title: A two-step pH-dependent liquid-liquid extraction combined with HPLC-fluorescence method for the determination of 10-hydroxycamptothecin in mouse liver tissue.
abstract: BACKGROUND
  Liquid-liquid extraction (LLE) shows high efficiency in the plasma sample preparation. However, this extraction method is not optimal for the biological samples containing complex organic interferences, such as liver and brain tissues. Some plant secondary metabolites can be converted between water-insoluble and water-soluble forms by pH adjustment.
  
  
  OBJECTIVE
  A two-step pH-dependent LLE method was introduced in this study to eliminate both water-soluble and lipidic interferences using the properties of pH-dependent interconvertible forms of analytes during sample preparation. A sensitive and reliable method using a reverse-phase HPLC coupled with a fluorescence detector was developed and validated.
  
  
  METHODS
  10-Hydroxycamptothecin (HCPT) with internal standard camptothecin and liver tissues were used as model compounds and biological samples. The lactone form of HCPT was converted to the water-soluble carboxylate form under moderate alkaline conditions, and the water-insoluble interferences were extracted with a nonpolar solvent. Afterward, the water-insoluble lactone form of HCPT was regenerated by acidification and then extracted using an organic solvent in a second LLE step.
  
  
  RESULTS
  The calibration curve was linear (r² > 0.999) for HCPT concentrations ranging from 2.5 to 160 ng/mL. The mean recoveries of HCPT were 114.94 ± 3.98, 104.30 ± 2.44 and 95.90 ± 1.40% (n = 6) at concentrations of 2.5, 10 and 80 ng/mL, respectively. The stability determination data showed that no significant degradation occurred under the experimental conditions. This method was successfully applied to liver tissue distribution study of HCPT in mice.
  
  
  CONCLUSIONS
  This two-step LLE can be applied to distribution studies of compounds with pH-dependent interconvertible forms in other biological matrices.
authors.affiliation: Alkali Soil Natural Environmental Science Center, Northeast Forestry University/Key Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil Field, Ministry of Education, Harbin, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Zheng
authors.forename: Honghong
authors.initials: H
authors.lastname: Guo
authors.forename: Na
authors.initials: N
authors.lastname: Guo
authors.forename: Wei
authors.initials: W
authors.lastname: Ma
authors.forename: Lijia
authors.initials: L
authors.lastname: Jing
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.forename: Zhifei
authors.initials: Z
authors.lastname: Dai
authors.forename: Xiufeng
authors.initials: X
authors.lastname: Yan
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.forename: Zhenyue
authors.initials: Z
authors.lastname: Wang
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000032:analysis* / Q000737:chemistry / Q000493:pharmacokinetics; D002138:Calibration; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D004355:Drug Stability; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007783:Lactones / Q000032:analysis / Q000737:chemistry; D057230:Limit of Detection; D059625:Liquid-Liquid Extraction; D008099:Liver / Q000737:chemistry*; D008297:Male; D051379:Mice; D015203:Reproducibility of Results; D012995:Solubility; D013050:Spectrometry, Fluorescence; D014018:Tissue Distribution
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D007783:Lactones; C028098:10-hydroxycamptothecin; D002166:Camptothecin