the analysis and quantitation of a variety of dmgs and metabolites in complex matrices. LC-MS has turned out to be a particularly sensitive and selective technique for the analysis and quantitation of pharmaceuticals. It is a fundamental analytical tool for the studies of drug metabolism of new dmg candidates and the identification and characterization of impurities and degradents in pharmaceuticals. Further transfer of the routine activity of immunosuppressants from immunoassays to LC-MS, which allows the automation of sample preparation, shortens analytical run times and is probably cost-effective despite heavy investment costs. 

An example of the application of these features to separation of some closely-related marine toxins (Vohner 2002) is shown in Figures 3.13 and 3.14. Many examples of the application of monolithic columns to biomedical applications can be cited. For example, it was shown (Dear 2001) that the use of short monolithic silica columns coupled to mass spectrometry dramatically reduced analytical run times for metabolite identification from in vitro samples with no loss in chromatographic performance. Six hydroxylated metabolite isomers were separated in one minute, with resolution and selectivity comparable to conventional analytical chromatography resulting in reduction of analysis time per sample from 30 to 5 minutes. Similar results were reported (Wu 2001 Hsieh 2002) for rapid qualitative and quantitative analyses in drug discovery programs (i.e., not fully validated). 

Question 1 Reasonable interactions, acceptable analytical run time ... 

In this chapter, urinary steroid profiling is discussed first, followed by a description of the state of the art concerning clinical profiling of steroid hormones in plasma by stable isotope dilution (ID) GC-MS. The reason is merely a historical one the art of urinary steroid profiling matured earlier and has found more widespread use. The early attempts to measure plasma steroids by GC-MS were rapidly surpassed by the introduction of immunoassay techniques with which MS could not compete, especially with respect to analytical run time and cost. However, it was not until recently that it was realized that the lack of specificity of immunoassays might initiate a renaissance for clinical MS techniques in steroid analysis. 

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