Application Brief 129 Simultaneous Analysis of Norepinephrine, Dopamine, and Serotonin in Microdialysis Perfusates Using hplc-ecd

Application Brief 129

Simultaneous Analysis of Norepinephrine,  

Dopamine, and Serotonin in Microdialysis  

Perfusates Using HPLC-ECD

IntroductIon

The analysis of norepinephrine (NE) continues to  

be a problem, even more so when measured in 

conjunction with dopamine (DA) and serotonin (5-HT). 

Until now, numerous attempts to develop such a method 

have met with limited success. The usual approach 

requires use of multiple analytical systems, thereby 

wasting precious sample.

Neuroscientists using microdialysis perfusion 

continuously demand greater temporal and spatial 

resolution. This can be achieved by placing smaller probes 

in more discrete brain regions, and by increasing the 

sampling rate. However, the resulting low-volume and 

dilute samples can be challenging to analyze, requiring a 

highly sensitive analytical system.

Today, a single chromatographic system provides 

simultaneous quantitation of NE, DA, and 5-HT in a 

single sample. This makes more efficient use of each 

sample and lets researchers get information about multiple 

pathways in a single analysis. It also provides a superior 

method for NE measurement.

This work presents an example of how the method 

can be used to measure prefrontal cortical levels of  

NE, DA, and 5-HT under basal and amphetamine 

stimulated conditions.

EquIpmEnt and condItIons

Liquid Chromatography 

An isocratic high-performance liquid chromatography 

(HPLC) system similar to the Thermo Scientific 

Dionex UltiMate

®

 3000 system including a pump, 

autosampler, and Thermo Scientific ESA  

Coulochem

®

 III detector

Coulochem III Organizer Module with Temperature 

Control (P/N 70-9121TA)

Column: 

MD160, 1.5 × 250 mm, 5 µm  

(P/N 70-7277)

Mobile Phase:

1

 MD-3MA

Flow Rate:  

0.20 mL/min

Temperature:  

32 °C

Inj. Volume:  

10 µL (partial loop)

Detector 

Coulochem III (P/N 70-9143)

Analytical Cell (P/N 70-4131)

Guard Cell (P/N 55-0417)

Cell Potentials:  Analytical Cell, E = +220 mV

 

Guard Cell, EGC = +275 mV

2 

Simultaneous Analysis of Norepinephrine, Dopamine, and Serotonin in Microdialysis Perfusates  

 

Using HPLC-ECD

Figure 1. Chromatography of NE, DA, and 5-HT standards  

in aCSF.

Microdialysis 

Probe: 

CMA11, 3 mm membrane length

Perfusion  

Flow Rate: 

2.0 µL/min

Artificial Cerebrospinal Fluid (aCSF) Composition: 

Sodium: 

147 mM 

Potassium: 

2.7 mM 

Calcium: 

1.2 mM 

Magnesium: 

1.0 mM 

Chloride: 

150 mM 

Phosphate: 

(pH 7.4 + 2) 2 mM

Collection Period:   20 min into 10 µL 0.2 M perchloric 

acid (containing 0.2 µM ascorbic 

acid and 0.2 µM EDTA)

Animal  Model: 

 Anesthetized  rat   

(urethane: 1.5g/kg i.p.)

Post Surgery  

Recovery: 

2–4 h

Probe Coordinates:   AP +3.2, LR +0.8, DV –5.0  

(from Bregma)

rEsults and dIscussIon

This method resolved all three monoamines in under 

16 min (Figure 1), showed excellent linearity (Figure 2), 

and extreme sensitivity (~100–150 fg on column).

Figure 2. Linear curves for NE, DA, and 5-HT.

Figure 3. Microdialysis samples obtained from a 3 mm  

concentric microdialysis probe placed in the prefrontal  

cortex of an anesthetized rat, before and after amphetamine  

(2 mg/kg i.p.) stimulation.

The method simultaneously measured all three 

monoamines under basal conditions, even in the urethane 

anesthetized animal where levels are expected to be 

depressed (Figure 3). Typical basal levels (uncorrected for 

in vitro recovery) were ~500, 250, and 160 fg/10 µL, for 

NE, DA, and 5-HT, respectively (data not shown). 

28709

0

2

4

8

12

15 16

6

10

14

1

3

5

9

13

7

11

18 19

17

-0.02

0.14

10 pg

Minutes

Volts

5 pg

2 pg

1 pg

0.5 pg

15.061 5-HT

7.083 DA

5.375 NE

0

20000

40000

60000

80000

100000

120000

0

2

4

6

8

10

12

Amount (pg)

Peak Height

NE

DA

5-HT

y = 10304x + 2147.4

R

2

 = 0.999

y = 9935.6x + 2621.9

R

2

 = 0.999

y = 6121.9x + 634.75

R

2

 = 0.996

28710

28711

5-HT

DA

NE

Post Stimulation with Amphetamine

Pre-Stimulation

0

2

4

8

12

15 16

6

10

14

1

3

5

9

13

7

11

-0.02

0.10

Minutes

Volts

Application Brief 129 

3

 

 

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LPN 2861  PDF  6/11

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Stimulation with amphetamine (2 mg/kg i.p.) 

resulted in significant increases of all three monoamine 

neurotransmitters to ~1500, 960, and 290 fg/10 µL, for 

NE, DA, and 5-HT, respectively (Figure 3). 

This method will be of great benefit to any researcher 

interested in studying the regional interplay of monoamine 

neurotransmitters during pharmacological manipulation  

or behavior.

acknowlEdgEmEnt

Thanks to Professors Timothy Maher and S. 

Phattanarudee of the Department of Pharmaceutical 

Sciences, Massachusetts College of Pharmacy, Boston, 

MA, for providing the microdialysis samples used during 

this study.

rEFErEncEs

1.  Dionex Corporation, Simultaneous Analysis of 

Monoamines. Technical Note 70-7661.  

Chelmsford, MA.