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Intracellular adenosine 5′‐triphosphate, adenosine 5′‐diphosphate, and adenosine 5′‐monophosphate detection by short‐end injection capillary electrophoresis using methylcellulose as the effective electroosmostic flow suppressor
Author(s) -
Zinellu Angelo,
Sotgia Salvatore,
Pasciu Valeria,
Madeddu Manuela,
Leoni Giovanni Giuseppe,
Naitana Salvatore,
Deiana Luca,
Carru Ciriaco
Publication year - 2008
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200800033
Subject(s) - adenosine triphosphate , adenosine , capillary electrophoresis , chemistry , adenosine diphosphate , adenosine monophosphate , chromatography , analyte , nucleotide , adenine nucleotide , capillary action , biochemistry , platelet , biology , materials science , platelet aggregation , gene , immunology , composite material
We present a new rapid CE method to measure adenine nucleotides adenosine 5′‐triphosphate (ATP), adenosine 5′‐diphosphate (ADP), and adenosine 5′‐monophosphate (AMP) in cells. The short‐end injection mode allows a decrease in the analysis time by injecting samples at the outlet end of a silica capillary closest to the detection window, reducing the migration distance. Moreover, the use of methylcellulose (MC) as run buffer additive to suppress EOF permits to further reduce the migration times of analytes. Thus, when a capillary with an effective length of 10.2 cm was used with a 60 mmol/L sodium acetate buffer pH 3.80 in the presence of 0.01% of MC, the migration time of analytes were 1.35 min for ATP, 1.85 min for ADP, and 4.64 min for AMP. These conditions gave a good reproducibility for intra‐ and interassay (CV <4 and 8%, respectively) and all the procedure demonstrated an excellent analytical recovery (from 98.3 to 99 %). The method suitability was proved both on red blood cells and in spermatozoa. We compared our proposed method to a spectrophotometric assay, by measuring ATP levels in 40 spermatozoa samples. The obtained data were analyzed by the Passing and Bablok regression and Bland–Altman test.