Analytical and Bioanalytical Chemistry

, Volume 400, Issue 2, pp 387–393 | Cite as

HPLC determination of methylphenidate and its metabolite, ritalinic acid, by high-performance liquid chromatography with peroxyoxalate chemiluminescence detection

  • Mitsuhiro Wada
  • Keisuke Abe
  • Rie Ikeda
  • Ruri Kikura-Hanajiri
  • Naotaka Kuroda
  • Kenichiro NakashimaEmail author
Original Paper


An HPLC–peroxyoxalate chemiluminescence (PO-CL) method for simultaneous determination of methylphenidate (MPH) and ritalinic acid (RA) was developed. The method was used to monitor MPH and RA after administration of MPH to rats. Deproteinized plasma spiked with 1-(3-trifluoromethylphenyl)piperazine (IS) was dried and labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F). The labeled sample was cleaned with two kinds of solid-phase extraction cartridge, and the DBD-labels were separated on an ODS column with gradient elution using a mixture of CH3CN and imidazole–HNO3 buffer. Separation of MPH and RA can be achieved within 33 min. The LODs of MPH and RA at a signal-to-noise ratio of 3 were 2.2 and 0.4 ng mL−1, respectively. Moreover, monitoring of MPH and RA after MPH administration (10 mg kg−1) to rat could be performed. The concentration of RA 480 min after administration was eight times higher than that of MPH. The proposed HPLC–PO-CL method was useful for determination of MPH and RA in rat plasma and was successfully used to monitor these substances after MPH administration.


Methylphenidate Ritalinic acid HPLC Peroxyoxalate chemiluminescence 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole 


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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Mitsuhiro Wada
    • 1
  • Keisuke Abe
    • 1
  • Rie Ikeda
    • 1
  • Ruri Kikura-Hanajiri
    • 2
  • Naotaka Kuroda
    • 1
  • Kenichiro Nakashima
    • 1
    Email author
  1. 1.Graduate School of Biomedical SciencesNagasaki UniversityNagasakiJapan
  2. 2.National Institute of Health SciencesSetagayaJapan

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