Simultaneous Analysis of Norepinephrine, Dopamine and Serotonin in 15 Minutes

Transcription

1 pplication Guide Simultaneous nalysis of Norepinephrine, Dopamine and Serotonin in 15 Minutes 1. Introduction Our customers have been seeking a separation method for norepinephrine (, noradrenalin), dopamine (D) and serotonin () in a single sample of brain microdialysate. It has been difficult to separate and on the same chromatogram due to the difference in the retention times of these compounds on the reverse phase column. Until now, it has been necessary to split the sample and apply each half to different methods in order to measure, D and with high specificity. There is a method which allows you to detect these neurotransmitters and their metabolites using a mobile phase with a lower ph. However, the specificity obtained by this method is sometimes less than that obtained by the method used to detect the monoamine neurotransmitters alone. Now, we introduce the EICOMPK CX (cation exchange mode, 2. 2 mm) to allow for the detection of, D and without their metabolites in a single sample injection. This method can be applied using single and constantly applied voltage electrochemical detection. The required analysis time is only 15 min. and the obtained lower sensitivity is as low as 1 fg/injection. NOTE: The procedure described in this manual cannot be applied for the separation of epinephrine and. Please do not apply a sample that contains epinephrine at more than negligible levels. n example of such a sample would be one that is not from the central nervous system (brain/spinal cord). 1

2 2. Principles The EICOMPK CX is a 5 μm silica-based cation-exchange column designed for the separation of cationic monoamines in a neutral or weak acid solution. Most of the metabolites of monoamines contained in brain/spinal cord microdialysis samples behave as anions in a mobile phase of neutral ph and will not be retained by the EICOMPK CX. Thus, the EICOMPK CX offers excellent selectivity and resolution for the separation of the monoamine neurotransmitters, D and in their cationic conditions. The setup necessary to achieve the best performance from the EICOMPK CX column has been incorporated into the HTEC-5. high precision pulse-free pump, an elution degasser, a column temperature controller and a highly sensitive and stable electrochemical detector are built into the compact cabinet of the HTEC-5. In this technical guide, we show the data obtained using the HTEC-5 with the online microdialysis configuration system. The system setup is shown in Fig. 1. Fig. 1 2

3 3. nalytical Condition, Mobile Phase and Standard Solution 3-1. nalytical Condition nalytical Instrument: HTEC-5 Separation Column: EICOMPK CX (2. id 2 mm) Column Temperature: 35 C Mobile Phase: 7%.1 mol/l acetate ammonium buffer including 5 mg/l EDT-2Na and.5 mol/l sodium sulfate, 3% methanol Flow Rate: 25 μl/min Detecting Condition: +45 mv vs. g/gcl Working Electrode: Graphite (WE-3G) with Gasket #GS-25 Time Constant: 3. sec Data cquisition: EPC-5 at 4 points/sec sampling speed * The EICOMPK CX and this mobile phase condition do not allow for the separation of and epinephrine. Only use this condition for samples which do not contain or contain minimum levels of epinephrine Mobile Phase Preparation Step 1 Preparation of.1 mol/l acetate ammonium buffer (ph6.). Dissolve 7.7 g acetate ammonium (FW 77.8) in 1 liter of ultrapure water. This gives a.1 mol/l acetic ammonium solution. dd 5.56 ml acetic acid to ultrapure water and make up to 1 liter with ultrapure water. This gives a.1 mol/l acetic acid solution. dd.1 mol/l of this acetic acid solution to the.1 mol/l acetic ammonium solution at the following mixing ratio and adjust the ph to 6. using a ph meter:.1 mol/l acetate ammonium:.1 mol/l acetic acid = 5 : 2 Simple Protocol: dd 5.18 g acetate ammonium and 164 μl acetic acid to ultrapure water and make up the total volume to 7 ml with ultrapure water. cetate ammonium is difficult to dissolve. Make sure you dissolve it completely. Step 2 Measure 3 ml HPLC grade methanol in a dry graduated cylinder. dd this 3 ml methanol to 7 ml.1 ml/l acetic ammonium buffer (ph6.). Dissolve 7.1 g sodium sulfate and add 1 ml 5 mg/ml EDT- 2Na solution to the obtained 1 liter solution. NOTE: Measure the 3 ml methanol separately and then add to the buffer solution. NOTE: Keep this solution in a clean glass bottle. Shake well. Do not refill the solution into used glassware. 3

4 3-3. Standard Solution 1 ng/μl Stock Solutions Prepare 1 ng/μl D and 1 ng/μl separately in.1 mol/l HCl including 1 mg/l EDT-2Na*. Prepare 1 ng/μl in.1 mol/l acetic acid** including 1 mg/l EDT-2Na*. * The 1 mg/l EDT-2Na will precipitate at a lower temperature. Please use the supernatant for the next steps. **See section 3-2 for how to prepare.1 mol/l acetic acid. Final Concentration The external standard needs to be a similar concentration to the real samples. Prepare the lower concentration standards before you inject. The solutions need to be diluted using.1 mol/l phosphate buffer at ph3.5 including.1 mmol/l EDT-2Na in order to prevent oxidation of monoamines. Strong acids used to prepare the 1 ng/μl stock solution above will interfere with the separation so do not use them. It is difficult to prevent oxidation of monoamines at concentrations lower than.1 pg/μl. To prepare.1 mol/l phosphate buffer at ph3.5, dilute 1 ml phosphoric acid in 9 ml ultrapure water to give a total volume of 1 ml. When using a pipette to measure 1 ml phosphoric acid, aspirate very slowly and use a wider tip mouth which can be made by simply cutting the tip. This is because the viscosity of phosphoric acid is very high. This gives 1% phosphoric acid. Dissolve 13.6 g potassium dihydrogenphosphate in ultrapure water and add 2.3 ml 1% phosphoric acid. Then make the total volume 1 liter by adding more ultrapure water New Columns The EICOMPK CX is shipped with analytical mobile phase (shown in 3-2) which has undergone quality tests. Deliver the analytical mobile phase directly to the column without connecting the detector to the outlet of the column in order to start the analysis. Keep the pump running to wash the column for at least one hour. Then connect the detector. If you connect the detector immediately after opening the package, it may result in contamination of the surface of the working electrode. fter opening a new EICOMPK CX, run the pump under the analytical condition for 12 hours or more to lower the background current (BGC) on the ECD. The optimal condition of the system should display 5 n or lower as its BGC. 4

5 4. Standard Chromatogram Peak height (mv,.1 n = 1 mv) min D 7.1 min 13.1 min min Fig. 2 Chromatogram obtained by 1 pg/1 μl injection of a standard solution of, D and 5. Calibration Curve, Lower Detection Limit, Reproducibility and Injection Capacity 5-1. Calibration R2 = Peak height (mv) mount (pg/injection) 2 R2 = R2 =.998 D Fig. 3 Calibration curve for D, and Fig. 3 in the range of.1-5 pg/injection Fig. 3 with the Eicom HTEC-5 Peak height (mv) mount (pg/injection) D Fig. 4 Calibration curve for D, and Fig. 3 in the range of 1-1 pg/injection Fig. 3 with the Eicom HTEC-5 5

6 5-2. Lower Detection Limit When using the EICOMPK CX on the Eicom HTEC-5, the lower detection limit for D is.5 pg/ injection and for and, it is.1 pg/injection at a signal to noise ratio (S/N) equal to pg/injection mv.2 D Blank min Fig. 5 Chromatogram obtained by a.1 pg/injection of a Fig. 5 mixture of, D and and one obtained from Fig. 5 a blank solution 5-3. Reproducibility For 1 injections of the mixture of, D and at a concentration of 1 pg/injection, the CV% was 2.51, 2.63 and 2.98 respectively Injection Volume When using the 5 μl loop, linearity is obtained in the range of an injection volume of 25 μl. The column can accept up to a 4 μl injection. 6. Brain Microdialysis pplication Condition nimal: Microdialysis Probe: Probe Location: Perfusate: Injection Volume: Male Wister Rat, BW g Eicom -I-4-3 (membrane length 3 mm) Prefrontal Cortex ( = +2.5 mm, L =.6 mm, V = 3 mm) Ringer s solution 1 μl/min 1 μl/min 15 min. via an automated online injector (Eicom ES-2) 6

7 min after Imipramine (1 mg/kg, i.p.) administration mv.4.2 D Basal level min Fig. 6 Simultaneous analysis of, D and obtained from a prefrontal cortex microdialysis sample using the EICOMPK CX Fig. 7 This figure shows the basal condition and the condition Fig. 7 9 min. after imipramine administration mv.6 Basal level min after Tetrodotoxin (TTX, 1 μmol/l) perfusion min Fig. 7 Simultaneous analysis of, D and obtained from a Fig. 7 prefrontal cortex microdialysis sample using the Fig. 7 EICOMPK CX Fig. 7 This figure shows the basal condition and the condition Fig. 7 6 min. after perfusion with tetrodotoxin at 1 μmol/l. Online/Offline nalysis The Eicom microfraction collector (EFC-82) is available and allows you to collect microdialysis samples automatically. The collected samples can then be injected automatically using the 719L/D autosampler or manually into the HTEC-5. Dispense.1 mol/l phosphate buffer (ph3.5) including.1 mmol/l EDT-2Na into the microdialysis collection vials before starting the collection so that its volume is 1/4 of the microdialysate to be collected. Collect the samples at 4 C. The sample analysis needs to be completed within 24 hours or if this is not possible, the samples can be stored at 8 C for up to one week. 7

8 Data Converter EPC-5 utoinjector ES-2 Sample Loop Microsyringe Pump ESP-64 Microdialysis Probe HPLC-ECD System HTEC-5 Fig. 8 System configration with the online autoinjector ES-2 Data Converter EPC-5 Microsyringe Pump ESP-64 utosampler 719 Fraction Collector EFC-82 Microdialysis Probe HPLC-ECD System HTEC-5 Fig. 9 System configration with the fraction collector (EFC-82) Fig. 9 and the autosampler (719L or D) 8

9 7. Mobile Phase Condition and Its Influence on the nalysis There are several factors that effect the retention time of monoamines. These include the salt concentration of the mobile phase, the concentration of the organic solution, the ph of the buffer, the buffer concentration, the type of salt used for the buffer solution, the column temperature and the mobile phase flow rate. The influence the sodium sulfate concentration and the methanol concentration in the mobile phase have is shown in figures 1 and 11, respectively. If you need to alter or modify the mobile phase condition due to difficulty in achieving separation, take these data into consideration Standard Condition 25 D Retention time (min) Retention time (min) Standard Condition 4 2 Na2SO4 concentration (mol/l) D Fig. 1 Sodium sulfate concentration in the Fig. 1 mobile phase vs. the retention time Methanol concentration (v/v%) Fig. 11 Methanol level in the mobile phase Fig. 1 vs. the retention time Note: If you use higher than a 35% methanol concentration, please use a glassy carbon working electrode (WE-GC) or a pure graphite working electrode (WE-PG) instead of a graphite working electrode (WE-3G). 8. Care and Maintenance 8-1. Column Care void physical damage to the column. Handle gently. The highest pressure capacity is 2 MPa (= 29 psi). The recommended ph to use is in the range of ph The mobile phase needs to always be prepared with fresh ultrapure water whose resistance is at 18.2 MΩ/cm or higher. Completely dissolve the salt in the mobile phase. To protect the EICOMPK CX from contamination by the mobile phase or the samples, it is highly recommended that you connect the C-ODS packed precolumn before the injectors and connect the inline filter (PP-LF) just before the EICOMPK CX. Do not inject proteins or solid compounds into the EICOMPK CX. The EICOMPK CX comes with two special fitting parts (FX-5). This is because the HTEC-5 is just big enough to contain the 2 cm column. Please use these fittings so that the column can be held by the stainless steel column holder in the HTEC-5. The EICOMPK CX has a flow direction. Please use the arrow printed on the column to determine the flow direction. 9

10 8-2. Storage To store the EICOMPK CX, please leave some analytical mobile phase (described in section 3) in the column and put a plug in each end. Store at room temperature where there is no risk of radical temperature changes (15 C 25 C degree). Do NOT store the column with a solution that does not contain any salt such as a methanol/water solution How to Wash the Column It is not required that you wash the column when it is in general use. If you run the pump with the analytical mobile phase overnight, the column will become clean. If an intensive cleaning is required for some reason, deliver 1% ultrapure water for an hour, then switch to 1% methanol and then switch back to 1% ultrapure water. ll of these washes should be performed at a flow rate of 2 μl/min. These washing solutions should not flow through the detector. Place a drain after the column to collect these washing solutions. Now you can switch back to the analytical mobile phase. Do NOT leave water in the column for several hours. This cleaning routine is very harsh on the column. Do NOT apply this intensive cleaning routine frequently. 9. Dealing with Unknown Peaks and Possible Causes of these Peaks The EICOMPK CX may show unknown peaks on the chromatogram. We have observed several unknown peak patterns. The following will help you to deal with these unknown peaks. The chromatogram shown in figure 12 is an image and was not obtained from real samples. This is probably derived from the brain but not specific to any brain area. The size of this peak is unstable. This is mainly derived from some contamination of the gastight syringe. Clean the syringe to avoid this peaks reappearance. D These may be derived from blood. By removing probe from the brain, the peak disappears. When blooding was observed during the surgical procedure to implant the microdialysis guide canula, this peak appeared min This peak came from a previous injection and its retention time is about 17 min. This became larger when bleeding was observed on the surface of the skull. The 17 min peak is probably derived from blood or tissue. This peak gets bigger sometimes from injection to injection. It is mainly derived from a damaged rotor seal of the injector. Washing and cleaning may not be a complete solution. Please replace it with a new one. Fig. 12 Possible unknown peaks and their causes 1 Most of these unknown peaks, except for the one appearing at a retention time of 4 min., are related to system contamination or unsuccessful microdialysis surgery.

11 1. Column Choice Eicom carries four types of separation columns for the analysis of monoamines in microdialysis samples. The EICOMPK CX does not give the best level of sensitivity. However, you can load a larger sample volume into this column compared to the PP-ODS column described below. This will eliminate the detection limit issue if you can collect samples for a longer time. Typical Chromatogram Time Length, D,, D, D D, Metabolites CX 15 min. PP-ODS 5 min. C-5ODS 2 min. B B B C SC-5ODS 2 min. D D D D D B B B : Excellent (Lower senstivity:.3 pg on the column) : Excellent (Lower sensitivity:.5 pg.1 pg on the column) B: Good C: vailable but no detailed information provided D: In most cases it is difficult : Very difficult EICOMPK CX, D and in brain/spinal cord microdialysis samples can be separated in 15 min. The lower detection limit is.5 to 1 pg on the column. You can load up to 4 μl in volume into the column. EICOMPK PP-ODS D and in brain/spinal cord microdialysis samples can be separated in 5 min. The lower detection limit is.3 pg on the column. You can load up to 25 μl in volume into the column. EICOMPK C-5ODS and D, or D and in brain/spinal cord microdialysis samples can be separated and it takes about 2 min. The lower detection limit is.1 pg on the column. It is excellent for catecholamine analysis in blood or tissue samples. EICOMPK SC-5ODS This is not good for microdialysis samples. For the analysis of monoamines and monoamine metabolites in tissue homogenate or microdialysate, it works well. The selectivity for is not good. 11

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