A sensitive HPLC-based method to quantify adenine nucleotides in primary astrocyte cell cultures

In mono-layered primary cell cultures baseline AMP and ADP levels are found nominally in the mid to low picomolar range and are thus difficult to measure with conventional HPLC methods that often require the pooling of samples or require indirect detection methods using radiotracers or enzyme coupled assays. To address this issue, we developed a highly sensitive and selective ion-pairing HPLC method with fluorescence detection to quantify adenine nucleotides and the adenylate energy charge in primary astrocyte cell cultures. To accomplish this, we optimized the fluorescence derivatization conditions and the HPLC parameters to achieve baseline separation and quantification of all adenine nucleotides. Nucleotides were converted to their respective 1, N ⁶-etheno derivatives by incubating with chloroacetaldehyde at pH 4.5 and 60°C for 60min. Under these conditions, the loss of the adenine nucleotides due to hydrolysis was minimized with a derivatization yield of 94.1% for 1, N ⁶-ethenoadenosine. The optimal concentration of tetrabutylammonium phosphate, the ion-pairing reagent, required to achieve a reproducible separation of the adenine nucleotides was found to be 0.8mM. Calibration curves of nucleotide standards were linear within the range of 0.16-10.4pmol for adenosine, 0.16-20.6pmol for AMP, 0.15-19.2pmol for ADP, and 0.15-19.5pmol for ATP. The limits of detection and quantification for all adenine nucleotides were approximately 0.08 and 0.16pmol, respectively. The intra- and inter-day variability for this method was less than 5.1 and 3.4%, respectively. This method was successfully used to measure all adenine nucleotides and an adenylate energy charge of 0.92±0.02 in primary astrocyte cell cultures.