Quantification of 5-azacytidine in plasma by electrospray tandem mass spectrometry coupled with high-performance liquid chromatography

Ming Zhao, Michelle Rudek-Renaut, Ping He, Carol Hartke, Steve Gore, Michael A Carducci, Sharyn D. Baker

Research output: Contribution to journalArticle

Abstract

5-Azacytidine (5AC), a nucleoside analogue and hypomethylating agent, has anticancer properties and has been utilized in the treatment of various malignancies. 5AC is unstable and rapidly hydrolyzed to several by-products, including 5-azacytosine and 5-azauracil. A sensitive, reliable method was developed to quantitate 5AC using LC/MS/MS to perform pharmacokinetic and pharmacodynamic studies on 5AC combination therapy trials. Blood samples were collected in a heparinized tube and immediately processed for storage. To increase the stability of 5AC in plasma, 25 ng/mL tetrahydrouridine was added to the plasma and snap frozen. Plasma samples were extracted using acetonitrile then cleaned up by Oasis MCX ion exchange solid-phase extraction cartridges. 5AC was separated on an YMC Jsphr M80™ C 18 column with gradient elution of ammonium acetate (2 mM) with 0.1% formic acid and methanol mobile phase. 5AC elutes at 5.0 ± 0.2 min with a total run time of 30 min. Identification was through positive-ion mode and multiple reaction monitoring mode at m/z+ 244.9 → 113.0 for 5AC and m/z+ 242.0 → 126.0 for 5-methyl-2′-deoxycytidine, the internal standard. The lower limit of quantitation of 5AC was 5 ng/mL in human plasma, and linearity was observed from 5 to 500 ng/mL fitted by linear regression with 1/x weight. This method is 50 times more sensitive than previously published assays and successfully allows studies to characterize the pharmacokinetics and pharmacodynamics of 5AC.

Original languageEnglish (US)
Pages (from-to)81-88
Number of pages8
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume813
Issue number1-2
DOIs
StatePublished - Dec 25 2004

Fingerprint

Azacitidine
High performance liquid chromatography
Tandem Mass Spectrometry
Mass spectrometry
High Pressure Liquid Chromatography
Plasmas
Pharmacodynamics
formic acid
5-methyldeoxycytidine
Pharmacokinetics
Tetrahydrouridine
Plasma (human)
Ion Exchange
Solid Phase Extraction
Nucleosides
Linear regression
Antineoplastic Agents
Byproducts
Methanol
Linear Models

Keywords

  • 5-Azacytidine
  • LC/MS/MS
  • Pharmacokinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Quantification of 5-azacytidine in plasma by electrospray tandem mass spectrometry coupled with high-performance liquid chromatography",
abstract = "5-Azacytidine (5AC), a nucleoside analogue and hypomethylating agent, has anticancer properties and has been utilized in the treatment of various malignancies. 5AC is unstable and rapidly hydrolyzed to several by-products, including 5-azacytosine and 5-azauracil. A sensitive, reliable method was developed to quantitate 5AC using LC/MS/MS to perform pharmacokinetic and pharmacodynamic studies on 5AC combination therapy trials. Blood samples were collected in a heparinized tube and immediately processed for storage. To increase the stability of 5AC in plasma, 25 ng/mL tetrahydrouridine was added to the plasma and snap frozen. Plasma samples were extracted using acetonitrile then cleaned up by Oasis MCX ion exchange solid-phase extraction cartridges. 5AC was separated on an YMC Jsphr M80™ C 18 column with gradient elution of ammonium acetate (2 mM) with 0.1{\%} formic acid and methanol mobile phase. 5AC elutes at 5.0 ± 0.2 min with a total run time of 30 min. Identification was through positive-ion mode and multiple reaction monitoring mode at m/z+ 244.9 → 113.0 for 5AC and m/z+ 242.0 → 126.0 for 5-methyl-2′-deoxycytidine, the internal standard. The lower limit of quantitation of 5AC was 5 ng/mL in human plasma, and linearity was observed from 5 to 500 ng/mL fitted by linear regression with 1/x weight. This method is 50 times more sensitive than previously published assays and successfully allows studies to characterize the pharmacokinetics and pharmacodynamics of 5AC.",
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author = "Ming Zhao and Michelle Rudek-Renaut and Ping He and Carol Hartke and Steve Gore and Carducci, {Michael A} and Baker, {Sharyn D.}",
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T1 - Quantification of 5-azacytidine in plasma by electrospray tandem mass spectrometry coupled with high-performance liquid chromatography

AU - Zhao, Ming

AU - Rudek-Renaut, Michelle

AU - He, Ping

AU - Hartke, Carol

AU - Gore, Steve

AU - Carducci, Michael A

AU - Baker, Sharyn D.

PY - 2004/12/25

Y1 - 2004/12/25

N2 - 5-Azacytidine (5AC), a nucleoside analogue and hypomethylating agent, has anticancer properties and has been utilized in the treatment of various malignancies. 5AC is unstable and rapidly hydrolyzed to several by-products, including 5-azacytosine and 5-azauracil. A sensitive, reliable method was developed to quantitate 5AC using LC/MS/MS to perform pharmacokinetic and pharmacodynamic studies on 5AC combination therapy trials. Blood samples were collected in a heparinized tube and immediately processed for storage. To increase the stability of 5AC in plasma, 25 ng/mL tetrahydrouridine was added to the plasma and snap frozen. Plasma samples were extracted using acetonitrile then cleaned up by Oasis MCX ion exchange solid-phase extraction cartridges. 5AC was separated on an YMC Jsphr M80™ C 18 column with gradient elution of ammonium acetate (2 mM) with 0.1% formic acid and methanol mobile phase. 5AC elutes at 5.0 ± 0.2 min with a total run time of 30 min. Identification was through positive-ion mode and multiple reaction monitoring mode at m/z+ 244.9 → 113.0 for 5AC and m/z+ 242.0 → 126.0 for 5-methyl-2′-deoxycytidine, the internal standard. The lower limit of quantitation of 5AC was 5 ng/mL in human plasma, and linearity was observed from 5 to 500 ng/mL fitted by linear regression with 1/x weight. This method is 50 times more sensitive than previously published assays and successfully allows studies to characterize the pharmacokinetics and pharmacodynamics of 5AC.

AB - 5-Azacytidine (5AC), a nucleoside analogue and hypomethylating agent, has anticancer properties and has been utilized in the treatment of various malignancies. 5AC is unstable and rapidly hydrolyzed to several by-products, including 5-azacytosine and 5-azauracil. A sensitive, reliable method was developed to quantitate 5AC using LC/MS/MS to perform pharmacokinetic and pharmacodynamic studies on 5AC combination therapy trials. Blood samples were collected in a heparinized tube and immediately processed for storage. To increase the stability of 5AC in plasma, 25 ng/mL tetrahydrouridine was added to the plasma and snap frozen. Plasma samples were extracted using acetonitrile then cleaned up by Oasis MCX ion exchange solid-phase extraction cartridges. 5AC was separated on an YMC Jsphr M80™ C 18 column with gradient elution of ammonium acetate (2 mM) with 0.1% formic acid and methanol mobile phase. 5AC elutes at 5.0 ± 0.2 min with a total run time of 30 min. Identification was through positive-ion mode and multiple reaction monitoring mode at m/z+ 244.9 → 113.0 for 5AC and m/z+ 242.0 → 126.0 for 5-methyl-2′-deoxycytidine, the internal standard. The lower limit of quantitation of 5AC was 5 ng/mL in human plasma, and linearity was observed from 5 to 500 ng/mL fitted by linear regression with 1/x weight. This method is 50 times more sensitive than previously published assays and successfully allows studies to characterize the pharmacokinetics and pharmacodynamics of 5AC.

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JF - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences

SN - 1570-0232

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