In situ derivatization-liquid liquid extraction as a sample preparation strategy for the determination of urinary biomarker prolyl-4-hydroxyproline by liquid chromatography-tandem mass spectrometry.

Polar analytes that possess protic functional groups have often been treated with alkyl chloroformates to decrease their polarity and increase their volatility prior to gas chromatography-mass spectrometry analysis. This derivatization reaction has two distinct advantages. It proceeds smoothly in aqueous media, and the desired reaction products are efficiently separated from interfering ionic components by their extraction into a water-immiscible organic phase. In the present work, the derivatization-liquid liquid sample preparation was examined in detail for analysis of a potential urinary dipeptide biomarker L-prolyl-4-L-hydroxyproline (PHP) by downstream liquid chromatography coupled to electrospray mass spectrometry. PHP was treated with a series of alkyl and fluoroalkyl chloroformates in aqueous media, and the detected reaction products were investigated. Smooth conversion of PHP into the N-isobutyloxycarbonyl isobutyl ester was accomplished by the coupled action of isobutanol, isobutyl chloroformate and the pyridine catalyst. This derivative afforded a highest detector response from all the derivatized forms examined, including the nonderivatized PHP. A simple isocratic elution on a common RP-C18 HPLC column coupled with tandem mass spectrometry, and use of the synthesized heptadeuterated analog (D7-PHP) as an internal standard, enabled validation of the method and determination of PHP in human urine in less than 5 min. The in situ derivatization-liquid liquid extraction has thus been demonstrated to be a useful sample preparation strategy for the analysis of polar metabolites by liquid chromatography-tandem mass spectrometry in the complex urine matrix.