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. 2008 Sep;32(7):478-84.
doi: 10.1093/jat/32.7.478.

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 Nikolaou  1 , Ioannis I Papoutsis, Constantinos P Maravelias, Chara A Spiliopoulou, Constantinos M Pistos, Antony C Calokerinos, Julia Atta-Politou
Affiliations

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 Nikolaou et al. J Anal Toxicol. 2008 Sep.

Abstract

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-d(9) as internal standard for the determination of methadone and EMDP, and EDDP-d(3) for the determination of EDDP. Calibration curves were linear within the range of 2.00-1000 microg/L for methadone (R(2) > 0.995) and 1.00-500 microg/L for EDDP (R(2) >0.997) and EMDP (R(2) > 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 degrees 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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