In Vitro Ketamine CYP3A Mediated Metabolism Study using Mammalian Liver S9 Fractions, cDNA Expressed Enzymes and Liquid Chromatography Tandem Mass Spectrometry
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Biomedical chromatography ; vol. 28, no. 12, pp. 1660-1669.Abstract(s)
Ketamine is widely used in medicine in combination with several benzodiazepines including
midazolam. The objectives of this study were to develop a novel HPLC-MS/SRM method
capable of quantifying ketamine and norketamine using an isotopic dilution strategy in biological
matrices and study the formation of norketamine, the principal metabolite of ketamine with and
without the presence of midazolam, a well-known CYP3A substrate. The chromatographic
separation was achieved using a Thermo Betasil Phenyl 100 x 2 mm column combined with an
isocratic mobile phase composed of acetonitrile, methanol, water and formic acid (60:20:20:0.4)
at a flow rate of 300 μL/min. The mass spectrometer was operating in selected reaction
monitoring mode and the analytical range was set at 0.05–50 μM. The precision (%CV) and
accuracy (%NOM) observed were ranging from 3.9–7.8 and 95.9.2–111.1% respectively. The
initial rate of formation of norketamine was determined using various ketamine concentration and
Km values of 18.4 μM, 13.8 μM and 30.8 μM for rat, dog and human liver S9 fractions were
observed respectively. The metabolic stability of ketamine on liver S9 fractions was significantly
higher in human (T1/2 = 159.4 min) compared with rat (T1/2 = 12.6 min) and dog (T1/2 = 7.3 min)
liver S9 fractions. Moreover significantly lower IC50 and Ki values observed in human compared
with rat and dog liver S9 fractions. Experiments with cDNA expressed CYP3A enzymes showed
the formation of norketamine is mediated by CYP3A but results suggest an important
contribution from others isoenzymes, most likely CYP2C particularly in rat.
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