Development and validation of LC-MS/MS method for quantification of ATP, ADP and AMP in dried blood spot, liver and brain of neonate mice pups

Abstract The objective of this study was to develop and validate a method using liquid chromatography coupled with a mass spectrometer (LC–MS/MS) for simultaneous analysis of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) levels in dry blood spot (DBS), liver, brain (parietal and occipital regions) of BALB/c neonatal mice pups, using 13C10 ATP as internal standard (IS). Simultaneous nucleotides separation of the investigated samples was achieved in 5 min on a C18 column [Waters × Bridge C18, (4.6 × 50 mm, 3.5 μm)] using a gradient elution of 4 mM ammonium acetate and 100% acetonitrile as mobile phase A and mobile phase B, respectively. MS detection was performed in an ESI positive ionization mode. The method was validated according to the international council for harmonization guidelines. The standard calibration curve was linear over the concentration range of 0.1, 1, 5, 10, 15, 20 µg/ml of standard mixtures with known quantity of internal standards (IS) and the data showed good linearity (R2 0.999), with lower limit of quantification (LLOQ 0.1 µg/ml). The within run and between run precision of QCs (%CV) were ≤ 15% and accuracy (%) of measured concentration of QCs ranged between 88 and 110%. All the processed QCs were stable after applying the various storage conditions. The robustness study showed no significant variance in analyte peak quality and quantity with the smaller variation of temperature, pH and acetonitrile concentration. The matrix factor was in acceptable range. The present validation results show that the developed LC–MS/MS method is suitable to evaluate ATP, ADP and AMP levels in DBS, liver and brain tissues of neonatal mice pups.