**Funding**

The work was funded by National Foundation for Science and Technology Development (NAFOSTED), Research Project Code Number: 106.05-2010.35.

## **10. References**


[5] Bradford MM (1976) A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-54.

76 Chromatography – The Most Versatile Method of Chemical Analysis

analysed.

**Funding** 

**10. References** 

Nature 426(6966): 570-4.

Biotechnol. 22(9): 1139–45.

**Author details** 

**Acknowledgement** 

Phan Van Chi and Nguyen Tien Dung

*Department of Protein Biochemistry, Institute of Biotechnology (IBT), Vietnam Academy of Science & Technology (VAST), Hanoi, Vietnam* 

(IBT), Vietnam Academy of Science and Technology (VAST)

(NAFOSTED), Research Project Code Number: 106.05-2010.35.

trypsin-digested. The peptide mixtures extracted from each gel slice were fractionated by 2D-nanoLC coupled online with tandem mass spectrometry analysis (nanoESI-Q-TOF-MS/MS). The proteins were identified by MASCOT search against mouse protein database using a peptide and fragment mass tolerance of ±0.25 Da. Protein identification was carried out using a MOWSE scoring algorithm with a confidence level of 95% and processed by MSQuant software for further validation. In total, 298 identified membrane proteins from mouse brain tissues were verified by UniProt database, SOSUI and TMHMM prediction algorithms. Of these, 129 (43.3%) proteins have at least one transmembrane domain according to SOSUI and TMHMM. Furthermore, the function, subcellular location, molecular weight, post-translational modifications, transmembrane domains (TMD) and average of hydrophobicity of the identified membrane proteins might be categorized and

The present study was carried out at the National Key Laboratory of Gene Technology (NKLGT) and was approved by the Ethics Committee of the Institute of Biotechnology

The work was funded by National Foundation for Science and Technology Development

[1] Andersen JS, Wilkinson CJ, Mayor T, Mortensen P, Nigg EA and Mann M (2003) Proteomic characterization of the human centrosome by protein correlation proling.

[2] Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G (2000) Gene ontology: tool for the

unication of biology. The Gene Ontology Consortium, Nat. Genet. 25(1): 25-9. [3] Binh DV, Thanh TT and Chi PV (2010) Proteomic characterization of the thermostable toxins from Naja naja venom. J. Venom. Anim. Toxins incl. Trop. Dis. 16(4): 631-8. [4] Blagoev B, Ong SE, Kratchmarova I and Mann M (2004) Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nat.

	- [21] Rabilloud T, Chevallet M, Luche S, Lelong C (2010). Tow-dimensional gel electrophoresis in proteomics: past, present and future. J. Proteomics 73(11):2064-77.

**Chapter 4** 

© 2012 Roškar and Trdan Lušin, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Roškar and Trdan Lušin, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

The study of the metabolic fate of drugs is an essential and important part of the drug development process. During drug evaluation the research of drug metabolism is of high importance especially when metabolites are pharmacologically active or toxic or when a

**Analytical Methods for Quantification of** 

The study of the metabolic fate of drugs is an essential and important part of the drug development process, research of drug metabolism pathways, drug-drug interactions, drugherb interactions, influence of genetic polymorphisms and other factors that influence the phase I and/or II metabolism of a drug. Different *in vitro* methods, from subcelullar to organ range, and *in vivo* studies are applied for the clarification of drug metabolism. The analysis of metabolites in complex biological matrices is a challenging task therefore several analytical methods for qualification and quantification of drug metabolites are used. Liquid chromatography coupled with mass spectrometry (LC-MS) has become the most powerful analytical tool for screening and identification of drug metabolites in biological matrices. However, adequate sample preparation is a key prerequisite aspect of successful quantitative and qualitative bioanalysis. Different approaches for metabolite quantification in biological samples from direct quantification, indirect quantification through parent drug after metabolite hydrolysis to quantification supported by using response factors between drug and their metabolites are often used. The most frequently used method for quantification is liquid chromatography coupled to different detectors such as mass spectrometer or UV detector. The LC-MS/MS methods are considered as most appropriate for determination of drugs and their metabolites and are also best suited for high throughput analysis. However, in LC-MS/MS assays, matrix effect and selection of suitable

**Drug Metabolites in Biological Samples** 

Robert Roškar and Tina Trdan Lušin

http://dx.doi.org/10.5772/51676

**1. Introduction** 

Additional information is available at the end of the chapter

internal standards should be adequately addressed.

**2. Background of drug metabolism** 


**Chapter 4** 
