Quantitative determination of fentanyl in newborn pig plasma and cerebrospinal fluid samples by HPLC‐MS/MS

Abstract

In this study, a selective and sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method requiring low sample volume (≤100 μL) was developed and validated for the quantitative determination of the opioid drug fentanyl in plasma and cerebrospinal fluid (CSF). A protein precipitation extraction with acetonitrile was used for plasma samples whereas CSF samples were injected directly on the HPLC column. Fentanyl and (13) C6 -fentanyl (Internal Standard) were analyzed in an electrospray ionization source in positive mode, with multiple reaction monitoring (MRM) of the transitions m/z 337.0/188.0 and m/z 337.0/105.0 for quantification and confirmation of fentanyl, and m/z 343.0/188.0 for (13) C6 -fentanyl. The respective lowest limits of quantification for plasma and CSF were 0.2 and 0.25 ng/mL. Intra- and inter-assay precision and accuracy did not exceed 15%, in accordance with bioanalytical validation guidelines. The described analytical method was proven to be robust and was successfully applied to the determination of fentanyl in plasma and CSF samples from a pharmacokinetic and pharmacodynamic study in newborn piglets receiving intravenous fentanyl (5 µg/kg bolus immediately followed by a 90-min infusion of 3 µg/kg/h).

Lipemia in the Plasma Sample Affects Fentanyl Measurements by Means of HPLC-MS2 after Liquid-Liquid Extraction

“…The effective plasma concentration for this compound can be as low as 1.0 µg/L, which requires a very sensitive analytical method to adequately measure this drug concentration in the clinical conditions [26]. The most frequent methods used for FEN determination are those based on either liquid chromatography combined with UV-VIS detection [31][32][33][34] or liquid and gas chromatography combined with mass spectrometry [20,24,26,32,[35][36][37][38][39][40][41][42][43]. Due to lower sensitivity and precision of UV-VIS detectors, this approach to the drug analysis is notably less frequent [42].…”

“…However, this approach requires chemical derivatisation and is relatively time-consuming as compared with liquid chromatography [42]. Therefore, determination of FEN is dominated by liquid chromatography-mass spectrometry methods offering a convenient, sensitive and reliable analysis [20,24,26,32,[35][36][37][38][39][40]42,44]. For liquid chromatography-mass spectrometry methods different authors consistently reported ESI as the best ionization mode [20,24,26,32,[35][36][37][38][39][40]42,44].…”

“…Therefore, determination of FEN is dominated by liquid chromatography-mass spectrometry methods offering a convenient, sensitive and reliable analysis [20,24,26,32,[35][36][37][38][39][40]42,44]. For liquid chromatography-mass spectrometry methods different authors consistently reported ESI as the best ionization mode [20,24,26,32,[35][36][37][38][39][40]42,44]. Based on the literature data, we also decided to use it in our study.…”

“…High prevalence of the drug in forensic toxicology cases called for effective methods for its detection in biological samples. A number of methods developed for FEN are reported in available literature [20][21][22][23][24][25]. Most of them use HPLC coupled with tandem mass spectrometry (HPLC-MS 2 ) that provides specificity, accuracy and sensitivity during the drug analysis as well as short analytical run time, relatively small sample size required and simplicity of the sample preparation [26].…”

Confirmation and Analysis of Fentanyl and Its Analogues Without the Standards

“…At the beginning of 2014, the availability of fentanyl in the United States had started to increase exponentially [4], with crime laboratories reporting more than 4,500 fentanyl cases in 2014 and over 14,400 Numerous analysis methods for fentanyl and its analogues have been published in the scientific literatures. Analysis technique such as liquid chromatography (HPLC) [5][6][7][8][9], gas chromatography/mass spectrometry (GC/MS) [10][11][12][13][14][15][16][17][18], liquid chromatography/high resolution mass spectrometry (LC-HRMS) [19][20], liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) [21][22][23][24][25][26][27][28][29][30] has been reported to apply in the precision determination and quantification of fentanyl and its analogues. GC/MS analysis method has been considered a gold standard for decades due to its combination of good selectivity and high specificity.…”