Precolumn derivatization of reducing carbohydrates with 4-(3-Methyl-5-oxo-2-pyrazolin-1-yl) benzoic acid. Study of reaction, high-performance liquid chromatographic separation and quantitative performance of method

Abstract

SummaryA simple method for quantitative determination of carbohydrates by reversed-phase, highperformance liquid chromatography after pre-column clerivatization and UV detection has been developed. Reducing sugars are condensed with 4-(3-methyl-5-oxo-2-pyrazohn-Lyl) benzoic acid (PMPA) to yield UVaclducts absorbing at 271 nm, which are resolved under typical reversed-phase conditions. After clerivatization, excess PMPA is easily removed from the reaction mixture by precipitation with mineral acids at pH < 4. The influence of experimental conditions on reaction yield, as well as chromatographic separation of derivatives, were investigatecl. The quantitative performancewas evaluatd by means of a protocol comprising replicate measurements at several analyte levels. The calibration curves obtained for 8 sugars showed excellent hnearity over 10 -5000 pmol. Limits of detection and quantification for several monosaccharicles were ca. 10 and 50 picomoles, respectively. Optimized conditions were successfully used for quantitative determination of monosaccharicles released after hydrolysis from fetuin, mucin, ~l-acid glycoprotein, ovalbumin and transferrin.

Decline of cell viability and mitochondrial activity in mouse skeletal muscle cell in a hypomagnetic field

“…Concentrations of glucose and lactic acid in the conditioned media (CM) were measured by Reverse‐Phase High Performance Liquid Chromatography (RP‐HPLC) (Shimadzu, Kyoto, Japan) as described (for glucose detection, see Castells et al []; for lactic acid detection, see Tusseau and Benoit []). Analytical grade glucose (Sigma–Aldrich) and lactic acid (Sigma–Aldrich) were used as standards.…”

Two-step derivatization for determination of sugar phosphates in plants by combined reversed phase chromatography/tandem mass spectrometry

“…To overcome this problem, different types of derivatization techniques have been used to improve retention in LC–MS. For example, 2-aminopyridine [21], p -amino benzoic ethyl ester [22] or 4-(3-methyl-5-oxo-2-pyrazolin-1-yl) benzoic acid [23] reagents have been used in different studies to improve chromatographic retention of highly polar compounds. However, these derivatization methods are tedious and not reliable due to incomplete derivatization.…”

Derivatization of carbohydrates for analysis by chromatography; electrophoresis and mass spectrometry

“…Related derivatives have been formed with 1-(4-methoxyphenyl)-3-methyl-5-pyrazolone (PMPMP, 115) [265] and 4-(3-methyl-5-oxo-2-pyrazon-1-yl) benzoic acid (PMPA, 116) [266]. The methoxy reagent showed higher reactivity towards carbohydrates and both derivatives had a higher UV absorbance than PMP derivatives.…”