The Simultaneous Analysis of Norepinephrine, Dopamine and Serotonin

  • Simultaneous analysis in 15 minutes
  • 260 amol (50 fg) detection limit for DA and 580 amol (0.1 pg) for NE and 5-HT
  • Allows for higher sample temporal resolution (15 min)

*Find the best catecholamine analysis for your samples here.

In-vivo brain-microdialysis users have been in search of a separation method for norepinephrine (NE, noradrenalin), dopamine (DA), and serotonin (5-HT) with both high sensitivity and specificity from a single brain microdialysate sample. Historically, it has been difficult to detect NE and 5-HT in the same chromatogram using a reverse-phase column due to the difference in the retention times of these compounds. Until now, it has been necessary to split the sample and apply two different analytical methods in order to measure NE, DA, and 5-HT with high specificity and sensitivity.

Now, Eicom introduces the EICOMPAK CAX (cation exchange mode, 2.0 × 200 mm) which allows for the detection of NE, DA, and 5-HT from a single sample injection. This method can be applied using a single constant applied voltage (DC mode) electrochemical detection. The required analysis time is only 15 min with a sensitivity of 50–100 fg/injection using EICOM’s HTEC-510. The system requirements necessary to achieve optimal performance from the EICOMPAK CAX column has been incorporated into the HTEC-510 (HTEC-600 is the newest model).

​Analytical Conditions

can be found in the download file

Microdialysis Conditions

Animal Freely moving male Wister rat, BW 240–270 g
Microdialysis Probe Eicom A-I-4-03 (membrane length 3 mm)
Probe Location Prefrontal Cortex (A = +2.5 mm, L = 0.6 mm, V = –3 mm)
Perfusate Ringer’s solution 1 μL/min
Injection Volume 1 μL/min × 15 min via automated online injector Eicom EAS-20

Conclusion

This new cutting edge methodology is user friendly and easily applied to microdialysis samples. The high sensitivity and selectivity were exemplified by TTX perfusion. This methodology enhances the efficiency of monoamine neurotransmitter analysis and allows for greater sample resolution when conducting pharmacological and behavioral tests.

Eicom provides a more detailed protocol including troubleshooting and mobile phase conditions. If higher sensitivity is required, Eicom also provides the PP-ODS column for the analysis of DA and 5-HT. The analysis time is only 5 min with a sensitivity level of 30 fg/injection. The EICOMPAK CAX with the current separation conditions does not allow the complete separation of NE and epinephrine. The difference in their retention time is only half a minute. Epinephrine levels in the brain are minimal and usually are below the sensitivity level. If detection of epinephrine levels is required, use EICOMPAK CA-5ODS.

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Fig. 1 – Calibration curve for DA, NE, and 5-HT in the range of 0.1 -5 pg/injection with the Eicom HTEC-500.

See in the download file

Fig. 2 – Chromatogram obtained by a 100 fg/injection of a standard solution of NE, DA, and 5-HT in 0.1 M phosphate buffer (ph3.5) and blank matrix.

See in the Download File

Fig. 3 – Simultaneous analysis of NE, DA, and 5-HT obtained from a prefrontal cortex microdialysis sample using the Eicompak CAX. This figure shows the basal condition and the condition 90 min after imipramine administration. Basal levels for NE, DA, and 5-HT are 1.71, 0.54, 0.83 pg in 15 μl respectively. Following drug administration, NE, DA, and 5-HT levels increased to 5.49, 1.09 2.10 pg in 15 μL, respectively.

See in the download file

Fig. 4 – The basal condition and the condition 60 min after perfusion with tetrodotoxin at 1 μM in the rat prefrontal cortex. Basal levels for NE, DA, and 5-HT are 5.11, 0.37, 0.34 pg in 15 μl, respectively. Following drug administration, NE, DA, and 5-HT levels decreased to 0.62, 0.06, 0.19 pg in 15 μl, respectively.

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