title: Revival of physostigmine - a novel HPLC assay for simultaneous determination of physostigmine and its metabolite eseroline designed for a pharmacokinetic study of septic patients.
abstract: BACKGROUND
  Physostigmine, commonly used as an antidote in anticholinergic poisoning, is reported to have additional pharmacological effects, such as activation of the cholinergic anti-inflammatory pathway in sepsis models. Due to the narrow therapeutic range of physostigmine and its metabolite eseroline, however, the plasma concentrations of these substances need to be determined so as to understand their effect and ensure safety in the treatment of septic patients.
  
  
  METHODS
  To determine physostigmine and its metabolite eseroline, a rapid and sensitive high performance liquid chromatography (HPLC) method has been developed and validated. Spiked plasma samples were cleaned up and concentrated using a simple liquid-liquid extraction (LLE) procedure with N-methylphysostigmine as internal standard. Separation was achieved using reversed-phase HPLC on a Kinetex C18 column with gradient elution and fluorescence detection (254 nm excitation/355 nm emission).
  
  
  RESULTS
  LLE produced clean extracts and a mean recovery of 80.3% for eseroline and 84.9% for physostigmine. The HPLC assay revealed a limit of detection (LOD) of 0.025 ng/mL and a lower limit of quantification (LLOQ) of 0.05 ng/mL for both analytes. Linearity was observed at 0.05-10.0 ng/mL (r²>0.999). Intra- and inter-day precision ranged from 0.7% to 6.6%, and intra- and inter-day accuracy 97.5%-110.0%.
  
  
  CONCLUSIONS
  The presented method is useful for human drug level monitoring of physostigmine and eseroline in accordance with current guidelines. Remarkably low plasma concentrations can be quantified after LLE with gradient elution and fluorescence detection, making this a suitable method for pharmacokinetic studies in a clinical setting.
authors.forename: Nadine
authors.initials: N
authors.lastname: Pinder
authors.forename: Johannes B
authors.initials: JB
authors.lastname: Zimmermann
authors.forename: Stefan
authors.initials: S
authors.lastname: Hofer
authors.forename: Thorsten
authors.initials: T
authors.lastname: Brenner
authors.forename: Markus A
authors.initials: MA
authors.lastname: Weigand
authors.forename: Ute
authors.initials: U
authors.lastname: Gubbe
authors.forename: Torsten
authors.initials: T
authors.lastname: Hoppe-Tichy
authors.forename: Stefanie
authors.initials: S
authors.lastname: Swoboda
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation; D064368:Healthy Volunteers; D006801:Humans; D007211:Indoles / Q000097:blood* / Q000378:metabolism / Q000493:pharmacokinetics*; D059625:Liquid-Liquid Extraction; D010830:Physostigmine / Q000097:blood* / Q000378:metabolism / Q000493:pharmacokinetics*; D012772:Shock, Septic / Q000097:blood*
chemical_list: D007211:Indoles; D010830:Physostigmine; C022594:eseroline