Fast Screening of Fentanyl in Urine

This article introduces a basic technique for fentanyl screening in urine by employing the Advion Touch Express Open Port Sampling Interface (OPSI) coupled with the Advion expression® Compact Mass Spectrometer (CMS).

Introduction

OPSI was produced by Vilmos Kertesz and Gary Van Berkel from the Oak Ridge National Laboratory[2,3,4]. The Touch Express OPSI, when paired with the electrospray ionization (ESI) source of the Advion expression CMS, provides an efficient assay benchtop solution in a simple-to-use system with a small footprint.

Fentanyl (1.0 mg/mL in methanol) and fentanyl-D5 (100 µg/mL in methanol) were utilized as analytical standards. Samples were calibrated into control urine that was free from fentanyl with concentrations of 2.5, 5, 10, 25, 50, 100, 250, 500, and 1000 ng/mL and fentanyl-D5 at 100 ng/mL.

Liquid-liquid extraction (LLE) was then used to prepare the samples. 1.5 mL of 1:1 ethyl acetate/hexane was introduced to 0.5 mL of a urine sample and vortexed for 20 seconds after being left to stand for 2 minutes.

1 mL of supernatant was transported to a clean 2 mL centrifuge vial and evaporated to dryness in a centrifuge vacuum evaporator. 250 µL methanol was used to reconstitute the dried extract and was subsequently utilized for the OPSI/CMS investigation.

Experimental setup and schematic of the CMS and OPSI.

Figure 1. Experimental setup and schematic of the CMS and OPSI. Image Credit: Advion

Method

Through the outer pair of concentric tubes at ~250 µL/minute, the solvent is transported to the open port sampling area. The solvent produces a meniscus at the open port before being controlled down the inner path into the mass spectrometer’s ion source as a result of the Venturi Effect of the nebulization gas in the ion source.

The fentanyl-spiked and control urine samples are touched in series to the OPSI sampling port (Figure 1) and the results are acquired in less than 30 seconds.

 

Results

The mass spectra of fentanyl and fentanyl-D5 (Figure 2) display the detection of protonated molecules at m/z 337.2 for fentanyl and 342.2 for fentanyl-D5. These ions are employed in a Selected Ion Monitoring (SIM) technique for the measurement of fentanyl in the urine.

Mass spectra of Fentanyl and Fentanyl-D5 in control urine.

Figure 2. Mass spectra of Fentanyl and Fentanyl-D5 in control urine. Image Credit: Advion

The extracted ion currents (XIC) of protonated fentanyl and fentanyl-D5 (Figure 3) demonstrated that no interferences were identified at the SIM m/z values in the control urine that was free from fentanyl after the LLE cleanup.

The signal-to-noise ratio (S/N) for 2.5 ng/mL of fentanyl is approximately 135, so the limit of quantitation (S/N = 10) is predicted to be less than 1 ng/mL. The urine sample that was laced with 2.5 ng/mL of fentanyl was studied through OPSI-ESI-SIM investigations and the XIC displays a signal to noise ratio of around 133.2 (Figure 4).

The limit of quantitation is the concentration with S/N at 10, which means fentanyl’s quantitation level is predicted to be less than 1 ng/ml.

Figure 5a shows that the investigation time for every sample can be completed in the range of 30 seconds with a peak width of around 12 seconds. No carryover was identified with the current arrangement which promotes a high throughput environment for the screening of fentanyl.

XIC of protonated fentanyl and fentanyl-D5 in control urine.

Figure 3. XIC of protonated fentanyl and fentanyl-D5 in control urine. Image Credit: Advion

Touch Express SIM analysis of fentanyl, 2.5 ng/mL (left) and fentanyl-D5, 100 ng/mL in control of urine (right).

Figure 4. Touch Express SIM analysis of fentanyl, 2.5 ng/mL (left) and fentanyl-D5, 100 ng/mL in control of urine (right). Image Credit: Advion

The calibration curve, over a linear dynamic range of 2.5 to 500 ng/mL, demonstrates a linear regression R2 value of 0.997.

The statistical overview (Table 1) displays the average precision and accuracy (CV%) of concentrations from 2.5 to 500 ng/mL, which are determined to be 87.5 ±0.17% to 106.5% ±3.36%, respectively. This is in line with the desired range of 0 to 15% for precision and 85 to 115% for accuracy.

Calibration curve of fentanyl in urine.

Figure 5. Calibration curve of fentanyl in urine. Image Credit: Advion

 

Table 1. Statistical Summary of Fentanyl in Urine Analysis. Source: Advion

Samples TypeT Samples Included Concentration (ng/mL) Measured Concentration (ng/mL) Averaged Accuracy (%) Analyte/
ISTD (Fentanyl/
F-D5)
Calculated Analyte/
ISTD (Fentanyl/
F-D5)
CV (%)
Cal 1 3/6 2.5 2.2 87.5 0.045 0.048 0.17
Cal 2 3/3 5 3.8 96.7 0.076 0.077 0.40
Cal 3 3/3 10 10.1 100.7 0.137 0.136 2.21
Cal 4 3/3 25 24.8 99.1 0.309 0.311 0.78
Cal 5 3/3 50 56.0 112.0 0.674 0.604 3.36
Cal 6 3/3 100 102.2 102.2 1.214 1.188 2.02
Cal 7 3/3 250 266.1 106.5 3.131 2.943 0.55
Cal 8 3/3 500 476.3 95.3 5.589 5.866 1.41

 

Conclusion

The Advion Touch Express OPSI offers:

  • A robust and simple operation requiring no mechanical components
  • Efficient analysis with results obtained in less than 30 seconds
  • Direct samples, touch and assay
  • No contamination or carryover is facilitated as the port is constantly self-cleaning
  • A high throughput and affordable screening method

References and Further Reading

[1] “Fentanyl And Analogues”. LiverTox. 14 December 2017.

[2] Van Berkel G., Vilmos K.; An open port sampling interface for liquid introduction atmospheric pressure ionization mass spectrometry; Rapid Comm. Mass Spec., 2015 29(19) 1749-1756

[3] Gary J V. B., Vilmos K.; Rapid sample classification using an open port sampling interface coupled with liquid introduction atmospheric pressure ionization mass spectrometry, Rapid Comm. Mass Spec. 2017 31(3) 281–291

[4] Gary J V. B., Vilmos K., Harry B.; Immediate drop on demand technology (I-DOT) coupled with mass spectrometry via an open port sampling interface; Bioanalysis, 2017, 9(21),1667-1679

Acknowledgments

Produced from materials originally authored by Changtong Hao from Advion, Inc.

About Advion

Advion’s nearly three-decade dedication to serving scientists yields customer-focused life science solutions. Our deep scientific, engineering and customer workflow knowledge spawns an unrivaled solution portfolio. We work directly with, train, and passionately advocate for our customers to ensure their success.

The Advion product portfolio includes the expression Compact Mass Spectrometer, the AVANT (U)HPLC, the SOLATION ICP-MS and many other novel sample introduction systems including the Plate Express TLC Plate reader and the TriVersa NanoMate nano-electrospray ionization technique.

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Last updated: Dec 5, 2019 at 11:02 AM

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