Application Note

Histamine Analysis: A Convenient HPLC Method

Introduction

Histamine(HA) participates in a wide variety of physiologic

Analytical Conditions

funcions, and therefore, is the focus of considerable research interest.

However, its a often a difficult molecule to study, owing to its low

HPLC
Detection

concentrations and instability.

Common methods of detection, such as capillary

Em, Ex Settings

electrophoresis and mass spectrometry, require specialized personel

and expensive equipment. And when you add to that frequent down

Separation Column
Precolumn

time for maintenance, a biological sample that easily degrades, and

technicians unaccustomed to handling them, the results are often less

Temperature
Flow Rate

Derivitization

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

Eicom 700 series

Fluorescence detector
Ex 340 nm, Em 450 nm
Online 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)

Basic System Configuration

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

1.0

0.8

System Components

HA

(brain microdialysate, histamine only)

0.6

Pump

0.4

Oven
Detector
Flow cell volume
Data Collection

mV

0.2

Eicom EP-700 pump

Pump 1: OPA solution
Pump 2: Mobile phase
Eicom ATC-700 column oven
Fluorescence detector
12 l
Eicom EPC-500

Additional System Components:

0.0

(only required for tissue homogenates and histamine metabolites)

0

min

10

12

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

injection

14

Switching Valve
Pump
Flow Rate

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

www.eicom-usa.com

Application Note

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/

10

or detect histamine along with a major metabolite, 1-methyl-histamine.

The additional components function to trap and divert late eluting

HA
8

compounds in a precolumn before enter the separation column. The

precolumn is automatically washed clean during each analytical run. This

Hypothalamus

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

mV

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!

Cortex
0
0

10

12

14

min

Fig.2 Rat Brain Homogenate 10l injection.

Real World Data:

Histamine and Metabolites
5

3MHA(IS)

mV
2

HA
1

1MHA

0
0

10

12

14

16 18
min

20

22

24

26

28

30

32

34

Fig.3 Rat Brain Homogenate from the Hypothalamus.

Eicom Corp. USA Office

Eicom Corporation HQ

7313 Carroll Rd Ste F

San Diego, CA 92121
Phone: 1-888-680-7775
Fax:
858-560-8040

113 Kita Enmenden-cho

Shimotoba, Fushimi-ku
Kyoto, Japan 612-8497

www.eicom-usa.com