Ultra-short columns and ballistic gradients: considerations for ultra-fast chromatographic liquid chromatographic-tandem mass spectrometric analysis.

Ultra-fast chromatographic separations has enabled fast chromatographic method development and rapid analysis for sample quantification. Decreasing over-all analytical time has become a factor of major importance for all aspects of drug discovery. However, merely decreasing chromatographic analysis time by decreasing k' can lead to inconsistent quantitative or qualitative results due to ineffective separations in complex matrices. We have found that by changing column length and gradient slope we can maintain chromatographic integrity of chemically diverse analytes and achieve the analytical speed required for bioanalytical drug discovery quantitative analysis. We have optimized method development strategy by performing separations on 2x20 mm HPLC columns at flow-rates of 1.5 ml/min to 2 ml/min with full linear gradients achieved in 1 min for the quantification of pharmaceuticals and their metabolites from biological matrices. This method development strategy can be readily adapted to other matrices. This paper will discuss the effects of column length and gradient time in ultra-fast chromatographic resolution.

Determination of valproate in the presence of felbamate in human plasma by narrow bore capillary gas chromatography.

A narrow bore capillary gas chromatographic method with one extraction step has been developed for quantitation of valproate (VPA) in the presence of felbamate (FBM) in human plasma. The method uses 0.250-ml aliquots of human plasma and one internal standard (IS). Chromatographic conditions include a DBWAX, 0.25 mm ID x 15 m, 0.25-micron film thickness column; splitless injection; and flame ionization detection. The linear quantitation range for VPA is 1.00-256 micrograms/ml.

Simultaneous determination of felbamate and three metabolites in human plasma by high-performance liquid chromatography.

An isocratic liquid-chromatographic method employing one extraction step and a 4.6 mm x 150 mm Spherisorb ODS2, 3 microns high-performance liquid chromatography (HPLC) column using ultraviolet (UV)-absorbance detection at 210 nm has been developed for quantitation of felbamate (FBM) and three felbamate metabolites in 0.100-ml aliquots of human plasma. The linear quantitation range for FBM and the two hydroxy metabolites is 0.781-200 micrograms/ml, and that for the monocarbamate metabolite is 0.391-200 micrograms/ml.

Simultaneous determination of felbamate, primidone, phenobarbital, carbamazepine, two carbamazepine metabolites, phenytoin, and one phenytoin metabolite in human plasma by high-performance liquid chromatography.

An isocratic liquid-chromatographic method employing one extraction step has been developed for the quantitation of five drugs and three metabolites in human plasma. The method uses 0.100-ml aliquots of human plasma and two internal standards. Chromatographic conditions include a 4.6 mm x 150 mm Spherisorb ODS2, 3 microns a high-performance liquid chromatography, (HPLC) column, a phosphate buffer-acetonitrile-methanol (700:160:140) mobile phase, and ultraviolet (UV) absorbance detection at 210 nm. Analytes and linear quantitation ranges (microgram/ml) were felbamate (FBM) 0.391-200; primidone (PRIM), 0.098-100; phenobarbital (PHENO), 0.195-100; carbamazepine (CBZ), 0.195-100; phenytoin (PHT), 0.195-200. For CBZ-transdiol (CBZ-TR) CBZ-epoxide (CBZ-EP), and the PHT metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), the range was 0.049-25.0 micrograms/ml. Ethosuximide, methsuximide, 2-methyl-2-phenyl-succinimide (methsuximide metabolite), 2-ethyl-2-phenyl malonamide (PRIM metabolite, 5-ethyl-5-(4-hydroxyphenyl)-barbituric acid (PHENO metabolite), and mephenytoin do not interfere with quantitation of the above compounds.

Simultaneous determination of felbamate and four metabolites in rat cerebrospinal fluid by high-performance liquid chromatography.

An isocratic liquid chromatographic method for direct sample injection has been developed for the quantitation of felbamate and four metabolites in rat cerebrospinal fluid. The method uses 0.050- or 0.025-ml aliquots of cerebrospinal fluid diluted with equal volumes of internal standard. Chromatography is performed on a 150 mm x 4.6 mm I.D. Spherisorb ODS2, 3-microns HPLC column eluted with a phosphate buffer-acetonitrile-methanol (820:120:60, v/v/v) mobile phase and ultraviolet absorbance detection at 210 nm. The linear quantitation ranges are: felbamate and the 2-hydroxy metabolite 0.195-200 micrograms/ml, the propionic acid metabolite 0.195-50.0 micrograms/ml, the p-hydroxy metabolite 0.781 to 50.0 micrograms/ml, and the monocarbamate metabolite 0.098-50.0 micrograms/ml.

Simultaneous determination of felbamate and three metabolites in rat and dog plasmas by high-performance liquid chromatography.

An isocratic liquid chromatographic method employing one extraction step and a 150 mm x 4.6 mm I.D. Spherisorb ODS2, 3-microns HPLC column using UV-absorbance detection at 210 nm has been developed for the quantitation of felbamate and three felbamate metabolites in 0.100-ml aliquots of rat and dog plasmas. The linear quantitation range in rat plasma is 0.195-200 micrograms/ml for felbamate; 1.563-200 micrograms/ml for the p-hydroxy metabolite; 0.391-200 micrograms/ml for the 2-hydroxy metabolite; and 0.098-200 micrograms/ml for the monocarbamate metabolite. The linear quantitation range in dog plasma is 0.195-200 micrograms/ml for felbamate; 0.781-200 micrograms/ml for the p-hydroxy metabolite; 0.195-200 micrograms/ml for the 2-hydroxy metabolite; and 0.098-200 micrograms/ml for the monocarbamate metabolite.

Isolation and identification of 3-carbamoyloxy-2-phenylpropionic acid as a major human urinary metabolite of felbamate.

A new metabolite of felbamate, isolated from the polar metabolite fraction of a human urine sample, was identified as 3-carbamoyloxy-2-phenylpropionic acid (CPPA) by electron impact and CI/MS of the methyl ester in the isolated fraction. Its presence in the unconjugated free form in two different human urine samples was confirmed by HPLC comparison of retention times with authentic synthetic CPPA and by on-line negative ion HPLC thermospray tandem mass spectrometric techniques. The amount of CPPA in 0-4 hr postdose human urine was estimated to be approximately 12% of the drug-derived substances present in the urine. Some CPPA was also found to be present in dog urine.