Quantitative determination of the enantiomers of methadone and its metabolite (EDDP) in human saliva by enantioselective liquid chromatography with mass spectrometric detection

Maria Esther Rodriguez Rosas, Kenzie L. Preston, David H. Epstein, Eric T. Moolchan, Irving W. Wainer

Research output: Contribution to journalArticle

Abstract

A sensitive enantioselective liquid chromatographic assay with mass spectrometric detection (LC-MS) has been developed and validated for the simultaneous determination of saliva concentrations of (R)- and (S)-methadone (Met) and (R)- and (S)-2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine (EDDP, a primary metabolite of Met). Saliva specimens were collected using Salivette devices (Sarsedt), and centrifuged; collected saliva was then spiked with deuterated internal standards, D3-Met and D3-EDDP, and directly injected into the LC-MS. Enantioselective separations were achieved on a liquid chromatographic chiral stationary phase (CSP) based upon immobilized α1-acid glycoprotein (AGP) using a mobile phase composed of acetonitrile: ammonium acetate buffer (10mM, pH 7.0) in a ratio of 18:82 (v/v), a flow rate of 0.9ml/min and a temperature of 25°C. Under these conditions, enantioselective separations were observed for methadone (α=1.30) and EDDP (α=1.17) within 15min. Met, EDDP, D3-Met and D3-EDDP were detected using selected ion monitoring at m/z 310.20, 278.20, 313.20 and 281.20, respectively. Linear relationships between peak height ratio and drug-enantiomer concentrations were obtained for methadone in the range of 5.0-600.0ng/ml, and for EDDP from 0.5 to 15.0ng/ml per enantiomer with correlation coefficients better than 0.9994, where lower limit of quantification (LLOQ) for Met was 5ng/ml and for EDDP 0.5ng/ml. Acceptable intra- and inter-day precision of the method (CVs

Original languageEnglish (US)
Pages (from-to)355-370
Number of pages16
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume796
Issue number2
DOIs
StatePublished - Nov 5 2003
Externally publishedYes

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Enantiomers
Methadone
Liquid chromatography
Metabolites
Saliva
Liquid Chromatography
Liquids
Assays
Glycoproteins
Buffers
Flow rate
Ions
Equipment and Supplies
Temperature
Acids
Monitoring
Pharmaceutical Preparations

Keywords

  • 2-Ethylene-1,5-dimethyl-3,3-diphenyl-pyrrolidine
  • Chiral stationary phases, LC
  • Enantiomer separation
  • Methadone

ASJC Scopus subject areas

  • Biochemistry

Cite this

Quantitative determination of the enantiomers of methadone and its metabolite (EDDP) in human saliva by enantioselective liquid chromatography with mass spectrometric detection. / Rodriguez Rosas, Maria Esther; Preston, Kenzie L.; Epstein, David H.; Moolchan, Eric T.; Wainer, Irving W.

In: Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, Vol. 796, No. 2, 05.11.2003, p. 355-370.

Research output: Contribution to journalArticle

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abstract = "A sensitive enantioselective liquid chromatographic assay with mass spectrometric detection (LC-MS) has been developed and validated for the simultaneous determination of saliva concentrations of (R)- and (S)-methadone (Met) and (R)- and (S)-2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine (EDDP, a primary metabolite of Met). Saliva specimens were collected using Salivette devices (Sarsedt), and centrifuged; collected saliva was then spiked with deuterated internal standards, D3-Met and D3-EDDP, and directly injected into the LC-MS. Enantioselective separations were achieved on a liquid chromatographic chiral stationary phase (CSP) based upon immobilized α1-acid glycoprotein (AGP) using a mobile phase composed of acetonitrile: ammonium acetate buffer (10mM, pH 7.0) in a ratio of 18:82 (v/v), a flow rate of 0.9ml/min and a temperature of 25°C. Under these conditions, enantioselective separations were observed for methadone (α=1.30) and EDDP (α=1.17) within 15min. Met, EDDP, D3-Met and D3-EDDP were detected using selected ion monitoring at m/z 310.20, 278.20, 313.20 and 281.20, respectively. Linear relationships between peak height ratio and drug-enantiomer concentrations were obtained for methadone in the range of 5.0-600.0ng/ml, and for EDDP from 0.5 to 15.0ng/ml per enantiomer with correlation coefficients better than 0.9994, where lower limit of quantification (LLOQ) for Met was 5ng/ml and for EDDP 0.5ng/ml. Acceptable intra- and inter-day precision of the method (CVs",
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AU - Rodriguez Rosas, Maria Esther

AU - Preston, Kenzie L.

AU - Epstein, David H.

AU - Moolchan, Eric T.

AU - Wainer, Irving W.

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AB - A sensitive enantioselective liquid chromatographic assay with mass spectrometric detection (LC-MS) has been developed and validated for the simultaneous determination of saliva concentrations of (R)- and (S)-methadone (Met) and (R)- and (S)-2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine (EDDP, a primary metabolite of Met). Saliva specimens were collected using Salivette devices (Sarsedt), and centrifuged; collected saliva was then spiked with deuterated internal standards, D3-Met and D3-EDDP, and directly injected into the LC-MS. Enantioselective separations were achieved on a liquid chromatographic chiral stationary phase (CSP) based upon immobilized α1-acid glycoprotein (AGP) using a mobile phase composed of acetonitrile: ammonium acetate buffer (10mM, pH 7.0) in a ratio of 18:82 (v/v), a flow rate of 0.9ml/min and a temperature of 25°C. Under these conditions, enantioselective separations were observed for methadone (α=1.30) and EDDP (α=1.17) within 15min. Met, EDDP, D3-Met and D3-EDDP were detected using selected ion monitoring at m/z 310.20, 278.20, 313.20 and 281.20, respectively. Linear relationships between peak height ratio and drug-enantiomer concentrations were obtained for methadone in the range of 5.0-600.0ng/ml, and for EDDP from 0.5 to 15.0ng/ml per enantiomer with correlation coefficients better than 0.9994, where lower limit of quantification (LLOQ) for Met was 5ng/ml and for EDDP 0.5ng/ml. Acceptable intra- and inter-day precision of the method (CVs

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