Quantitative Determination of Norepinephrine in Human Plasma by SPE and Derivatization followed by
LC-MS/MS Analysis
David Delinsky, Kaitlyn Hipp, Fola Omofoye, Leslie Freeman, Mary Barringer, Valerie Belcher, Brian Nofsinger and Mike Allen
Overview
Methods
This work describes the validation of a sensitive method
For extraction, norepinephrine is complexed with 2-aminoethyl
diphenylborinate (Reference 2, Figure 1C) followed by solid
phase extraction. The eluent was pH adjusted, and derivatized
by an ethylation reaction with deuterated acetaldehyde using
cyanoborohydride as a catalyst. Heptafluorobutyric acid was
added to the sample as an ion pair agent for chromatographic
purposes. Chromatographic separation was performed on
an Waters Acquity® C18 column with a gradient separation
using 20 mM ammonium formate with 0.1% formic acid and
methanol for the mobile phase (Table 1). Detection of derivatized
norepinephrine and d6-norepinephrine (internal standard) was
on a AB Sciex® API 5000 using MRM in the positive ion mode
(Tables 2-3).
for the quantitation of endogenous concentrations of
norepinephrine (also known as noradrenaline) in human
plasma using UPLC-MS/MS.
Norepinephrine is an endogenous compound that functions
both as a neurotransmitter and as a hormone. Norepinephrine
is released into the blood by the adrenal glands as part of the
body’s response to stress.
Norepinephrine is sensitive to photolytic and oxidative
degradation and as a result, an anti-oxidant must be used to
stabilize plasma shortly after collection. Sodium metabisulfite
(NaMBS) is used in this method. Blank plasma for use in
Table 1. UPLC Chromatographic Conditions
standard and QC preparations was generated by placing
normal, K 2EDTA human plasma under fluorescent lights
Results (continued)
Table 3. MRM Mass Transitions
Results
Due to norepinephrine’s susceptibility to photolytic and
oxidative degradation, plasma samples must be stabilized
with an antioxidant. The method was validated over the linear
calibration range of 10.0 to 1000 pg/mL (Table 4). Sensitivity at
the LLOQ of 10 pg/mL was acceptable, with a signal to noise
ratio > 5:1 achieved (Figure 3). Accuracy (<±15%) and precision
(<15%) of the method was demonstrated (Table 5). The lack of
instrument response at analyte and internal standard retention
times in ten individual photolyzed plasma lots proves method
specificity (Figure 6). No significant ionization effects were
observed. A number of stability tests were performed to show
that norepinephrine is stable in matrix when NaMBS is present.
while gently mixing for approximately 48 hours to degrade
endogenous norepinephrine. Photolyzed plasma was then
stabilized with sodium metabisulfite to prevent degradation
of norepinephrine once it is spiked into the plasma. Due to
the polarity of norepinephrine, it is not amenable to reversed
phase HPLC and is not efficiently ionized. Ethylating the amine
on norepinephrine greatly reduces the polarity (Reference
1), thereby improving RP-HPLC characteristics and improving
ionization. Combining SPE cleanup with derivatization
improved sensitivity by approximately 100-fold as compared
to SPE alone (using a UPLC-MS/MS platform).
Table 4. Back-Calculated Concentrations of
Calibration Standards for Norepinephrine
Table 6. Norepinephrine Validation Results
Figure 3. Representative Low Standard (10.0 pg/mL)
Figure 7. Representative QC0
Figure 4. Representative High Standard (1000 pg/mL)
Table 5. Quality Control Samples for Norepinephrine
Conclusions
A validated method for the quantitation of
norepinephrine in stabilized human plasma has
been established (Table 6). The method requires
the addition of NaMBS to study samples at the
collection site. The method is to be used for the
analysis of clinical study samples.
Figure 5. Representative Endogenous Blank
References
Figure 2. Representative Calibration Curve
1. Ji et al., Simultaneous determination of plasma
epinephrine and norepinephrine using an integrated
strategy of a fully automated protein precipitation
technique, reductive ethylation labeling and UPLC–MS/
MS, Analytica Chimica Acta, 670, 2010, 84–91
Figure 1. Chemical Structures
Table 2. Mass Spectrometer Tune Parameters
Figure 6. Representative Matrix Blank
Covance is the drug development business of Laboratory Corporation of America® Holdings (LabCorp®). Content of this material was
developed by scientists who at the time were affiliated with LabCorp Clinical Trials or Tandem Labs, now part of Covance.
2. Talwar et al., Extraction and separation of urinary
catecholamines as their diphenyl boronate complexes
using C18 solid-phase extraction sorbent and highperformance liquid chromatography, Journal of
Chromatography B, 769 2002, 341-349