title: Determination of flumazenil in serum by liquid chromatography-mass spectrometry: Application to kinetics study in acute diazepam overdose.
abstract: OBJECTIVE
  Flumazenil is benzodiazepine receptor antagonist. It has been studied for a various indications, including reversal of sedation after surgery or diagnostic procedures, awakening of comatose patients in benzodiazepine overdose, or for symptomatic treatment of hepatic encephalopathy. Some drugs, like theophylline, may prolong its elimination half-life. Considering the long half-life of diazepam and its metabolites, concomitant use of theophylline may reduce the need for repeated dosing of flumazenil in patients with acute diazepam poisoning. The aim of this study was to introduce a reliable and accurate method for determining the concentration of flumazenil after therapeutic application in patients with acute poisoning, and using that method to assess whether the kinetics of flumazenil change in the presence of aminophylline (combination of theophylline and ethylenediamine in a 2:1 ratio) applied as concomitant therapy.
  
  
  METHODS
  Blood samples from patients with acute diazepam poisoning that received flumazenil at the dose of 0.5 mg, or the same dose with 3 mg/kg of body weight of aminophylline, were collected 1, 3, 10, 30, 60, 120 and 240 min after its intravenous administration. Samples were prepared by solid-phase extraction on Oasis HLB cartridges with ethylacetate as extracting agens. Flumazenil was determined by liquid chromatography with mass spectrometry (LC-MS) in single ionmonitoring mode at m/z 304. Separation of flumazenil from matrix compound was performed on Lichrospher RP-8 column usingthe mixture of acidic acetonitrile and 20 mM of ammonium acetatein water (55 : 45) as a mobile phase.
  
  
  RESULTS
  The applied analitycal method showed excellent recovery (94.65%). The obtained extracts were much cleaner than the extracts obtained by the sameextractant in the process of liquid-liquid extraction. The limit ofdetection of the LC-MS method described in this paper was 0.5 ng/mL and the limit of quantitation was 1 ng/mL. In the patientstreated with both flumazenil and aminophylline, the eliminationconstant for flumazenil was significantly lower and the elimination half-life was longer (p < 0.05) in comparison with the same parameters in.the patients who received flumazenil alone.
  
  
  CONCLUSIONS
  The applied LC-MS method for the determination of flumazenil in serum samples of patients with acute diazepam poisoning is rapid, sensitive, precise and specific. Concomitant use with theophylline significantly prolonged elimination of flumazenil during the treatment of acute poisonings with diazepam.
authors.forename: Snezana
authors.initials: S
authors.lastname: Djordjević
authors.forename: Jasmina
authors.initials: J
authors.lastname: Jović-Stosić
authors.forename: Vesna
authors.initials: V
authors.lastname: Kilibarda
authors.forename: Zoran
authors.initials: Z
authors.lastname: Segrt
authors.forename: Natasa
authors.initials: N
authors.lastname: Perković-Vukcević
mesh_terms: D000628:Aminophylline / Q000493:pharmacokinetics*; D000931:Antidotes / Q000032:analysis / Q000378:metabolism / Q000493:pharmacokinetics; D002853:Chromatography, Liquid; D003975:Diazepam / Q000009:adverse effects*; D061827:Dimensional Measurement Accuracy; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D062787:Drug Overdose / Q000188:drug therapy / Q000209:etiology; D005442:Flumazenil / Q000032:analysis / Q000097:blood / Q000493:pharmacokinetics; D006207:Half-Life; D006801:Humans; D006993:Hypnotics and Sedatives / Q000009:adverse effects; D013058:Mass Spectrometry; D010726:Phosphodiesterase Inhibitors / Q000493:pharmacokinetics; D015203:Reproducibility of Results
chemical_list: D000931:Antidotes; D006993:Hypnotics and Sedatives; D010726:Phosphodiesterase Inhibitors; D000628:Aminophylline; D005442:Flumazenil; D003975:Diazepam