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.
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.
Department of Biomedical Sciences, University of Sassari, Sassari, Italy. angelozinellu@libero.it
Angelo
A
Zinellu
Salvatore
S
Sotgia
Valeria
V
Pasciu
Manuela
M
Madeddu
Giovanni Giuseppe
GG
Leoni
Salvatore
S
Naitana
Luca
L
Deiana
Ciriaco
C
Carru
D000244:Adenosine Diphosphate / Q000097:blood*; D000249:Adenosine Monophosphate / Q000097:blood*; D000255:Adenosine Triphosphate / Q000097:blood*; D019075:Electrophoresis, Capillary / Q000379:methods*; D008747:Methylcellulose / Q000737:chemistry*; D009995:Osmosis
D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D008747:Methylcellulose