Validation of a Fast and Simple HPLC-UV Method for the Quantification of Adenosine Phosphates in Human Bronchial Epithelial Cells

Abstract

A new HPLC method for the simultaneous quantitative analysis of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) was developed and validated. ATP, ADP, and AMP were extracted from human bronchial epithelial cells with a rapid extraction procedure and separated with a C18 column (3 × 150 mm, 2.7 µm) using isocratic elution with a mobile phase consisting of 50 mM of potassium hydrogen phosphate (pH 6.80). The absorbance was monitored at 254 nm. The calibration curves were linear in 0.2 to 10 µM, selective, precise, and accurate. This method allowed us to quantify the nucleotides from two cell models: differentiated NHBE primary cells grown at the air–liquid interface (ALI) and BEAS-2B cell line. Our study highlighted the development of a sensitive, simple, and green analytical method that is faster and less expensive than other existing methods to measure ATP, ADP, and AMP and can be carried out on 2D and 3D cell models.

Effects of DEHP, DEHT and DINP Alone or in a Mixture on Cell Viability and Mitochondrial Metabolism of Endothelial Cells In Vitro

“…Intracellular adenosine amounts (ATP, ADP and AMP respectively) were measured on the supernatant of the cellular extract after PCA extraction, as described above. The quantification of cellular adenosines was made using a high-performance liquid chromatography coupled with diode array detector (HPLC-DAD) method previously developed and validated in our laboratory for the simultaneous determination of ATP, ADP and AMP in biological samples [ 48 ]. Analyses were performed using the 1260 Infinity II LC System (Agilent Technologies, Santa Clara, CA, USA).…”

Ultrafine Particles Issued from Gasoline-Fuels and Biofuel Surrogates Combustion: A Comparative Study of the Physicochemical and In Vitro Toxicological Effects

“…ATP and ADP levels were determined on perchloric extracts as previously described [ 34 ]. Briefly, after 3 h and 24 h exposure, inserts were gently rinsed with PBS-1X.…”

Detection of Biomarker Using Aptasensors to Determine the Type of Diabetes

“…Several biomarkers are used as tests for DM patients, such as oral glucose tolerance tests (OTGG), fasting glucose level, insulin levels, glycated hemoglobin (HbA1c), and glycated albumin (GHSA) levels [ 3 , 4 ]. Various techniques have been developed to detect diabetes, including the HPLC-UV technique [ 5 ], fluorescence [ 6 , 7 , 8 , 9 ], chemiluminescence [ 10 ], ion exchange chromatography [ 11 ], immunoassay [ 12 ], and capillary electrophoresis [ 13 ]. However, measuring these biomarkers with instruments is not suitable for on-site monitoring [ 13 , 14 ].…”