Comparison of two automated solid phase extractions for the detection of ten fentanyl analogs and metabolites in human urine using liquid chromatography tandem mass spectrometry

Abstract

Two types of automated solid phase extraction (SPE) were assessed for the determination of human exposure to fentanyls in urine. High sensitivity is required to detect these compounds following exposure because of the low dose required for therapeutic effect and the rapid clearance from the body for these compounds. To achieve this sensitivity, two acceptable methods for the detection of human exposure to seven fentanyl analogs and three metabolites were developed using either off-line 96-well plate SPE or on-line SPE. Each system offers different advantages: off-line 96-well plate SPE allows for high throughput analysis of many samples, which is needed for large sample numbers, while on-line SPE removes almost all analyst manipulation of the samples, minimizing the analyst time needed for sample preparation. Both sample preparations were coupled with reversed phase liquid chromatography and isotope dilution tandem mass spectrometry (LC-MS/MS) for analyte detection. For both methods, the resulting precision was within 15%, the accuracy within 25%, and the sensitivity was comparable with the limits of detection ranging from 0.002-0.041ng/mL. Additionally, matrix effects were substantially decreased from previous reports for both extraction protocols. The results of this comparison showed that both methods were acceptable for the detection of exposures to fentanyl analogs and metabolites in urine.

Quantitation of fentanyl analogs in dried blood spots by flow-through desorption coupled to online solid phase extraction tandem mass spectrometry

“…To address this issue, unique quantitation transitions were selected for each compound to prevent signal contribution between all analytes, including the isomers as previously documented. 42 Because baseline resolution from detectable matrix interferences was achieved with SPE alone, no additional separation was deemed necessary for this method.…”

“…Comparable matrix effects have been noted in urine for reverse phase SPE followed by LC-MS/MS. 42 Additionally, an in-house evaluation of plasma using similar SPE-LC-MS/MS parameters 42 resulted in matrix effects ranging 19–93% for the same fentanyls and normetabolites. The combination of SPE and HPLC has not significantly decreased matrix effects for these analytes from clinical samples.…”

“…While past urinary methods had a lower limit of quantitation of 0.050 ng/mL, these methods required a 100-fold increase in sample volume (500 μL) plus a concentration step. 42 For a 1.0 ng/mL sample the DBS method evaluated here injected 0.005 ng on cartridge, whereas 0.1 ng were injected on column for the urinary SPE-LC-MS/MS method. This significant difference confirms that sample volume plays a critical role in sensitivity and is the most limiting factor for this DBS method.…”

Analysis of fentanyl derivatives by ultra high performance liquid chromatography with diode array ultraviolet and single quadrupole mass spectrometric detection

“…Any fentanyl approved for medical use has been scheduled by the Drug Enforcement Administration. To avoid potential legal prosecution, derivatives of scheduled substances are being synthesized, as these are structurally different and not scheduled . The Drug Enforcement Administration has temporarily placed most illicit fentanyl analogues, homologues, and positional isomers into Schedule I to avoid scheduling each individual fentanyl derivative .…”

Utilizing liquid chromatography tandem mass spectrometry and direct analysis in real time to assess the metabolism of fentanyl derivatives

“…Synthetic urine is a substance that simulates the appearance and chemical properties of urine, and is used in toxicology or as a calibrant. The SPE approach will likely be based off of methods in literature, and will selectively isolate metabolites of opioid adulterants 28,29 .…”