Determination of lipoic acid, Trolox methyl ether and tocopherols in human plasma by liquid‐chromatography and ion‐trap tandem mass spectrometry

Abstract

A method for the simultaneous determination of lipoic acid and/or Trolox methyl ether, along with α-, γ- and δ-tocopherol was developed using liquid chromatography-tandem mass spectrometry with negative electrospray ionization (HPLC-ESI-MS/MS) in an ion-trap mass spectrometer. Detection and quantification were accomplished by a multiple reaction monitoring method, using specific transitions from precursor ion to product ion for each analyte. Chromatographic separation was achieved in a 12 min run using a C(18) -bonded phase and methanol-aqueous ammonium acetate elution gradient. Linear correlations of the chromatographic peak area (r.u. × s(-1) ) to the injected amount (ng) gave the slope values (r.u. × s(-1) × ng(-1) ) 2.34 × 10(4) for α-tocopherol, 5.05 × 10(4) for γ-tocopherol, 1.27 × 10(5) for δ-tocopherol, 8.86 × 10(5) for lipoic acid and 1.23 × 10(5) for Trolox methyl ether. The lower limit of quantification ranged between 0.02 and 1.22 ng for Trolox methyl ether and lipoic acid. MS(3) experiments of γ- and δ-tocopherol suggest ion-radical reactions and dependence of the tocopherol fragmentation pattern on the phenolic ring methylation degree. The method is shown to be applicable to measurement of these metabolites in human serum after extraction.

Detection and quantification of vitamins in microliter volumes of biological samples by LC‐MS for clinical screening

“…Fat‐soluble vitamins are considerably less polar than water‐soluble vitamins and lack functional groups that readily accept or donate electrons, so mobile phase additives were necessary to facilitate their ionization with adduct formation using the hydroxyl (vitamin A, D 3 , 25[OH]D 3 , and E) or oxygen (vitamin K 1 ) groups in their structures. Mobile phase additives that have been used to promote ion formation for fat‐soluble vitamins include formic acid, silver perchlorate, ammonium acetate, ammonium formate, or cesium acetate . We systematically tested these additives at different concentrations.…”

A review of chromatographic methods for the determination of water‐ and fat‐soluble vitamins in biological fluids

“…For saponification, KOH or isopropanol are used . For further sample purification, LLE is additionally applied using heptane , hexane , ethyl acetate , chloroform , methyl tert ‐butyl ether , or a mixture of hexane/ethanol/acetone/toluene . Deproteinization and LLE resulted in endogenous compounds of the matrix passing into the supernatant, which may affect the separation and determination of the analytes.…”

The issue of HPLC determination of endogenous lipoic acid in human plasma

“…Teichert and Preiss (1992) published values in the range of 4.8-121.0 nmol/L. Montero, Ramírez, Sánchez, and Gonzáles (2012) gave undetectable contents for LA in human plasma. Montero, Ramírez, Sánchez, and Gonzáles (2012) gave undetectable contents for LA in human plasma.…”