A direct injection capillary electrophoretic technique for miniaturized high-throughput metabolic screening of the CYP 3A4 enzyme using quinidine as a probe

Bhoopathy, Siddhartha; Sarkar, Mohamadi; Karnes, H. Thomas

doi:10.1016/s0731-7085(01)00347-8

Cited by 9 publications

(3 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For a method to effectively screen for drug interactions it should be capable of screening for high potency, medium potency and low potency inhibitors of a particular enzyme. Previously we had developed a direct injection CE method for 3OHQ in the absorbance mode (233 nm; Bhoopathy and Karnes, 2001). This method was used to calculate the K i values for three inhibitors of the CYP 3A4 isozyme, ketoconazole, fluconazole and erythromycin in decreasing order of potency.…”

Section: Methodsmentioning

confidence: 99%

“…This probe is metabolized by the CYP 3 A4 isozyme to form 3OHQ (Nielsen et al, 1999) and hence this metabolite can be used as a biomarker for CYP 3A4 activity in liver microsomes and to study drug interactions with this isozyme in screening studies. Previously we had used absorbance detection (233 nm) for the determination of 3OHQ (Bhoopathy and Karnes, 2001).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Determination of (3S)‐3‐hydroxy quinidine for metabolism screening experiments using direct injection capillary electrophoresis and laser‐induced fluorescence detection

Bhoopathy

Karnes

2001

Biomedical Chromatography

View full text Add to dashboard Cite

Capillary electrophoresis (CE) has been used with collinear laser-induced fluorescence detection (LIF) to determine the amount of (3S)-3-hydroxy quinidine (3OHQ) formed on direct injection of microsomal incubation mixtures. 3OHQ is the CYP 3A4 metabolite of quinidine sulfate (QS) and is therefore useful for metabolism screening studies. The method was validated analytically and tested for its capability of screening for a weak inhibitor of the CYP 3A4 isozyme. A linear calibration was found to provide the best fit for the standard curve with a correlation of 0.9950 and all concentration residuals less than 15%. The percentage relative standard deviations (RSDs) of two controls, 175 and 2250 ng/mL, were 9.29 and 5.68% and the percentage differences from normal (DFN) were 6.87 and -4.37%, respectively. The concentration limit of detection (LOD) for 3OHQ in the incubation matrix was 52.11ng/mL and the mass LOD was approximately 521.1 fg (injection volume 10 nL). The effectiveness of the method to screen for the weak inhibitor erythromycin has been shown by calculating percentage inhibition when incubating with different concentrations of QS. Sensitive detection coupled with the convenience of the direct injection technique makes this an attractive approach for metabolism screening. The small sample size capability of CE will further reduce the quantities of probe drug, microsomes and other reagents required for incubation studies.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Determination of (3S)‐3‐hydroxy quinidine for metabolism screening experiments using direct injection capillary electrophoresis and laser‐induced fluorescence detection

Bhoopathy

Karnes

2001

Biomedical Chromatography

View full text Add to dashboard Cite

show abstract

“…Only few studies used capillary electrophoresis (CE) for the drug metabolism investigations. These studies focused on the analysis of incubation mixtures of drugs or model substances (coumarine, phenol, quinidine, mephenytoin, methadone, methaqualone, pyrazoloacridine, itraconazole) with human liver microsomes or cDNA-expressed human CYPs, by looking at the formation of metabolites [9][10][11][12][13][14][15][16][17][18]. Verapamil belongs to the pharmacological class of calcium channel blockers.…”

Section: Introductionmentioning

confidence: 99%

Kinetic study of CYP3A4 activity on verapamil by capillary electrophoresis

Zhang

Lou

et al. 2005

Journal of Pharmaceutical and Biomedical Analysis

View full text Add to dashboard Cite

Screening of drug metabolism by CE

et al. 2006

View full text Add to dashboard Cite

The use of CE for rapid assessment of metabolic stability of drugs with cytochrome P450 (CYP) enzymes, based on relative rates of reduced nicotinamide adenine dinucleotide phosphate (NADPH) consumption and nicotinamide adenine dinucleotide phosphate (NADP) production, was investigated. The separation conditions were as follows: capillary, 80.5 cm (75 microm id, 72 cm effective length for UV detection, 58 cm effective length for fluorescence detection); 25 mM sodium phosphate buffer (pH 8.8); 28 kV (80 microA) applied voltage; UV, 260 nm; fluorescence detection, excitation wavelength, 310 nm, emission wavelength, 418 nm; capillary temperature, 25 degrees C. For UV detection, the incubation conditions were as follows: CYP3A4: 20 pmol/mL; NADPH: 1 mM; EDTA: 1 mM; concentration of the substrate: 5-10 times its reported literature K(m) value; temperature: 37 degrees C; incubation time: 15 min. For fluorescence detection, the concentrations were reduced to CYP3A4: 4 pmol/mL, NADPH: 20 microM, EDTA: 20 microM and substrate: 10 microM. Blank incubations were performed in the absence of substrate. Compared with the blank, significant differences were found for the consumption of NADPH and the production of NADP. The development of this assay system allows rapid assessment of metabolic stability relative to standard compounds, as well as potential identification of the major CYP involved in the metabolism. It would reduce the backlog of compounds that require LC/MS analysis, and thereby expedite the process of metabolic stability screening.

show abstract

A direct injection capillary electrophoretic technique for miniaturized high-throughput metabolic screening of the CYP 3A4 enzyme using quinidine as a probe

Cited by 9 publications

References 12 publications

Determination of (3S)‐3‐hydroxy quinidine for metabolism screening experiments using direct injection capillary electrophoresis and laser‐induced fluorescence detection

Determination of (3S)‐3‐hydroxy quinidine for metabolism screening experiments using direct injection capillary electrophoresis and laser‐induced fluorescence detection

Kinetic study of CYP3A4 activity on verapamil by capillary electrophoresis

Screening of drug metabolism by CE

Contact Info

Product

Resources

About