Navigation Links
Simultaneous Detection of CYP3A4, CYP2D6 and CYP2C9 Metabolites with a Single, Sensitive, LC/MS/MS Method

Jason S. Wood, Varian, Inc.

Introduction

Cytochrome P450s (CYPs) are heme-containing enzymes responsible for initial metabolism of xenobiotics within the human body1. CYPs are part of the Phase I (oxidative) metabolism within the body, which introduces a hydrophilic site on the metabolized drug enabling either easy elimination of the drug or further conjugation by Phase II enzymes, such as UDPGTs, sulfotransferases or amino acid-conjugation (among others)2. Recently there has been increased interest in understanding CYPs to discover the underlying mechanisms of drug-drug interactions3, drug side effect(s)4 and toxicity (including hepatotoxicity). Also relevant to CYP testing is the discovery that polymorphisms in the genetic sequences expressing these enzymes leads to populations of people whose ability to metabolize certain classes of drugs may be attenuated in comparison to other races/populations5.

In this application note LC-ESI-MS/MS is used to simultaneously characterize three drug metabolites for the detection of three different cytochrome P450s enzymatic activities; the hydroxylation of midazolam (CYP3A4), hydroxylation of tolbutamide (CYP2C9) and Odemethylation of dextromethorphan (CYP2D6), see Figure 1. This assay could be used to analyze CYP3A4, CYP2D6 and CYP2C9 activity in vitro for kinetic studies (e.g., liver microsome inhibition studies) or possibly for detection of these metabolites in more complex matrices (plasma or urine) after SPE.

Instrumentation

Varian ProStar 210 Solvent Delivery Modules (2)

Varian 1200L LC/MS equipped with an ESI source

Varian ProStar 430 AutoSampler

Materials and Reagents

All chemicals were reagent or HPLC grade from Sigma- Aldrich Corporation (St. Louis, MO) with the exception of midazolam and α-hydroxymidazolam standard solutions (1 mg/mL and 100 μg/mL in methanol, respectively) from Cerilliant (Round Rock, TX).

Sample Preparation

Stock solutions were prepared in HPLC grade water (dextrorphan) or methanol (4-hydroxytolbutamide) at 1 mg/ mL. All diluted samples were prepared in 50:50 methanol: water (HPLC grade).

Discussion

Testing of drug metabolizing enzymes (such as cytochrome P450s) is becoming increasingly important for the evaluation of new chemical entities (NCEs) before evaluation of the new drug in the clinic. Newly synthesized compounds that display proper biological effects on the target of choice in vitro, can and should be evaluated for their effects on drug metabolizing enzymes before an expensive scale-up of the new drug takes place for clinical testing. CYP3A4, 2D6 and 2C9 were chosen for our evaluation because 45-60% of the drugs currently on todays market are metabolized by CYP3A isoforms alone6, and CYP2D6 and CYP2C family members are subject to polymorphisms leading to populations of people that are poor metabolizers7. Having a combined method for all three would, therefore, be useful for testing samples from all of these enzymes at one time, eliminating the need to switch solvents and/or whole methods to evaluate these enzymes.

Good separation of the metabolites is obtained, in less than 5 minutes, on a Varian MonoChrom MS column with 0.1% formic acid (Solvent A) and acetonitrile (Solvent B) with the gradient mentioned (see LC Method, above). Figure 2A C, shows the individual peaks as detected by single reaction mode (SRM) in the 1200L. Limit of detection (LOD) for each of these metabolites was determined to be: 0.5, 0.25 and 0.25 pg (4-hydroxytolbutamide, -hydroxymidazolam and dextrorphan, respectively) and the limit of quantitation for each of these metabolites was determined to be: 1.0, 0.5 and 0.5 pg, respectively.

Calibration curves for the three metabolites were run in triplicate over the range of 1000 to 1 pg and are shown in Figures 3 - 5. The original xenobiotics can also be quantified, if desired, to determine percent usage of the drug (data not shown). Excellent linearity is seen within this range with the average correlation coefficient being 0.993. Accuracy in retention times of the metabolites is also excellent with an average 1.15% RSD, the dextrorphan being slightly more variable (2.74% RSD).

Conclusion

With this application note we have demonstrated a single method for the separation and quantification of the enzymatic activities of the three most important Cytochrome P450 enzymes (3A4, 2D6 and 2C9). This method can be used for pre-clinical evaluation of NCEs as inhibitors of CYPs or quantification of these metabolites from clinical samples after SPE.

References

1. Wrighton, S. A., Stevens, J. C. Crit. Rev. Toxicol. 1, 1-21 (1992).

2. Kwak, M. K. et al. Mutat. Res. 480-481, 305-15 (2001).

3. Honig, P. K. et al. J. Am. Med. Assoc. 269, 1513- 1518 (1993).

4. Kuntzman, R., et al. J. Pharmacol. Exp. Ther. 152, 151 (1996).

5. Lee, C.R., Goldstein, J.A., Pieper, J.A. Pharmacogenetics 3, 251-63 (2002).

6. Wojnowski, L. Ther. Drug. Monit. 26, 192-9 (2004).

7. Brockmoller J., et al. Pharmacogenomics 1, 125-151 (2000).


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Simultaneous Detection of 8 Cytokines in Mouse or Human Sera
2. Simultaneous Determination of Residues of Approximately 100 Pesticides and Metabolites in Fruit and Vegetables by LC/MS/MS
3. Simultaneously characterizing and quantifying chloramphenicol and its metabolite using LC/MS/MS
4. Simultaneous Screening of 23 Drugs of Abuse in Oral Fluid Using an LC/MS/MS Method
5. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
6. A New PCR-based Mycoplasma Detection Method
7. HSVision Molecular Beacon Detection Module Rapidly Detects Herpes Simplex Virus DNA
8. Detection and Identification of Phosphorylation Sites in Proteins Using LC/MS/MS with Neutral Fragment Loss Mapping
9. Detection of mRNAs on Cryosections of the Cardiovascular System Using DIG-Labeled RNA Probes
10. Gene Expression Arrays: Highly Sensitive Detection of Expression Patterns with Improved Tools for Target Amplification
11. The DIG System Nonradioactive and Highly Sensitive Detection of Nucleic Acids
Post Your Comments:
(Date:9/29/2014)... 29, 2014 Transparency Market Research, in ... SPPS, LPPS, HPPS, by Application - Cancer, Metabolic, CVS, ... and by Delivery) - Global Industry Analysis, Size, Share, ... the global peptide therapeutics market was worth US$14.1 billion ... by 2018 at an 8.7% CAGR. , The two ...
(Date:9/29/2014)... YORK , Sept. 29, 2014 ... and The Channel Group ("TCG"), a leader in ... advisors, have formed an alliance to expand the ... prospective clients. With the alliance, Burnham Securities will ... the scientific, clinical medicine and management in healthcare ...
(Date:9/29/2014)... Latest study suggests ... network connectivity as customers put quality and price ... , the leading independent provider of global optical ... their survey conducted at the 2014 European Conference ... professionals from across the globe. "The ...
(Date:9/29/2014)... , Sept. 29, 2014  Perrigo Company ... today that it has transferred a portfolio of ... December 2013 acquisition of Elan Corporation plc, to ... terms of the agreement, Perrigo will receive an ... payments and royalties on any future sales derived ...
Breaking Biology Technology:Global Peptide Therapeutics Market to be Worth US$25.4 Billion by 2018: Transparency Market Research 2Global Peptide Therapeutics Market to be Worth US$25.4 Billion by 2018: Transparency Market Research 3Burnham Securities Inc. and The Channel Group Form Healthcare Alliance 2Burnham Securities Inc. and The Channel Group Form Healthcare Alliance 3End of an Era: OEM Dominance - Could it be a Thing of the Past? 2Perrigo Announces the Out-Licensing of Preclinical Assets to Imago Pharmaceuticals 2Perrigo Announces the Out-Licensing of Preclinical Assets to Imago Pharmaceuticals 3Perrigo Announces the Out-Licensing of Preclinical Assets to Imago Pharmaceuticals 4
... analysis results expected in Q3 2008 -, ... Therapeutics Inc.,(TSX:FXI) ("Fralex" or the "Company"), a ... today announced the,randomization of the 100th patient ... double-blind placebo-controlled randomized clinical trial,designed to assess ...
... Calif., March 10 AutoGenomics, a leader in,developing ... be among the leading healthcare and biotechnology companies ... that will be held at the,Boston Marriott Copley ... Executive Officer will present an overview of the,company, ...
... Nuclear Security Administration has awarded a $17 million cooperative ... Park to develop advanced simulations for commercial and defense ... center will focus on the behavior and reliability of ... of Excellence chosen by the NNSA. , About 35 ...
Cached Biology Technology:Fralex reaches 100th patient milestone in fibromyalgia study 2Fralex reaches 100th patient milestone in fibromyalgia study 3Purdue leads center to simulate behavior of micro-electromechanical systems 2Purdue leads center to simulate behavior of micro-electromechanical systems 3Purdue leads center to simulate behavior of micro-electromechanical systems 4
(Date:9/29/2014)... to a new Food and Brand Lab pilot study, ... participation in the National School Lunch Program (NSLP) by ... 16%! , Chefs Move to Schools (CMTS), an initiative ... schools in order to provide nutrition instruction to students ... , A CMTS event was held in an Upstate ...
(Date:9/29/2014)... team says recently identified radiation detection properties of a ... for homeland security and medical advances. , In a ... of Optics Letters , UT Arlington Physics Professor ... to fabricate transparent nanoscintillators by heating nanoparticles composed of ... formed. A scintillator refers to a material that ...
(Date:9/29/2014)... bad rap, and understandably so. The grain-of-rice-sized insects are ... of acres in the Western U.S. and Canada over ... belief, these pests may not be to blame for ... through the region. Instead, weather and topography play a ... these bark-boring beetles. , New research led by the ...
Breaking Biology News(10 mins):Chefs move to schools can increase school meal participation and vegetable intake among students 2UT Arlington researchers develop new transparent nanoscintillators for radiation detection 2UT Arlington researchers develop new transparent nanoscintillators for radiation detection 3Tree killers, yes, fire starters, no: Mountain pine beetles get a bad rap, study says 2Tree killers, yes, fire starters, no: Mountain pine beetles get a bad rap, study says 3Tree killers, yes, fire starters, no: Mountain pine beetles get a bad rap, study says 4
... both crop and ornamental plants. Each year, powdery mildew and ... intense research, little is known of the plant molecules that ... European research group lead by Ueli Grossniklaus, a plant geneticist ... Science shedding a new light on mildew susceptibility ...
... parent that takes their kid out for a walk knows ... that? Do kids and small adults walk differently from taller ... Peter Weyand from Southern Methodist University, USA, is fascinated by ... goes back to Max Kleiber,s work on resting metabolic rates ...
... are getting warmer. Conservative estimates suggest that tropical areas ... the end of this century. Does global warming spell ... at the Smithsonian Tropical Research Institute, and colleagues report ... million years ago rainforests prospered at temperatures that were ...
Cached Biology News:Fertility or powdery mildew resistance? 2Fertility or powdery mildew resistance? 3New equation calculates cost of walking for first time 2New equation calculates cost of walking for first time 3Tropical forest diversity increased during ancient global warming event 2
Porcine Aortic Endothelial Cells (PAOEC) (>500,000 cells)...
Bovine Pulmonary Artery Smooth Muscle Cells (BPASMC) (>500,000 cells)...
Image Acquisition and documentation system for fluorescent and non-fluorescent gels, membranes, blots, film, plates and assays...
... ChromaDoc-It features a 4.1 mega pixel ... capturing brilliant TLC color images., The ... shortwave and longwave UV tubes positioned on ... illumination for fluorescence studies , Interior ...
Biology Products: