Monoamines and Metabolites in Tissue Homogenate

  • 11 peaks in 24 minutes
  • Analyze MHPG, NE, Epi, DOPAC, NM, DA, 5-HIAA, ISO, HVA, 3MT, 5-HT in one injection
  • Special condition for detection of L-DOPA in addition to above peaks (30 minutes run time)
  • Highly sensitive down to 0.5 fmol

We present an application for highly sensitive detection of monoamines and their metabolites from tissue homogenate. The analysis utilizes an ion pair reagent in conjunction with reverse-phase liquid chromatography and electrochemical detection. This method includes a simple easy-to-follow sample preparation protocol including homogenization of tissue samples.

Sample Preparation

  1. Dissect the sample area from the animal and weight.
  2. Add 0.5 mL of 0.2 M perchloric acid to the sample per 100 mg wet tissue.
  3. Add 100 ng isoproterenol/100 mg wet tissue as an internal standard (Using 1 ng/μL ISO in Solution A is preferable).
  4. Homogenize at a constant speed and duration.
  5. Denature the protein by keeping the homogenate in an ice bath for 30 min.
  6. Spin at 20,000 G for 15 min at 4°C.
  7. Remove the supernatant.
  8. Modify the pH of the supernatant to become pH3.0 by using 1 M sodium acetate.
  9. Filter using a 0.45 μm syringe filter (after filtering, please store at -80°C if it is required).
  10. Analyze by HPLC.

Analytical Conditions

Column Eicompak SC5-0DS
Precolumn AC-ODS packing materials in ID 3.0 x 4.0 mm
Flow Rate 500 µL/min
Working Electrode Graphite WE-3G (Gasket GS-25)
Applied Potential +750 mV vs. Ag/AgCl
Temperature 25ºC

Detection of MHPG, NE, Epi, DOPAC, NM, DA, 5-HIAA, ISO, HVA, 3MT, 5-HT in 24 minutes.

The analysis can be modified for detection of the above compounds with the addition of L-DOPA, however this extends the runtime to 30 minutes

Detection of the same compounds above with the addition of L-DOPA in 30 minutes.

Fig. 1 – Rat Cerebrum (10 µL injection)

Fig. 2 – Rat Hippocampus (10 µL injection)

Fig. 3 – Rat Hypothalamus (10 µL injection)

Fig. 4 – Rat Striatum (10 µL injection)