Lillsunde P., Seppälä J., Mykkänen S., Ariniemi K., Korte T., Seppälä T.

National Public Health Institute, Mannerheimintie 166 Fin-00300 Helsinki, Finland

A method using gas chromatograph with electron ionization detector (GCD) for simultaneous determination of morphine, codeine, dihydrocodeine, 6-monoacetylmorphine and ethylmorphine in blood after propionic acid anhydride derivatization is described.
The opioids were extracted in blood (1 mL) using butylacetate as an extraction solvent (flurazepam as Internal Standard) after adjusting pH to 8.9-9.0 with 0.5 M Na₂HPO₄ x H₂O. After transferring to a clean test tube, organic solvent was evaporated to dryness. To the dry residue, 100 µl of triethylamine and 100 µl of propionic acid anhydride were added. The tubes were capped tightly and placed in 80° C for 30 minutes. As a cleaning step, buffer and organic solvent were added to the tubes and the extraction procedure was repeated. After transferring to a clean tube, the extraction solvent was evaporated to dryness. The dry residue was dissolved in organic solvent, which was then injected into the GCD (HP). A HP-5 column (30 m x 0.25 mm x 0.25 µm) was used. The initial temperature was 150° C for 1 min and was then increased with the rate of 15° C per min to 220° C and, finally, with the rate of 10° C/min to 320° C where it was held for 1 min.
The ions followed were m/z 357 and 284 for dihydrocodeine (RT 9.9), m/z 282 and 355 for codeine (RT 10.2), m/z 296 and 369 for ethylmorphine (RT 10.4), m/z 327 and 268 for 6-acetylmorphine (RT 11.1) as well as m/z 341 and 324 for morphine (RT 11.7).
The intraday RSDs varied from 7.2 to 10% at a level of 0.5 mg/l and day to day RSDs varied from 7.5 to 11% at level of 0.85 mg/l. The limits of quantitation were from 0.05 mg/l to 0.10 mg/l.