MAGNETIC RESONANCE IN CHEMISTRY Magn. Reson. Chem. 2001; 39: 773–776 Short Communication Temperature calibration of a high-resolution magic-angle spinning NMR probe for analysis of tissue samples Andrew W. Nicholls 1∗ and Russell J. Mortishire-Smith 2 1 Biological Chemistry, Imperial College of Science, Technology and Medicine, Sir Alexander Fleming Building, South Kensington, London SW7 2AZ, UK 2 Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, UK Received 9 April 2001; Revised 13 July 2001; Accepted 13 July 2001 The increase in tissue sample temperature caused by the spinning of a rotor in high-resolution magic- angle spinning (HRMAS) NMR spectroscopy was investigated using two literature NMR thermometers. The classical methanol thermometer was used to calibrate the temperature difference between the set temperature and the actual sample temperature. At spin speeds of 2, 4, 6 and 8 kHz, a mean increase in sample temperature over the set temperature of 0.8 ± 0.1, 2.2 ± 0.1, 5.0 ± 0.1 and 7.9 ± 0.2 K, respectively, was noted. This calibration was used to gauge the accuracy of the literature glucose thermometer, which was shown to have a mean increase over the set temperature of -0.3 ± 0.1, 1.2 ± 0.2, 3.0 ± 0.2 and 6.1 ± 0.1K at 2, 4, 6 and 8 kHz, respectively. By correcting for the errors measured in this study, a more accurate determination and adjustment of sample temperature in HRMAS NMR spectroscopic analyses of intact tissues is now possible. Copyright  2001 John Wiley & Sons, Ltd. KEYWORDS: NMR; 1 H NMR; magic-angle spinning; temperature calibration; methanol; glucose INTRODUCTION Recent studies have shown the utility of high-resolution magic-angle spinning (HRMAS) NMR spectroscopy to investigations of biochemical variation in intact tissues, such as liver, 1 kidney, 2 prostate, 3 cardiac muscle, 4 brain, 5 and testis. 6 Spinning of the tissue sample, needed to induce mechanically sufficient molecular tumbling to overcome anisotropic effects, is typically of the order of 2–10 kHz. These relatively slow speeds stop the sample being subjected to high shearing forces, which might be expected to result in loss of cellular structure. However, some tissue types, notably brain tissue, start to undergo enzymatic degradation relatively quickly once removed from the animal. 5 This situation permits only the simplest of NMR experiments to be acquired on viable tissue. Such samples require analysis at reduced temperature, but to do so requires knowledge of the extent of sample heating caused by spinning of the HRMAS rotor. In this work, we calibrated the temperature increase in a typical HRMAS rotor caused by spinning of the sample Ł Correspondence to: A. W. Nicholls, Biological Chemistry, Imperial College of Science, Technology and Medicine, Sir Alexander Fleming Building, South Kensington, London SW7 2AZ, UK. E-mail: a.nicholls@ic.ac.uk Contract/grant sponsor: Merck Sharp and Dohme Ltd. using two literature methods, the methanol 7 and glucose thermometers. 8 EXPERIMENTAL All measurements were made on a Bruker DPX 400 spectrometer ( 1 H observation frequency 400.13 MHz) using a 4 mm 1 Hf 13 Cg HRMAS probe with a single-gradient coil aligned to the magic angle and a BVT3300 variable- temperature unit. This unit allowed the probe temperature to be set in integer intervals with subsequent control to within š0.2 ° C. The bearing gas for the HRMAS rotor was cooled using a heat exchanger placed in a mixture of acetone and dry-ice. Temperature measurements were made between 277 and 312 K in 5 K increments at spin speeds of 2, 4, 6 and 8 kHz. At each measurement point a temperature equilibration period of 4 min was left before NMR spectra were acquired. Farrant et al. 8 showed that this period was sufficient to achieve thermal equilibrium. For calibration of the probe temperature, an air-saturated methanol sample containing a trace level of methanol-d 4 to act as the field frequency lock was placed in a 4 mm zirconium oxide rotor, resulting in an active volume of ¾80 μl. Single-pulse NMR spectra were acquired using an initial 32 dummy scans followed by the collection of eight free induction decays (FIDs) into 16 K data points with a DOI: 10.1002/mrc.924 Copyright  2001 John Wiley & Sons, Ltd.