Rapid method for monitoring muscle protein metabolites by LC‐MS

Abstract

America's growing elderly population has led to an increase in research on the aging process, including the relationship of muscle retention to health. Monitoring muscle development at the molecular level is an excellent complement to traditional whole‐muscle assessment methods. 3‐Methylhistidine (3‐MH) is an amino acid derivative and breakdown product of muscle protein. Urinary 3‐MH levels vary with diet and exercise, meriting its use as a molecular marker of muscle turnover. A method to perform rapid quantitation of 3‐MH in urine was developed using single‐ion monitoring (SIM) LC‐MS. After being spiked with an added internal standard and desalted by solid‐phase extraction, samples were analyzed by SIM LC‐MS, quantified versus the added internal standard and normalized versus a ubiquitous internal standard, creatinine. The use of LC‐MS eliminates the need for derivatization as required in established HPLC fluorescence and GC‐MS techniques. Constructed calibration curves plotting the ratio of 3‐MH to internal standard show a strong linear relationship with 3‐MH concentrations, with R‐squared values as high as 0.9983. The method presented is not only effective for monitoring 3‐MH, but holds potential for simultaneous monitoring of additional urine metabolites with run times of approximately 13 minutes.