Myriocin is a potent inhibitor of serine-palmitoyl-transferase, the first and rate-determining enzymein 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 analog14-OH-myriocin was used as the internal standard. The two molecules were extracted from the tis-sue homogenate by solid-phase extraction, separated by gradient reversed-phase liquid chromatog-raphy and measured by negative ion electrospray mass spectrometry in the triple quadrupole.Detection was accomplished by multiple reaction monitoring, employing the most representativetransitions, 400@104 and 402@104 for myriocin and 14-OH-myriocin, respectively. The typicallimit 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/mLrange (r2= 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 administra-tion 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 thedifferent formulations used. Myriocin was also measured in retinas of mice treated by intravitrealinjection and ranged from 0.045 (less than the limit of detection) to 0.35 pmol/retina.
Determination of the serine palmitoyl transferase inhibitor myriocin by electrospray and Q-trap mass spectrometry
Strettoi E;
2017
Abstract
Myriocin is a potent inhibitor of serine-palmitoyl-transferase, the first and rate-determining enzymein 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 analog14-OH-myriocin was used as the internal standard. The two molecules were extracted from the tis-sue homogenate by solid-phase extraction, separated by gradient reversed-phase liquid chromatog-raphy and measured by negative ion electrospray mass spectrometry in the triple quadrupole.Detection was accomplished by multiple reaction monitoring, employing the most representativetransitions, 400@104 and 402@104 for myriocin and 14-OH-myriocin, respectively. The typicallimit 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/mLrange (r2= 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 administra-tion 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 thedifferent formulations used. Myriocin was also measured in retinas of mice treated by intravitrealinjection and ranged from 0.045 (less than the limit of detection) to 0.35 pmol/retina.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.