ABSTRACT
High-performance liquid chromatography-solid phase extraction-NMR spectroscopy (HPLC-SPE-NMR) has recently become commercially available and has been evaluated with regard to its applicability in a pharmaceutical environment. The addition of an automated SPE unit to an HPLC-NMR system for peak trapping results in an improved NMR signal-to-noise ratio (S/N) and also has other practical advantages. The trapping efficiency is shown to depend on compound polarity and is highest for compounds eluting late on reversed-phase HPLC systems. Multiple peak trapping further increases the S/N, again with the best results for less polar compounds. For polar compounds, multiple peak trapping resulted in no S/N gain as the amount of material retained on the SPE cartridge was equivalent to that from a single injection. When compared with conventional HPLC-NMR, a S/N gain of up to five-fold could be achieved for some compounds in a single trapping step. A major advantage of the technique is the independence of the chromatographic step from the NMR step, resulting in greater versatility than conventional HPLC-NMR in the HPLC solvents and NMR solvents that can be used. Practical applications from both drug metabolite and drug impurity identification are presented.
Subject(s)
Chromatography, High Pressure Liquid/methods , Magnetic Resonance Spectroscopy/methods , Pharmaceutical Preparations/analysis , Acetonitriles/chemistry , Acids, Carbocyclic/analysis , Acids, Carbocyclic/chemistry , Animals , Bile/chemistry , Bile/metabolism , Dogs , Ketones/analysis , Ketones/chemistry , Pharmaceutical Preparations/chemistry , Rats , Sensitivity and Specificity , Solvents/chemistryABSTRACT
Starting from a high throughput screening hit, a series of 3,4-dihydro-2H-benzoxazinones has been identified with both high affinity for the 5-HT(1A) receptor and potent 5-HT reuptake inhibitory activity. The 5-(2-methyl)quinolinyloxy derivative combined high 5-HT(1A/1B/1D) receptor affinities with low intrinsic activity and potent inhibition of the 5-HT reuptake site (pK(i)8.2). This compound also had good oral bioavailability and brain penetration in the rat.
Subject(s)
Benzoxazines/chemical synthesis , Selective Serotonin Reuptake Inhibitors/chemical synthesis , Serotonin 5-HT1 Receptor Antagonists , Animals , Benzoxazines/pharmacology , Biological Availability , Brain/metabolism , Cell Line , Drug Stability , Humans , Radioligand Assay , Rats , Selective Serotonin Reuptake Inhibitors/pharmacology , Structure-Activity Relationship , Synaptosomes/metabolismABSTRACT
In combining the high peak concentrations of capillary liquid chromatography (CapLC) with the high mass sensitivity of micro scale nuclear magnetic resonance (NMR) the hyphenation of CapLC to micro NMR offers a substantial gain in overall sensitivity. This paper deals with our experiences gained using a commercial CapLC-NMR system which has very recently become available. The limits of detection (SNR > 3) for a test compound of a molecular weight of M 318 were found to be approximately 100 ng (0.35 nmol) within an hour acquisition time and approximately 25 ng over night (85 pmol). Practical aspects such as the feasibility of stopped-flow experiments and sample handling issues are discussed in detail and first possible drug metabolite applications to hepatocyte incubations and direct analysis of plasma samples are presented.
