Analysis of Synthetic Opiates and Novel Analgesics in Urine Using SPE and HPLC–MS/MS

Danielle Mackowsky, UCT, LLC

Summary

Opiate abuse is drastically on the rise in the United States. In addition to traditional naturally occurring opiate compounds, forensic toxicologists also need to be able to rapidly identify synthetic opioid-like drugs. A rapid, three-step solid-phase extraction (SPE) procedure for the identification and quantification of fentanyl and its major urinary metabolite norfentanyl, in addition to four “designer” compounds, U-47700, W-18, W-15, and furanyl fentanyl, is presented here. As a result of the rapid use and abuse of fentanyl in medical and recreational settings, respectively, it is important to develop a method that can accurately extract this Schedule II drug from any other novel compounds that may be present.

Procedure

1.        Sample Pretreatment:

            To 1 mL of urine sample, add 1 mL of 100 mM phosphate buffer (pH = 6) and an appropriate amount of internal standard.

2.         Sample Extraction:

            Apply the sample to the SPE cartridge (if required, use a low vacuum to draw the sample through at ≤3 mL/min).

3.         Wash Cartridge:

            a)         1 × 3 mL D.I. H2O

            b)         1 × 3 mL 100 mM acetic acid

            c)         Dry column under full vacuum or pressure for 10 min.

4.         Elution:

            a)         1 × 3 mL methanol containing 2% ammonium hydroxide (methanol:NH4OH, 98:2 v/v).

            b)        Evaporate the sample to dryness under a gentle stream of nitrogen.

            c)         Reconstitute in 100 µL 95:5 D.I. H2O:methanol and vortex for 1 min.

            d)        Transfer sample to autosampler vial containing a low volume insert.

Instrumental

LC–MS/MS: Thermo Scientific™ Dionex™ Ultimate™ 3000 UHPLC and TSQ Vantage™ (MS/MS)

Column: 100 × 2.1 mm, 3-μm UCT Selectra® DA HPLC column

Guard Column: 10 × 2.1 mm, 3-μm UCT Selectra® DA guard column

Injection Volume: 5 μL

Mobile Phase A: D.I. H2O 0.1% formic acid

Mobile Phase B: Methanol 0.1% formic acid

Column Flow Rate: 0.30 mL/min

Results

Conclusion

A fast and effective method was developed for the determination of six designer opiates in urine samples. All analytes of interest were extracted using a Clean Screen® XCEL I column. Analysis of the samples was performed by LC–MS/MS utilizing a Selectra® DA HPLC column, which allowed for improved separation of furanyl fentanyl and fentanyl, when compared to other column phases. Absolute recoveries ranged from 72.9–125.3% for all three control levels tested. With the unfortunate (and often unaware) abuse of synthetic opiates throughout the United States, it is critical that forensic laboratories have accurate and rapid SPE methods for the identification of this class of compounds. This method will be of great use as drugs with similar structures start to be found in casework.

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