Selective Analysis of Dopamine Receptor Antagonist LE300 and its N-Methyl Metabolite in Mouse Sera at the Trace Level by HPLC–Fluorescence Detection
A highly selective, sensitive, and reliable high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of a novel type of dopamine receptor antagonist LE300 and its N-methyl metabolite in mouse sera. LE300, its N-methyl metabolite, and verapam...
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Published in | Chromatographia Vol. 78; no. 9-10; pp. 655 - 661 |
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Main Authors | , , , , , , , , |
Format | Journal Article |
Language | English |
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Springer-Verlag
01.05.2015
Springer Berlin Heidelberg |
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Abstract | A highly selective, sensitive, and reliable high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of a novel type of dopamine receptor antagonist LE300 and its N-methyl metabolite in mouse sera. LE300, its N-methyl metabolite, and verapamil (an internal standard) were detected using excitation and emission wavelengths of 275 and 340 nm, respectively. HPLC analysis using a deproteinization procedure was performed by injecting an aliquot of the supernatant into the chromatographic system. Chromatographic separation was achieved on a reversed-phase Spherisorb Cyano (CN) column with a mobile phase consisting of acetonitrile:50 mM phosphate buffer pH 3.5 (70:30, v/v) pumped at a flow rate of 1.0 mL min⁻¹. Regression analyses showed excellent linearity (r = 0.999) for concentrations of LE300 ranging from 4 to 500 ng mL⁻¹and for concentrations of its N-methyl metabolite of 6–600 ng mL⁻¹. The HPLC-FLD method had limits of detection of 1.6 ng mL⁻¹for LE300 and 2.4 ng mL⁻¹for its N-methyl metabolite in mouse sera. The precision results, expressed as the intraday and interday relative standard deviation (RSD) values, ranged from 0.65 to 2.85 % (repeatability) and from 0.37 to 2.62 % (intermediate precision) for LE300 and its N-methyl metabolite, respectively; these values are in line with ICH guidelines. The assay was successfully applied in a pharmacokinetic study. The mean values of Tₘₐₓand Cₘₐₓwere 2 h and 25.03 ± 5.60 ng mL⁻¹for LE300 and 3 h and 19.92 ± 2.88 ng mL⁻¹for its N-methyl metabolite, respectively. |
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AbstractList | A highly selective, sensitive, and reliable high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of a novel type of dopamine receptor antagonist LE300 and its
N
-methyl metabolite in mouse sera. LE300, its
N
-methyl metabolite, and verapamil (an internal standard) were detected using excitation and emission wavelengths of 275 and 340 nm, respectively. HPLC analysis using a deproteinization procedure was performed by injecting an aliquot of the supernatant into the chromatographic system. Chromatographic separation was achieved on a reversed-phase Spherisorb Cyano (CN) column with a mobile phase consisting of acetonitrile:50 mM phosphate buffer pH 3.5 (70:30, v/v) pumped at a flow rate of 1.0 mL min
−1
. Regression analyses showed excellent linearity (
r
= 0.999) for concentrations of LE300 ranging from 4 to 500 ng mL
−1
and for concentrations of its
N
-methyl metabolite of 6–600 ng mL
−1
. The HPLC-FLD method had limits of detection of 1.6 ng mL
−1
for LE300 and 2.4 ng mL
−1
for its
N
-methyl metabolite in mouse sera. The precision results, expressed as the intraday and interday relative standard deviation (RSD) values, ranged from 0.65 to 2.85 % (repeatability) and from 0.37 to 2.62 % (intermediate precision) for LE300 and its
N
-methyl metabolite, respectively; these values are in line with ICH guidelines. The assay was successfully applied in a pharmacokinetic study. The mean values of
T
max
and
C
max
were 2 h and 25.03 ± 5.60 ng mL
−1
for LE300 and 3 h and 19.92 ± 2.88 ng mL
−1
for its
N
-methyl metabolite, respectively. A highly selective, sensitive, and reliable high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of a novel type of dopamine receptor antagonist LE300 and its N-methyl metabolite in mouse sera. LE300, its N-methyl metabolite, and verapamil (an internal standard) were detected using excitation and emission wavelengths of 275 and 340 nm, respectively. HPLC analysis using a deproteinization procedure was performed by injecting an aliquot of the supernatant into the chromatographic system. Chromatographic separation was achieved on a reversed-phase Spherisorb Cyano (CN) column with a mobile phase consisting of acetonitrile:50 mM phosphate buffer pH 3.5 (70:30, v/v) pumped at a flow rate of 1.0 mL min⁻¹. Regression analyses showed excellent linearity (r = 0.999) for concentrations of LE300 ranging from 4 to 500 ng mL⁻¹and for concentrations of its N-methyl metabolite of 6–600 ng mL⁻¹. The HPLC-FLD method had limits of detection of 1.6 ng mL⁻¹for LE300 and 2.4 ng mL⁻¹for its N-methyl metabolite in mouse sera. The precision results, expressed as the intraday and interday relative standard deviation (RSD) values, ranged from 0.65 to 2.85 % (repeatability) and from 0.37 to 2.62 % (intermediate precision) for LE300 and its N-methyl metabolite, respectively; these values are in line with ICH guidelines. The assay was successfully applied in a pharmacokinetic study. The mean values of Tₘₐₓand Cₘₐₓwere 2 h and 25.03 ± 5.60 ng mL⁻¹for LE300 and 3 h and 19.92 ± 2.88 ng mL⁻¹for its N-methyl metabolite, respectively. |
Author | Mostafa, Gamal Mohammed, Mostafa El-Subbagh, Hussein Lehmann, Jochen Abounassif, Mohammed Al-Swaidan, Ibrahim Alanazi, Amer Attia, Sabry Hefnawy, Mohamed |
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Cites_doi | 10.1016/j.chroma.2014.03.077 10.1007/s00210-002-0641-z 10.1002/ardp.201000121 10.1016/j.jpba.2007.06.013 10.4155/bio.11.240 10.1021/jm050846j 10.1016/j.ejmech.2004.02.001 10.1016/S0163-7258(99)00029-7 |
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SubjectTerms | Analytical Chemistry antagonists Chemistry Chemistry and Materials Science Chromatography detection limit dopamine receptors guidelines high performance liquid chromatography Laboratory Medicine metabolites mice Original pharmacokinetics Pharmacy phosphates Proteomics regression analysis verapamil wavelengths |
Title | Selective Analysis of Dopamine Receptor Antagonist LE300 and its N-Methyl Metabolite in Mouse Sera at the Trace Level by HPLC–Fluorescence Detection |
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