Quantitative high-performance liquid chromatography–tandem mass spectrometry method for the analysis of free desmosines in plasma and urine

• For the first time quantification of free desmosines in human plasma is described.
• Utilization of a three step orthogonal sample preparation procedure was key.
• Sensitive, reproducible and accurate quantification was achieved.
• Avoiding acid hydrolysis of samples reduced the total analysis time significantly.
• A simplified method can be used to quantify free desmosines in urine samples.

A rapid method for the determination of the sum of free desmosine and isodesmosine in human plasma and urine is described. Efficient sample clean-up prior to LC–MS/MS analysis is mandatory for detection of free desmosines in plasma samples. The combination of ultra-filtration and a two-step solid phase extraction minimizes the sample complexity and ion suppression effects. The flow through from the ultra filtration is passed through a C18 resin and then the target analytes are trapped and enriched on a mixed mode solid phase extraction material. The combination of these three orthogonal sample preparation steps allows detection of endogenous free desmosines in plasma from healthy individuals. An analytical column packed with porous graphitic carbon material enables the retention of the polar desmosine analytes, which are measured by electrospray ionization tandem mass spectrometry. Deuterium labeled isodesmosine is added as internal standard and a linear calibration curve was constructed in the range of 0.1–2.0 nmol/L for plasma samples and 5–200 nmol/L for urine samples. These results demonstrate that the described LC–MS/MS method provides sensitive, repeatable and accurate quantification of free desmosines in plasma and urine samples.