A Sensitive Method for Simultaneous Determination of Histamine and Noradrenaline with High-Performance liquid Chromatography/Electrochemistry
XINQIANG HAN AND MAN M. VOHRA
A simple and reliable high-performance liquid chromatographic method is described for the simultaneous determination of histamine (His), which cannot be directly oxidized, and noradrenaline (NA), which can be directly oxidized within the useful working potential range. The isoindole products formed by precolumn derivatization of His and NA with o-phthalaldehyde (OPA) and Z-mercaptoethanol (2-ME) yielded a linear relationship of detection between the electrochemical signal and the compound content to a minimum detectable limit of 50 pg (signalto-noise ratio = 3:l) for both compounds at 0.5 nA of detector range. Without 2-ME, OPA derivatives of both His and NA were not detectable electrochemically at the oxidation potential range from 0 to +I V. Although the peak potential was +0.85 V for both His and NA, we used +0.7 V for both compounds to keep background noise minimal. The capacity factors of some electrochemically interfering compounds were also determined. The significance of OPA/2-ME derivative of NA is discussed relative to the direct oxidation of catecholamines. An example of a practical application of the method to the determination of His and NA in rat cardiac tissue is presented. Key Words. HPLC; Electrochemical detection; column derivatization
Histamine;
Noradrenaline;
Pre-
INTRODUCTION Both
histamine
and
noradrenaline
ischemia/reperfusion-induced al., 1980). In animal hearts, aline (Giotti
are important
arrhythmogenic
mediators
in
cardiac dysfunctions (Masini et al., 1987; Rochette et the distribution of histamine parallels that of noradren-
et al., 1966; Harvey,
1978). There
is evidence
that histamine
can evoke
the release of catecholamines in vivo (Staszewska-Barczak and Vane, 1965) and in vitro (Macintosh and Vohra, 1982). On the other hand, catecholamines can stimulate both the synthesis (Rothschild and Comes, 1984) and the release (Giotti et al., 1966) of histamine, and can decrease its uptake and metabolism (Moroni et al., 1977). Also, stimulating the sympathetic nerves evokes the release of histamine (Gross et al., 1984). Simultaneous would,
therefore,
From the Department Address Medical
reprint Building,
Received
determination
of the tissue contents
be a useful approach
of Pharmacology,
requests Dalhousie
April 26, 1990;
to:
Dr.
Man
University, revised
for studying
Dalhousie M.
Vohra,
Halifax,
and accepted
University, Department
Nova Scotia,
of the two compounds
their roles in the mechanisms
Halifax,
Nova Scotia,
of Pharmacology, Canada
Canada.
Sir Charles
Tupper
B3H 4H7.
July 23, 1990. 29
Journalof Pharmacological Methods 25, 29-40 (1991) 0 1991 Elsevier Science Publishing
Co., Inc., 655 Avenue of the Americas, New York. NY 1M)lO
0160.5402/91/13.50
30
X. Han and M. M. Vohra
underlying many pathophysiological conditions. Previous reports about the determination of histamine and noradrenaline concentrations were all performed separately in different experimental setups and by different methods. The sensitivity for histamine determination was usually not high enough, and this, in turn, required tissue pooling from several animals. In recent years, high-performance liquid chromatography (HPLC) coupled with electrochemical (EC) detection has been applied to the determination of biogenic amines and their metabolites (Kissinger et al., 1981). These amines include noradrenaline but not histamine, as this primary amine cannot be directly oxidized within the currently useful potential range due to its low level or lack of intrinsic electrochemical activity. Several researchers have reported that o-phthalaldehyde (OPA)/2mercaptoethanol (2-ME) derivatives of certain primary amines and amino acids undergo anodic oxidation at moderate potential, permitting the use of HPLC/EC for detection (Joseph and Davies, 1983; Allison et al., 1984; Leroy et al., 1984). In view of the established OPA/2-ME chemistry, this study was designed to provide a sensitive and practical method for the simultaneous determination of histamine and noradrenaline contents in biological tissues. MATERIALS AND METHODS Apparatus Isocratic liquid chromatographic experiments on OPA/2-ME derivatives were performed on a Waters (Mississauga, Ontario, Canada) system. The system utilized a Waters 501 solvent delivery pump with a Bioanalytical LC-4B detector and a KippZonen BD 40 recorder. All chromatography was performed with a Nucleosil C-18 (Caltech Associates, Deerfield, IL, USA) column (100 x 4.6 mm, 5+m particles). Flow rate was 1 mUmin. A Bioanalytical Systems (Lafayette, IN) prototype detector cell was used which placed a glassy carbon working electrode, reference electrode, and auxiliary electrode in the vicinity of the thin-layer region. The potential of the working electrode was maintained at +0.7 V vs. Ag/AgCI, unless otherwise indicated. Filter constant was 0.1 in oxidation mode. Reagents The mobile phase was composed of two major components: acetate buffer and acetonitrile. The acetate buffer was prepared with deionized millipore water and the following chemicals (in mM): Na2EDTA, 0.149; octyl sodium sulphate, 0.431; NaAcetate.3Hz0, 100; citric acid, 20. The final pH of the buffer was between 4.85 and 5.00. The buffer was filtered through a 0.45~pm HA membrane (millipore) and then was mixed with acetonitrile. The concentration of acetonitrile in the mixture was 30% (v/v). The mobile phase was degassed prior to use. OPA (0.25%, w/v) and 2-ME (0.25%, v/v) were dissolved in methanol separately and kept in light-protected bottles at room temperature. Standard stock solutions of histamine, noradrenaline, and other compounds were prepared with 0.1 M perchloric acid (containing 0.1% EDTA) and stored in a refrigerator. Fresh dilutions of the compounds were made with the same perchloric acid solution as needed.
HPLC Method for Determining Biogenic Amines
Precolumn
Derivatization
of Compounds
with OPA/Z-ME
For derivatization of histamine, noradrenaline, and other compounds, reagents were mixed in the following sequence: 15 FL OPA were added to 390 PL of sodium acetate/acetonitrile histamine mediately reaction
buffer.
This solution
was mixed with 15 PL 2-ME and then 15 FL
or noradrenaline or other compounds (0.1-10 @mL) were added. Imthereafter, we added 2 M NaOH (usually 15 FL) to bring the pH of the mixture
to 10, then
for 1 min. We injected the reaction.
put the mixture
20 u.L of the mixture
For the detection
of tissue extracts
instead
in a light-protected
of His and NA in biological
of standard
tube
and stirred
into the HPLC system 2 min after starting
solutions
samples,
and adjusted
we used 40 FL
the amount
of NaOH
to keep the pH at 10.
Tissue Extraction of Histamine and Noradrenaline Rats were
decapitated
and the hearts were
dissected
quickly
from
the thoracic
cavity. The tissues were blotted on filter paper, weighed, and homogenized in 4.0 mL of 0.4 M perchloric acid containing 0.05% Na2EDTA, using a Polytron apparatus (Brinkmann Instruments Co., Westbury, NY) and an ice bath. The samples were then centrifuged at 27,000 g for IO min, the supernatant was collected, and the pellets
were
further
the two supernatants
extracted were
by adding
combined
The pH of 1 mL of the supernatant
2 mL of the 0.4 M perchloric
and stored at -70°C
was adjusted
to 6 and then mixed with Amberlite
CC 50 (H+) (BDH, Toronto, Canada), which was prepared al. (1962). After shaking the Amberlite-sample-containing pirated
the supernatant.
acid. Then
until they were assayed.
We used 1 mL of millipore
as described by Oats et tube for IO min, we as-
water to wash out the impurities
in the Amberlite and then aspirated the water. Histamine and noradrenaline were eluted with 2 M HCI, vortexed for IO min, and 40 P,L of the supernatant were used to derivatize with OPA/2-ME. 3-Methylhistamine (3-M-His) and 3,4_dihydroxybenzylamine (DHBA) were used as the internal standards to monitor the recoveries of histamine
and noradrenaline,
respectively,
homogenate just before centrifugation. In preliminary experiments, known were
mixed with standard
samples were the calculated
histamine
and these were
amounts
of 3-M-His
and noradrenaline
subjected to the procedure recoveries from the internal
described standards,
added
to the prepared
and DHBA
(I pg each),
(5 kg each)
and then these
above. In these experiments, 3-M-His and DHBA were 74.0
? 4.7% and 75.9 + 3.0%, respectively, and those for histamine and noradrenaline were 70.5 & 4.7% and 77.6 + 6.3%, respectively (mean + SE for five experiments). This indicates
that the recoveries
those of the internal
of histamine
and noradrenaline
were
similar
to
standards.
RESULTS OPA/2-ME
Derivatives
Using the isocratic conditions described istry was evaluated for use with HPLC/EC.
above, the established OPA/2-ME Both histamine and noradrenaline
chemderiv-
31
32
X. Han and M. M. Vohra
atives and their corresponding internal standards were well separated on the chromatograms. A representative example of HPLC/EC recording is shown in Figure 1 under the following conditions: oxidation potential = +0.7 V vs. Ag/AgCI; detector range = 2 nA; flow rate = 1 mL/min; loop size = 20 FL; pH of the derivitization mixture = 10. In A (Figure I), the injected reaction mixture contains standard solutions of histamine and noradrenaline with final amounts of 1 ng each, and the corresponding internal standards, 3-M-His and DHBA, with final amounts of 5 ng each. We consistently found that the noradrenaline peak was larger than that of histamine, suggesting that the sensitivities for both compounds are different. In B, no histamine or noradrenaline, but rather 0.1 M perchloric acid was included in the reaction mixture (blank), so no peak was being recorded within 60 min of observation except the solvent front which was the same as in A. Without 2-ME, neither
I
10
Time
FIGURE 1. adrenaline
HPLC chromatograms (NA). 3-M-His
L
20 0
of precolumn
= 3-methylhistamine;
(min) derivatization of histamine (His) and norDHBA
= 3,4-dihydroxybenzylamine.
HPLC Method for Determining
Biogenic Amines
compound was detectable within 60 min of observation even when they were added in the reaction mixture (C). Scanning of the applied oxidation potentials from 0 to +I V revealed no histamine or noradrenaline peak within the derivatization pH range of 5-12. The solvent front of 2-ME-free derivatives was different from that of blank with 0.1 M perchloric acid (B), indicating that the derivatives of histamine and noradrenaline formed by OPA only are not oxidized under the conditions described. Direct oxidation of histamine and noradrenaline was tried in 3 runs and no signal was found for either compound under the same conditions (data not shown). The retention times were 6.0 + 0.4 min for histamine and 11.2 + 0.7 min for noradrenaline (mean * SE, n = 6 each). In D, a biological sample from rat heart extracts was tested, and the histamine and noradrenaline peaks were easily identified by referring to the retention times and the external standards, that is, adding known concentrations of standard histamine and noradrenaline to the extracted sample. Electrochemical
Properties
Electrochemical properties of the isoindole derivatives of histamine and noradrenaline with OPA/2-ME were characterized chromatographically. As shown in Figure 2, the electrochemical signals of histamine and noradrenaline derivatives were obtained in a potential range of +0.4 to +0.85 V. The half-peak potentials for his-
100
60
0.2
0.4 Oxidation
0.6 potential
0.8
1 .O
(V)
FIGURE 2 Hydrodynamic voltammograms for precolumn OPA/2-ME derivatives of histamine (His) and noradrenaline (NA). Flow rate = 1 mUmin; pH = 10.
33
34
X. Han and M. M. Vohra
tamine and noradrenaline derivatives were +0.59 and +0.52 V, respectively, whereas both peaked at +0.85 V. When the holding potential was below +0.4 V, no response could be observed in the chromatogram. On the other hand, when the potential was above +0.85 V, not only were the amplitudes of the peak signals decreased, but also the noise and the background current were simultaneously increased to an undesired extent. We found that +0.7 V was the most appropriate oxidation potential for both compounds. Because the precolumn derivatization required a critical pH condition in the reaction mixture, we also determined the influence of pH changes on the amplitudes of the electrochemical signals being generated. As illustrated in Figure 3, no signal could be detected if the precolumn derivatization pH was below 5. A linear increase of the detector response was seen between pH 5 and 8, and the signal peaked between pH 8 and 10 then decreased above pH 10 for both derivitives, suggesting a possible source of experimental error if proper care was not taken to maintain the correct pH. Therefore, in the following experiments care was taken to maintain the pH between 9.5 and 10.5. Figure 4 shows that under the electrochemical conditions described above, the relationship between the amplitudes of electrochem-
100
60
4
5
9
10
Derivatization
pH
6
7
8
11
12
13
FIGURE 3 HPLC response as a function of reaction pH for precolumn OPA/2-ME derivatives of histamine (His) and noradrenaline (NA). Flow rate = 1 mUmin; oxidation potential = +0.7 V vs. AglAgCI.
HPLC Method for Determining
Amount injected
Biogenic Amines
(n9)
16 IS:
8
y-0.746x
0 NA: y-l
.364x
f 0.129 + 0.246
0 0
7
14
Amount injected
21
28
(ng)
FIGURE 4 HPLC response as a function of amount of histamine (His) and noradrenaline (NA) being injected. Standard curves were obtained under the following conditions: oxidation potential = +0.7 V vs. Ag/AgCI, flow rate = 1 mUmin, pH of derivatization = 10.0, loop size = 20 uL. Note that the slopes of the respective lines are not significantly different and that the intercepts are close to zero, indicating a linear increase over the concentration ranges tested for both compounds.
ical signals and the amount of compounds being injected was strictly linear to a minimum detectable limit of 50 pg at a signal-to-noise ratio of 3:l for both histamine and noradrenaline at 0.5 nA of detector range (in fact, both signals produced by 5 pg of compounds were easily identified at 0.1 nA of detector range}. The relationship between the precolumn derivatization time and the amplitude of electrochemical signals at a injected amount of 0.5 ng for both histamine and noradrenaline is illustrated in Figure 5. It can be seen that the signal amplitudes remain relatively constant 2 min after the reaction started up to a 30-min period. Resolution
from Other Amines and Amino Acids
The isoindole derivatives of histamine and noradrenaline can be well separated chromatographically from those of the other structurally similar amines or amino acids (Table 1). Various amounts of compounds were derivatized and injected until the optimal responses were obtained. It can be seen from Table 1 that all the methylated metabolites of histamine as well as the precursor amino acid can be well differentiated from histamine. Whereas noradrenaline was easily detected, adrenaline was totally undetectable, because even at 1 pg, there was no peak being
35
36
2.0 NA
A-A
B-W 1.5
1.0 o-o
0.0
His
A-A
0.5
D-0
’
I
1
5
0
I
10
15 TIME
I
I
20
25
I
30
(min)
FIGURE 5 Relationship between the HPLC response and the precolumn derivatization time for histamine (His) and noradrenaline (NA). pH = 10; oxidation potential = +0.7 V vs. Ag/AgCI. Results of three separate experiments are denoted by different symbols. Derivatizing pH = 10.
TABLE 1 Capacity Factors (K’) of Some Electrochemically Interfering Compounds Derivatized With o-Phthalaldehyde (OPA)/2Mercaptoethanol (2-ME) Under the Experimental Conditions Described in the Text K’=
COMPOUND
COMPOUND
K’
4.6
Histamine
2.2
I-Methylhistamine
2-Methylhistamine
2.7
3-Methylhistamine
3.0
4-Methylhistamine
3.6
Histidine
NDb
Spermine
ND
Spermidine
ND
Noradrenaline
6.2
DHBA
7.2
Adrenaline
ND
5-HT
4.0
Dopamine
8.9
a K’ was defined
as (V, -
ume of compound
tested
V,)/V,, and V,
where
Vt = retention
vol-
= void volume.
b Not detectable. Abbreviation:
DHBA
5hydroxytryptamine.
= 3,4-dihydrobenzylamine.
5-HT
=
HPLC Method for Determining TABLE 2
Age-Related
AGE
WEIGHT
(WEEK)
Cd
Histamine and Noradrenaline
Biogenic Amines
Changes in Rat Hearts
HISTAMINE
NORADRENALINE
piHEART
pG/Cwr
PC/HEART
pc/cwl
IO-13
1.35
2
0.06
2.76
? 0.04
2.04
2
0.35
1.11
? 0.36
0.82
? 0.30
23-26
1.60
-+ 0.06
4.59
*
1.03a
2.86
2
0.66
1.88
+ 0.36”
1.17
+ 0.20
62-65
1.85
2
5.33
+ 1.06”
2.88
? 0.41
1.69
4
0.28a
0.97
2
0.25
Data are presented of variance
as mean
and Tukey
a Significantly
different cwr
resolved
within
SE with n = 5 for each mean.
Statistics were done using one-way
analysis
test.
Abbreviation:
sensitivities
f
0.10
from the IO- to 13-week
group
(p < 0.05).
= gram wet tissue.
60 min of observation.
of most other compounds
We want to mention were
here that the detection
lower than those of noradrenaline
and
histamine.
Application
to the Determination
By means of the method described noradrenaline in five adult rats were + 0.44 kg/g wet tissue
of Biological Samples above, the cardiac contents determined. The histamine
and the noradrenaline
content
of histamine and content was 2.69
was 1.03
2 0.21 pg/g wet
tissue. In another series of experiments, we also determined the age-related changes of histamine and noradrenaline contents and concentrations in rat hearts. As summarized
in Table 2, the cardiac
and noradrenaline
increased
wet weight
and cardiac
with age; however,
content
of both
the concentration
remained unchanged. These results are in accordance ously (Wolff and Levi, 1986; Howes et al., 1980).
with
histamine
of both amines
those
reported
previ-
DISCUSSION Although
many authors
by means of HPLUEC of histamine
have described
detection,
by this technique
methods
there are relatively (Mine
for noradrenaline
determination
few studies on the measurement
et al., 1986; Harsing et al., 1986). Noradrenaline
can be directly oxidized at +0.5 to +0.70 V of applied potential (Mefford, Chaudhry and Vohra, 1984), and this renders easy an HPLC/EC determination content
in biological
samples.
Therefore,
precolumn
derivatization
1981; of its
may not be re-
quired if the detection is only directed to noradrenaline. Histamine, on the other hand, cannot be directly oxidized within the useful working potential range due to its low intrinsic electrochemical activity. Other methods-such as OPA derivatization
coupled
with
HPLC-fluorometry/fluorescence
detection
(RGnnberg
et al.,
1984b), liquid chromatography or thin-layer chromatography after dansylation (Yamatodani et al., 1977; Schippert et al., 1979), gas-liquid chromatography (Wollin and Navert, 1985), gas chromatography-mass spectrometry (Mita et al., 19801, and radioenzymatic assay (Verburg et al., 1983)-have been used for the detection of histamine, but many of them have problems of derivative instability, lack of sensitivity, and high cost or complexity of manipulations. Also, the measurement may
37
38
X. Han and M. M. Vohra sometimes be complicated fering agents. Although
the OPA/2-ME
OPA-histamine reaction method was introduced of histamine product
are probably
workers
(Allison
be formed
established,
inter-
the structures
of
(Ronnberg
highly linear, this suggests that either
stereoisomers
that coelute
is further
et al., 1984;
Furthermore,
et al., 1984a).
Because
under
products of histamine and noradrenaline detecting peaks and the standard curves
in each case or, if multiple
This reasoning
conditions.
well
or other
with OPA, it has been shown that more than one reaction might
were
is being formed
constant.
has been
conditions, the reaction only single well-defined
for both compounds they
chemistry
of the metabolites
products have not been clearly defined even though this 30 years ago (Shore et al., 1959). Under certain conditions
derivatization
(stereoisomers)
our experimental with OPA yielded product
due to the presence
reaction
and
one major reaction
products
are generated,
have proportions
that
remain
supported
Harsing
by the findings reported by other et al., 1986) who used similar derivatization
the reproducibility
of the derivatization
step under
our
experimental conditions was found to be highly consistent (Figure 5). The reductive substance, 2-ME, may merely be used to stabilize the isoindole derivatives produced by OPA-primary fluorescence
amines
reaction
that are detected
by both
electrochemical
and
techniques.
Based on the above principle, the method described determination of histamine and noradrenaline, without
here allows simultaneous sacrificing detection sen-
sitivity or changing experimental setup, because both compounds are optimally detected at the same applied potential (+0.7 V), under the same pH conditions. The
minimum
detectable
quantity,
which
further improved by slightly increasing can be achieved within 2 hr, including use for constant
routine
laboratory
for more
applications.
is 50 pg for both
compounds,
may be
the acetonitrile concentration. A single run the sample purifications, which permits its Moreover,
than 30 min after derivatization
the
detection
(Figure
signals
5), suggesting
remain that de-
composition should not be a major concern for this system as both compounds and their internal standards can be eluted within 20 min (Figure 1, retention time). I-Methylhistamine, the major methylated metabolite, also can be determined with histamine, although the sensitivity may be slightly lower. The minimum detectable limit for 5-HT determinations simultaneously
is 0.2 ng (data not shown), which may satisfy many tissue sample of this biological amine, especially when it is to be determined with histamine and/or noradrenaline. It is also interesting to note
that the OPA/2-ME derivative of noradrenaline can be eluted at high solvent acetonitrile concentration (30%). The electrochemical signal of direct noradrenaline oxidation can only be separated from the solvent front at low concentrations of acetonitrile (less than 10%) in our setup. This indicates that the chromatographic profile (characteristics) of noradrenaline also has been changed by the derivatization procedure, although it can easily be oxidized directly. Although the K’ factors shown in Table 1 clearly indicate that many electrochemically interfering compounds can be separated chromatographically, one limitation of this procedure is that adrenaline (which is a secondary amine) cannot be detected by this method because, as Benson and Hare (1975) stated, it will not derivatize with OPA/2-ME. The recovery
HPLC Method for Determining
Biogenic Amines
values reported for histamine and noradrenaline in this paper may appear to be relatively low (-70%) and variable (coefficient of variation = 16-20%). These can be improved further (Harsing et al., 19861, but this would likely increase the duration of the assay. As every sample is spiked with the internal standards, these apparent low recoveries should not affect the precision of the measurements. Although changing the solvent components (such as acetonitrile concentration) may be a practical way to achieve a good chromatographic resolution, this study suggests that derivatization of compounds with some special agents, resulting in electrochemically active derivatives, may also be an approach sometimes required to achieve satisfactory separation without sacrificing detection sensitivity. The authors thank Miss Leslie lngraham for her excellent technical assistance and Mr. Shaun Black for providing some biological samples. This study was supported in part by grants from the Canadian Heart Foundation and the Medical Research Council of Canada.
REFERENCES Allison LA, Mayer CS, Shoup RE (1984) o-Phthalaldehyde derivatives of amines for high-speed liquid chromatography/electrochemistry. Anal Chem 56:1089-1096. Benson JR, Hare PE (1975) o-Phthalaldehyde: fluorogenic detection of primary amines in the picomole range. Comparison with fluorescamine and ninhydrin. Proc Nat Acad Sci USA 72:619622.
Chaudhry A, Vohra MM (1984) Depletion of cardiac noradrenaline stores by calcium channel blocker D-600. Can / Physiol Pharmaco/62:640-644. Giotti A, Cuidotti A, Mannaioni PF, Zilletti L (1966) The influences of adrenotropic drugs and noradrenaline on the histamine release in cardiac anaphylaxis in vitro. / Physiol (Lond) 184:924-941. Gross SS, Guo Z-G, Levi R, Bailey WH, Chenouda AA (1984) Release of histamine by sympathetic nerve stimulation in the guinea pig heart and modulation of adrenergic responses: A physiological role for cardiac histamine? Circ Res 54:516-526. Harsing LG Jr., Nagashima H, Vizi ES, Duncalf D (1986) Electrochemical determination of histamine derivatized with o-phthalaldehyde and 2mercaptoethanol. / Chromatogr 383:19-26. Harvey SC (1978) Studies on myocardial histamine. Effects of catecholamine-depleting drugs. Arch Int Pharmacodyn
Ther 232:141-149
Howes LG, Summers RJ, Louis WJ (1980) The influence of age and sex on cardiac, renal and caudal
artery catecholamine content in spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats. 1 Auton Pharmacol6:171-180. Joseph MH, Davies P (1983) Electrochemical activity of o-phthalaldehyde-mercaptoethanol derivatives of amino acids: Application to high-performance liquid chromatographic determination of amino acids in plasma and other biological materials. / Chromatogr 277:125-136. Kissinger PT, Bruntlett CS, Shoup RE (1981) Neurochemical applications of liquid chromatography with electrochemical detection. Life Sci 28~455-465.
Leroy P, Nicolas A, Moreau A (1984) Electrochemical detection of sympatomimetic drugs, following pre-column o-phthalaldehyde derivatization and reversed-phase high-performance liquid chromatography. / Chromatogr 282:561-567. Macintosh CM, Vohra MM (1982) Effect of histamine and H,- and Hz-agonists on the fractional outflow of radioactivity from the rat vas deferens preloaded with [3H]-noradrenaline. Life Sci 31: 2819-2826. Masini E, Giannella (1987) Histamine (LDH) release in guinea-pig. Agents
E, Bianchi S, Mannaioni PF and lactate dehydrogenase ischemic myocardium of the Actions
20:281-283.
Mefford IN (1981) Application of high performance liquid chromatography with electrochemical analysis: measurement of catecholamines, serotonin and metabolites in rat brain. / Neurosci Methods
3~207-224.
39
40
X. Han and M. M. Vohra Mine K, Jacobson KA, Kirk KL, Kitajima Y, Linnoila M (1986) Simultaneous determination of histamine and NT-methylhistamine with high-performance liquid chromatography using electrochemical detection. Anal Biochem 152:127-135. Mita H, Yasueda H, Shida T (1980) Simultaneous determination of histamine and NT-methylhistamine in human plasma and urine by gas chromatography-mass spectrometry. 1 Chromatogr 221:1-7. Moroni F, Fantozzi R, Masini E, Maunaionie PF (1977) The influence of catecholamines and serotonin on histamine uptake and metabolism by guinea pig atrium. fur) Pharmaco/41:59-63. Oats ]A, Marsh E, Sjoerdsma (1962) Studies on histamine in human urine using a fluorometric method of assay. C/in Chim Acta 71488-497. Rochette L, Didier JP, Moreau D, Bralet J (1980) Effect of substrate on release of myocardial norepinephrine and ventricular arrhythmias following reperfusion of the ischemic isolated working rat heart. j Cardiovasc Phatmacol2:267-279. Rannberg
AL, Hansson C, DrakenbergT, Hakanson R (1984a) Reaction of histamine with o-phthalaldehyde: isolation and analysis of the fluorophore. Anal Biochem 139:329-337.
Rijnnberg AL, Hansson C, Hakanson R (19&b) High-performance liquid chromatographic determination of histamine in biological samples
after derivation with Biochem 139:338-344.
o-phthalaldehyde.
Anal
Rothschild AM, Comes ELT (1984) Stimulation by epinephrine of histamine synthesis by rat peritoneal fluid mast cells in vitro. Life Sci 35:16451651. Schippert B, Kovar K-A, Sewing K-F (1979) Determination of histamine and its metabolic products in the pig gastric mucosa. Pharmacology 19:8695. Shore PA, Burkhalter A, Cohn VH Jr. (1959) A method for the fluorometric assay of histamine in tissues. / Pharmacol Exp Thet 127:182-196. Staszewska-Barczak J, Vane JR (1965) The release of catechol amines from the adrenal medulla by histamine. Br / Pharmaco/25:728-742. Verburg KM, Bowsher RR, Henry DP (1983) A new radioenzymatic assay for histamine using purified histamine N-methyltransferase. Life Sci 32 :2855-2867. Wolff AA, Levi R (1986) Histamine and cardiac arrhythmias. Circ Res 58:1-16. Wollin A, Navert H (1985) Quantitation of histamine and some of its basic methylated metabolites in biological materials by gas-liquid chromatography. Anal Biochem 145:73-79. Yamatodani A, Seki T, Taneda M, Wada H (1977) Determination of histamine and methylhistamines by dansylation and its application to biological specimens. / Chtomatogt 144:141-145.
XINQIANG HAN AND MAN M. VOHRA
A simple and reliable high-performance liquid chromatographic method is described for the simultaneous determination of histamine (His), which cannot be directly oxidized, and noradrenaline (NA), which can be directly oxidized within the useful working potential range. The isoindole products formed by precolumn derivatization of His and NA with o-phthalaldehyde (OPA) and Z-mercaptoethanol (2-ME) yielded a linear relationship of detection between the electrochemical signal and the compound content to a minimum detectable limit of 50 pg (signalto-noise ratio = 3:l) for both compounds at 0.5 nA of detector range. Without 2-ME, OPA derivatives of both His and NA were not detectable electrochemically at the oxidation potential range from 0 to +I V. Although the peak potential was +0.85 V for both His and NA, we used +0.7 V for both compounds to keep background noise minimal. The capacity factors of some electrochemically interfering compounds were also determined. The significance of OPA/2-ME derivative of NA is discussed relative to the direct oxidation of catecholamines. An example of a practical application of the method to the determination of His and NA in rat cardiac tissue is presented. Key Words. HPLC; Electrochemical detection; column derivatization
Histamine;
Noradrenaline;
Pre-
INTRODUCTION Both
histamine
and
noradrenaline
ischemia/reperfusion-induced al., 1980). In animal hearts, aline (Giotti
are important
arrhythmogenic
mediators
in
cardiac dysfunctions (Masini et al., 1987; Rochette et the distribution of histamine parallels that of noradren-
et al., 1966; Harvey,
1978). There
is evidence
that histamine
can evoke
the release of catecholamines in vivo (Staszewska-Barczak and Vane, 1965) and in vitro (Macintosh and Vohra, 1982). On the other hand, catecholamines can stimulate both the synthesis (Rothschild and Comes, 1984) and the release (Giotti et al., 1966) of histamine, and can decrease its uptake and metabolism (Moroni et al., 1977). Also, stimulating the sympathetic nerves evokes the release of histamine (Gross et al., 1984). Simultaneous would,
therefore,
From the Department Address Medical
reprint Building,
Received
determination
of the tissue contents
be a useful approach
of Pharmacology,
requests Dalhousie
April 26, 1990;
to:
Dr.
Man
University, revised
for studying
Dalhousie M.
Vohra,
Halifax,
and accepted
University, Department
Nova Scotia,
of the two compounds
their roles in the mechanisms
Halifax,
Nova Scotia,
of Pharmacology, Canada
Canada.
Sir Charles
Tupper
B3H 4H7.
July 23, 1990. 29
Journalof Pharmacological Methods 25, 29-40 (1991) 0 1991 Elsevier Science Publishing
Co., Inc., 655 Avenue of the Americas, New York. NY 1M)lO
0160.5402/91/13.50
30
X. Han and M. M. Vohra
underlying many pathophysiological conditions. Previous reports about the determination of histamine and noradrenaline concentrations were all performed separately in different experimental setups and by different methods. The sensitivity for histamine determination was usually not high enough, and this, in turn, required tissue pooling from several animals. In recent years, high-performance liquid chromatography (HPLC) coupled with electrochemical (EC) detection has been applied to the determination of biogenic amines and their metabolites (Kissinger et al., 1981). These amines include noradrenaline but not histamine, as this primary amine cannot be directly oxidized within the currently useful potential range due to its low level or lack of intrinsic electrochemical activity. Several researchers have reported that o-phthalaldehyde (OPA)/2mercaptoethanol (2-ME) derivatives of certain primary amines and amino acids undergo anodic oxidation at moderate potential, permitting the use of HPLC/EC for detection (Joseph and Davies, 1983; Allison et al., 1984; Leroy et al., 1984). In view of the established OPA/2-ME chemistry, this study was designed to provide a sensitive and practical method for the simultaneous determination of histamine and noradrenaline contents in biological tissues. MATERIALS AND METHODS Apparatus Isocratic liquid chromatographic experiments on OPA/2-ME derivatives were performed on a Waters (Mississauga, Ontario, Canada) system. The system utilized a Waters 501 solvent delivery pump with a Bioanalytical LC-4B detector and a KippZonen BD 40 recorder. All chromatography was performed with a Nucleosil C-18 (Caltech Associates, Deerfield, IL, USA) column (100 x 4.6 mm, 5+m particles). Flow rate was 1 mUmin. A Bioanalytical Systems (Lafayette, IN) prototype detector cell was used which placed a glassy carbon working electrode, reference electrode, and auxiliary electrode in the vicinity of the thin-layer region. The potential of the working electrode was maintained at +0.7 V vs. Ag/AgCI, unless otherwise indicated. Filter constant was 0.1 in oxidation mode. Reagents The mobile phase was composed of two major components: acetate buffer and acetonitrile. The acetate buffer was prepared with deionized millipore water and the following chemicals (in mM): Na2EDTA, 0.149; octyl sodium sulphate, 0.431; NaAcetate.3Hz0, 100; citric acid, 20. The final pH of the buffer was between 4.85 and 5.00. The buffer was filtered through a 0.45~pm HA membrane (millipore) and then was mixed with acetonitrile. The concentration of acetonitrile in the mixture was 30% (v/v). The mobile phase was degassed prior to use. OPA (0.25%, w/v) and 2-ME (0.25%, v/v) were dissolved in methanol separately and kept in light-protected bottles at room temperature. Standard stock solutions of histamine, noradrenaline, and other compounds were prepared with 0.1 M perchloric acid (containing 0.1% EDTA) and stored in a refrigerator. Fresh dilutions of the compounds were made with the same perchloric acid solution as needed.
HPLC Method for Determining Biogenic Amines
Precolumn
Derivatization
of Compounds
with OPA/Z-ME
For derivatization of histamine, noradrenaline, and other compounds, reagents were mixed in the following sequence: 15 FL OPA were added to 390 PL of sodium acetate/acetonitrile histamine mediately reaction
buffer.
This solution
was mixed with 15 PL 2-ME and then 15 FL
or noradrenaline or other compounds (0.1-10 @mL) were added. Imthereafter, we added 2 M NaOH (usually 15 FL) to bring the pH of the mixture
to 10, then
for 1 min. We injected the reaction.
put the mixture
20 u.L of the mixture
For the detection
of tissue extracts
instead
in a light-protected
of His and NA in biological
of standard
tube
and stirred
into the HPLC system 2 min after starting
solutions
samples,
and adjusted
we used 40 FL
the amount
of NaOH
to keep the pH at 10.
Tissue Extraction of Histamine and Noradrenaline Rats were
decapitated
and the hearts were
dissected
quickly
from
the thoracic
cavity. The tissues were blotted on filter paper, weighed, and homogenized in 4.0 mL of 0.4 M perchloric acid containing 0.05% Na2EDTA, using a Polytron apparatus (Brinkmann Instruments Co., Westbury, NY) and an ice bath. The samples were then centrifuged at 27,000 g for IO min, the supernatant was collected, and the pellets
were
further
the two supernatants
extracted were
by adding
combined
The pH of 1 mL of the supernatant
2 mL of the 0.4 M perchloric
and stored at -70°C
was adjusted
to 6 and then mixed with Amberlite
CC 50 (H+) (BDH, Toronto, Canada), which was prepared al. (1962). After shaking the Amberlite-sample-containing pirated
the supernatant.
acid. Then
until they were assayed.
We used 1 mL of millipore
as described by Oats et tube for IO min, we as-
water to wash out the impurities
in the Amberlite and then aspirated the water. Histamine and noradrenaline were eluted with 2 M HCI, vortexed for IO min, and 40 P,L of the supernatant were used to derivatize with OPA/2-ME. 3-Methylhistamine (3-M-His) and 3,4_dihydroxybenzylamine (DHBA) were used as the internal standards to monitor the recoveries of histamine
and noradrenaline,
respectively,
homogenate just before centrifugation. In preliminary experiments, known were
mixed with standard
samples were the calculated
histamine
and these were
amounts
of 3-M-His
and noradrenaline
subjected to the procedure recoveries from the internal
described standards,
added
to the prepared
and DHBA
(I pg each),
(5 kg each)
and then these
above. In these experiments, 3-M-His and DHBA were 74.0
? 4.7% and 75.9 + 3.0%, respectively, and those for histamine and noradrenaline were 70.5 & 4.7% and 77.6 + 6.3%, respectively (mean + SE for five experiments). This indicates
that the recoveries
those of the internal
of histamine
and noradrenaline
were
similar
to
standards.
RESULTS OPA/2-ME
Derivatives
Using the isocratic conditions described istry was evaluated for use with HPLC/EC.
above, the established OPA/2-ME Both histamine and noradrenaline
chemderiv-
31
32
X. Han and M. M. Vohra
atives and their corresponding internal standards were well separated on the chromatograms. A representative example of HPLC/EC recording is shown in Figure 1 under the following conditions: oxidation potential = +0.7 V vs. Ag/AgCI; detector range = 2 nA; flow rate = 1 mL/min; loop size = 20 FL; pH of the derivitization mixture = 10. In A (Figure I), the injected reaction mixture contains standard solutions of histamine and noradrenaline with final amounts of 1 ng each, and the corresponding internal standards, 3-M-His and DHBA, with final amounts of 5 ng each. We consistently found that the noradrenaline peak was larger than that of histamine, suggesting that the sensitivities for both compounds are different. In B, no histamine or noradrenaline, but rather 0.1 M perchloric acid was included in the reaction mixture (blank), so no peak was being recorded within 60 min of observation except the solvent front which was the same as in A. Without 2-ME, neither
I
10
Time
FIGURE 1. adrenaline
HPLC chromatograms (NA). 3-M-His
L
20 0
of precolumn
= 3-methylhistamine;
(min) derivatization of histamine (His) and norDHBA
= 3,4-dihydroxybenzylamine.
HPLC Method for Determining
Biogenic Amines
compound was detectable within 60 min of observation even when they were added in the reaction mixture (C). Scanning of the applied oxidation potentials from 0 to +I V revealed no histamine or noradrenaline peak within the derivatization pH range of 5-12. The solvent front of 2-ME-free derivatives was different from that of blank with 0.1 M perchloric acid (B), indicating that the derivatives of histamine and noradrenaline formed by OPA only are not oxidized under the conditions described. Direct oxidation of histamine and noradrenaline was tried in 3 runs and no signal was found for either compound under the same conditions (data not shown). The retention times were 6.0 + 0.4 min for histamine and 11.2 + 0.7 min for noradrenaline (mean * SE, n = 6 each). In D, a biological sample from rat heart extracts was tested, and the histamine and noradrenaline peaks were easily identified by referring to the retention times and the external standards, that is, adding known concentrations of standard histamine and noradrenaline to the extracted sample. Electrochemical
Properties
Electrochemical properties of the isoindole derivatives of histamine and noradrenaline with OPA/2-ME were characterized chromatographically. As shown in Figure 2, the electrochemical signals of histamine and noradrenaline derivatives were obtained in a potential range of +0.4 to +0.85 V. The half-peak potentials for his-
100
60
0.2
0.4 Oxidation
0.6 potential
0.8
1 .O
(V)
FIGURE 2 Hydrodynamic voltammograms for precolumn OPA/2-ME derivatives of histamine (His) and noradrenaline (NA). Flow rate = 1 mUmin; pH = 10.
33
34
X. Han and M. M. Vohra
tamine and noradrenaline derivatives were +0.59 and +0.52 V, respectively, whereas both peaked at +0.85 V. When the holding potential was below +0.4 V, no response could be observed in the chromatogram. On the other hand, when the potential was above +0.85 V, not only were the amplitudes of the peak signals decreased, but also the noise and the background current were simultaneously increased to an undesired extent. We found that +0.7 V was the most appropriate oxidation potential for both compounds. Because the precolumn derivatization required a critical pH condition in the reaction mixture, we also determined the influence of pH changes on the amplitudes of the electrochemical signals being generated. As illustrated in Figure 3, no signal could be detected if the precolumn derivatization pH was below 5. A linear increase of the detector response was seen between pH 5 and 8, and the signal peaked between pH 8 and 10 then decreased above pH 10 for both derivitives, suggesting a possible source of experimental error if proper care was not taken to maintain the correct pH. Therefore, in the following experiments care was taken to maintain the pH between 9.5 and 10.5. Figure 4 shows that under the electrochemical conditions described above, the relationship between the amplitudes of electrochem-
100
60
4
5
9
10
Derivatization
pH
6
7
8
11
12
13
FIGURE 3 HPLC response as a function of reaction pH for precolumn OPA/2-ME derivatives of histamine (His) and noradrenaline (NA). Flow rate = 1 mUmin; oxidation potential = +0.7 V vs. AglAgCI.
HPLC Method for Determining
Amount injected
Biogenic Amines
(n9)
16 IS:
8
y-0.746x
0 NA: y-l
.364x
f 0.129 + 0.246
0 0
7
14
Amount injected
21
28
(ng)
FIGURE 4 HPLC response as a function of amount of histamine (His) and noradrenaline (NA) being injected. Standard curves were obtained under the following conditions: oxidation potential = +0.7 V vs. Ag/AgCI, flow rate = 1 mUmin, pH of derivatization = 10.0, loop size = 20 uL. Note that the slopes of the respective lines are not significantly different and that the intercepts are close to zero, indicating a linear increase over the concentration ranges tested for both compounds.
ical signals and the amount of compounds being injected was strictly linear to a minimum detectable limit of 50 pg at a signal-to-noise ratio of 3:l for both histamine and noradrenaline at 0.5 nA of detector range (in fact, both signals produced by 5 pg of compounds were easily identified at 0.1 nA of detector range}. The relationship between the precolumn derivatization time and the amplitude of electrochemical signals at a injected amount of 0.5 ng for both histamine and noradrenaline is illustrated in Figure 5. It can be seen that the signal amplitudes remain relatively constant 2 min after the reaction started up to a 30-min period. Resolution
from Other Amines and Amino Acids
The isoindole derivatives of histamine and noradrenaline can be well separated chromatographically from those of the other structurally similar amines or amino acids (Table 1). Various amounts of compounds were derivatized and injected until the optimal responses were obtained. It can be seen from Table 1 that all the methylated metabolites of histamine as well as the precursor amino acid can be well differentiated from histamine. Whereas noradrenaline was easily detected, adrenaline was totally undetectable, because even at 1 pg, there was no peak being
35
36
2.0 NA
A-A
B-W 1.5
1.0 o-o
0.0
His
A-A
0.5
D-0
’
I
1
5
0
I
10
15 TIME
I
I
20
25
I
30
(min)
FIGURE 5 Relationship between the HPLC response and the precolumn derivatization time for histamine (His) and noradrenaline (NA). pH = 10; oxidation potential = +0.7 V vs. Ag/AgCI. Results of three separate experiments are denoted by different symbols. Derivatizing pH = 10.
TABLE 1 Capacity Factors (K’) of Some Electrochemically Interfering Compounds Derivatized With o-Phthalaldehyde (OPA)/2Mercaptoethanol (2-ME) Under the Experimental Conditions Described in the Text K’=
COMPOUND
COMPOUND
K’
4.6
Histamine
2.2
I-Methylhistamine
2-Methylhistamine
2.7
3-Methylhistamine
3.0
4-Methylhistamine
3.6
Histidine
NDb
Spermine
ND
Spermidine
ND
Noradrenaline
6.2
DHBA
7.2
Adrenaline
ND
5-HT
4.0
Dopamine
8.9
a K’ was defined
as (V, -
ume of compound
tested
V,)/V,, and V,
where
Vt = retention
vol-
= void volume.
b Not detectable. Abbreviation:
DHBA
5hydroxytryptamine.
= 3,4-dihydrobenzylamine.
5-HT
=
HPLC Method for Determining TABLE 2
Age-Related
AGE
WEIGHT
(WEEK)
Cd
Histamine and Noradrenaline
Biogenic Amines
Changes in Rat Hearts
HISTAMINE
NORADRENALINE
piHEART
pG/Cwr
PC/HEART
pc/cwl
IO-13
1.35
2
0.06
2.76
? 0.04
2.04
2
0.35
1.11
? 0.36
0.82
? 0.30
23-26
1.60
-+ 0.06
4.59
*
1.03a
2.86
2
0.66
1.88
+ 0.36”
1.17
+ 0.20
62-65
1.85
2
5.33
+ 1.06”
2.88
? 0.41
1.69
4
0.28a
0.97
2
0.25
Data are presented of variance
as mean
and Tukey
a Significantly
different cwr
resolved
within
SE with n = 5 for each mean.
Statistics were done using one-way
analysis
test.
Abbreviation:
sensitivities
f
0.10
from the IO- to 13-week
group
(p < 0.05).
= gram wet tissue.
60 min of observation.
of most other compounds
We want to mention were
here that the detection
lower than those of noradrenaline
and
histamine.
Application
to the Determination
By means of the method described noradrenaline in five adult rats were + 0.44 kg/g wet tissue
of Biological Samples above, the cardiac contents determined. The histamine
and the noradrenaline
content
of histamine and content was 2.69
was 1.03
2 0.21 pg/g wet
tissue. In another series of experiments, we also determined the age-related changes of histamine and noradrenaline contents and concentrations in rat hearts. As summarized
in Table 2, the cardiac
and noradrenaline
increased
wet weight
and cardiac
with age; however,
content
of both
the concentration
remained unchanged. These results are in accordance ously (Wolff and Levi, 1986; Howes et al., 1980).
with
histamine
of both amines
those
reported
previ-
DISCUSSION Although
many authors
by means of HPLUEC of histamine
have described
detection,
by this technique
methods
there are relatively (Mine
for noradrenaline
determination
few studies on the measurement
et al., 1986; Harsing et al., 1986). Noradrenaline
can be directly oxidized at +0.5 to +0.70 V of applied potential (Mefford, Chaudhry and Vohra, 1984), and this renders easy an HPLC/EC determination content
in biological
samples.
Therefore,
precolumn
derivatization
1981; of its
may not be re-
quired if the detection is only directed to noradrenaline. Histamine, on the other hand, cannot be directly oxidized within the useful working potential range due to its low intrinsic electrochemical activity. Other methods-such as OPA derivatization
coupled
with
HPLC-fluorometry/fluorescence
detection
(RGnnberg
et al.,
1984b), liquid chromatography or thin-layer chromatography after dansylation (Yamatodani et al., 1977; Schippert et al., 1979), gas-liquid chromatography (Wollin and Navert, 1985), gas chromatography-mass spectrometry (Mita et al., 19801, and radioenzymatic assay (Verburg et al., 1983)-have been used for the detection of histamine, but many of them have problems of derivative instability, lack of sensitivity, and high cost or complexity of manipulations. Also, the measurement may
37
38
X. Han and M. M. Vohra sometimes be complicated fering agents. Although
the OPA/2-ME
OPA-histamine reaction method was introduced of histamine product
are probably
workers
(Allison
be formed
established,
inter-
the structures
of
(Ronnberg
highly linear, this suggests that either
stereoisomers
that coelute
is further
et al., 1984;
Furthermore,
et al., 1984a).
Because
under
products of histamine and noradrenaline detecting peaks and the standard curves
in each case or, if multiple
This reasoning
conditions.
well
or other
with OPA, it has been shown that more than one reaction might
were
is being formed
constant.
has been
conditions, the reaction only single well-defined
for both compounds they
chemistry
of the metabolites
products have not been clearly defined even though this 30 years ago (Shore et al., 1959). Under certain conditions
derivatization
(stereoisomers)
our experimental with OPA yielded product
due to the presence
reaction
and
one major reaction
products
are generated,
have proportions
that
remain
supported
Harsing
by the findings reported by other et al., 1986) who used similar derivatization
the reproducibility
of the derivatization
step under
our
experimental conditions was found to be highly consistent (Figure 5). The reductive substance, 2-ME, may merely be used to stabilize the isoindole derivatives produced by OPA-primary fluorescence
amines
reaction
that are detected
by both
electrochemical
and
techniques.
Based on the above principle, the method described determination of histamine and noradrenaline, without
here allows simultaneous sacrificing detection sen-
sitivity or changing experimental setup, because both compounds are optimally detected at the same applied potential (+0.7 V), under the same pH conditions. The
minimum
detectable
quantity,
which
further improved by slightly increasing can be achieved within 2 hr, including use for constant
routine
laboratory
for more
applications.
is 50 pg for both
compounds,
may be
the acetonitrile concentration. A single run the sample purifications, which permits its Moreover,
than 30 min after derivatization
the
detection
(Figure
signals
5), suggesting
remain that de-
composition should not be a major concern for this system as both compounds and their internal standards can be eluted within 20 min (Figure 1, retention time). I-Methylhistamine, the major methylated metabolite, also can be determined with histamine, although the sensitivity may be slightly lower. The minimum detectable limit for 5-HT determinations simultaneously
is 0.2 ng (data not shown), which may satisfy many tissue sample of this biological amine, especially when it is to be determined with histamine and/or noradrenaline. It is also interesting to note
that the OPA/2-ME derivative of noradrenaline can be eluted at high solvent acetonitrile concentration (30%). The electrochemical signal of direct noradrenaline oxidation can only be separated from the solvent front at low concentrations of acetonitrile (less than 10%) in our setup. This indicates that the chromatographic profile (characteristics) of noradrenaline also has been changed by the derivatization procedure, although it can easily be oxidized directly. Although the K’ factors shown in Table 1 clearly indicate that many electrochemically interfering compounds can be separated chromatographically, one limitation of this procedure is that adrenaline (which is a secondary amine) cannot be detected by this method because, as Benson and Hare (1975) stated, it will not derivatize with OPA/2-ME. The recovery
HPLC Method for Determining
Biogenic Amines
values reported for histamine and noradrenaline in this paper may appear to be relatively low (-70%) and variable (coefficient of variation = 16-20%). These can be improved further (Harsing et al., 19861, but this would likely increase the duration of the assay. As every sample is spiked with the internal standards, these apparent low recoveries should not affect the precision of the measurements. Although changing the solvent components (such as acetonitrile concentration) may be a practical way to achieve a good chromatographic resolution, this study suggests that derivatization of compounds with some special agents, resulting in electrochemically active derivatives, may also be an approach sometimes required to achieve satisfactory separation without sacrificing detection sensitivity. The authors thank Miss Leslie lngraham for her excellent technical assistance and Mr. Shaun Black for providing some biological samples. This study was supported in part by grants from the Canadian Heart Foundation and the Medical Research Council of Canada.
REFERENCES Allison LA, Mayer CS, Shoup RE (1984) o-Phthalaldehyde derivatives of amines for high-speed liquid chromatography/electrochemistry. Anal Chem 56:1089-1096. Benson JR, Hare PE (1975) o-Phthalaldehyde: fluorogenic detection of primary amines in the picomole range. Comparison with fluorescamine and ninhydrin. Proc Nat Acad Sci USA 72:619622.
Chaudhry A, Vohra MM (1984) Depletion of cardiac noradrenaline stores by calcium channel blocker D-600. Can / Physiol Pharmaco/62:640-644. Giotti A, Cuidotti A, Mannaioni PF, Zilletti L (1966) The influences of adrenotropic drugs and noradrenaline on the histamine release in cardiac anaphylaxis in vitro. / Physiol (Lond) 184:924-941. Gross SS, Guo Z-G, Levi R, Bailey WH, Chenouda AA (1984) Release of histamine by sympathetic nerve stimulation in the guinea pig heart and modulation of adrenergic responses: A physiological role for cardiac histamine? Circ Res 54:516-526. Harsing LG Jr., Nagashima H, Vizi ES, Duncalf D (1986) Electrochemical determination of histamine derivatized with o-phthalaldehyde and 2mercaptoethanol. / Chromatogr 383:19-26. Harvey SC (1978) Studies on myocardial histamine. Effects of catecholamine-depleting drugs. Arch Int Pharmacodyn
Ther 232:141-149
Howes LG, Summers RJ, Louis WJ (1980) The influence of age and sex on cardiac, renal and caudal
artery catecholamine content in spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats. 1 Auton Pharmacol6:171-180. Joseph MH, Davies P (1983) Electrochemical activity of o-phthalaldehyde-mercaptoethanol derivatives of amino acids: Application to high-performance liquid chromatographic determination of amino acids in plasma and other biological materials. / Chromatogr 277:125-136. Kissinger PT, Bruntlett CS, Shoup RE (1981) Neurochemical applications of liquid chromatography with electrochemical detection. Life Sci 28~455-465.
Leroy P, Nicolas A, Moreau A (1984) Electrochemical detection of sympatomimetic drugs, following pre-column o-phthalaldehyde derivatization and reversed-phase high-performance liquid chromatography. / Chromatogr 282:561-567. Macintosh CM, Vohra MM (1982) Effect of histamine and H,- and Hz-agonists on the fractional outflow of radioactivity from the rat vas deferens preloaded with [3H]-noradrenaline. Life Sci 31: 2819-2826. Masini E, Giannella (1987) Histamine (LDH) release in guinea-pig. Agents
E, Bianchi S, Mannaioni PF and lactate dehydrogenase ischemic myocardium of the Actions
20:281-283.
Mefford IN (1981) Application of high performance liquid chromatography with electrochemical analysis: measurement of catecholamines, serotonin and metabolites in rat brain. / Neurosci Methods
3~207-224.
39
40
X. Han and M. M. Vohra Mine K, Jacobson KA, Kirk KL, Kitajima Y, Linnoila M (1986) Simultaneous determination of histamine and NT-methylhistamine with high-performance liquid chromatography using electrochemical detection. Anal Biochem 152:127-135. Mita H, Yasueda H, Shida T (1980) Simultaneous determination of histamine and NT-methylhistamine in human plasma and urine by gas chromatography-mass spectrometry. 1 Chromatogr 221:1-7. Moroni F, Fantozzi R, Masini E, Maunaionie PF (1977) The influence of catecholamines and serotonin on histamine uptake and metabolism by guinea pig atrium. fur) Pharmaco/41:59-63. Oats ]A, Marsh E, Sjoerdsma (1962) Studies on histamine in human urine using a fluorometric method of assay. C/in Chim Acta 71488-497. Rochette L, Didier JP, Moreau D, Bralet J (1980) Effect of substrate on release of myocardial norepinephrine and ventricular arrhythmias following reperfusion of the ischemic isolated working rat heart. j Cardiovasc Phatmacol2:267-279. Rannberg
AL, Hansson C, DrakenbergT, Hakanson R (1984a) Reaction of histamine with o-phthalaldehyde: isolation and analysis of the fluorophore. Anal Biochem 139:329-337.
Rijnnberg AL, Hansson C, Hakanson R (19&b) High-performance liquid chromatographic determination of histamine in biological samples
after derivation with Biochem 139:338-344.
o-phthalaldehyde.
Anal
Rothschild AM, Comes ELT (1984) Stimulation by epinephrine of histamine synthesis by rat peritoneal fluid mast cells in vitro. Life Sci 35:16451651. Schippert B, Kovar K-A, Sewing K-F (1979) Determination of histamine and its metabolic products in the pig gastric mucosa. Pharmacology 19:8695. Shore PA, Burkhalter A, Cohn VH Jr. (1959) A method for the fluorometric assay of histamine in tissues. / Pharmacol Exp Thet 127:182-196. Staszewska-Barczak J, Vane JR (1965) The release of catechol amines from the adrenal medulla by histamine. Br / Pharmaco/25:728-742. Verburg KM, Bowsher RR, Henry DP (1983) A new radioenzymatic assay for histamine using purified histamine N-methyltransferase. Life Sci 32 :2855-2867. Wolff AA, Levi R (1986) Histamine and cardiac arrhythmias. Circ Res 58:1-16. Wollin A, Navert H (1985) Quantitation of histamine and some of its basic methylated metabolites in biological materials by gas-liquid chromatography. Anal Biochem 145:73-79. Yamatodani A, Seki T, Taneda M, Wada H (1977) Determination of histamine and methylhistamines by dansylation and its application to biological specimens. / Chtomatogt 144:141-145.
