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Published:
Journal of Analytical Toxicology,
ISSN 0146-4760,
Volume 32, Number 7, September,
pp.478-484
Development
and Validation of an EI-GC–MS Method for the Determination
of Methadone and its Major Metabolites (EDDP and EMDP) in Human
Breast Milk
Panagiota D. Nikolaou1,2, Ioannis I. Papoutsis1, Constantinos
P. Maravelias1, Chara A. Spiliopoulou1, Constantinos M. Pistos1,
Antony C. Calokerinos2, and Julia Atta-Politou2
1Department of Forensic Medicine and Toxicology, Medical School,
University of Athens, Athens 115 27, Greece and
2Laboratory of
Analytical Chemistry, Department of Chemistry, University of Athens,
Athens 157 71, Greece
Methadone is used extensively for the maintenance
of opioid-addicted pregnant women. Because methadone and the
two major metabolites, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine
(EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP), are
excreted into breast milk, a sensitive and specific gas chromatographic–mass
spectrometric method has been developed, optimized, and validated
for their quantitative determination in human breast milk. The
procedure combined protein precipitation with acetonitrile and
solid-phase extraction, using Isolute Confirm HCX mixed-mode
SPE columns, with minimal matrix effect. The optimum extraction
conditions for all three analytes were evaluated using spiked
human breast milk, and the recovery exceeded 93.0%. This assay
uses methadone-d9 as internal standard for the determination
of methadone and EMDP, and EDDP-d3 for the determination
of EDDP. Calibration curves were linear within the range of 2.00–1000 µg/L
for methadone (R2 > 0.995) and 1.00–500 µg/L
for EDDP (R2 >0.997) and EMDP (R2 > 0.991).
Intra- and interday accuracy and precision were within the range
of 0.8–5.7%
and 1.3–5.2%, respectively, for all analytes. The stability
study was assessed by fortifying human breast milk with methadone
and its metabolites at two different concentrations and keeping
the samples at different temperature conditions. The analytes
were found to be stable in breast milk at room temperature for
at least 4 h and at –20°C for at least one month. The
method was used for the determination of methadone and its major
metabolites in human breast milk samples obtained from women
in the postpartum period participating in a methadone maintenance
program.
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