Validated liquid chromatography-tandem mass spectrometry method for determination of totally nine probe metabolites of cytochrome P450 enzymes and UDP-glucuronosyltransferases

Jr Ting Lee, Li Heng Pao, Cheng Huei Hsiong, Pei Wei Huang, Tung Yuan Shih, Oliver Yoa-Pu Hu

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

A simplified, rapid, and selective liquid chromatography-tandem mass spectrometry method for the determination of the activities of cytochrome P450 (CYP) enzymes and UDP-glucuronosyltransferases (UGTs) in two separate settings was developed and successfully applied to 8 CYP isoenzymes and UGT2B7 enzyme activities in rat liver microsomes. The triple-quadrupole mass spectrometric detection was operated in positive mode for the probe metabolites: CYP1A2 (resorufin), CYP2B6 (hydroxybupropion), CYP2C19 (5-hydroxyomeprazole), CYP2D6 (dextrophan), CYP3A4 (6β-hydroxytestosterone), and UGT2B7 (morphine-3-glucuronide); also in negative mode for CYP2C9 (4- hydroxytolbutamide), CYP2E1 (6-hydroxychloroxazone), and CYP4A (hydroxylauric acid). The metabolic reactions were terminated with acetonitrile, containing metoprolol and acetaminophen as the internal standard for positive and negative ion electrospray ionization, respectively. The method was validated over the concentration range of 25-2500 ng/mL for 5-hydroxyomeprazole, dextrophan, hydroxylauric acid, and morphine-3-glucuronide; 5-500 ng/mL for resorufin; 3-300 ng/mL for hydroxybupropion; 10-1000 ng/mL for 4-hydroxytolbutamide; 40-4000 ng/mL for 6-hydroxychloroxazone; and 63-6300 ng/mL for 6β- hydroxytestosterone. All of the extraction recoveries of these analytes were greater than 85%, except for hydroxylauric acid at mid-concentration with a recovery of 83.2%±3.2%. The matrix effects were between 85.8% and 119.9%; the respective within- and between-run precisions were 0.9-12.0% and 2.0-13.9%; and the within- and between-run accuracy levels were 0.6-17.2% and 0.1-15.1%, respectively, for all these analytes. All of the analytes were stable during the assay and storage in the liver microsomes of Sprague-Dawley rats. The measurement activity of multiple enzymes was feasible using a cocktail approach. This method proved to be a robust, fast, accurate, specific and sensitive assay, and was successfully used to investigate in vivo enzyme activities of 8 major CYP isoenzymes and UGT2B7 in Sprague-Dawley rats with fatty livers. By the end of the eighth week, the CD-fed induced fatty liver rats showed a significant decrease in the activities of CYP1A2 and UGT2B7 as compared to the standard diet group.

Original languageEnglish
Pages (from-to)220-228
Number of pages9
JournalTalanta
Volume106
DOIs
Publication statusPublished - Jan 29 2013
Externally publishedYes

Fingerprint

Glucuronosyltransferase
Liquid chromatography
Metabolites
Liver
Cytochrome P-450 Enzyme System
Mass spectrometry
Rats
Hydroxytestosterones
Cytochrome P-450 CYP1A2
Enzyme activity
Isoenzymes
Acids
Assays
Cytochrome P-450 CYP4A
Cytochrome P-450 CYP3A
Recovery
Cytochrome P-450 CYP2E1
Electrospray ionization
Cytochrome P-450 CYP2D6
Metoprolol

Keywords

  • Cocktail assay
  • Cytochrome P450
  • Fatty liver
  • Liquid chromatography-tandem mass spectrometry
  • Morphine-3-glucuronide
  • UDP-glucuronosyltransferases 2B7

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Validated liquid chromatography-tandem mass spectrometry method for determination of totally nine probe metabolites of cytochrome P450 enzymes and UDP-glucuronosyltransferases. / Lee, Jr Ting; Pao, Li Heng; Hsiong, Cheng Huei; Huang, Pei Wei; Shih, Tung Yuan; Yoa-Pu Hu, Oliver.

In: Talanta, Vol. 106, 29.01.2013, p. 220-228.

Research output: Contribution to journalArticle

Lee, Jr Ting ; Pao, Li Heng ; Hsiong, Cheng Huei ; Huang, Pei Wei ; Shih, Tung Yuan ; Yoa-Pu Hu, Oliver. / Validated liquid chromatography-tandem mass spectrometry method for determination of totally nine probe metabolites of cytochrome P450 enzymes and UDP-glucuronosyltransferases. In: Talanta. 2013 ; Vol. 106. pp. 220-228.
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AU - Pao, Li Heng

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AU - Shih, Tung Yuan

AU - Yoa-Pu Hu, Oliver

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N2 - A simplified, rapid, and selective liquid chromatography-tandem mass spectrometry method for the determination of the activities of cytochrome P450 (CYP) enzymes and UDP-glucuronosyltransferases (UGTs) in two separate settings was developed and successfully applied to 8 CYP isoenzymes and UGT2B7 enzyme activities in rat liver microsomes. The triple-quadrupole mass spectrometric detection was operated in positive mode for the probe metabolites: CYP1A2 (resorufin), CYP2B6 (hydroxybupropion), CYP2C19 (5-hydroxyomeprazole), CYP2D6 (dextrophan), CYP3A4 (6β-hydroxytestosterone), and UGT2B7 (morphine-3-glucuronide); also in negative mode for CYP2C9 (4- hydroxytolbutamide), CYP2E1 (6-hydroxychloroxazone), and CYP4A (hydroxylauric acid). The metabolic reactions were terminated with acetonitrile, containing metoprolol and acetaminophen as the internal standard for positive and negative ion electrospray ionization, respectively. The method was validated over the concentration range of 25-2500 ng/mL for 5-hydroxyomeprazole, dextrophan, hydroxylauric acid, and morphine-3-glucuronide; 5-500 ng/mL for resorufin; 3-300 ng/mL for hydroxybupropion; 10-1000 ng/mL for 4-hydroxytolbutamide; 40-4000 ng/mL for 6-hydroxychloroxazone; and 63-6300 ng/mL for 6β- hydroxytestosterone. All of the extraction recoveries of these analytes were greater than 85%, except for hydroxylauric acid at mid-concentration with a recovery of 83.2%±3.2%. The matrix effects were between 85.8% and 119.9%; the respective within- and between-run precisions were 0.9-12.0% and 2.0-13.9%; and the within- and between-run accuracy levels were 0.6-17.2% and 0.1-15.1%, respectively, for all these analytes. All of the analytes were stable during the assay and storage in the liver microsomes of Sprague-Dawley rats. The measurement activity of multiple enzymes was feasible using a cocktail approach. This method proved to be a robust, fast, accurate, specific and sensitive assay, and was successfully used to investigate in vivo enzyme activities of 8 major CYP isoenzymes and UGT2B7 in Sprague-Dawley rats with fatty livers. By the end of the eighth week, the CD-fed induced fatty liver rats showed a significant decrease in the activities of CYP1A2 and UGT2B7 as compared to the standard diet group.

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KW - Liquid chromatography-tandem mass spectrometry

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