title: Analysis of remifentanil with liquid chromatography-tandem mass spectrometry and an extensive stability investigation in EDTA whole blood and acidified EDTA plasma.
abstract: BACKGROUND
  Remifentanil is a μ-opioid receptor agonist that was developed as a synthetic opioid for use in anesthesia and intensive care medicine. Remifentanil is rapidly metabolized in both blood and tissues, which results in a very short duration of action. Even after blood sampling, remifentanil is unstable in whole blood and plasma through endogenous esterases and chemical hydrolysis. The instability of remifentanil in these matrices makes sample collection and processing a critical phase in the bioanalysis of remifentanil.
  
  
  METHODS
  We have developed a fast and simple sample preparation method using protein precipitation followed by liquid chromatography-tandem mass spectrometry analysis. To improve the stability of remifentanil, citric acid, ascorbic acid, and formic acid were investigated for acidification of EDTA plasma. The stability of remifentanil was investigated in stock solution, EDTA whole blood, EDTA plasma, and acidified EDTA plasma at ambient temperature, 4 °C, 0 °C, and at -20 °C.
  
  
  RESULTS
  The analytical method was fully validated based on the Food and Drug Administration guidelines for bioanalytical method validation with a large linear range of 0.20 to 250 ng/mL remifentanil in EDTA plasma acidified with formic acid. The stability results of remifentanil in EDTA tubes, containing whole blood placed in ice water, showed a decrease of approximately 2% in 2 hours. EDTA plasma acidified with citric acid, formic acid, and ascorbic acid showed 0.5%, 4.2%, and 7.2% remifentanil degradation, respectively, after 19 hours at ambient temperature. Formic acid was chosen because of its volatility and thus liquid chromatography-tandem mass spectrometry compatibility. The use of formic acid added to EDTA plasma improved the stability of remifentanil, which was stable for 2 days at ambient temperature, 14 days at 4 °C, and 103 days at -20 °C.
  
  
  CONCLUSIONS
  The analytical method we developed uses a simple protein precipitation and maximal throughput by a 2-point calibration curve and short run times of 2.6 minutes. Best sample stability is obtained by placing tubes containing EDTA whole blood in ice water directly after sampling, followed by centrifugation and transfer of the EDTA plasma to tubes with formic acid. The stability of remifentanil in EDTA plasma was significantly improved by the addition of 1.5 μL formic acid per milliliter of EDTA plasma. This analytical method and sample pretreatment are suitable for remifentanil pharmacokinetic studies.
authors.affiliation: From the *Department of Clinical Pharmacy and Pharmacology, Laboratory for Clinical and Forensic Toxicology and Drugs Analysis, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands; and †Department of Anesthesiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Remco A
authors.initials: RA
authors.lastname: Koster
authors.forename: Hugo E M
authors.initials: HE
authors.lastname: Vereecke
authors.forename: Ben
authors.initials: B
authors.lastname: Greijdanus
authors.forename: Daan J
authors.initials: DJ
authors.lastname: Touw
authors.forename: Michel M R F
authors.initials: MM
authors.lastname: Struys
authors.forename: Jan Willem C
authors.initials: JW
authors.lastname: Alffenaar
mesh_terms: D000701:Analgesics, Opioid / Q000097:blood* / Q000493:pharmacokinetics; D001800:Blood Specimen Collection / Q000379:methods*; D002138:Calibration; D011232:Chemical Precipitation; D002853:Chromatography, Liquid / Q000592:standards; D016903:Drug Monitoring / Q000379:methods*; D004355:Drug Stability; D004492:Edetic Acid / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016014:Linear Models; D010880:Piperidines / Q000097:blood* / Q000493:pharmacokinetics; D011237:Predictive Value of Tests; D000077208:Remifentanil; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000592:standards; D053719:Tandem Mass Spectrometry / Q000592:standards; D013696:Temperature; D013997:Time Factors
chemical_list: D000701:Analgesics, Opioid; D010880:Piperidines; D004492:Edetic Acid; D000077208:Remifentanil