Quantification of modified nucleotides and nucleosides by isotope dilution mass spectrometry.

Epigenetic modifications are closely related to certain disorders of the organism, including the development of tumors. One of the main epigenetic modifications is the methylation of DNA cytosines, 5-methyl-2'-deoxycycytidine. Furthermore, 5-mdC can be oxidized to form three new modifications, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, and 5-carboxy-2'-deoxycytidine. The coupling of liquid chromatography with tandem mass spectrometry has been widely used for the total determination of methylated DNA cytosines in samples of biological and clinical interest. These methods are based on the measurement of the free compounds (e.g., urine) or after complete hydrolysis of the DNA (e.g., tissues) followed by a preconcentration, derivatization, and/or clean-up step. This review highlights the main advances in the quantification of modified nucleotides and nucleosides by isotope dilution using isotopically labeled analogs combined with liquid or gas chromatography coupled to mass spectrometry reported in the last 20 years. The different possible sources of labeled compounds are indicated. Special emphasis has been placed on the different types of chromatography commonly used (reverse phase and hydrophilic interaction liquid chromatography) and the derivatization methods developed to enhance chromatographic resolution and ionization efficiency. We have also revised the application of bidimensional chromatography and indicated significant biological and clinical applications of these determinations.

Comprehensive Isotope Ratio Metabolomics: Gas chromatography Isotope Ratio Mass Spectrometry of urinary metabolites and exhaled breath.

We have developed an analytical procedure to measure the carbon isotopic composition of multiple compounds even when there is a partial overlap in the chromatographic profiles and applied this procedure to measure the carbon isotopic composition of different metabolites in human urine and exhaled breath. Method development and validation was performed with CRM IAEA-600 caffeine after calibration of the reference CO2 gas using a mixture of certified undecane, pentadecane and eicosane δ(13C) standards. The alternative data treatment procedure included the correction of time-lag between Faraday cup amplifiers (44 ms at mass 45 and -160 ms at mass 46), the calculation and correction of chromatographic isotope effects on each peak (isotope shifts) and the calculation of the isotope ratio for each compound using the linear regression slope procedure with data only at the top of the chromatographic peak. In that way, partial chromatographic overlap between different metabolites can be tolerated (resolution equal or higher than 1). The reproducibility (SD) of the carbon isotope composition of 93 metabolites in human urine (n = 8) from one volunteer was typically better than 0.5 δ(13C) (range 0.1-2.0 δ(13C), median 0.4 δ(13C)). The method was applied to follow the carbon isotope composition of different metabolites in human urine and exhaled breath after the oral administration of 100 mg of universally labelled 13C-glucose to another human volunteer. It was demonstrated that isotopically labelled compounds could be detected in both samples even 2 h after administration. So, the developed methodology can be applied to multiple types of samples containing a large number of partially overlapping analytes including environmental applications, anti-doping control or metabolomics studies, including the use of enriched isotope tracers.