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

31 P magnetic resonance spectroscopy ( 31 P MRS) can measure intracellular pH (pH i ) using the chemical shift difference between pH‐dependent inorganic phosphate (P i ) 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 1 H 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 P i –PCr method was best for those spectra with a very well‐defined PCr, such as muscle (pH=7.05 ± 0.02). In liver, the P i –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 P i –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.