The Detection and Analytical Confirmation of Synthetic Fentanyl Analogues in Human Urine & Serum using an UltivoLC/TQ

Significance of the Topic

This study addresses the urgent need for rapid and sensitive detection of synthetic fentanyl analogs and related opioids in biological fluids. Given the rising prevalence of fentanyl-related overdoses and the emergence of novel analogs, reliable quantitation in human serum and urine is critical for clinical toxicology, forensic investigations and public health monitoring.

Objectives and Study Overview

The primary goal was to develop and validate a targeted liquid chromatography–tandem mass spectrometry (LC-MS/MS) method capable of quantifying twelve synthetic fentanyl analogs, the precursor 4-ANPP and the potent W-18 analog in human serum and urine. The study aimed to demonstrate method speed, robustness, sensitivity and analytical performance across multiple sample batches.

Methodology and Instrumentation

A simple protein precipitation and dilution workflow was applied to serum and urine samples, followed by analysis on an Agilent 1290 Infinity II UHPLC coupled to the Ultivo triple quadrupole mass spectrometer with electrospray ionization in positive mode. Chromatographic separation used a Poroshell EC-C18 column (2.1×50 mm, 2.7 µm) and a seven-minute gradient with aqueous 0.01% formic acid/5 mM ammonium formate (mobile phase A) and 0.01% formic acid in methanol (mobile phase B). Two multiple reaction monitoring transitions per analyte and a single transition per isotopically labeled internal standard ensured selectivity. Sample preparation steps included:

• Human serum (250 µL) protein precipitation with acetonitrile/internal standard mixture, centrifugation and 1:2 dilution in methanol:water.
• Human urine spiked with calibrators/internal standards, centrifugation and dilution to 1 mL with water (resulting in 1:10 dilution).

Results and Discussion

The method achieved low limits of quantitation for serum (10–100 pg/mL) and urine (50–100 pg/mL), corresponding to femtogram-level on-column sensitivity. Calibration curves spanned 1 pg/mL to 500 ng/mL, with linearity coefficients above 0.996 for all analytes. Precision across three independent batches yielded relative standard deviations below 7% in serum and below 9% in urine for all calibration levels. The seven-minute cycle time supports high-throughput sample analysis while maintaining robust chromatographic resolution and mass spectrometric sensitivity.

Benefits and Practical Applications

The described LC-MS/MS workflow offers:

• Rapid turnaround for toxicology and forensic laboratories handling high sample volumes.
• High analytical sensitivity suitable for early detection of fentanyl analog exposure.
• Reliable quantitation across a broad dynamic range to support clinical and regulatory decision-making.

Future Trends and Possibilities

Future work may focus on expanding the panel to include emerging analogs, integrating high-resolution mass spectrometry for enhanced selectivity, and automating sample preparation to further increase throughput. Addressing potential matrix interferences and incorporating additional internal standards will strengthen method robustness in complex biological samples.

Conclusion

This research demonstrates that the Agilent Ultivo LC/TQ system, combined with a streamlined sample preparation protocol, delivers sensitive, precise and high-throughput quantitation of synthetic fentanyl analogs in human serum and urine. The method is well-suited for forensic, clinical and quality-control applications where accurate detection of ultra-trace opioids is essential.

References

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