Clinica chimica acta; international journal of clinical chemistry · 2015
Targeted arginine metabolomics: A rapid, simple UPLC-QToF-MS(E) based approach for assessing the involvement of arginine metabolism in human disease.
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van Dyk Madelé, Mangoni Arduino A, McEvoy Mark, Attia John R, Sorich Michael J, Rowland Andrew
Аннотация
BACKGROUND: Nitric oxide synthase (NOS) mediated conversion of arginine (ARG) to citrulline (CIT) is a key pathway for nitric oxide synthesis. ARG is also metabolised by alternate pathways to ornithine (ORN), homoarginine (HMA), N(G)-monomethyl-L-arginine (MMA), N(G),N(G)-dimethyl-L-arginine (ADMA) and N(G),N(G)'-dimethyl-L-arginine (SDMA), all of which have the capacity to alter NOS activity. Simultaneous assessment of these analytes, when assessing the impact of arginine metabolism in human disease states, is desirable.
METHODS: Analytes (ARG, ADMA, SDMA, MMA, HMA, CIT and ORN) were isolated from human plasma by solvent extraction, evaporated and reconstituted. Ultra-performance liquid chromatography (UPLC) was performed on a 150mm×2.1mm T3 HSS column using a gradient mobile phase comprising ammonium formate (10mM, pH3.8) in methanol (1% to 63%). Analytes were detected by time-of-flight mass spectrometry (Q-ToF-MS) in positive ion mode with electrospray ionisation (ESI+). Data were collected using MS(E).
RESULTS: Solvent extraction provided high recovery (>95%). UPLC-QToF-MS(E) facilitated the separation and quantification of the 7 analytes in an analysis time of 6min. The approach has high sensitivity; LOQ range from 0.005μM (NMMA) to 0.25μM (ARG and ORN), and good precision; intra- and inter-day %RSD are <6% for all analytes.
CONCLUSIONS: This approach provides the capacity to quantify 7 key compounds involved in ARG metabolism in a small sample volume, with a short total analysis time. These characteristics make this approach ideal for undertaking a comprehensive characterisation of this pathway in large data sets (e.g. population studies).
METHODS: Analytes (ARG, ADMA, SDMA, MMA, HMA, CIT and ORN) were isolated from human plasma by solvent extraction, evaporated and reconstituted. Ultra-performance liquid chromatography (UPLC) was performed on a 150mm×2.1mm T3 HSS column using a gradient mobile phase comprising ammonium formate (10mM, pH3.8) in methanol (1% to 63%). Analytes were detected by time-of-flight mass spectrometry (Q-ToF-MS) in positive ion mode with electrospray ionisation (ESI+). Data were collected using MS(E).
RESULTS: Solvent extraction provided high recovery (>95%). UPLC-QToF-MS(E) facilitated the separation and quantification of the 7 analytes in an analysis time of 6min. The approach has high sensitivity; LOQ range from 0.005μM (NMMA) to 0.25μM (ARG and ORN), and good precision; intra- and inter-day %RSD are <6% for all analytes.
CONCLUSIONS: This approach provides the capacity to quantify 7 key compounds involved in ARG metabolism in a small sample volume, with a short total analysis time. These characteristics make this approach ideal for undertaking a comprehensive characterisation of this pathway in large data sets (e.g. population studies).