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Analysis of Fentanyl and Its Analogues in Human Urine by LC-MS/MS

By Shun-Hsin Liang and Frances Carroll


Synthetic opioid drugs, such as fentanyl and sufentanil, have very high analgesic potency. Abuse of these prescription painkillers—along with a rapidly growing list of illicit analogues—is a significant public health problem. In this study, we developed a simple dilute-and-shoot method that provides a fast 3.5 minute analysis of fentanyl and related compounds (norfentanyl, acetyl fentanyl, alfentanil, butyryl fentanyl, carfentanil, remifentanil, and sufentanil) in human urine by LC-MS/MS using a Raptor Biphenyl column.


In recent years, the illicit use of synthetic opioids has skyrocketed, and communities worldwide are now dealing with an ongoing epidemic. Of the thousands of synthetic opioid overdose deaths per year, most are related to fentanyl and its analogues. With their very high analgesic properties, synthetic opioid drugs such as fentanyl, alfentanil, remifentanil, and sufentanil are potent painkillers that have valid medical applications; however, they are also extremely addictive and are targets for abuse. For example, carfentanil is a very powerful anesthetic used as a tranquilizer for large animals, primarily elephants. It is 10,000 times more potent than morphine, making it one of the most powerful synthetic opioids available. The increase in its illicit use, most commonly by mixing with heroin, has been linked to a significant number of overdose deaths since 2016. In addition to abuse of these prescription drugs, the current opioid crisis is fueled by a growing number of illicit analogues, such as acetyl fentanyl and butyryl fentanyl, which have been designed specifically to evade prosecution by drug enforcement agencies.

As the number of opioid drugs and deaths increases, so does the need for a fast, accurate method for the simultaneous analysis of fentanyl and its analogues. Therefore, we developed this LC-MS/MS method for measuring fentanyl, six analogues, and one metabolite (norfentanyl) in human urine (Figure 1). A simple dilute-and-shoot sample preparation procedure was coupled with a fast (3.5 minutes) chromatographic analysis using a Raptor Biphenyl column. This method provides accurate, precise identification and quantitation of fentanyl and related compounds, making it suitable for a variety of testing applications including clinical toxicology, forensic analysis, workplace drug testing, and pharmaceutical research.

Figure 1: Chemical Structures



Sample Preparation

The analytes were fortified into pooled human urine. An 80 µL urine aliquot was mixed with 320 µL of 70:30 water:methanol solution (five-fold dilution) and 10 µL of internal standard (40 ng/mL in methanol) in a Thomson SINGLE StEP filter vial (Restek cat.# 25895). After filtering through the 0.2 µm PVDF membrane, 5 µL was injected into the LC-MS/MS.

Calibration Standards and Quality Control Samples

The calibration standards were prepared in pooled human urine at 0.05, 0.10, 0.25, 0.50, 1.00, 2.50, 5.00, 10.0, 25.0, and 50.0 ng/mL. Three levels of QC samples (0.75, 4.0, and 20 ng/mL) were prepared in urine for testing accuracy and precision with established calibration standard curves. Recovery analyses were performed on three different days. All standards and QC samples were subjected to the sample preparation procedure described above.

LC-MS/MS analysis of fentanyl and its analogues was performed on an ACQUITY UPLC instrument coupled with a Waters Xevo TQ-S mass spectrometer. Instrument conditions were as follows, and analyte transitions are provided in Table I.

Analytical column: Raptor Biphenyl (5 µm, 50 mm x 2.1 mm; cat.# 9309552)
Guard column: Raptor Biphenyl EXP guard column cartridge, (5 µm, 5 mm x 2.1 mm; cat.# 930950252)
Mobile phase A: 0.1% Formic acid in water
Mobile phase B: 0.1% Formic acid in methanol
Gradient Time (min) %B
  0.00 30
  2.50 70
  2.51 30
  3.50 30
Flow rate: 0.4 mL/min
Injection volume: 5 µL
Column temp.: 40 °C
Ion mode: Positive ESI


Table I: Analyte Transitions
Analyte Precursor Ion Product Ion
Product Ion
Internal Standard
Norfentanyl 233.27 84.15 56.06 Norfentanyl-D5
Acetyl fentanyl 323.37 188.25 105.15 Acetyl fentanyl-13C6
Fentanyl 337.37 188.26 105.08 Fentanyl-D5
Butyryl fentanyl 351.43 188.20 105.15 Carfentanil-D5
Remifentanil 377.37 113.15 317.30 Norfentanyl-D5
Sufentanil 387.40 238.19 111.06 Sufentanil-D5
Carfentanil 395.40 113.14 335.35 Carfentanil-D5
Alfentanil 417.47 268.31 197.23 Acetyl fentanyl-13C6
Norfentanyl-D5 238.30 84.15
Acetyl fentanyl-13C6 329.37 188.25
Fentanyl-D5 342.47 188.27
Sufentanil-D5 392.40 238.25
Carfentanil-D5 400.40 340.41

Results and Discussion

Chromatographic Performance

All eight analytes were well separated within a 2.5-minute gradient elution (3.5-minute total analysis time) on a Raptor Biphenyl column (Figure 2). No significant matrix interference was observed to negatively affect quantification of the five-fold diluted urine samples. The 5 µm particle Raptor Biphenyl column used here is a superficially porous particle (SPP) column. It was selected for this method in part because it provides similar performance to a smaller particle size fully porous particle (FPP) column, but it generates less system backpressure.

Figure 2: The Raptor Biphenyl column effectively separated all target compounds in urine with no observed matrix interferences.

Blank Urine


10 ng/mL Urine Standard

PeakstR (min)Conc.
Precursor IonProduct IonProduct Ion
1.Norfentanyl-d5 1.031238.3084.15-
4.Acetyl fentanyl-13C61.691329.37188.25-
5.Acetyl fentanyl1.6910323.37188.25105.15
11.Butyryl fentanyl2.1510351.43188.20105.15
ColumnRaptor Biphenyl (cat.# 9309552)
Dimensions:50 mm x 2.1 mm ID
Particle Size:5 µm
Pore Size:90 Å
Guard Column:Raptor Biphenyl EXP guard cartridge 5 mm, 2.1 mm ID, 5 µm (cat.# 930950252)
Temp.:40 °C
Diluent:70:30 Water:methanol
Inj. Vol.:5 µL
Mobile Phase
A:0.1% Formic acid in water
B:0.1% Formic acid in methanol
Time (min)Flow (mL/min)%A%B
Ion Mode:ESI+
Sample PreparationHuman urine was fortified at 10 ng/mL with target analytes. An 80 μL urine aliquot was mixed with 320 μL of 70:30 water:methanol solution (5-fold dilution) and 10 μL of internal standard solution (40 ng/mL in methanol) in a Thomson SINGLE StEP filter vial (Restek cat.# 25895). After filtering through the 0.2 µm PVDF membrane, 5 μL was injected for analysis.


Linear responses were obtained for all compounds and the calibration ranges encompassed typical concentration levels monitored for both research and abuse. Using 1/x weighted linear regression (1/x2 for butyryl fentanyl), calibration linearity ranged from 0.05 to 50 ng/mL for fentanyl, alfentanil, acetyl fentanyl, butyryl fentanyl, and sufentanil; from 0.10 to 50 ng/mL for remifentanil; and from 0.25 to 50 ng/mL for norfentanyl and carfentanil. All analytes showed acceptable linearity with r2 values of 0.996 or greater (Figure 3) and deviations of <12% (<20% for the lowest concentrated standard).

Figure 3: Calibration Curves

Accuracy and Precision

Based on three independent experiments conducted on multiple days, method accuracy for the analysis of fentanyl and its analogues was demonstrated by the %recovery values, which were within 10% of the nominal concentration for all compounds at all QC levels. The %RSD range was 0.5-8.3% and 3.4-8.4% for intraday and interday comparisons, respectively, indicating acceptable method precision (Table II).

Table II: Accuracy and Precision Results for Fentanyl and Related Compounds in Urine QC Samples.

  QC Level 1 (0.750 ng/mL) QC Level 2 (4.00 ng/mL) QC Level 3 (20.0 ng/mL)
Analyte Average Conc. (ng/mL) Average Accuracy (%) %RSD Average Conc. (ng/mL) Average Accuracy (%) %RSD Average Conc. (ng/mL) Average Accuracy (%) %RSD
Acetyl fentanyl 0.761 102 1.54 3.99 99.7 2.08 19.9 99.3 0.856
Alfentanil 0.733 97.6 3.34 3.96 98.9 8.38 20.9 104 6.73
Butyryl fentanyl 0.741 98.9 6.29 3.77 94.3 6.01 20.8 104 4.95
Carfentanil 0.757 101 7.34 3.76 94.0 4.64 20.6 103 4.24
Fentanyl 0.761 102 1.98 3.96 99.1 2.31 19.9 99.6 1.04
Norfentanyl 0.768 103 6.50 4.04 101 1.84 20.1 101 2.55
Remifentanil 0.765 102 3.42 3.97 99.2 3.68 20.8 104 4.14
Sufentanil 0.752 100 1.67 3.93 98.3 1.28 20.1 100 0.943


A simple dilute-and-shoot method was developed for the quantitative analysis of fentanyl and its analogues in human urine. The analytical method was demonstrated to be fast, rugged, and sensitive with acceptable accuracy and precision for urine sample analysis. The Raptor Biphenyl column is well suited for the analysis of these synthetic opioid compounds and this method can be applied to clinical toxicology, forensic analysis, workplace drug testing, and pharmaceutical research.


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