Information about the intracellular concentration of dNTPs and NTPs is important for studies of the mechanisms of DNA replication and repair, but the low concentration of dNTPs and their chemical similarity to NTPs present a challenge for their measurement. Here, we describe a new rapid and sensitive method utilizing hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry for the simultaneous determination of dNTPs and NTPs in biological samples. The developed method showed linearity (R2 > 0.99) in wide concentration ranges and could accurately quantify dNTPs and NTPs at low pmol levels. The intra-day and inter-day precision were below 13%, and the relative recovery was between 92% and 108%. In comparison with other chromatographic methods, the current method has shorter analysis times and simpler sample pre-treatment steps, and it utilizes an ion-pair-free mobile phase that enhances mass-spectrometric detection. Using this method, we determined dNTP and NTP concentrations in actively dividing and quiescent mouse fibroblasts.
BACKGROUND: Measurement of methylmalonic acid (MMA) in serum or plasma is useful for diagnosing cobalamin deficiency. We developed a method for quantifying MMA in plasma based on hydrophilic interaction liquid chromatography (HILIC) and single-stage negative electrospray ionization (ESI) mass spectrometry.
A new dansylation reaction, where trifluoromethanesulfonic acid is used as catalyst, has been characterized for six ketosteroids by employing experimental design followed by multivariate data analysis. The molar ratio between the steroid and the derivatization reagent was found to be the factor most strongly affecting the reaction. Faster reaction kinetics was achieved when the molar ratio between dansylhydrazine and the steroid was increased. Mass spectroscopic analysis showed that the dual peaks observed when derivatized progesterone was separated on an octadecyl silica stationary phase were due to the syn and anti hydrazones formed. We furthermore conclude that the dansylation reaction is subject to alkyl catalysis rather than acid catalysis, since methyl trifluoromethanesulfonate showed a strong catalytic action, while the catalytic action of trifluoromethanesulfonic acid was lower when diluted in other alcohols and disappeared in aprotic solvents. A sensitivity to water in the reaction mixture strengthens the evidence for alkyl catalysis. When optimal experimental conditions were used, derivatization of picomole amounts of ketosteroids could be accomplished in 25 min. Analysis of spiked plasma containing 0.2-2.0 nmol each of progesterone and 3α-hydroxy-5β-pregnan-20-one showed overall recoveries of 69-76% and 40-55%, respectively. The corresponding 3σ detection limits estimated from calibration curve data were 12 and 15 pmol (n = 4, 500 μL injected).
Separation science is an art of obtaining adequate resolution of the desired compounds in minimum time, and with minimum effort in terms of sample preparation and data evaluation. In LC, where selectivity is a main driving force for separation, the availability of different separation modes capable of operating at high flow rates is a way to make combined optimal use of selectivity, efficiency, and speed. The separation of polar and hydrophilic compounds is problematic in RP LC due to the poor retention. Hydrophilic interaction liquid chromatography (HILIC) is a more straightforward separation mode to address this problem. Herein, it is shown that separations in HILIC mode are equally efficient as for RP, providing a potential for very fast separations on short columns. This is not only facilitated by the low viscosity of the mobile phase compositions used, compared to typical RP eluents, but also due to higher column permeability. To exemplify this, baseline separations of uracil and cytosine are shown in less than 4 s and of Tamiflu and its main metabolite in less than 40 s, both under isocratic conditions. HILIC must therefore be considered having potential for high throughput purposes, and being an attractive candidate as the second separation dimension in 2-D HPLC.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.