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 is used as the internal standard. The two molecules are extracted from the tissue homogenate by solid-phase extraction, separated by gradient reverse-phase liquid chromatography and measured by negative ion electrospray mass spectrometry in the triple quadrupole. Detection is accomplished by Multiple Reaction Monitoring, employing the most representative transitions: 400@104 and 402@104 for Myriocin and 14-OH-Myriocin, respectively. The typical LoD and LLoQ of the optimized method are 0.9 pmoles/mL (approx. 0.016 pmoles injected) and 2.3 pmoles/mL, respectively, and the method is linear up to 250 pmoles/mL range (r2= 0.9996). The intra-and between-day repeatability affords a CV% ≤ 7.0. Applications included quantification of Myriocin in mouse lungs after 24 hrs from administration of ~4 nmoles by intra-trachea delivery. Measured levels ranged from 4.11 (median; 2.3-7.4 IQR, n=4) to 11.7 (median; 7.6-22.7 IQR, n=6) pmoles/lung depending on the different formulations used. Myriocin was also measured in retinas of mice treated by intravitreal injection and ranged from 0.045 (<LoD) to 0.35 pmoles/retina.
Determination of the serine palmitoyl transferase inhibitor Myriocin by electrospray and Q-trap mass spectrometry / G.M. Campisi, P. Signorelli, J. Rizzo, C. Ghilardi, J. Antognetti, A. Caretti, J.S. Lazarevic´, E. Strettoi, E. Novelli, R. Ghidoni, F.M. Rubino, R.C. Paroni. - In: BIOMEDICAL CHROMATOGRAPHY. - ISSN 1099-0801. - (2017). [Epub ahead of print] [10.1002/bmc.4026]
Determination of the serine palmitoyl transferase inhibitor Myriocin by electrospray and Q-trap mass spectrometry
G.M. CampisiPrimo
;P. SignorelliSecondo
;J. Rizzo;A. Caretti;R. Ghidoni;F.M. RubinoPenultimo
;R.C. ParoniUltimo
2017
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 is used as the internal standard. The two molecules are extracted from the tissue homogenate by solid-phase extraction, separated by gradient reverse-phase liquid chromatography and measured by negative ion electrospray mass spectrometry in the triple quadrupole. Detection is accomplished by Multiple Reaction Monitoring, employing the most representative transitions: 400@104 and 402@104 for Myriocin and 14-OH-Myriocin, respectively. The typical LoD and LLoQ of the optimized method are 0.9 pmoles/mL (approx. 0.016 pmoles injected) and 2.3 pmoles/mL, respectively, and the method is linear up to 250 pmoles/mL range (r2= 0.9996). The intra-and between-day repeatability affords a CV% ≤ 7.0. Applications included quantification of Myriocin in mouse lungs after 24 hrs from administration of ~4 nmoles by intra-trachea delivery. Measured levels ranged from 4.11 (median; 2.3-7.4 IQR, n=4) to 11.7 (median; 7.6-22.7 IQR, n=6) pmoles/lung depending on the different formulations used. Myriocin was also measured in retinas of mice treated by intravitreal injection and ranged from 0.045 (File | Dimensione | Formato | |
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