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:11/27/2014)... November 27, 2014 First ...   Elsevier , a world-leading provider ... is pleased to announce the launch of a new ... The launch of Extreme Mechanics ... at the forefront of applied sciences such as micro ...
(Date:11/27/2014)... BELLINGHAM, Washington, and PITTSFIELD, Massachusetts, USA (PRWEB) November 26, ... pinpoint cancer cells with more accuracy, additive systems to ... improve highway safety by analyzing road conditions are among ... Awards for Photonics Innovation . The awards are sponsored ... photonics , and Photonics Media . , Winners ...
(Date:11/26/2014)... 2014 The report on the Global ... the concerned market for revenue forecasts and market trends. ... of 51.9% from 2013 to 2018. At the projected ... of $1,415.6 million by 2018. , Browse through the ... for information about the market segmentation and in-depth analysis ...
(Date:11/26/2014)... The North America Thermal Protective Clothing Market ... analysis of the growth trends and revenue forecasts. ... by 2018, growing at a CAGR of 7.1% ... TOC of the North America Thermal Protective Clothing ... in-depth analysis and industry segmentation supported by various ...
Breaking Biology Technology:Elsevier Announces the Launch of a New Journal: Extreme Mechanics Letters 2Elsevier Announces the Launch of a New Journal: Extreme Mechanics Letters 3Finalists Named for 2015 Prism Awards for Photonics Innovation 2Finalists Named for 2015 Prism Awards for Photonics Innovation 3Finalists Named for 2015 Prism Awards for Photonics Innovation 4Finalists Named for 2015 Prism Awards for Photonics Innovation 5Global Smart Sensor Market in Building Automation is expected to reach a value of $1,415.6 million by 2018 - Report by MicroMarket Monitor 2Global Smart Sensor Market in Building Automation is expected to reach a value of $1,415.6 million by 2018 - Report by MicroMarket Monitor 3Global Smart Sensor Market in Building Automation is expected to reach a value of $1,415.6 million by 2018 - Report by MicroMarket Monitor 4The North America Thermal Protective Clothing Market is estimated to grow up to $616.3 million by 2018 - Report by MicroMarket Monitor 2The North America Thermal Protective Clothing Market is estimated to grow up to $616.3 million by 2018 - Report by MicroMarket Monitor 3
... Silence Therapeutics plc (London AIM:,SLN), a leading ... the successful opposition of a fundamental Alnylam,Pharmaceuticals, Inc. ... its entirety., The Opposition Division of the ... its decision to revoke European Patent EP 1,230 ...
... First-in-Man study success, Ovalum Ltd. launches a multicenter study using ... privately held Israeli medical device company, has announced today successful ... in the coronary arteries. , ... ...
... European Molecular Biology Laboratory (EMBL) discovered a new way to ... computational method that compares how similar the side effects of ... on the same target molecule. The study, published in ... drugs. , Similar drugs often share target proteins, modes of ...
Cached Biology Technology:Silence Therapeutics Announces Successful Opposition of Glover Patent 2Silence Therapeutics Announces Successful Opposition of Glover Patent 3Ovalum Ltd. Announces Success in Coronary Cases Using CiTop™ ExPander™ Guidewire 2Teaching old drugs new tricks 2
(Date:11/7/2014)... Debra Auguste, associate professor, biomedical engineering, in the ... of New York, have identified a molecule that ... the most aggressive forms of breast cancer. , ... mortality rate owing to aggressive proliferation and metastasis ... Professor Auguste,s team, discovered the overexpression of intercellular ...
(Date:11/7/2014)... November 6, 2014 Leading Biometric companies ... mobile security revolutionizing online transactions.  Companies in focus today are: ... (NYSE: BABA ), Google Inc. (NASDAQ: ... Inc. (NASDAQ: EBAY ) and MasterCard Inc. (NYSE: ... and NXTDW), a biometric authentication company focused on the growing ...
(Date:11/6/2014)... occur because of metastasis, yet progress in preventing ... , "It,s been particularly challenging to design drugs ... fellow in systems biology at Harvard Medical School. ... after they,ve already metastasized." , Gujral and colleagues ... help researchers better understand how metastasis begins. Their ...
Breaking Biology News(10 mins):Universal Authentication Methods Provide More Secure Operations for Biometrics Devices - Company Joins FIDO Alliance for Consumer Protection 2Universal Authentication Methods Provide More Secure Operations for Biometrics Devices - Company Joins FIDO Alliance for Consumer Protection 3Universal Authentication Methods Provide More Secure Operations for Biometrics Devices - Company Joins FIDO Alliance for Consumer Protection 4Universal Authentication Methods Provide More Secure Operations for Biometrics Devices - Company Joins FIDO Alliance for Consumer Protection 5Universal Authentication Methods Provide More Secure Operations for Biometrics Devices - Company Joins FIDO Alliance for Consumer Protection 6Migration negation 2Migration negation 3
... Motorola Launches New Multi-Modal Biometric Identification Solution ... Delivering Enhanced Performance and Interoperability, SCHAUMBURG, Ill., ... today announced that it has been selected by ... Printrak Biometric Identification,Solution (BIS). Motorola,s latest BIS solution ...
... demonstrate both genetic and pharmaceutical evidence for the role ... multiple sclerosis (MS), providing a potential new way to ... member of a protein family called matrix metalloproteinases (MMPs, ... break down collagen and other components of the body,s ...
... Medical, environmental and chemical conundrums including the mystery of ... create clouds will be unravelled at a conference hosted ... UCLs Materials Simulation Laboratory on Monday 31 March 2008. The ... such as nanosensors that could one day be used for ...
Cached Biology News:Motorola Biometric Identification System Selected by West Virginia State Police 2New horizons in modelling surface processes 2
...
... is a genetically engineered Klenow polymerase in which ... removed, leaving only the 5 to 3 polymerase ... Klenow Fragment the enzyme of choice for isotopic ... random primed method, and for DNA sequencing by ...
Request Info...
supplied with 10x reaction buffer...
Biology Products: