Application Note The Simultaneous Analysis of Norepinephrine, Dopamine & Serotonin in Microdialysis Samples AN03-0815 In vivo brain-microdialysis users have been in search of a separation method 1.0 for norepinephrine (NE, noradrenalin), dopamine (DA) and serotonin (5-HT) 0.9 with both high sensitivity and specificity from a single brain microdialysate 2 R2 = 0.999 sample. Historically, it has been difficult to detect NE and 5-HT in the same 0.8 chromatogram using a reverse phase column due to the difference in the 0.7 retention times of these compounds. Until now, it has been necessary to 0.6 2 split the sample and apply two different analytical methods in order to R2 = 0.999 measure NE, DA and 5-HT with high specificity and sensitivity. 0.5 0.4 Now, Eicom introduces the EICOMPAK CAX (cation exchange mode, 2.0 × 2 R2 = 0.998 200 mm) which allows for the detection of NE, DA and 5-HT from a single Peak height (mV) 0.3 sample injection. This method can be applied using a single constant 0.2 DA applied voltage 5-HT 0.1 NE (DC mode) electrochemical detection. The required analysis time is only 15 0.0 min with a sensitivity of 50–100 fg/injection using EICOM’s HTEC-500. The 0 1 2 3 4 5 system requirements necessary to achieve optimal performance from the Amount (pg/injection) EICOMPAK CAX column has been incorporated into the HTEC-500. Fig. 1 Calibration curve for DA, NE and 5-HT in the range. 3 of 0.1-5 pg/ injectionFig. 3 with the Eicom HTEC-500. 0 Analytical Conditions Analytical Instrument Eicom HTEC-500 Separation Column EICOMPAK CAX (2.0 id × 200 mm) 0.6 Column Temperature 35°C Flow Rate 250 μl/min 0.4 Detecting Condition +450 mV vs. Ag/AgCl 0.1 pg/injection mV 0.2 Working Electrode Graphite (WE-3G) with Gasket #GS-25 NE DA Time Constant 3.0 sec 5-HT 0 Data Acquisition Eicom EPC-500 Blank 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 When using the EICOMPAK CAX on the Eicom HTEC-500, the lower limit min of detection for DA is 50 fg/injection and 100 fg/injection for both NE and Fig. 2 Chromatogram obtained by a 100 fg/injection of a standard solution 5-HT with a S/N = 3. Using a 10 μl injection volume for the 10 pg/injection of NE, DA and 5-HT in 0.1 M phosphate buffer (pH3.5) and blank matrix. standard solution containing NE, DA and 5-HT, the CV% was 2.51, 2.63 and 2.98 respectively. When using a 50 μl loop, linearity is obtained within the injection volume range of 0–25 μl. The column can tolerate up to a 40 μl injection volume. US: www.eicom-usa.com - [email protected] | EU: www.eicomeurope.com - [email protected] Application Note 1.0 Microdialysis Conditions 0.8 90 min after Imipramine (10 mg/kg, i.p.) Animal Freely moving male Wister rat, BW 240–270 g NE administration 0.6 Microdialysis Probe Eicom A-I-4-03 (membrane length 3 mm) mV 0.4 DA Probe Location Prefrontal Cortex 5-HT (A = +2.5 mm, L = 0.6 mm, V = –3 mm) 0.2 Perfusate Ringer’s solution 1 μl/min 0 Basal level Injection Volume 1 μl/min × 15 min. via automated online injector 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 (Eicom EAS-20) min This new cutting edge methodology is user friendly and easily applied to Fig. 3 Simultaneous analysis of NE, DA and 5-HT obtained from a microdialysis samples. The high sensitivity and selectivity was exemplified prefrontal cortex microdialysis sample using the EICOMPAK CAX. This by TTX perfusion. This methodology enhances the efficiency of monoamine 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 neurotransmitter analysis and allows for greater sample resolution when 15 μl, respectively. Following drug administration NE, DA and 5-HT levels conducting pharmacological and behavioral tests. increased to 5.49, 1.09, 2.10 pg in 15 μl, respectively. 1.0 0.8 Basal level 0.6 mV 0.4 0.2 Eicom will provide a more detailed protocol including trouble shooting 0 60 min after Tetrodotoxin (TTX, 1 µmol/L) perfusion and mobile phase conditions. If higher sensitivity is required, Eicom also 012345678910 11 12 13 14 15 provides the PP-ODS column for the analysis of DA and 5-HT. The analysis min Fig. 4 Simultaneous analysis of NE, DA and 5-HT obtained from a time is only 5 min with a sensitivity level of 30 fg/injection. The EICOMPAK Fig. 7 CAX with the current separation conditions does not allow the complete prefrontal Fig. 7cortex microdialysis sample using the EICOMPAK CAX. This separation of NE and epinephrine. The difference in their retention time is figureFig. shows 7 the basal condition and the condition 60 min. after perfusion with tetrodotoxinFig. 7 at 1 μM. Basal levels for NE, DA and 5-HT are 5.11, 0.37, only half a minute. Epinephrine levels in the brain are minimal and usually Fig. 7 0.34 pg in 15 μl, respectively. Following drug administration NE, DA and 5-HT are below the sensitivity level. If detection of epinephrine levels is required, Fig. 7 levelsFig. decreased 7 to 0.62, 0.06, 0.19 pg in 15 μl, respectively. use EICOMPAK CA-5ODS. Fig. 7 US: www.eicom-usa.com - [email protected] | EU: www.eicomeurope.com - [email protected].