Application Note

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Histamine Analysis: A Convenient HPLC Method

Introduction Histamine(HA) participates in a wide variety of physiologic funcions, and therefore, is the focus of considerable research interest. However, its a often a difficult molecule to study, owing to its low concentrations and instability. Common methods of detection, such as capil lary electrophoresis and mass spectrometry, require specialized personel and expensive equipment. And when you add to that frequent down time for maintenance, a biological sample that easily degrades, and technicians unaccustomed to handling them, the results are often less than satisfactory, or simply out of reach for some labs. Eicom sought to simplify the detection of histamine and make it accessible to more researchers. The HPLC-based system that we describe here allows for more consistent results with excellent sensitivity. The method relies on post column derivitization with ortho-phthaldialdehyde (OPA) and subsequence fluorometric detection. The simplest version of this method detects 5 fmol of histamine per injection of microdialysis samples. The system can also be configured for analysis of histamine in tissue homogenates. (Additional system components required.) In addition, the simultaneous detection of histamine and its metabolite, 1-methyl-histamine, is possible with detection limits of 100 and 200 fmol, respectively.

Real World Data: Brain Microdialysis

HPLCDetectionEm, Ex SettingsDerivitization Separation ColumnPrecolumn TemperatureFlow Rate

Basic System Configuration

Pump

OvenDetectorFlow cell volume Data Collection

Fig. 1 Rat Hippocampus microdialysate (2 uL/min x 10 min) 20 uL injection

Eicom 700 seriesFluorescence detectorEx 340 nm, Em 450 nmOnline post-column OPA EICOMPAK SC-5ODS (3.0 ID x 150 mm) PREPAKSET CA-ODS (3.0 ID x 4 mm)40C (column and reaction loop)500 l/min (mobile phase)200 l/min (OPA)

Eicom EP-700 pumpPump 1: OPA solution Pump 2: Mobile phase Eicom ATC-700 column ovenFluorescence detector12 l Eicom EPC-500

Analytical Conditions

System Components(brain microdialysate, histamine only)

Additional System Components: (only required for tissue homogenates and histamine metabolites)

Switching ValvePump

Flow Rate

Eicom EAS-20Eicom EP-700Pump 3: precolumn washing500 l/min

mV

0.0

0.2

0.4

0.6

0.8

1.0

HA

0 2 4 6 8 10 12 14min

Application Note

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Fig.2 Rat Brain Homogenate 10l injection.

Real World Data: Brain Tissue Homogenates

Discussion The system described here represents a simple and reliable method for analyzing histamine in various biological sample types. The standard configuration is appropriate for in vivo microdialysis samples and provides the greatest sensitivity. With the addition of another pump and switching valve, the system can handle tissue samples and/or detect histamine along with a major metabolite, 1-methyl-histamine. The additional components function to trap and divert late eluting compounds in a precolumn before enter the separation column. The precolumn is automatically washed clean during each analytical run. This method depends on post-column derivitization with OPA. The working OPA reagent is not stable and must be made fresh every day. If maximum convenience is a priority, individual components can be mixed with another pump. In this case , the stocks reagents are stable for a week. This is a great application for the researcher who wants to take control of his own histamine analysis, or simply wants a quick and reliable method. Its fully supported and guaranteed by Eicom which means that you get results; no HPLC experience necessary!

Fig.3 Rat Brain Homogenate from the Hypothalamus.

Real World Data: Histamine and Metabolites

mV

0

2

4

6

8

10

HA

0 2 4 6 8 10 12 14min

Hypothalamus

Cortex

mV

0

1

2

3

4

5

3MHA(IS)

HA

1MHA

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34min