Analysis of Fentanyl and its Metabolite, Norfentanyl by CESI-MSJohn C. Hudson, Beckman Coulter Canada
Regina, Saskatchewan, S4V 0R4 Canada

Introduction

Biological Fluid Extraction Protocol

There is an on-going requirement in clinical and forensic
casework to detect and quantify low levels of potent drugs andtheir metabolites. This need arises especially when there has
been extended periods of time between administration of thedrug and collection of the sample for analysis. Forensic cases
include Drug-Facilitated Sexual Assault (DFSA) and Driving
Under the Influence of Drugs (DUID) where there may be alengthy delay in the assault being reported or samples from an
impaired driver being obtained.
In this work, one such drug, fentanyl, and its metabolite,
norfentanyl, were analyzed at challenging sub-therapeuticlevels with both samples and unknowns processed as a blindtrial. For this purpose, we have used a new techniquedeveloped in our laboratory, called CESI-MS, where capillaryelectrophoresis (CE) and electrospray ionization (ESI) havebeen integrated into a single dynamic process, to significantlyimprove the efficiency of analyte ionization.
Fentanyl (Figure 1), a potent synthetic narcotic analgesic,
surgical anesthetic and recreational drug, is commonlyadministered at low dosages of 25 µg/patch, resulting in very
low therapeutic levels of 0.3 to 1.2 ng/mL of serum after 24hours. It is reported to be 50-100 times more potent thanmorphine (1). Its main metabolite, norfentanyl can be detected
for up to 72 hours after administration.

HNNOO

H3C

H3C

Fentanyl

1. Add 0.2 mL conc. NH4OH and vortex.
2. Add 5 mL of 1-chlorobutane and shake for 10 min.
3. Centrifuge at 0-4oC for 10 min. at 3000 rpm.
4. Evaporate carefully just to dryness with N2 or in a SpeedVacŦ.
5. Add 100 µL of 5 mM BGE to tube, vortex, heat to dissolve extract.
6. Transfer to a 200 µL Microfuge tube.
7. Centrifuge at 14,000 rpm for 20 min.
8. Pressure-inject the sample at 10 seconds at 5 psi.
Figure 2: Liquid-Liquid Extraction Protocol for Biofluids

Results
1. Serum calibration samples for fentanyl and norfentanyl using
doxapram as the internal standard were prepared and analyzed using
a liquid-liquid extraction protocol (Figure 2).

Figure 5: Serum Calibration with Bracketed Blind Controls

2. An OptiMS CESI interface (Figure 3) was used to interface CE andMS, providing the required sensitivity on injections of only 7 nL of
extract dissolved in 100 µL.
3. Samples were analyzed by CESI-MS and the separation is shownin Figure 4.
4. Multiple Reaction Monitoring (MRM) was used in the quantitative
processing (Fentanyl: 337.4  188.2, Norfentanyl: 233.3  84.1,
Doxapram IS: 379.4  292.4).
4. Samples including blanks prepared by the external agency, wereextracted in duplicate bracketing the blind controls (Figure 5).

NN

To 1 mL of whole blood, serum or urine:

Norfentanyl

5. The calibrations were linear with R2 >0.99 for both fentanyl andnorfentanyl from 0.1 to 50 ng/mL (Figure 6).
6. LOD and LOQ calculations showed that the protocol can provide
detection of fentanyl and norfentanyl at low picogram per milliliter
levels in serum (Figure 7).
7. The results for the externally prepared blind controls compared tothe target values were acceptable at less than 15% (Figure 8).

Figure 6: Linear Regression for Fentanyl and Norfentanyl

Figure 1: Fentanyl Metabolism in Biological Fluids

OptiMS ESI Interface
Material and Methods
Chemicals:
All chemicals were reagent grade and were purchased on-linefrom VWR International.

Surface+ Coating

Drug and Metabolite Standards:
Standards were purchased from Cerilliant Corporation, Round
Rock, TX, USA. Stock solutions were prepared at aconcentration of 1 mg/mL and further diluted for spiking serumwith methanol. Standard solutions for mass spectrometry andextractions were prepared at 5, 1 or 0.1 ng/µL in 5 to 50 mM
Ammonium Formate (pH 2.85).

Figure 7: Calculated LOD and LOQ

Figure 3: OptiMS CESI Interface Schematic

Serum Calibration Standards:
Serum was provided and the samples, including blind controls,
were prepared by an external agency (Pharmalytics Inc.,
Saskatoon, SK, Canada). Fentanyl and norfentanyl solutions
were prepared, spiked into blank human serum and extracted
as per the protocol (Figure 2). The samples were kept frozen
until the time of analysis.

Figure 8: Blind Control ResultsConclusions
Doxapram

Fentanyl
Norfentanyl

CESI-MS Conditions
OptiMS* Porous
Capillary Interface

Separation
Temperatures
Background
Electrolyte (BGE)
SampleIntroduction
CE Instrument
MS Instrument
Conductive
LiquidESI Voltage
CapillaryConditioning

92.4 cm Surface+ (covalent cation)coated prototype 150 µm OD, 30 µm ID
with porous emitter tip
30kV, 216 V/cm, 1.6 µamps
Capillary and Sample Storage = 20oC
50 mM Ammonium Formate, pH 2.85

References
Figure 4: Separation of Fentanyl, Norfentanyl and Doxapram IS

Hydrodynamic 5 psi for 10 s
CESI 8000* PrototypeWaters Xevo TQ with MassLynx 4.1
0.7% Formic Acid

Acknowledgements
Dr. Gordon McKay, CEO, Pharmalytics Inc., Saskatoon, SK, Canada.
Jeff Chapman, Chitra Ratnayake and Scott Mack ofBeckman Coulter Inc., Brea, CA, USA.

1.25 kVThe capillaries were initially conditioned
with MeOH, water, 1N NaOH, water andBGE.

CESI, which is the integration of CE and ESI into a single dynamicprocess, interfaced to mass spectrometry provides the resolution andsensitivity required to analyze one of the most challenging drugs andits metabolite, fentanyl and norfentanyl. The small sample volumes
required, in this case, 7 nL injected, create a significant advantage toforensic scientists who, very often, deal with inadequate specimen
submission. This important advancement in separation and ionization
technology permits replicate quantitative analysis on less than 100
microliters of biofluid and detection of many challenging drugs andtheir metabolites at sub-therapeutic levels. The process is robust andautomated and will be of great value to analytical laboratories thatwork with charged analytes.

1. Baselt, R.C. Disposition of Toxic Drugs and Chemicals in Man,
pp. 616-619, 8th Edition, Biomedical Publications, Foster City,
California, 94404 (2008).
2. H. Copley, “Validation of Capillary Electrophoresis in Forensic
Toxicological Analyses: Screening Whole Blood for Common
Basic Drugs”, A Thesis Submitted to the College of Graduate
Studies and Research in Partial Fulfilment of the Requirements
for the Degree of Master of Science in the Toxicology Graduate
Program University of Saskatchewan, Saskatoon. (2004).

* In development.
Ŧ All trademarks are the property of its respective owners.

For Laboratory Use Only; not for use in diagnostic procedures.
Beckman Coulter, the stylized logo and Microfuge are trademarks of Beckman Coulter, Inc. and are registered with the USPTO.
CESI and OptiMS are trademarks of Beckman Coulter, Inc.

3. Shah, V.P. et.al., “Bioanalytical Method Validation – A Revisit witha Decade of Progress”, Pharmaceutical Research17(12) 1551-57 2000.
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