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.Efavirenz is a potentNNRTI that has been approved by regulatory agencies worldwide to treat AIDS.Extensive use of efavirenz has demonstrated a durable, long-lasting reduction inHIV RNA after once-a-day dosing in combination with other drugs such as AZTand 3-TC.Efavirenz is metabolized extensively by rats, cynomolgus monkeysand humans with the major metabolite being the 8-OH efavirenz glucuronide.LC NMR MS analyses of bile and urine samples from rats showed the presenceof glutathione-derived adducts formed by enzymatic addition of glutathioneacross the triple bond of the acetylene moiety [38,39].The formation of suchconjugates has also been linked to the species-specific nephrotoxicity observed inrats [40]. On-Line LC NMR in Drug Metabolism Studies 994.3.1 EXPERIMENTALSample PreparationThe sample for analysis was prepared by solid-phase extraction of 5 ml of 0 24 hurine obtained from rats after dosing with 800 mg/kg of efavirenz.The extractwas dried and reconstituted with 100 ml of 80 % D2O and 20 % acetonitrile-d3.HyphenationThe control of the hyphenated system, comprised of an Agilent 1100 HPLC, aBruker Avance 600 MHz NMR spectrometer and a Bruker Daltonics Esquireion-trap mass spectrometer, was achieved by using HYSTAR software fromBruker Analytik.Peaks were selected for analysis using both UV and massdetection and were stored in the Bruker BPSU-36 loop storage unit prior toNMR analysis.A schematic diagram of the system is shown in Figure 4.9.14.1 tesla(600 MHz)Stray fieldShielded Magnet(radial) 5 gauss1.8 mLC/flowprobeGlison 215 forflow injectionFraction collectorBPSU36Esquire Ion-TrapInjectionDiode-Mass SpectrometerHPLCvalvearraysystemdetectorBNMILC columnSplitter 20:1AutosamplerBruker AvanceDRX 600HPLC Pentium PC Silicon GraphicsConsole/RF systemFigure 4.9 A schematic diagram of an LC NMR MS system 100 LC NMR and Related TechniquesLiquid ChromatographyAn LC system was used with a Bruker diode-array detector set at 254 nm.A 3.9× 150 mm Waters Symmetry® C18 column was used.A gradient from 75 %D2O and 25 % acetonitrile-d3 to 50 % D2O and 50 % acetonitrile-d3 over 20 minat a flow rate of 0.8 ml/min was employed for separation.Both solvents con-tained 0.05 % trifluoroacetic acid (TF A).Mass SpectrometryThe mass spectrometry portion of the analysis was carried out by coupling aBruker Esquire ion-trap mass spectrometer to the LC NMR system with a 20:1splitter.The major portion of the flow was directed to the NMR systemwhile the minor fraction went to the mass spectrometer.The system wasplumbed such that the sample reached the mass spectrometer and the UVdetector at the same time.In this configuration, it is possible to use the massspectrometer as an intelligent detector, thus allowing stop-flow experiments tobe initiated on the basis of observed molecular ions or daughter ion fragments.Data were acquired with electro-spray ionization (ESI) in the positive-ionmode.NMR SpectroscopyAll NMR data were obtained on a Bruker Avance 600 MHz NMR spectrom-191eter equipped with a 4 mm H F dual LC NMR flow-probe, (cell volume/of 120 "l).Chemical shifts were referenced to acetonitrile at & 2.0 ppm for1 19H NMR, while F NMR chemicals shifts were referenced to TFA at 78.5 ppm.1H LC NMR1The H LC NMR spectra were obtained on peaks stored in the BPSU-36 storage loops.Data were acquired with WET [41] solvent suppressionon the residual water and acetonitrile signals.A composite 90 observepulse, (% (% 2) x (% (% 2)x, was employed.Spectra were collectedy y/2) /2)/ /into 32K data points over a width of 12 019 Hz, giving an acquisition time of1.36 s, with an additional relaxation delay of 1.5 s.The data were multiplied bya line-broadening function of 1 Hz to improve the signal-to-noise ratio andzero-filled by a factor of two before Fourier transformation. On-Line LC NMR in Drug Metabolism Studies 10119F Continuous-Flow LC NM R19Continuous-flow F LC NMR spectra were acquired for 16 transientsusing 60 pulses into 8192 data points over a spectral width of 11 364 Hz, givingan acquisition time of 0.36 s.A relaxation delay of 0.64 s was added togive a total acquisition time for each spectrum of 16 s.The data weremultiplied by a line-broadening function of 3 Hz to improve the signal-to-noise ratio and zero-filled by a factor of two before Fourier transformation [ Pobierz caÅ‚ość w formacie PDF ]
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.Efavirenz is a potentNNRTI that has been approved by regulatory agencies worldwide to treat AIDS.Extensive use of efavirenz has demonstrated a durable, long-lasting reduction inHIV RNA after once-a-day dosing in combination with other drugs such as AZTand 3-TC.Efavirenz is metabolized extensively by rats, cynomolgus monkeysand humans with the major metabolite being the 8-OH efavirenz glucuronide.LC NMR MS analyses of bile and urine samples from rats showed the presenceof glutathione-derived adducts formed by enzymatic addition of glutathioneacross the triple bond of the acetylene moiety [38,39].The formation of suchconjugates has also been linked to the species-specific nephrotoxicity observed inrats [40]. On-Line LC NMR in Drug Metabolism Studies 994.3.1 EXPERIMENTALSample PreparationThe sample for analysis was prepared by solid-phase extraction of 5 ml of 0 24 hurine obtained from rats after dosing with 800 mg/kg of efavirenz.The extractwas dried and reconstituted with 100 ml of 80 % D2O and 20 % acetonitrile-d3.HyphenationThe control of the hyphenated system, comprised of an Agilent 1100 HPLC, aBruker Avance 600 MHz NMR spectrometer and a Bruker Daltonics Esquireion-trap mass spectrometer, was achieved by using HYSTAR software fromBruker Analytik.Peaks were selected for analysis using both UV and massdetection and were stored in the Bruker BPSU-36 loop storage unit prior toNMR analysis.A schematic diagram of the system is shown in Figure 4.9.14.1 tesla(600 MHz)Stray fieldShielded Magnet(radial) 5 gauss1.8 mLC/flowprobeGlison 215 forflow injectionFraction collectorBPSU36Esquire Ion-TrapInjectionDiode-Mass SpectrometerHPLCvalvearraysystemdetectorBNMILC columnSplitter 20:1AutosamplerBruker AvanceDRX 600HPLC Pentium PC Silicon GraphicsConsole/RF systemFigure 4.9 A schematic diagram of an LC NMR MS system 100 LC NMR and Related TechniquesLiquid ChromatographyAn LC system was used with a Bruker diode-array detector set at 254 nm.A 3.9× 150 mm Waters Symmetry® C18 column was used.A gradient from 75 %D2O and 25 % acetonitrile-d3 to 50 % D2O and 50 % acetonitrile-d3 over 20 minat a flow rate of 0.8 ml/min was employed for separation.Both solvents con-tained 0.05 % trifluoroacetic acid (TF A).Mass SpectrometryThe mass spectrometry portion of the analysis was carried out by coupling aBruker Esquire ion-trap mass spectrometer to the LC NMR system with a 20:1splitter.The major portion of the flow was directed to the NMR systemwhile the minor fraction went to the mass spectrometer.The system wasplumbed such that the sample reached the mass spectrometer and the UVdetector at the same time.In this configuration, it is possible to use the massspectrometer as an intelligent detector, thus allowing stop-flow experiments tobe initiated on the basis of observed molecular ions or daughter ion fragments.Data were acquired with electro-spray ionization (ESI) in the positive-ionmode.NMR SpectroscopyAll NMR data were obtained on a Bruker Avance 600 MHz NMR spectrom-191eter equipped with a 4 mm H F dual LC NMR flow-probe, (cell volume/of 120 "l).Chemical shifts were referenced to acetonitrile at & 2.0 ppm for1 19H NMR, while F NMR chemicals shifts were referenced to TFA at 78.5 ppm.1H LC NMR1The H LC NMR spectra were obtained on peaks stored in the BPSU-36 storage loops.Data were acquired with WET [41] solvent suppressionon the residual water and acetonitrile signals.A composite 90 observepulse, (% (% 2) x (% (% 2)x, was employed.Spectra were collectedy y/2) /2)/ /into 32K data points over a width of 12 019 Hz, giving an acquisition time of1.36 s, with an additional relaxation delay of 1.5 s.The data were multiplied bya line-broadening function of 1 Hz to improve the signal-to-noise ratio andzero-filled by a factor of two before Fourier transformation. On-Line LC NMR in Drug Metabolism Studies 10119F Continuous-Flow LC NM R19Continuous-flow F LC NMR spectra were acquired for 16 transientsusing 60 pulses into 8192 data points over a spectral width of 11 364 Hz, givingan acquisition time of 0.36 s.A relaxation delay of 0.64 s was added togive a total acquisition time for each spectrum of 16 s.The data weremultiplied by a line-broadening function of 3 Hz to improve the signal-to-noise ratio and zero-filled by a factor of two before Fourier transformation [ Pobierz caÅ‚ość w formacie PDF ]