C Morphine and its known and postulated metabolites and congeners, i.r.. morphine N-oxide, normorphine, pseudomorphine, morphine-N-methyl iodide, codeine, norcodeine. morphine-3-glucuronide, and morphine ethereal sulfate. were separated by TLC and GLC. The R j values of nalorphine and nicotine and its metabolites and the retention times of nalorphine and seven other chemicals commonly used for GLC quantitative determination of morphine and codeine are also presented.
Keyphrases 0 Morphine and metabolites-separation, identification, TLC and GLC 0 TLC-separation, identification, morphine and metabolites 0 GLC separation, identification, morphine and metabolites Studies in various animal species have shown that morphine is biotransformed to morphine-3-and 6glucuronide ( 1 3 , morphine-3-ethereal sulfate (6-8), morphine N-oxide (9, lo), normorphine (1 1-14), and codeine (15). Pseudomorphine ( ), an oxidation product of morphine, and morphine glutathione ( ) have also been postulated to be metabolites.
While investigating the pharmacokinetics and metabolism of morphine in man, need arose for the development of suitable methods for separation and identification of morphine and its metabolites. Although many procedures, including TLC and GLC, have been described for separation of morphine from its congeners, as mentioned in recent reviews (18, 19), none has been described for separation of morphine from its metabolites. This report describes the application of TLC and GLC for the separation and identification of these compounds.
EXPERIMENTAL
Morphine sulfate USPl, codeine phosphate USP', and normorphine hydrochloridez were obtained. Pseudomorphine, morphine N-oxide, and morphine-N-methyl iodide were prepared by previously described methods (20-22). Morphine-3-glucuronide and morphine-3-ethereal sulfate were isolated from the urine of dog and cat, respectively (4, 8).
TLC3-The compounds were localized after devclopment by spraying with potassium iodoplatinate reagent or ninhydrin reagent.
CLC-A chromatograph' equipped with dual-flame-ionization detectors and dual-pen recorders5 was used for GLC. The columns (Table ) were conditioned at 275' under nitrogen, 30 ml./min., for 1 hr.; then at 330" without nitrogen for 4 hr.; and finally at 290" with nitrogen, 16 ml./min., for 72 hr. The temperatures of injector and detector were set at 255 and 295". respcctively: gas flow rates were 30 ml./min. for nitrogen and hydrogen and 300-400 ml./min. for compressed dried air.
1 Merck Sharp & Dohme.
2 Through the courtesy of