Fast HPLC

Other methods employ a microplate format followed by fast HPLC. Some researchers approach the determination from a different perspective. For example, an alternative method for ionizable substances is the pSol determination based on an acid-base titration.25 26 Kinetic solubility determinations involve determining the concentration of the compound in the buffer of interest when an induced precipitate first appears. 

Wainhaus et al.116 provided a good overview of UPLC/MS/MS and showed how it could be used to enhance discovery PK assays. Their report indicated that PK screening assays be performed more quickly because of shorter run times with UPLC for the separation mode, and assay LOQs were often lower with UPLC. In one example, the LOQ for the fast HPLC/MS/MS assay was 1.5 ng/ml. The LOQ for the same compound using UPLC/MS/MS was 0.04 ng/ml. Wainhaus et al. also reported improved separation of metabolites of a test compound when using UPLC/MS/MS. 

We focus the following discussion on analysis techniques for small molecular entities. The reader should be aware that there are also a number of solutions for analytical tools that are useful for fast HPLC, GPC, or even DSC. The classification principles that apply for these techniques are the same as for the analytical tools described in this chapter. 

FIGURE I Typical fast HPLC chromatograms for a set of three dissolution samples at three time points. 

Tor further extensive discussion on the theoretical basis of fast HPLC, see Chapter 4. 

A commercial HPLC system and columns capable of performing ultra high-pressure LC were recendy introduced at PITTCON 2004 (ACQUITY Ultra Performance LC System by Waters). This HPLC system was designed to take full advantage of the potential of novel, sub-2-micron particles to give scientists chromatographic run times that are up to 9 times shorter than current fast HPLC systems, up to 2 times better peak capacity or resolution, and 3 times better routine sensitivity. 

Combinatorial chemistry techniques produce hundreds, if not thousands, of samples that need to be analyzed in a relatively short period of time. While fast HPLC analyses with short columns and fast gradients have increased throughput, there are still limits on the number of samples that can be processed. Some systems have evolved that incorporate multiple injectors, pumps, and UV detectors, but are still limited by having only one mass spectrometer available for detection and analyte identification. 

Hsieh, Y. Brisson, J. M. Wang, G. Ng, K. Korfmacher, W. A. Simultaneous fast HPLC-MS/MS analysis of drug candidates and hydroxyl metabolites in plasma. 

Smith, J. H. McNair, H. M. Fast HPLC with a Silica-Based Monolithic ODS Column. J Chromatogr Sci 2003, 41, 209-214. 

Ketamine, a general anesthetic producing hallucinations and exhibiting an addiction potential, and norketamine have been determined in urine using SPE combined with RPLC-ESI(+)-MS-MS [49]. Cheng and Mok [49] developed a fast HPLC-MS-MS method able to separate and detect ketamine in a 2.5 min chromatographic run with a LOD of 5ng/mL. The method was applied to routine ketamine urine screening to a maximum of 200 samples per day. 

Hertog et al. (119) developed a fast HPLC method for the identification and quantification of five major flavonoid aglycones (quercetin, kaempferol, myricetin, luteolin, and apigenin) in freeze-dried vegetable and fruits. However, due to the inadequate resolution of quercetin and luteolin on RP-HPLC on Nova-Pak C]8, two different eluents of different solvent strength and viscosity were utilized. The conditions for hydrolysis and extraction were tested based on different conditions of hydrochloric acid concentration (1.2-2.0 M), reaction period (0.5-6 h), and meth-... 

Ultra-fast HPLC—An HPLC system designed to use <2-,um spherical packing at high flow rate and pressure ( 12,000psi) to produce very rapid, high-... 

Trace acetone in water may be determined by a fast HPLC method (Takami et al., 1985) aqueous sample passed through a cartridge packed with a moderately sulfonated cation-exchange resin charged with 2,4-dinitrophenylhydrazine (DNPH) DNPH derivative eluted with acetonitrile and analyzed by HPLC with a 3-mm ODS column. 

Various advantages can be realized by varying the column dimensions from the conventional 4.6 mm ID x 25 cm. One approach has been to use shorter columns of 3-10 cm packed with 3-/ m or 5-fim particles. When operating at high flow rates (2-5 ml/min), this approach has become known as fast HPLC [41]. 

The introduction of "fast HPLC" has proven to be particularly valuable in protein analysis. As stated earlier, assay time in RP-HPLC analysis of proteins is typically long compared to that for smaller organic molecules. We have evaluated the use of 0.6-cm ID x 4-cm columns packed with 3-um particles in the analysis of insulin by RP-HPLC for potency determination, related substances, and in peptide mapping (7). The use of the "fast column" allows considerable savings (40-60%) in analysis time, compared to the regular (0.46-cm ID x 25-cm) columns, without loss in resolving power. 

For this reason, the performance of these instruments is less satisfactory in applications with high speed columns which allow the analysis of biopolymers to be carried out on a time scale of a minute or less. In order to expand the scope of HPLC to such applications, some of the considerations described below should be taken in to account in the design of fast HPLC analyzers for biological macromolecules. 

---