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. Lo uis, 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:
*Name:
*Comment:
*Email:


(Date:2/24/2017)... 2017 China Biologic Products, Inc. (NASDAQ: CBPO) ("China ... company in China, today announced its financial results for the ... Fourth Quarter 2016 Financial Highlights Total ... in RMB terms, or increased by 13.6% in USD terms ... of 2015. Gross profit increased by 13.3% ...
(Date:2/23/2017)... -- Financial Highlights ... unaudited)Three Months Ended December 31,Twelve Months Ended December 31,20162015% ... $           300$   ... Product Revenue 3539(10)%9498(4)%Kuvan Net Product ... 756025%297303(2)%Vimizim Net Product Revenue ...
(Date:2/23/2017)... Atlanta, it seems everyone has a chance to express their ... expressive and dynamic community unlike any other. The businesses that ... With their newest salon in ... on that tradition with a unique, fresh approach to head ... the newest of 13 nationwide locations, each of them well-situated ...
(Date:2/23/2017)... ... February 23, 2017 , ... ... evaluation of multiple immunoassay-based threat detection technologies by researchers from the Pacific ... biosensor threat detection technology was found to have the best level of ...
Breaking Biology Technology:
(Date:2/3/2017)... , Feb. 3, 2017  Texas Biomedical Research Institute ... Larry Schlesinger as the Institute,s new President ... Biomed effective May 31, 2017. He is currently the Chair ... of the Center for Microbial Interface Biology at Ohio State ... as the new President and CEO of Texas Biomed," said ...
(Date:2/2/2017)... , Feb. 1, 2017  Central to its ... meaningful advances worldwide, The Japan Prize Foundation today ... who have pushed the envelope in their respective ... Communication. Three scientists are being recognized with the ... that not only contribute to the advancement of ...
(Date:1/26/2017)... Jan. 26, 2017  Crossmatch, a leading provider of ... solution aimed at combatting fraud, waste and abuse in ... at the Action on Disaster Relief conference in ... for UN agencies and foreign assistance organizations throughout ... and abuse are a largely unacknowledged problem in the ...
Breaking Biology News(10 mins):