A Forensic Toxicology Analysis of Synthetic Fentanyl Compounds in Whole Blood Using Oasis Prime MCX and Waters TQ-S Micro

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Jonathan P. Danaceau, Scott Freeto, Kim Haynes and Lisa J. Calton
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Novel Aspect:

A novel SPE sorbent enabled the near elimination of residual phospholipids from whole blood samples.


Overdose deaths from opiates and synthetic opioids have risen dramatically over the past few years, with the largest increases attributed to synthetic opioids such as fentanyl and its analogs.  Due to their high potency, the concentrations of these compounds are often in the sub ng/mL range.  In addition, forensic samples are often analyzed from whole blood, presenting challenges not seen in plasma or urine samples.  This work details a rapid UPLC-MS/MS method for the analysis of synthetic fentanyl compounds in whole blood employing a novel mixed-mode SPE strategy designed to minimize residual phospholipids.  The resulting method minimizes ion suppression while producing linear and accurate quantification over the entire expected analytical range of these compounds.


Whole blood samples were diluted 1:1 with ZnSO4/NH4OAc followed by precipitation with 1:1 ACN:MeOH.  After centrifugation, the samples were diluted with 4% H3PO4 and loaded directly onto Waters Oasis PRiME MCX µElution plates.  The sorbent was washed with 2% formic acid:NH4COOH followed by MeOH.  Samples were eluted with 2 x 25 µL aliquots of 50:50 ACN:MeOH and diluted with 50 µL of 97:2:1 water:ACN:formic acid.  Samples were analyzed on a Waters Xevo TQ-S micro triple quadrupole MS system after UPLC separation on a 1.7 µm BEH C18 column (2.1 x 100 mm). 

Preliminary Data:  

13 synthetic fentanyl analogues including carfentanyl, furanyl fentanyl and acetyl fentanyl were extracted and analyzed from whole blood with recoveries that ranged from 50-84% with an average of 74%.  All %CVs were >10%.   Matrix effects were all less than 20% with an average of 12%.  The MCX PRiME protocol was able to eliminate >95% of residual phospholipids compared to traditional mixed-mode cation exchange SPE.  The UPLC method was rapid, with a 3 minute analysis and a total cycle tim of less than 4 minutes.  All analytes demonstrated linearity from 20 pg/mL to 10 ng/mL, easily encompassing previously documented levels in whole blood samples.  Quality control samples were accurate and precise, ranging from 92-109% with averages of 97%, 101% and 104% at low, medium and high levels, respectively.  The use of the Oasis PRiME MCX µElution plates enabled direct analysis without any evaporation or reconstitution.  The TQ-S micro MS, with Step Wave technology and extended dynamic range (XDR) detection enabled highly sensitive detection with a linear dynamic range that includes documented impaired driving cases.  The combination of novel SPE cleanup and UPLC-MS/MS analysis resulted in a rapid, clean method capable of rapidly and accurately analyzing a wide variety of synthetic fentanyl analogues from whole blood.

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