Application

note

M E Derivatization
Amino Acids - OPA/

Amino acids are the essential build-
ing blocks of all proteins and pep-
tides, take part in intermediary SCH2 CH2OH
metabolism, and act as precursors CHO
COOH HOCH 2
CH 2
SH COOH
to the common amine neurotrans- + N
mitters. Additionally, physiological R
CHO H N R
-MERCAPTO-
and anatomical evidence suggests 2
H ETHANOL H
that certain amino acids act as neu- ALKYLTHIOL-
O -PHTHALDIALDEHYDE 
-ME)
ISOINDOLE
rotransmitters of the major excita- (OPA) AMINO ACID
DERIVATIVE
tory (aspartate and glutamate) or
inhibitory (GABA and taurine)
commands in the central nervous Figure 1. Derivatization Of Amino Acids With OPA/
ME Produces
system (McGeer and McGeer Electrochemically Active Isoindoles.
(1989)). Due to of the number of
roles amino acids play, it has been difficult to definitively prove using conventional HPLC-UV or HPLC-ECD techniques. How-
their action as true neurotransmitters, neurohormones, or neu- ever, a very sensitive and selective alternative to direct analysis is
romodulators. Therefore in order to assess their neurochemical the precolumn derivatization of amino acids with
roles, it is important to study these compounds in response to o-phthaldialdehyde (OPA)/ 2-mercaptoethanol (
ME) prior to
several stimuli in living systems. detection by the Coulochem® II electrochemical detector
(Figure 1). An isocratic chromatographic method was developed
Low level analysis of free amino acids is not easily achieved to achieve the stability and sensitivity required for this analysis
(Donazanti and Yamamoto (1988)). As
low as 200 femtomoles of the twelve
1 Aspartate
2 Glutamate amino acids can be detected in under
3 Serine 22 minutes (Figure 2), making it ideal
4 Glutamine for the analysis of both tissue extracts
5 Glycine and microdialysis perfusate samples
950 6 Threonine (Figure 3).
7 Phenylethanolamine
4
8 Taurine
Response (mV)

9 Alanine
10 GABA Materials and Methods
I.S. 11 Tyrosine The isocratic analytical system consisted
1 2
I.S. Homoserine of a pump, an autosampler and a
6
(internal standard) Coulochem II electrochemical detector.
5
250 7 LC Conditions:
3 9 Column: HR-80 (4.6 x
8 10 11 80mm; 3µm)
Mobile Phase: 0.1M Sodium
Phosphate in 25%
Methanol, pH 6.75
0 10 20
with Phosphoric
Acid.
Time (minutes) Flow Rate: 1.2mL/min.
Temperature: Ambient.
Figure 2. Chromatogram Of OPA/
ME Derivatized Amino Acid Standards Injection Volume: 20µL.
(1.11ng each on column).
 Application
note
M E Derivatization
Amino Acids - OPA/

with 3mL tetraborate buffer. This solution

1 must be prepared fresh daily.

Automated precolumn amino acid derivati-

950 zation was performed by mixing either
4 11µL of the working derivatizing reagent
with 20-25µL microdialysis perfusate, or
Response (mV)

50µL of the working derivatizing reagent

I.S.
with 20-25µL tissue extract (typically
deproteinized in 0.1M perchloric acid; 1:10
tissue w/v (wet weight: acid)). Derivatiza-
tion was allowed to proceed for 2 minutes
250 6 before injection onto the analytical system.
2 7 9 10 11
5
Results and Discussion
3
Since the amino acid derivatives them-
0 10 20 selves are inherently unstable, having vary-
Time (minutes) ing half-lives, the system is most accurate
and precise when the derivatization reac-
tion is fully automated. The autosampler
dispenses the reagent into the microdialysis
Figure 3. Chromatogram Of OPA/
ME Derivatized Amino Acids In A perfusate or extracted tissue sample vial
Microdialysis Sample. For Analyte Identification See Figure 2. and mixes it thoroughly. The injection is
made precisely after 2 minutes of reaction
time.
Detector and Conditions:
Detector: Coulochem II, Model 5200A The Coulochem detector is unique in that its electrodes respond
Guard Cell, Model 5020 to 100% of the analyte at the electrode surface. By selective choice
Analytical Cell, Model 5010 of potentials, the typically large solvent front (due to excess
Cell Potentials E1 = +400mV OPA/
ME) is minimized by the screening action of the first elec-
E2 = +600mV trode so that all the amino acid derivatives can be easily detected
EGC = +700mV at the second electrode without interference. Excellent linearity is
observed for each amino acid derivative from 20pg to 10ng on
column (Figure 3A and B). This large dynamic range demonstrates
Derivatization Procedure the advantage of using electrochemical over fluorescence
detection since no quenching problems are associated with the
Stock solutions of amino acid standards were prepared at a level electrochemical techniques.
of 1mg/mL in 50% methanol. Working solutions were prepared
in either 0.05M perchloric acid or diluent (189mM sodium chlo- Multiple electrode electrochemical detection offers the most
ride, 3.9mM potassium chloride, 3.4mM calcium chloride; selective, sensitive, and reproducible detection technique for
pH 7.2). OPA/
ME derivatized amino acids.

Stock solutions of the OPA/
ME derivatizing reagent were

prepared by dissolving 27mg OPA (Kodak) in 1mL methanol.
5µL of
ME and 9mL tetraborate buffer (0.1M sodium tetrabo-
rate; pH 9.3) were then added. The solution is stable for up to
five days.
The working OPA/
ME derivatizing reagent was prepared by
diluting 1mL of the working OPA/
ME derivatizing reagent
References
Donzanti, B. A., and Yamamoto, B. K. (1988). An improved and
rapid HPLC-EC method for the isocratic separation of amino
acid neurotransmitters from brain tissue and microdialysis per-
fusates. Life Sci., 43, 913-922.
 3500
McGeer, P. L., and McGeer, B. G. (1989). Amino acid neuro-
ASN
transmitters. In: Basic Neurochemistry. Siegel, G., Agranoff, B.,
GLU
3000
TAU
Albers, R. W., and Molinoff, P. (Eds.). Raven Press, New York.
GABA Pp. 311-332.
2500
Response (mV)

2000

Ordering Information
1500

1000 Description Part Number

Coulochem II, Model 5200A 70-0650
500
Cell, Analytical, Model 5011 55-0434
0
Cell, Guard, Model 5020 55-0417
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 Pump, Model 582 70-4049
Amount On Column (ng) Autosampler, Model 540 70-1484
Column, HR-80 70-0021
Figure 3A. Linear Regression Curves For Amino Acid
Standards (r2 > 0.999). For further information, please contact your local ESA
representative

4000
Ser
3500 Gln
Gly
3000 Thr
Response (mV)

Ala
2500 Tyr

2000

1500

1000

500

0
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

Amount On Column (ng)

Figure 3B. Linear Regression Curves For Amino Acid

Standards (r2 > 0.999).

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Protected under U.S. and international patents. Specifications subject to change without notice. ESA and CoulArray are registered
trademarks of ESA, Inc. All other trademarks are property of their respective owners. The data shown in this Application Note are
intended to illustrate the utility of ESA’s instruments for the determination of the selected analyte(s). Optimal performance may be
affected by variables beyond ESA’s control and may only be achieved by careful consideration of all materials, instruments and 70-0160F
procedures as recommended by ESA. For additional information please contact ESA or its representatives. IA - 1