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Reverse-phase separation and electrochemical detection of neuropeptides
Peptides, Vol. 6, pp. 1173-1178. 1985. ©Ankho InternationalInc. Printed in the U.S.A.
0196-9781/85 $3.00 + .00
Reverse-Phase Separation and Electrochemical Detection of Neuropeptides RALPH DAWSON, JR.,'* JOANNE
P. S T E V E S , t
J O A N F. L O R D E N t
AND SUZANNE
OPARIL*
Department of Medicine and * Cardiovascular Research and Training Center, School of Medicine University of Alabama at Birmingham, Birmingham, AL 35294 and tDepartment of Psychology, University College University of Alabama in Birmingham, Birmingham, AL 35294 R e c e i v e d 7 M a r c h 1985 DAWSON, R., JR., J. P. STEVES, J. F. LORDEN AND S. OPARIL. Reverse-phase separation and electrochemical detection of neuropeptides. PEPT1DES 6(6) 1173-1178, 1985.--The reverse-phase separation of neuropeptides using isocratic conditions is described. Each component of the mobile phase was examined for its ability to influence the separation of complex mixtures of neuropeptides. Manipulation of buffer strength, pH, organic modifier and column type provided sufficient flexibility to resolve closely related neuropeptides. Amperometric detection of oxidizable amino acids in the peptide sequence of a number of endogenous neuropeptides proved suitable for the identification of peptide standards and quantification of neural lobe arginine vasopressin and striatal methionine enkephalin. Neuropeptides HPLC Methionine enkephalin
Electrochemistry
Paraventricular nucleus
N E U R O P E P T I D E S have received increasing attention because of their possible roles in numerous pathologic conditions [24,27]. This has led to the development of techniques for the separation and quantitation of neuropeptides. Procedures based on radioimmunoassay techniques (RIA), high performance liquid chromatography (HPLC) coupled with UV absorbance or fluorescence detection and HPLC followed by RIA or radioreceptor assay quantitation have been described [12, 13, 26]. HPLC has also been used in the purification of neuropeptides from complex biological matrices [3, 18, 19]. While the use of HPLC in peptide research is increasing, there are few reports of systematic evaluations of parameters relevant to the reverse-phase separation of complex mixtures of neuropeptides. A number of reported separations of neuropeptide mixtures are based on the use of linear programmed gradients of solvents that require expensive instrumentation. Therefore, we attempted to develop and evaluate reliable and reproducible isocratic separations of neuropeptides. We also assessed the role of each component of the mobile phase to aid in the selection of a particular separation for specific analytical or purification application. A second aim of these studies was to apply a previously underutilized detection technique to quantify neur0peptides in tissues. The amino acids tyrosine, tryptophan, and cysteine are electroactive and can be oxidized and quantitated electrochemically [2]. Neuropeptides that contain tyrosine or tryptophan residues can also be oxidized and detected electrochemically [2, 6, 15, 20, 21, 23, 25, 28]. Electrochemical detection (ED) appears to be a reliable and perhaps more
Arginine vasopressin
sensitive detection method for neuropeptides than UV methods [20,25]. The HPLC-ED method has been successfully applied for the separation and quantitation of opiate peptides [6, 15, 20, 25]. Therefore, ED was employed in this study to quantitate endogenous levels of methionine enkephalin, arginine vasopressin and oxytocin, as well as other neuropeptides. Neuropeptide levels were determined in posterior pituitary and striatal tissue from rats. In addition, lesions were made in the paraventricular nucleus (PVN) of rats to specifically perturb endogenous levels of neural lobe vasopressin in order to further validate the technique. We have extended the applicability of HPLC-ED to a wide range of electroactive neuropeptides and have characterized the chromatographic behavior of a number of physiologically relevant neuropeptides. METHOD
lnstrttmenlalion A Waters M-45 solvent delivery system operated at a flow rate of 1.0 ml/min (2100 psi backpressure) was used throughout the study. Column temperature was maintained at 40°C. Detection of peptides was accomplished with an LC-4A electrochemical detector with a TL-5 glassy carbon working electrode (Bioanalytical Systems, West Lafayette, IN) using an applied potential of 0.9 V vs. a Ag/AgC1 reference electrode and a sensitivity of 5 or 10 nA. Chromatographic data were plotted and integrated by a Hewlett Packard 3390A recording integrator. Peak height was used to quantitate
'Requests for reprints should be addressed to Ralph Dawson, Jr., Ph.D., University of Florida, College of Pharmacy, Box J-4, Department of Pharmacodynamics, J. Hillis Miller Health Center, Gainesville, FL 32610.
1173
1174
DAWSON, STEVES, L O R D E N AND O P A R I L
neuropeptides and both the methods of internal and external standardization were used in computing the data. Separations were performed on either an IBM Octyl (C8) or Alltech Cyano (CN) column (4.5x250 mm, 5 /zm). The analytical columns were protected by a 4.6x30 mm C18 guard column (Brownlee Labs, Inc., Santa Clara, CA). Sample injections were performed using a Rheodyne 7125 fixed loop (50 tzl) injector.
19.0 o
18.0 17.0 16.0
~
o
15.0 14.0
Standards and Reagents
~ ' ~
- " .... • OXT
"h.
All peptide standards were purchased from either Calbiochem (San Diego, CA) or Sigma (St. Louis, MO). Standards were diluted in mobile phase and stored at 4°C as stock solutions of 100/zg/ml. Buffers (citric acid trisodium salt, sodium phosphate dibasic and monobasic) were purchased from Sigma and were of the highest purity. The organic modifiers propanol-I and tetrahydrofuran, methanol, butanol and acetonitrile were obtained from Burdick and Jackson Laboratories, Inc. (Muskegon, MI).
.c_ E
13.0
LU
12.0
I-Z
11.0
tZ UJ l-
10.0
o_
•
v
o
~ o
All
~ LE
~ o
'
~
~
MEA
cXEDH
9.0
UJ
8.0 7.0
Extraction Procedure Neural lobes were homogenized in a glass homogenizer in 2.0 ml of 1.0 M HC1 which contained 5% formic acid, 1.0% trifluroacetic acid (TFA) and 1.0% NaCi. The homogenates were centrifuged 30 minutes at 17,800xg (4°C). The supernatant was diluted by the addition of 1.0 ml of H._,O and aspirated through a C18 extraction column (Baker-10 Extraction System, J.T. Baker Chemical Co., Phillipsburg, N J). The column was washed with 2 ml of H._,O followed by a 2 ml wash with 5.0% methanol. The arginine vasopressin (AVP) and oxytocin (OXT) were eluted in 1.0 ml of 80% methanol and directly injected for HPLC analysis after filtration through 0.2 tzm filters (Nylon 66). Recovery of AVP standards (125 ng) after extraction was 61-+5% ( N = 11). Striatal tissue from SHR and W K Y rats was homogenized in 5.0 ml of the same homogenization media as used for AVP determination. The homogenate was centrifuged and poured onto C18 extraction columns as described above. The striatal samples were washed with 2.0 ml of H._,O, 2 ml of 0.1% T F A , 2.0 ml of 0.02 M NaH.,PO~ (pH=2.4) and finally with 2 ml of 5% methanol. Methionine enkephalin (ME) was eluted in 1.0 ml of 100% methanol and the methanol was then evaporated under vacuum. The striatal samples were reconstituted in 100/zl of mobile phase and 50/zl was injected onto the column after filtration. ME (25 ng) recovery after extraction was 63-+5% (N=5).
_ 0
~
5.0 --
~ 0 - -
© AVT
4.0 0 0.025
0 05
0 10
0 15
Na2 HPO 4 (M) FIG. 1. The effect of buffer strength on neuropeptide RT. The experiments were performed using a C8 column with 10% propanol as the organic modifier and mobile phase pH was maintained at 6.0.
anterior to the ear bar zero, 3.5 mm dorsal to the interaural line, and -+0.5 mm from the midline. Lesions were performed by passing 1 mA anodal current for 10 seconds through stainless steel electrodes (size 00 insect pins) insulated to 0.75-1.0 mm from the tip. Sham operated animals were treated similarly, except that the electrode was not lowered into the brain. The rats were decapitated on postsurgical day 30 and neural lobes were removed for assay of AVP and OXT. The brains of the rats with PVN lesions were fixed in neutral buffered formalin and later cut at 25 ~m in a cryostat. Lesion placement was verified in cresyl violet stained sections.
Animal Experiments Striatal and neural lobe tissue was obtained from male spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats which were purchased from Charles River Breeding Laboratories and maintained under standard laboratory conditions in our colony. Neural :lobes were also obtained from two adult male Brattleboro rats (a gift of Dr. K. H. Berecek). Lesions of the PVN of the hypothalamus were made in female Sprague-Dawley rats weighing 270-330 g which were anesthetized with 35 mg/kg of sodium pentobarbital and 0.2 ml ketamine HC1 (50 mg/ml) and received either bilateral electrolytic lesions or sham surgery. With the incisor bar of the stereotaxic instrument set 5 mm dorsal to the interaural line, the coordinates for the PVN lesion were: 6.2 mm
--,,~• AVPLvP
_
6.0
RESULTS
Neuropeptide HPLC The effects of increasing buffer strength on neuropeptide retention time (RT) are shown in Fig. 1. In general, increasing buffer strength decreased RT, with angiotension II (All) showing the most pronounced effects. The RT's of ME and LE were relatively unaffected by changes in buffer strength, whereas the presence of an amide group in MEA restored the effect of buffer strength on RT. The effects of pH on neuropeptide RT are presented in Fig. 2. pH had pronounced effects on neuropeptide RT over the range examined. The nonapeptides, AVT, LVP, AVP and OXT exhibited increases in RT with increases in pH whereas the opiate peptides, ME, LE, c~endorphin (c~EDH)
HPLC-ED OF NEUROPEPTIDES
1175 TABLE1
40.0
32.0
24.0
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.<
"v ACTH
"><
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\
20.0
E E
\Q, \
18.o
\ \\\
v'~ ~ ) X
\
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14o
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OXT
." - " / /
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o
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Chromatographic Conditions* (RT)
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/"
o MEA
~ - @ ~
v o(EDH
12.0
\\
10.0
\ I~
~e AVP
8.0
6.0
.....
:
. . . . .
z
/
4.0
I
I
3.0
4.0
I 5.0 APPARENT
I
I
6.0
7.0
pH
FIG. 2. Changes in neuropeptide RT as a function of alterations in mobile phase pH. The mobile phase was 0.1 M NaH2PO~ with 10% propanol as the organic modifier, pH adjustments were made with either NaOH or HCI. All separations were performed on a C8 column.
and ACTH 1-24 all showed substantial decreases in RT under these conditions. The amide group in MEA altered the chromatographic characteristics of this opiate peptide analog in relation to changes in pH of the mobile phase, pH was an important determinant in the reverse phase separation of neuropeptides and had differential effects dependent on the relevant functional group(s). The effects of organic modifiers, buffer composition and two types of reverse phase packings on neuropeptide separations were also examined. For isocratic separations, propanol-1 gave the best resolution and shortest run times. Methanol and acetonitrile had insufficient solvent strength to effectively elute highly hydrophobic peptides. Butanol had good eluting properties but its limitied water solubility made its use impractical. T H F (Table 1) did not prove satisfactory for most of our applications. Citrate buffer substituted well for phosphate buffer and at equal molarity (Table 1) was more effective in eluting all the peptides tested. We chose a C8 column for most of our studies since the C8 packing material provided both good resolution and favorable RT characteristics. In preliminary studies with C 18 columns we found that the highly retentive nature of C18 packing material resulted in inordinately long RT's for peptides under isocratic conditions. The more polar CN column (Table 1) gave very favorable (reduced) RT's for the larger molecular weight peptides. The resolution of smaller peptides, however, was poor with the CN column. Thus, depending on the desired application, either the C8 or CN col-
Neuropeptide Arg-Vasotocin (AVT) Lys-Vasopressin (LVP) Arg-Vasopressin (AVP) Met-Enkephalin (ME) Met-Enkephalin amide (MEA) a-Endorphin (aEDH) Leu-Enkephalin (LE) Angiotensin II (All) Angiotensin I (AI) Oxytocin (OXT) ACTH 1-24 Neurotensin (NT) LHRH c~-MSH
A
B
C
D
E
F
NC 7.17 5 . 5 7 4.79 NC 6.52 6.49 13.58 7 . 5 9 6 . 3 0 10.30 7.48 7.30 14.69 8 . 3 4 6 . 7 5 10.70 8.27 7.39 14.69 7 . 6 9 7 . 3 1 7 . 8 0 5.51 11.70
NC
11.22 12.65 14.48 NC 14.33 33.67 NC NC NC
22.07 21.69 NC NC 30.01 NC NC NC NC
12.35 10.32 11.81 13.16 15.89 NC 15.47 33.63 NC NC NC
10.75 12.27 13.39 NC 13.65 32.59 NC NC NC
NC NC NC 9.64 14.67 10.00 12.72 NC 15.77 20.46
NC NC 6.30 8.12 NC 8.63 12.92 13.50 16.84 35.77
*A=0.1 M NaeHPO4, pH=6.0, 10% propanol, C8 column; B=0.1 M Na._,HPO,, pH=6.0, 10% THF, C8 column; C=0.05 M Na2HPO4, pH=6.0, 10% propanol, C8 column; D=0.05 M Citrate Buffer, pH=6.0, 10% propanol, C8 column; E=0.05 M Citrate Buffer, pH=6.0, 15% propanol, CN column; F=0.1 M Na._,HPO4, pH=6.0, l(F/b propanol, CN column. NC=not chromatographed.
umn can resolve a large number of neuropeptides using simple isocratic separations. Oxidative electrochemistry was a reliable method for detection and quantification of neuropeptides that contain tyrosine or tryptophan as constituent amino acids. The major technical difficulty is the high potentials (0.90 V) necessary to effectively oxidize the reactive amino acids and consequent electrode passivation (inactivation) that occurs over time [ 1,6]. This can be dealt with by frequent polishing of the electrode, but this is at the expense of analysis time since it takes time for the baseline to return to a stability sufficient for analytical purposes. Higher potentials (1.0 V) result in more efficient oxidation and therefore a greater absolute amount of signal, but since baseline noise also increases, little is gained in terms of sensitivity [28]. ED was utilized for all these studies, but UV detection at 220 nM was also performed to confirm the identity of electrochemically identified peaks. A chromatogram of the separation and UV detection of neuropeptides is presented in Fig. 3. Animal Studies AVP and ME levels in neural lobe and striatum of SHR and WKY rats are presentedin Table 2. No significant strain effects were noted in either neural lobe AVP content or striatal ME levels. Chromatograms of standards and tissue samples are shown for the neural lobe and striatum in Figs. 4
1176
DAWSON, STEVES, LORDEN AND OPARII_ TABLE 2
'°°so-1I
A V P AND ME L E V E L S IN T H E S H R
Q
~
Group
60-
Age (days)
N
Amount
112 270 270
5 6 6
Ng/lobe ± SE 1819 ± 365 2286 ± 264 1987 ± 338
98 98
4 4
Ng/g± SE 610 ± 94 693 ± 177
_J
u~ 4 0
i..,~ x
o
.~
I
I
z ~
Neural Lobe AVP SHR SHR WKY
z
"~
'-~ 2 0 u.
0
i
0
10
i
210 TIME
i
3~0
4~0
[mini
StfiatalME SHR WKY
FIG. 3. UV detection of a 2/~g standard mixture of neuropeptides (0.01 AUFS at 220 nM). The mobile phase consisted of 0.1 M Na2HPO4 and 15% propanol (pH=6.0). The flow rate was 1.0 ml/min and a CN column was used. This separation was performed at ambient temperature. (A) STANDARDS
(A) ENKEPHALIN STANDARDS (25~ng)
lOnA -
I OnA
5-
5 l
0JF
~l ,4 \ I
(B)
E X T R A C T E D N E U R A L LOBE
(C)
lOnA
o
i
5
,'o
,'5
,o'
0
E X T R A C T E D N E U R A L LOBE FROM BRATTLEBORO RAT
5'
,'o Time (rain)
Time (rain)
(B)
lOnA -
0
I l EXTRACTED
5
,o
I I STRIATAL
• SAMPLE
,'5
25
2'0
Time (min)
FIG. 4. Chromatograms of (A) standards (125 ng of AVP and 200 ng of OXT) and solid-phase extractions of neural lobes from (B) Sprague-Dawley and (C) Brattleboro rats. Chromatographic conditions are the same as in condition C of Table 1.
FIG. 5. Chromatograms of (A) enkephalin standards and (B) an extracted striatal sample. Chromatographic conditions are similar to condition C in Table 1.
and 5, respectively. The Brattleboro rat (Fig. 4) (N=2) had undetectable levels of AVP, as expected. The effects of electrolytic lesions of the PVN on neural lobe content of AVP and OXT are shown in Fig. 6. The PVN lesions resulted in significant reductions in neural lobe AVP content (Fig. 6).
Manipulation of pH, buffer strength or organic modifier provided sufficient flexibility to successfully resolve complex mixtures of neuropeptides. The retention and separation of peptides on reverse phase columns is based primarily on hydrophobic interactions between the peptide and the bonded phase packing material. The use of extremely hydrophobic bonded phases such as octadecyi (C18) results in long retention times unless high concentrations or organic modifiers are used. The high degree of hydrophobicity exhibited by most peptides allows the use of more polar reverse phase packings such as octyl (C8) or cyano (CN). Low molecular weight neuropeptides (MW 500-1000) can easily be resolved on a C8 column, whereas a
DISCUSSION
Reverse phase high performance liquid chromatography (RP-HPLC) is a powerful and versatile analytical tool for the study of neuropeptides [5, 16, 22]. We were able to separate and detect a wide range of biologically active neuropeptides using isocratic conditions and electrochemical detection.
HPLC-ED O F N E U R O P E P T I D E S
1177
2200 2000 1800
SHAM PVN LESION
1600 1400 1200 1000 80O 600 40O r
0~
AVP
OXT
FIG. 6. The effect of electrolytic destruction of the PVN of the hypothalamus on neural lobe AVP and OXT levels. (*p<0.05).
CN column is useful for separations of larger peptides (MW >1000). Molecular weight, however, is not as important as the amino acid composition of the peptide, since the presence of aromatic or acidic amino acids greatly influences the relative hydrophobicity of a peptide [16,17]. Among factors examined in the isocratic separation of neuropeptides, pH had the most pronounced effect on selectivity. Other studies have also pointed out the importance of pH in the separation of peptides [6, 9, 12, 13]. pH influences the relative hydrophobicity of the constituent amino acids dependent on their pKas [16]. Thus the separation and retention of a particular peptide can be selectively manipulated by the judicious use of mobile phase pH. Buffer strength (ionic strength) has previously been shown to be an important variable in the separation of neuropeptides [6, 7, 12]. Increases in buffer strength were found to decrease the retention time of neuropeptides in this study. The use of ion pairing agents such as phosphoric acid [7,8] to decrease retention times of peptides and proteins has previously been described. Phosphate buffer has a similar action [7]. Citrate buffer was found to substitute well for phosphate buffer, so citric acid may also have ion pairing properties [11]. Buffer strength and type play an important role in the separation of complex mixtures of peptides, so care must be taken in selecting the proper concentration and type of buffer. Further, it is important to obtain the highest purity reagents. Organic modifiers are especially crucial in effecting isocratic separation of peptides [6]. We found propanol to be the most effective organic modifier. Methanol, acetonitrile and T H F had insufficient solvent strength to elute highly hydrophobic peptides at concentrations comparable to that of propanol. Mixtures of these solvents were not tested in this study but could be useful in situations where other mobile phase manipulations were unsuccessful in resolving particular peptides.
ED proved to be a useful means of quantifying neuropeptides containing tyrosine or tryptophan in their amino acid sequences. This method of detection was adequate for the determination of ME levels in striatal tissue and AVP and OXT levels in posterior pituitary. The animal studies confirmed the utility and specificity of the HPLC-ED method for determining tissue content of neuropeptides. Neural lobe AVP and OXT levels determined by HPLC-ED were in excellent agreement with values obtained by RIA techniques [4]. AVP was not detectable in the neural lobes of Brattleboro rats, and lesions of the PVN significantly reduced neural lobe AVP content. Striatal ME levels were slightly lower than previously reported values [21]. Differences in age and strain of the rats may account for this minor discrepancy. Thus, HPLC-ED can be utilized for the direct assay of endogenous neuropeptides. The detection limits of HPLC-ED do not approach the sensitivity of RIA methods, but the specificity is greatly improved. ED is more sensitive than UV detection and is gaining acceptance as a method for quantitation of endogenous peptides [6, 15, 20, 21, 23]. The major difficulty with ED is that at high working potentials (/>0.9 V) the working electrode loses sensitivity with time (passivation) [I]. This is particularly important for neuropeptides, since they exhibit relatively steep oxidation curves [2,6]. Oxidation of ME at 0.8 V gives only 29% of the signal generated when it is oxidized at 0.9 V (unpublished observations). Twelve hours of operation at 0.9 V resulted in a 31% loss of sensitivity for ME and LE; thus to maintain maximal sensitivity, the working electrode must be polished daily as previously noted [20]. pH is almost important in maintaining optimal sensitivity, since at higher pH the peak oxidation potential is reduced [2]. During the course of our pH studies, we noted a loss of sensitivity at low pH. Buffer type and concentration are also important factors [6], since the mobile phase must be a good electrical conductor, have a sufficiently high dielectric constant to permit ionization of the electrolyte and be electrochemically inert at the electrode surface. New developments in working electrode materials and ED instrumentation will broaden the applications of ED to peptide chemistry. Recent developments in pre-column derivation techniques for the ED of amino acids [10,141 may be applicable to peptides and dramatically increase the sensitivity of ED for peptides. This study has demonstrated the influence of pH, buffer strength, organic modifier and other variables pertinent to the isocratic separation and ED of neuropeptides. Future developments in ED methodology will improve the sensitivity of this technique and result in significant advances in our understanding of the role of neuropeptides in health and disease. ACKNOWLEDGEMENTS The authors wish to thank John Clos for technical assistance and Debra Roaden and Mary Edenhauser for secretarial assistance. The authors thank Dr. Steve Barker for the UV analysis of neuropeptide standards. The work was supported by grants NS14755 (J.F.L.), HL-22544, HL-25451 and 5T32 HL-07457.
REFERENCES
1. Anton, A. H. A simple, reliable and rapid method for increasing the responsiveness of the glassy carbon electrode (GCE) for the analysis of biogenic amines by high performance liquid chromatography with electrochemical detection (LCEC). Life Sci 35: 79-85, 1984.
2. Bennett, G. W., M. P. Brazell and C. A. Marsden. Electrochemistry of neuropeptides: A possible method for assay and in vivo detection. Life Sci 29: 1001-1007, 1981.
1178 3. Bennet, H. P. J., C. A. Browne and S. Solomon. Purification of the two major forms of rat pituitary corticotropin using only reversed-phase liquid chromatography. Biochemistry 20: 45334546, 1981. 4. Crowley, W. R., T. L. O'Donohue, J. M. George and D. M. Jacobowitz. Changes in pituitary oxytocin and vasopressin during the estrous cycle and after ovarian hormones: evidence for mediation by norepinephrine. Lift, Sci 23: 257%2586, 1978. 5. Davis, T. P., H. Schoemaker, A. Chen and H. I. Yamamura. High performance liquid chromatography of pharmacologically active amines and peptides in biological materials. Life Sci 30: 971-987, 1982. 6. Fleming, L. H. and N. C. Reynolds. LC-ED of endorphins. J Liq Chromatogr 7: 793-808, 1984. 7. Hancock, W. S., C. A. Bishop, R. L. Prestidge and D. R. K. Harding. High-pressure liquid chromatography of peptides and proteins. II. The use of phosphoric acid in the analysis of underivatised peptides by reversed-phase high-pressure liquid chromatography. J Chromatogr 153: 391-398, 1978. 8. Hancock, W. S., C. A. Bishop, R. L. Prestidge and D. R. K. Harding. Reversed-phase, high-pressure liquid chromatography of peptides and proteins with ion-pairing reagents. Science 200: 1168-1170, 1978. 9. Hernandez, O., K. Dermott and L. H. Lazarus. Highperformance liquid chromatography of amphibian peptides. Selectivity changes induced by pH. J Liquid Chromatogr 7: 893-905, 1984. 10. Joseph, M. H. and P. J. Davies. Electrochemical activity of O-phthalaldebyde-mercapto-ethanol derivatives of amino acids-application to high performance liquid chromatographic determination of amino acids in plasma and other biological materials. J Chromatogr 277: 125-136, 1983. 11. Kontur, P., R. Dawson and A. A. Monjan. Manipulation of mobile phase parameters for the HPLC separation of endogenous monoamines in rat brain tissue. Neurosci Methods 11: 5-18, 1984. 12. Krummen, K. and R. W. Frei. The separation of nonapeptides by reversed-phase high performance liquid chromatography. J Chromatogr 132: 27-36, 1977. 13. Lewis, R. V., S. Stein and S. Udenfriend. Separation of opioid peptides utilizing high performance liquid chromatography, lnt J Peptide Protein Res 13: 493-497, 1979. 14. Mahachi, J. J., R. M. Carlson and D. P. Poe. p-N, N-dimethylaminophenyl-isothiocyanate as an electrochemical label for high-performance liquid chromatographic determination of amino acids. J Chromatogr 298: 27%288, 1984. 15. Meek, J. L. and T. P. Bohan. Use of high pressure liquid chromatography (HPLC) to study enkephalins. Adv Biochem Psychapharmaeol 18: 141-147, 1978. 16. Meek, J. L. Separation of Neuropeptides by HPLC. In: Neural Peptides and Neuronal Communication. edited by E. Costa and M. Trabucchi. New York: Raven Press, 1980, pp. 145-151.
DAWSON, STEVES, LORDEN AND OPARIL 17. Molnar, I. and C. Horvath. Separation of amino acids and peptides on non-polar stationary phases by high-performance liquid chromatography. J Chromatogr 142: 623-640, 1977. 18. Moore, G. J., III. Reversed phase high pressure liquid chromatography for the identification and purification of neuropeptides. L(fe Sei 30: 995-1002, 1982. 19. Morris, H. R., A. T. Etienne, A. Dell and J. Albuquerque. A rapid and specific method for the high resolution purification and characterization of neuropeptides. Neuroehemistry 34: 574-582, 1980. 20. Mousa, S. and D. Couri. Analysis of enkephalins, B-endorphins and small peptides in their sequences by highly sensitive highperformance liquid chromatography with electrochemical detection: implications in opioid peptide metabolism. ./ Chromatog 267: 191-198, 1983. 21. Nabeshima, T., M. Hiramatsu, S. Noma, M. Ukai, M. Amano and T. Kameyama. Determination of methionine-enkephalin, norepinephrine, dopamine, 3, 4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxy-4-hydroxyphenylacetic acid (HVA) in brain by high-pressure liquid chromatography with electrochemical detector. Res Commun ('hem Pathol Pharmacol 35: 421442, 1982. 22. Rivier, J. and R. Burgus. Application of reverse phase high pressure liquid chromatography to peptides. In: Biological~Biomedical Applications of Liquid Chromatography, edited by G. L. Hawk. New York: Marcel Dekker, 1977, pp. 147-161. 23. Sauter, A. and W. Frick. Determination of neuropeptides in discrete regions of the rat brain by high-performance liquid chromatography with electrochemical detection. J Chromutogr 297: 215-223, 1984. 24. Scbolkens, 13. A., W. Jung, R. E. Lang, W. Rascher, Th. Unger and D. Ganten. The role of neuropeptides in central mechanisms of blood pressure regulation. In: Endocrinolo~,,y qf Hypertension, edited by F. Mantero, E. G. Biglieri and C. R. W. Edwards. New York: Academic Press, 1982, pp. 33%362. 25. Spatola, A. F. and D. E. Benovitz. Improved detection limits in the analysis of tyrosine-containing polypeptide hormones by using electrochemical detection..I Chromatogr 327: 165-171, 1985. 26. Spindel, E., D. Pettibone, L. Fisher, J. Fernstrom and Wurtman. Characterization of neuropeptides by reversedphase, ion-pair liquid chromatography with post-column detection by radioimmunoassay: application to thyrotropin-releasing hormone, substance P and vasopressin. J Chromatogr 222: 381-387, 1981. 27. Taylor, G. W. and H. R. Morris. High-performance liquid chromatography: purification and characterization of neuropeptides. In: Handbook ofPsyehopharmacology, vol 15, edited by S. D. Iversen and S. H. Snyder. New York: Plenum Press, 1982, pp. 271-297. 28. White, M. W. High-performance liquid chromatography of tyrosine-related peptides with electrochemical detection. J Chromatogr 262: 420--425, 1983.
0196-9781/85 $3.00 + .00
Reverse-Phase Separation and Electrochemical Detection of Neuropeptides RALPH DAWSON, JR.,'* JOANNE
P. S T E V E S , t
J O A N F. L O R D E N t
AND SUZANNE
OPARIL*
Department of Medicine and * Cardiovascular Research and Training Center, School of Medicine University of Alabama at Birmingham, Birmingham, AL 35294 and tDepartment of Psychology, University College University of Alabama in Birmingham, Birmingham, AL 35294 R e c e i v e d 7 M a r c h 1985 DAWSON, R., JR., J. P. STEVES, J. F. LORDEN AND S. OPARIL. Reverse-phase separation and electrochemical detection of neuropeptides. PEPT1DES 6(6) 1173-1178, 1985.--The reverse-phase separation of neuropeptides using isocratic conditions is described. Each component of the mobile phase was examined for its ability to influence the separation of complex mixtures of neuropeptides. Manipulation of buffer strength, pH, organic modifier and column type provided sufficient flexibility to resolve closely related neuropeptides. Amperometric detection of oxidizable amino acids in the peptide sequence of a number of endogenous neuropeptides proved suitable for the identification of peptide standards and quantification of neural lobe arginine vasopressin and striatal methionine enkephalin. Neuropeptides HPLC Methionine enkephalin
Electrochemistry
Paraventricular nucleus
N E U R O P E P T I D E S have received increasing attention because of their possible roles in numerous pathologic conditions [24,27]. This has led to the development of techniques for the separation and quantitation of neuropeptides. Procedures based on radioimmunoassay techniques (RIA), high performance liquid chromatography (HPLC) coupled with UV absorbance or fluorescence detection and HPLC followed by RIA or radioreceptor assay quantitation have been described [12, 13, 26]. HPLC has also been used in the purification of neuropeptides from complex biological matrices [3, 18, 19]. While the use of HPLC in peptide research is increasing, there are few reports of systematic evaluations of parameters relevant to the reverse-phase separation of complex mixtures of neuropeptides. A number of reported separations of neuropeptide mixtures are based on the use of linear programmed gradients of solvents that require expensive instrumentation. Therefore, we attempted to develop and evaluate reliable and reproducible isocratic separations of neuropeptides. We also assessed the role of each component of the mobile phase to aid in the selection of a particular separation for specific analytical or purification application. A second aim of these studies was to apply a previously underutilized detection technique to quantify neur0peptides in tissues. The amino acids tyrosine, tryptophan, and cysteine are electroactive and can be oxidized and quantitated electrochemically [2]. Neuropeptides that contain tyrosine or tryptophan residues can also be oxidized and detected electrochemically [2, 6, 15, 20, 21, 23, 25, 28]. Electrochemical detection (ED) appears to be a reliable and perhaps more
Arginine vasopressin
sensitive detection method for neuropeptides than UV methods [20,25]. The HPLC-ED method has been successfully applied for the separation and quantitation of opiate peptides [6, 15, 20, 25]. Therefore, ED was employed in this study to quantitate endogenous levels of methionine enkephalin, arginine vasopressin and oxytocin, as well as other neuropeptides. Neuropeptide levels were determined in posterior pituitary and striatal tissue from rats. In addition, lesions were made in the paraventricular nucleus (PVN) of rats to specifically perturb endogenous levels of neural lobe vasopressin in order to further validate the technique. We have extended the applicability of HPLC-ED to a wide range of electroactive neuropeptides and have characterized the chromatographic behavior of a number of physiologically relevant neuropeptides. METHOD
lnstrttmenlalion A Waters M-45 solvent delivery system operated at a flow rate of 1.0 ml/min (2100 psi backpressure) was used throughout the study. Column temperature was maintained at 40°C. Detection of peptides was accomplished with an LC-4A electrochemical detector with a TL-5 glassy carbon working electrode (Bioanalytical Systems, West Lafayette, IN) using an applied potential of 0.9 V vs. a Ag/AgC1 reference electrode and a sensitivity of 5 or 10 nA. Chromatographic data were plotted and integrated by a Hewlett Packard 3390A recording integrator. Peak height was used to quantitate
'Requests for reprints should be addressed to Ralph Dawson, Jr., Ph.D., University of Florida, College of Pharmacy, Box J-4, Department of Pharmacodynamics, J. Hillis Miller Health Center, Gainesville, FL 32610.
1173
1174
DAWSON, STEVES, L O R D E N AND O P A R I L
neuropeptides and both the methods of internal and external standardization were used in computing the data. Separations were performed on either an IBM Octyl (C8) or Alltech Cyano (CN) column (4.5x250 mm, 5 /zm). The analytical columns were protected by a 4.6x30 mm C18 guard column (Brownlee Labs, Inc., Santa Clara, CA). Sample injections were performed using a Rheodyne 7125 fixed loop (50 tzl) injector.
19.0 o
18.0 17.0 16.0
~
o
15.0 14.0
Standards and Reagents
~ ' ~
- " .... • OXT
"h.
All peptide standards were purchased from either Calbiochem (San Diego, CA) or Sigma (St. Louis, MO). Standards were diluted in mobile phase and stored at 4°C as stock solutions of 100/zg/ml. Buffers (citric acid trisodium salt, sodium phosphate dibasic and monobasic) were purchased from Sigma and were of the highest purity. The organic modifiers propanol-I and tetrahydrofuran, methanol, butanol and acetonitrile were obtained from Burdick and Jackson Laboratories, Inc. (Muskegon, MI).
.c_ E
13.0
LU
12.0
I-Z
11.0
tZ UJ l-
10.0
o_
•
v
o
~ o
All
~ LE
~ o
'
~
~
MEA
cXEDH
9.0
UJ
8.0 7.0
Extraction Procedure Neural lobes were homogenized in a glass homogenizer in 2.0 ml of 1.0 M HC1 which contained 5% formic acid, 1.0% trifluroacetic acid (TFA) and 1.0% NaCi. The homogenates were centrifuged 30 minutes at 17,800xg (4°C). The supernatant was diluted by the addition of 1.0 ml of H._,O and aspirated through a C18 extraction column (Baker-10 Extraction System, J.T. Baker Chemical Co., Phillipsburg, N J). The column was washed with 2 ml of H._,O followed by a 2 ml wash with 5.0% methanol. The arginine vasopressin (AVP) and oxytocin (OXT) were eluted in 1.0 ml of 80% methanol and directly injected for HPLC analysis after filtration through 0.2 tzm filters (Nylon 66). Recovery of AVP standards (125 ng) after extraction was 61-+5% ( N = 11). Striatal tissue from SHR and W K Y rats was homogenized in 5.0 ml of the same homogenization media as used for AVP determination. The homogenate was centrifuged and poured onto C18 extraction columns as described above. The striatal samples were washed with 2.0 ml of H._,O, 2 ml of 0.1% T F A , 2.0 ml of 0.02 M NaH.,PO~ (pH=2.4) and finally with 2 ml of 5% methanol. Methionine enkephalin (ME) was eluted in 1.0 ml of 100% methanol and the methanol was then evaporated under vacuum. The striatal samples were reconstituted in 100/zl of mobile phase and 50/zl was injected onto the column after filtration. ME (25 ng) recovery after extraction was 63-+5% (N=5).
_ 0
~
5.0 --
~ 0 - -
© AVT
4.0 0 0.025
0 05
0 10
0 15
Na2 HPO 4 (M) FIG. 1. The effect of buffer strength on neuropeptide RT. The experiments were performed using a C8 column with 10% propanol as the organic modifier and mobile phase pH was maintained at 6.0.
anterior to the ear bar zero, 3.5 mm dorsal to the interaural line, and -+0.5 mm from the midline. Lesions were performed by passing 1 mA anodal current for 10 seconds through stainless steel electrodes (size 00 insect pins) insulated to 0.75-1.0 mm from the tip. Sham operated animals were treated similarly, except that the electrode was not lowered into the brain. The rats were decapitated on postsurgical day 30 and neural lobes were removed for assay of AVP and OXT. The brains of the rats with PVN lesions were fixed in neutral buffered formalin and later cut at 25 ~m in a cryostat. Lesion placement was verified in cresyl violet stained sections.
Animal Experiments Striatal and neural lobe tissue was obtained from male spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats which were purchased from Charles River Breeding Laboratories and maintained under standard laboratory conditions in our colony. Neural :lobes were also obtained from two adult male Brattleboro rats (a gift of Dr. K. H. Berecek). Lesions of the PVN of the hypothalamus were made in female Sprague-Dawley rats weighing 270-330 g which were anesthetized with 35 mg/kg of sodium pentobarbital and 0.2 ml ketamine HC1 (50 mg/ml) and received either bilateral electrolytic lesions or sham surgery. With the incisor bar of the stereotaxic instrument set 5 mm dorsal to the interaural line, the coordinates for the PVN lesion were: 6.2 mm
--,,~• AVPLvP
_
6.0
RESULTS
Neuropeptide HPLC The effects of increasing buffer strength on neuropeptide retention time (RT) are shown in Fig. 1. In general, increasing buffer strength decreased RT, with angiotension II (All) showing the most pronounced effects. The RT's of ME and LE were relatively unaffected by changes in buffer strength, whereas the presence of an amide group in MEA restored the effect of buffer strength on RT. The effects of pH on neuropeptide RT are presented in Fig. 2. pH had pronounced effects on neuropeptide RT over the range examined. The nonapeptides, AVT, LVP, AVP and OXT exhibited increases in RT with increases in pH whereas the opiate peptides, ME, LE, c~endorphin (c~EDH)
HPLC-ED OF NEUROPEPTIDES
1175 TABLE1
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Chromatographic Conditions* (RT)
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I
3.0
4.0
I 5.0 APPARENT
I
I
6.0
7.0
pH
FIG. 2. Changes in neuropeptide RT as a function of alterations in mobile phase pH. The mobile phase was 0.1 M NaH2PO~ with 10% propanol as the organic modifier, pH adjustments were made with either NaOH or HCI. All separations were performed on a C8 column.
and ACTH 1-24 all showed substantial decreases in RT under these conditions. The amide group in MEA altered the chromatographic characteristics of this opiate peptide analog in relation to changes in pH of the mobile phase, pH was an important determinant in the reverse phase separation of neuropeptides and had differential effects dependent on the relevant functional group(s). The effects of organic modifiers, buffer composition and two types of reverse phase packings on neuropeptide separations were also examined. For isocratic separations, propanol-1 gave the best resolution and shortest run times. Methanol and acetonitrile had insufficient solvent strength to effectively elute highly hydrophobic peptides. Butanol had good eluting properties but its limitied water solubility made its use impractical. T H F (Table 1) did not prove satisfactory for most of our applications. Citrate buffer substituted well for phosphate buffer and at equal molarity (Table 1) was more effective in eluting all the peptides tested. We chose a C8 column for most of our studies since the C8 packing material provided both good resolution and favorable RT characteristics. In preliminary studies with C 18 columns we found that the highly retentive nature of C18 packing material resulted in inordinately long RT's for peptides under isocratic conditions. The more polar CN column (Table 1) gave very favorable (reduced) RT's for the larger molecular weight peptides. The resolution of smaller peptides, however, was poor with the CN column. Thus, depending on the desired application, either the C8 or CN col-
Neuropeptide Arg-Vasotocin (AVT) Lys-Vasopressin (LVP) Arg-Vasopressin (AVP) Met-Enkephalin (ME) Met-Enkephalin amide (MEA) a-Endorphin (aEDH) Leu-Enkephalin (LE) Angiotensin II (All) Angiotensin I (AI) Oxytocin (OXT) ACTH 1-24 Neurotensin (NT) LHRH c~-MSH
A
B
C
D
E
F
NC 7.17 5 . 5 7 4.79 NC 6.52 6.49 13.58 7 . 5 9 6 . 3 0 10.30 7.48 7.30 14.69 8 . 3 4 6 . 7 5 10.70 8.27 7.39 14.69 7 . 6 9 7 . 3 1 7 . 8 0 5.51 11.70
NC
11.22 12.65 14.48 NC 14.33 33.67 NC NC NC
22.07 21.69 NC NC 30.01 NC NC NC NC
12.35 10.32 11.81 13.16 15.89 NC 15.47 33.63 NC NC NC
10.75 12.27 13.39 NC 13.65 32.59 NC NC NC
NC NC NC 9.64 14.67 10.00 12.72 NC 15.77 20.46
NC NC 6.30 8.12 NC 8.63 12.92 13.50 16.84 35.77
*A=0.1 M NaeHPO4, pH=6.0, 10% propanol, C8 column; B=0.1 M Na._,HPO,, pH=6.0, 10% THF, C8 column; C=0.05 M Na2HPO4, pH=6.0, 10% propanol, C8 column; D=0.05 M Citrate Buffer, pH=6.0, 10% propanol, C8 column; E=0.05 M Citrate Buffer, pH=6.0, 15% propanol, CN column; F=0.1 M Na._,HPO4, pH=6.0, l(F/b propanol, CN column. NC=not chromatographed.
umn can resolve a large number of neuropeptides using simple isocratic separations. Oxidative electrochemistry was a reliable method for detection and quantification of neuropeptides that contain tyrosine or tryptophan as constituent amino acids. The major technical difficulty is the high potentials (0.90 V) necessary to effectively oxidize the reactive amino acids and consequent electrode passivation (inactivation) that occurs over time [ 1,6]. This can be dealt with by frequent polishing of the electrode, but this is at the expense of analysis time since it takes time for the baseline to return to a stability sufficient for analytical purposes. Higher potentials (1.0 V) result in more efficient oxidation and therefore a greater absolute amount of signal, but since baseline noise also increases, little is gained in terms of sensitivity [28]. ED was utilized for all these studies, but UV detection at 220 nM was also performed to confirm the identity of electrochemically identified peaks. A chromatogram of the separation and UV detection of neuropeptides is presented in Fig. 3. Animal Studies AVP and ME levels in neural lobe and striatum of SHR and WKY rats are presentedin Table 2. No significant strain effects were noted in either neural lobe AVP content or striatal ME levels. Chromatograms of standards and tissue samples are shown for the neural lobe and striatum in Figs. 4
1176
DAWSON, STEVES, LORDEN AND OPARII_ TABLE 2
'°°so-1I
A V P AND ME L E V E L S IN T H E S H R
Q
~
Group
60-
Age (days)
N
Amount
112 270 270
5 6 6
Ng/lobe ± SE 1819 ± 365 2286 ± 264 1987 ± 338
98 98
4 4
Ng/g± SE 610 ± 94 693 ± 177
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Neural Lobe AVP SHR SHR WKY
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0
i
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10
i
210 TIME
i
3~0
4~0
[mini
StfiatalME SHR WKY
FIG. 3. UV detection of a 2/~g standard mixture of neuropeptides (0.01 AUFS at 220 nM). The mobile phase consisted of 0.1 M Na2HPO4 and 15% propanol (pH=6.0). The flow rate was 1.0 ml/min and a CN column was used. This separation was performed at ambient temperature. (A) STANDARDS
(A) ENKEPHALIN STANDARDS (25~ng)
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(B)
E X T R A C T E D N E U R A L LOBE
(C)
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E X T R A C T E D N E U R A L LOBE FROM BRATTLEBORO RAT
5'
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Time (rain)
(B)
lOnA -
0
I l EXTRACTED
5
,o
I I STRIATAL
• SAMPLE
,'5
25
2'0
Time (min)
FIG. 4. Chromatograms of (A) standards (125 ng of AVP and 200 ng of OXT) and solid-phase extractions of neural lobes from (B) Sprague-Dawley and (C) Brattleboro rats. Chromatographic conditions are the same as in condition C of Table 1.
FIG. 5. Chromatograms of (A) enkephalin standards and (B) an extracted striatal sample. Chromatographic conditions are similar to condition C in Table 1.
and 5, respectively. The Brattleboro rat (Fig. 4) (N=2) had undetectable levels of AVP, as expected. The effects of electrolytic lesions of the PVN on neural lobe content of AVP and OXT are shown in Fig. 6. The PVN lesions resulted in significant reductions in neural lobe AVP content (Fig. 6).
Manipulation of pH, buffer strength or organic modifier provided sufficient flexibility to successfully resolve complex mixtures of neuropeptides. The retention and separation of peptides on reverse phase columns is based primarily on hydrophobic interactions between the peptide and the bonded phase packing material. The use of extremely hydrophobic bonded phases such as octadecyi (C18) results in long retention times unless high concentrations or organic modifiers are used. The high degree of hydrophobicity exhibited by most peptides allows the use of more polar reverse phase packings such as octyl (C8) or cyano (CN). Low molecular weight neuropeptides (MW 500-1000) can easily be resolved on a C8 column, whereas a
DISCUSSION
Reverse phase high performance liquid chromatography (RP-HPLC) is a powerful and versatile analytical tool for the study of neuropeptides [5, 16, 22]. We were able to separate and detect a wide range of biologically active neuropeptides using isocratic conditions and electrochemical detection.
HPLC-ED O F N E U R O P E P T I D E S
1177
2200 2000 1800
SHAM PVN LESION
1600 1400 1200 1000 80O 600 40O r
0~
AVP
OXT
FIG. 6. The effect of electrolytic destruction of the PVN of the hypothalamus on neural lobe AVP and OXT levels. (*p<0.05).
CN column is useful for separations of larger peptides (MW >1000). Molecular weight, however, is not as important as the amino acid composition of the peptide, since the presence of aromatic or acidic amino acids greatly influences the relative hydrophobicity of a peptide [16,17]. Among factors examined in the isocratic separation of neuropeptides, pH had the most pronounced effect on selectivity. Other studies have also pointed out the importance of pH in the separation of peptides [6, 9, 12, 13]. pH influences the relative hydrophobicity of the constituent amino acids dependent on their pKas [16]. Thus the separation and retention of a particular peptide can be selectively manipulated by the judicious use of mobile phase pH. Buffer strength (ionic strength) has previously been shown to be an important variable in the separation of neuropeptides [6, 7, 12]. Increases in buffer strength were found to decrease the retention time of neuropeptides in this study. The use of ion pairing agents such as phosphoric acid [7,8] to decrease retention times of peptides and proteins has previously been described. Phosphate buffer has a similar action [7]. Citrate buffer was found to substitute well for phosphate buffer, so citric acid may also have ion pairing properties [11]. Buffer strength and type play an important role in the separation of complex mixtures of peptides, so care must be taken in selecting the proper concentration and type of buffer. Further, it is important to obtain the highest purity reagents. Organic modifiers are especially crucial in effecting isocratic separation of peptides [6]. We found propanol to be the most effective organic modifier. Methanol, acetonitrile and T H F had insufficient solvent strength to elute highly hydrophobic peptides at concentrations comparable to that of propanol. Mixtures of these solvents were not tested in this study but could be useful in situations where other mobile phase manipulations were unsuccessful in resolving particular peptides.
ED proved to be a useful means of quantifying neuropeptides containing tyrosine or tryptophan in their amino acid sequences. This method of detection was adequate for the determination of ME levels in striatal tissue and AVP and OXT levels in posterior pituitary. The animal studies confirmed the utility and specificity of the HPLC-ED method for determining tissue content of neuropeptides. Neural lobe AVP and OXT levels determined by HPLC-ED were in excellent agreement with values obtained by RIA techniques [4]. AVP was not detectable in the neural lobes of Brattleboro rats, and lesions of the PVN significantly reduced neural lobe AVP content. Striatal ME levels were slightly lower than previously reported values [21]. Differences in age and strain of the rats may account for this minor discrepancy. Thus, HPLC-ED can be utilized for the direct assay of endogenous neuropeptides. The detection limits of HPLC-ED do not approach the sensitivity of RIA methods, but the specificity is greatly improved. ED is more sensitive than UV detection and is gaining acceptance as a method for quantitation of endogenous peptides [6, 15, 20, 21, 23]. The major difficulty with ED is that at high working potentials (/>0.9 V) the working electrode loses sensitivity with time (passivation) [I]. This is particularly important for neuropeptides, since they exhibit relatively steep oxidation curves [2,6]. Oxidation of ME at 0.8 V gives only 29% of the signal generated when it is oxidized at 0.9 V (unpublished observations). Twelve hours of operation at 0.9 V resulted in a 31% loss of sensitivity for ME and LE; thus to maintain maximal sensitivity, the working electrode must be polished daily as previously noted [20]. pH is almost important in maintaining optimal sensitivity, since at higher pH the peak oxidation potential is reduced [2]. During the course of our pH studies, we noted a loss of sensitivity at low pH. Buffer type and concentration are also important factors [6], since the mobile phase must be a good electrical conductor, have a sufficiently high dielectric constant to permit ionization of the electrolyte and be electrochemically inert at the electrode surface. New developments in working electrode materials and ED instrumentation will broaden the applications of ED to peptide chemistry. Recent developments in pre-column derivation techniques for the ED of amino acids [10,141 may be applicable to peptides and dramatically increase the sensitivity of ED for peptides. This study has demonstrated the influence of pH, buffer strength, organic modifier and other variables pertinent to the isocratic separation and ED of neuropeptides. Future developments in ED methodology will improve the sensitivity of this technique and result in significant advances in our understanding of the role of neuropeptides in health and disease. ACKNOWLEDGEMENTS The authors wish to thank John Clos for technical assistance and Debra Roaden and Mary Edenhauser for secretarial assistance. The authors thank Dr. Steve Barker for the UV analysis of neuropeptide standards. The work was supported by grants NS14755 (J.F.L.), HL-22544, HL-25451 and 5T32 HL-07457.
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2. Bennett, G. W., M. P. Brazell and C. A. Marsden. Electrochemistry of neuropeptides: A possible method for assay and in vivo detection. Life Sci 29: 1001-1007, 1981.
1178 3. Bennet, H. P. J., C. A. Browne and S. Solomon. Purification of the two major forms of rat pituitary corticotropin using only reversed-phase liquid chromatography. Biochemistry 20: 45334546, 1981. 4. Crowley, W. R., T. L. O'Donohue, J. M. George and D. M. Jacobowitz. Changes in pituitary oxytocin and vasopressin during the estrous cycle and after ovarian hormones: evidence for mediation by norepinephrine. Lift, Sci 23: 257%2586, 1978. 5. Davis, T. P., H. Schoemaker, A. Chen and H. I. Yamamura. High performance liquid chromatography of pharmacologically active amines and peptides in biological materials. Life Sci 30: 971-987, 1982. 6. Fleming, L. H. and N. C. Reynolds. LC-ED of endorphins. J Liq Chromatogr 7: 793-808, 1984. 7. Hancock, W. S., C. A. Bishop, R. L. Prestidge and D. R. K. Harding. High-pressure liquid chromatography of peptides and proteins. II. The use of phosphoric acid in the analysis of underivatised peptides by reversed-phase high-pressure liquid chromatography. J Chromatogr 153: 391-398, 1978. 8. Hancock, W. S., C. A. Bishop, R. L. Prestidge and D. R. K. Harding. Reversed-phase, high-pressure liquid chromatography of peptides and proteins with ion-pairing reagents. Science 200: 1168-1170, 1978. 9. Hernandez, O., K. Dermott and L. H. Lazarus. Highperformance liquid chromatography of amphibian peptides. Selectivity changes induced by pH. J Liquid Chromatogr 7: 893-905, 1984. 10. Joseph, M. H. and P. J. Davies. Electrochemical activity of O-phthalaldebyde-mercapto-ethanol derivatives of amino acids-application to high performance liquid chromatographic determination of amino acids in plasma and other biological materials. J Chromatogr 277: 125-136, 1983. 11. Kontur, P., R. Dawson and A. A. Monjan. Manipulation of mobile phase parameters for the HPLC separation of endogenous monoamines in rat brain tissue. Neurosci Methods 11: 5-18, 1984. 12. Krummen, K. and R. W. Frei. The separation of nonapeptides by reversed-phase high performance liquid chromatography. J Chromatogr 132: 27-36, 1977. 13. Lewis, R. V., S. Stein and S. Udenfriend. Separation of opioid peptides utilizing high performance liquid chromatography, lnt J Peptide Protein Res 13: 493-497, 1979. 14. Mahachi, J. J., R. M. Carlson and D. P. Poe. p-N, N-dimethylaminophenyl-isothiocyanate as an electrochemical label for high-performance liquid chromatographic determination of amino acids. J Chromatogr 298: 27%288, 1984. 15. Meek, J. L. and T. P. Bohan. Use of high pressure liquid chromatography (HPLC) to study enkephalins. Adv Biochem Psychapharmaeol 18: 141-147, 1978. 16. Meek, J. L. Separation of Neuropeptides by HPLC. In: Neural Peptides and Neuronal Communication. edited by E. Costa and M. Trabucchi. New York: Raven Press, 1980, pp. 145-151.
DAWSON, STEVES, LORDEN AND OPARIL 17. Molnar, I. and C. Horvath. Separation of amino acids and peptides on non-polar stationary phases by high-performance liquid chromatography. J Chromatogr 142: 623-640, 1977. 18. Moore, G. J., III. Reversed phase high pressure liquid chromatography for the identification and purification of neuropeptides. L(fe Sei 30: 995-1002, 1982. 19. Morris, H. R., A. T. Etienne, A. Dell and J. Albuquerque. A rapid and specific method for the high resolution purification and characterization of neuropeptides. Neuroehemistry 34: 574-582, 1980. 20. Mousa, S. and D. Couri. Analysis of enkephalins, B-endorphins and small peptides in their sequences by highly sensitive highperformance liquid chromatography with electrochemical detection: implications in opioid peptide metabolism. ./ Chromatog 267: 191-198, 1983. 21. Nabeshima, T., M. Hiramatsu, S. Noma, M. Ukai, M. Amano and T. Kameyama. Determination of methionine-enkephalin, norepinephrine, dopamine, 3, 4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxy-4-hydroxyphenylacetic acid (HVA) in brain by high-pressure liquid chromatography with electrochemical detector. Res Commun ('hem Pathol Pharmacol 35: 421442, 1982. 22. Rivier, J. and R. Burgus. Application of reverse phase high pressure liquid chromatography to peptides. In: Biological~Biomedical Applications of Liquid Chromatography, edited by G. L. Hawk. New York: Marcel Dekker, 1977, pp. 147-161. 23. Sauter, A. and W. Frick. Determination of neuropeptides in discrete regions of the rat brain by high-performance liquid chromatography with electrochemical detection. J Chromutogr 297: 215-223, 1984. 24. Scbolkens, 13. A., W. Jung, R. E. Lang, W. Rascher, Th. Unger and D. Ganten. The role of neuropeptides in central mechanisms of blood pressure regulation. In: Endocrinolo~,,y qf Hypertension, edited by F. Mantero, E. G. Biglieri and C. R. W. Edwards. New York: Academic Press, 1982, pp. 33%362. 25. Spatola, A. F. and D. E. Benovitz. Improved detection limits in the analysis of tyrosine-containing polypeptide hormones by using electrochemical detection..I Chromatogr 327: 165-171, 1985. 26. Spindel, E., D. Pettibone, L. Fisher, J. Fernstrom and Wurtman. Characterization of neuropeptides by reversedphase, ion-pair liquid chromatography with post-column detection by radioimmunoassay: application to thyrotropin-releasing hormone, substance P and vasopressin. J Chromatogr 222: 381-387, 1981. 27. Taylor, G. W. and H. R. Morris. High-performance liquid chromatography: purification and characterization of neuropeptides. In: Handbook ofPsyehopharmacology, vol 15, edited by S. D. Iversen and S. H. Snyder. New York: Plenum Press, 1982, pp. 271-297. 28. White, M. W. High-performance liquid chromatography of tyrosine-related peptides with electrochemical detection. J Chromatogr 262: 420--425, 1983.