A highly sensitive method for the simultaneous UHPLC-MS/MS analysis of clonidine, morphine, midazolam and their metabolites in blood plasma using HFIP as the eluent additive

J Chromatogr B Analyt Technol Biomed Life Sci. 2017 May 1;1052:150-157. doi: 10.1016/j.jchromb.2017.03.007. Epub 2017 Mar 10.


In intensive care units, the precise administration of sedatives and analgesics is crucial in order to avoid under- or over sedation and for appropriate pain control. Both can be harmful to the patient, causing side effects or pain and suffering. This is especially important in the case of pediatric patients, and dose-response relationships require studies using pharmacokinetic-pharmacodynamic modeling. The aim of this work was to develop and validate a rapid ultra-high performance liquid chromatographic-tandem mass spectrometric method for the analysis of three common sedative and analgesic agents: morphine, clonidine and midazolam, and their metabolites (morphine-3-glucuronide, morphine-6-glucuronide and 1'-hydroxymidazolam) in blood plasma at trace level concentrations. Low concentrations and low sampling volumes may be expected in pediatric patients; we report the lowest limit of quantification for all analytes as 0.05ng/mL using only 100μL of blood plasma. The analytes were separated chromatographically using the C18 column with the weak ion-pairing additive 1,1,1,3,3,3-hexafluoro-2-propanol and methanol. The method was fully validated and a matrix matched calibration range of 0.05-250ng/mL was attained for all analytes In addition, between-day accuracy for all analytes remained within 93-108%, and precision remained within 1.5-9.6% for all analytes at all concentration levels over the calibration range.

Keywords: Chromatographic separation; Clonidine; Hexafluoroisopropanol; Midazolam; Morphine; UHPLC–MS/MS.

Publication types

  • Validation Study

MeSH terms

  • Analgesics / blood*
  • Analgesics / isolation & purification
  • Analgesics / metabolism
  • Chromatography, High Pressure Liquid / methods*
  • Clonidine / blood*
  • Clonidine / isolation & purification
  • Clonidine / metabolism
  • Humans
  • Limit of Detection
  • Midazolam / blood*
  • Midazolam / isolation & purification
  • Midazolam / metabolism
  • Morphine / blood*
  • Morphine / isolation & purification
  • Morphine / metabolism
  • Propanols / chemistry
  • Reproducibility of Results
  • Tandem Mass Spectrometry / methods*


  • Analgesics
  • Propanols
  • hexafluoroisopropanol
  • Morphine
  • Clonidine
  • Midazolam