The opioid epidemic has become a serious public health problem in the United States. The increasing abuse of synthetic opioids has raised concerns in the society. Fentanyl is a synthetic opioid analgesic which has resulted in an increasing number of drug overdoses since 2013. In addition, fentanyl analogs, originally manufactured for use as analgesics or animal tranquilizers, have emerged in the United States drug market. Fentanyl and its analogs, similar to other opioids, work as full µ-agonists, binding with µ-receptors in the brain. Fentanyl and its analogs elicit more potent effects compared to the traditional opioids being abused such as morphine or heroin. With the emergence of fentanyl analogs in the drug market, identifying and differentiating those analogs becomes a challenge due to their structural similarities to fentanyl.
The purpose of this research was to develop a method of identifying and quantifying nine fentanyl analogs in urine and oral fluid using the QSight® Triple Quad LC-MS/MS, coupled with a Halo® C18, 2.7µm column. The method was validated based on AAFS Standards Board (ASB) Standard 036, Standard Practices for Method Validation in Forensic Toxicology. The analytes in this research included fentanyl, norfentanyl, acetyl fentanyl, carfentanil, cyclopropyl fentanyl, methoxyacetyl fentanyl, valeryl fentanyl, furanyl fentanyl and 4-anilino-N-phenethylpiperdine (4ANPP). All samples, calibrators, and quality controls (QC) were prepared by spiking certified reference standards into donated human urine or human oral fluid. Supported liquid extraction (SLE) was performed as the sample preparation method using ISOLUTE® SLE+ 1mL columns followed by evaporation. All samples were reconstituted with 200 µL methanol. The mobile phases used in this method were 5mM ammonium formate in Millipore water with 0.1% formic acid and methanol with 0.1% formic acid.
A 10-minute LC method achieved complete resolution of the analytes, with specific retention times ranging from 3.5 to 5.7 minutes. For urine and oral fluid analysis, the calibration range for all analytes was established from 1 to 70 ng/mL. The resulting r2 values were greater than 0.988 for all analytes. Bias and precision were evaluated at 3, 25 and 60 ng/mL, and bias and percent coefficient of variation (%CV) for within and between run precision had acceptable values within ±20%. The limit of detection (LOD) was 0.1 ng/mL for most fentanyl analogs, with a LOD of 0.01 ng/mL for valeryl fentanyl and furanyl fentanyl. Carryover was not detected for any analytes in either matrix. Recovery of all compounds following SLE for both urine and oral fluid was above 50%. For urine, the ion enhancement and suppression of all analytes was within 25%. For oral fluid, the ion enhancement and suppression of most analytes was within 25% except valeryl fentanyl, which experienced suppression of 35%. The matrices analyzed had no interference effect on the detection or quantitation of analytes in this method. The interference effects of different commonly encountered drugs were studied and showed minimal impacts on the results generated from this method. All analytes were stable for up to 72 hours at room temperature, except cyclopropyl fentanyl.
In conclusion, using the QSight® Triple Quad LC-MS/MS following SLE effectively identified and quantified fentanyl analogs present in both urine and oral fluid. This method has shown its potential to be applied to casework samples for fentanyl analogs detection.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/41277 |
| Date | 09 July 2020 |
| Creators | Ke, Yiling |
| Contributors | Botch-Jones, Sabra R. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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