Comparison of three reference methods for the measurement of intracellular pH using 31P MRS in healthy volunteers and patients with lymphoma

31P magnetic resonance spectroscopy (31P MRS) can measure intracellular pH (pHi) using the chemical shift difference between pH‐dependent inorganic phosphate (Pi) and a pH‐independent reference peak. This study compared three different frequency reference peaks [phosphocreatine (PCr), α resonance of adenosine triphosphate (αATP) and water (using 1H MRS)] in a cohort of 10 volunteers and eight patients with non‐Hodgkin's lymphoma (NHL). Well‐resolved chemical shift imaging (CSI) spectra were acquired on a 1.5T scanner for muscle, liver and tumour. The pH was calculated for all volunteers and patients using the available methods. The consistency of the resulting pH was evaluated. The direct Pi–PCr method was best for those spectra with a very well‐defined PCr, such as muscle (pH=7.05 ± 0.02). In liver, the Pi–αATP method gave more consistent results (pH=7.30 ± 0.06) than the calibrated water‐based method (pH=7.27 ± 0.11). In NHL nodes, the measured pH using the Pi–αATP method was 7.25 ± 0.12. Given that the measured range includes some biological variation in individual patients, treatment‐related changes of the order of 0.1 pH units should be detectable. © 2013 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.