Simultaneous determination of norethindrone and ethinyl estradiol in human plasma by high performance liquid chromatography with tandem mass spectrometry--experiences on developing a highly selective method using derivatization reagent for enhancing sensitivity

J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Oct 25;825(2):223-32. doi: 10.1016/j.jchromb.2005.01.012.


In the present work, for the first time, a liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the simultaneous analysis of norethindrone, and ethinyl estradiol, was developed and validated over the concentration range of 50-10000pg/ml and 2.5-500pg/ml, respectively, using 0.5 ml of plasma sample. Norethindrone, ethinyl estradiol, and their internal standards norethindrone-(13)C2, and ethinyl estradiol-d4, were extracted from human plasma matrix with n-butyl chloride. After evaporation of the organic solvent, the extract was derivatized with dansyl chloride and the mixture was injected onto the LC-MS/MS system. The gradient chromatographic elution was achieved on a Genesis RP-18 (50 mm x 4.6 mm, 3 microm) column with mobile phase consisted of acetonitrile, water and formic acid. The flow rate was 1.0 ml/min and the total run time was 5.0 min. Important parameters such as sensitivity, linearity, matrix effect, reproducibility, stability, carry-over and recovery were investigated during the validation. The inter-day precision and accuracy of the quality control samples at low, medium and high concentration levels were <6.8% relative standard deviation (RSD) and 4.4% relative error (RE) for norethindrone, and 4.2% RSD and 5.9% RE for ethinyl estradiol, respectively. Chromatographic conditions were optimized to separate analytes of interest from the potential interference peaks, arising from the derivatization. This method could be used for pharmacokinetic and drug-drug interaction studies in human subjects.

Publication types

  • Validation Study

MeSH terms

  • Chromatography, High Pressure Liquid / methods*
  • Dansyl Compounds / chemistry
  • Drug Stability
  • Ethinyl Estradiol / blood*
  • Ethinyl Estradiol / chemistry
  • Humans
  • Mass Spectrometry / methods*
  • Norethindrone / blood*
  • Reproducibility of Results
  • Sensitivity and Specificity


  • Dansyl Compounds
  • Ethinyl Estradiol
  • dansyl chloride
  • Norethindrone