Non-Invasive Monitoring of Corticosterone Levels in Mouse Urine with Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry

An innovative method for the quantification of corticosterone in the urine of C57BL/6J mice by liquid chromatography-electrospray ionization-tandem mass spectrometry was developed. Unconjugated and glucuronidated corticosterone was detected in the urine samples using enzymatic hydrolysis following liquid-liquid extraction. After optimization of the extraction protocol and LC-MS/MS parameters, we performed a validation study using a representative urine pool of C57BL/6J and Naval Medical Research Institute mice. The method shows good linearity (1-5000 fmol/µL) and the calculated limit of quantification amounts to 0.823 fmol/µL. Both intra-day and inter-day variation was ≤10%, while their recoveries amounted to 90.4-110.6% and 99.8%, respectively. Twenty-four hour urine collection of C57BL/6J mice restrained in two different metabolic cage types for two times was used to test the validated method. To control the hydration level of mice, the corticosterone concentration in their urine was normalized to urinary creatinine concentration. Our LC-MS/MS method represents a highly specific analytical tool for the quantification of corticosterone levels in urine samples, assisting in non-invasive monitoring of acute stress levels in laboratory mice.