A unique quantitative high-performance liquid chromatography-mass spectrometry (HPLC-MS) method to investigate the energy state in cells and tissues was developed and validated using a chromatographic method designed to (i) separate and quantify more than 11 nucleotides without the use of phosphate buffer and (ii) minimize the potential ion suppression common to other nucleotide methods. Several commonly used extraction methods were compared based on absolute recoveries and reproducibilities. Perchloric acid (PCA) extraction yielded the highest recoveries (75-86%) and showed the best reproducibility (coefficient of variation=2.5-9.5%). Our assay, which included PCA extraction, online desalting, separation of the high-energy phosphates on a C18 reversed-phase column using a methanol/dibutylammonium formate gradient, and detection of negative ions in the single ion mode, met all predefined acceptance criteria for the quantification of AMP, ADP, ATP, CDP, CTP, FAD, GDP, GTP, UDP, and UTP. Detection limits ranged from 0.25 pmol on-column (FAD) to 4 pmol (NAD+). Assay development also included validation of tissue sample collection procedures. ATP/ADP concentrations and the resulting energy charge in kidney tissues are very sensitive to hypoxia, with significant decreases occurring within seconds. Avoidance of hypoxia during tissue retrieval is critical, and in vivo freeze clamping compares favorably with other tissue collection techniques.