| Abstract: | Myriocin is a potent inhibitor of serine‐palmitoyl‐transferase, the first and rate‐determining enzyme in the sphingolipids biosynthetic pathway. This study developed, validated and applied a LC–MS/MS method to measure myriocin in minute specimens of animal tissue. The chemical analog 14‐OH–myriocin was used as the internal standard. The two molecules were extracted from the tissue homogenate by solid‐phase extraction, separated by gradient reversed‐phase liquid chromatography and measured by negative ion electrospray mass spectrometry in the triple quadrupole. Detection was accomplished by multiple reaction monitoring, employing the most representative transitions, 400@104 and 402@104 for myriocin and 14‐OH‐myriocin, respectively. The typical limit of detection and lower limit of quantitation of the optimized method were 0.9 pmol/mL (~0.016 pmol injected) and 2.3 pmol/mL, respectively, and the method was linear up to 250 pmol/mL range (r 2 = 0.9996). The intra‐ and between‐day repeatability afforded a coefficient of variation ≤7.0%. Applications included quantification of myriocin in mouse lungs after 24 h from administration of ~4 nmol by intra‐tracheal delivery. Measured levels ranged from 4.11 (median; 2.3–7.4 IQR, n = 4) to 11.7 (median; 7.6–22.7 interquartile range (IQR), n = 6) pmol/lung depending on the different formulations used. Myriocin was also measured in retinas of mice treated by intravitreal injection and ranged from 0.045 (less than the limit of detection) to 0.35 pmol/retina. |
