by increasing the accelerating voltage from 7.5 to 20 kV, the total ion transmission will increase by an order of magnitude. At the same time, we expect a significant improvement in the resolution and abundance sensitivity of the CID spectrum, since the peak width and broad shape in this spectrum is a function of the ratio of chemical bond energy to the kinetic energy of the primary ion. Using CI or low energy E1 as the ionization technique would minimize the problems of ion divergence and thereby increase the collection efficiency of the mass selected primary ions. High voltage acceleration (10 to 15 kV) is desired in this case also, to ensure adequate quality of the CID spectrum. In short, it is expected that by using improved FI or, alternatively, CI or low energy E1 in conjunction with higher accelerating voltage, the sensitivity of this technique may be increased by one to two orders of magnitude, and a precision of 1 to 3% a t the same sample size seems to be well within reach.
ACKNOWLEDGMENT T h e authors acknowledge John F. Burke and Vernon R. Young for stimulating discussions concerning potential applications of this methodology.
LITERATURE CITED (1) D. Rlttenberg, A. S. Keston, F. Rosebury. and R. Schoenheimer, J . Bioi. Chem., 127, 291 (1939). (2) K. Wetzel, H. Fawt, W. Hartig, “Proceedings of the Second International Conference on Stable Isotopes”, Oakbrook, Ill., October 1975, E. R. Klein,
P. D. Klein, Ed., ERDA Conf. 75-1027, 421 (1976). (3) R. A. Saunders, J . Sci. Insfrum., Ser. 2 , 1, 1053 (1966). (4) R. M. Caprioli in “Biochemical Applications of Mass Spectrometry”, G. R. Walier, Ed., Wiley-Interscience, New York, N.Y. 735 (1972). (5) A. M. Lawson, Clin. Chem. Winston-Salem, N . C . , 21, 803 (1975). (6) D. A. Schoeller and J. M. Hayes, Anal. Chem., 47, 408 (1975). (7) C. V . Lundeen, A. S.Viscomi, and F . H. Field, Anal. Chem., 45, 1288 (1973). (6) K. Biernann and G. G. J. Deffner, Biochem. Biophys. Res. Comm., 4 , 287 (1961). (9) G. R. Waller, R. Ryhage, and S. Meyerson, Anal. B k h e m . , 16, 277 (1966). (10) W. F. Haddon, H. C. Lukens, and R. H. Elsken, Anal. Chem., 45, 682 (1973). (11) J. H. Beynon, D. F. Brothers, and R. G. Cooks, Anal. Chem., 46, 1299 f 19741.
(12) .L:? Kruger, J. F Litton, R. W. Kondrat, and R. G. Cooks, Anal. Chem., 48. 2113 (1976). (13) J. H. McReynolds and M. Anbar, Int. J . Mass Spectrom. Ion Phys., 24, 37 (1977). (14) W. C. Turner, C. N. Biltz. J. H. McReynolds, and M . Anbar, Rev. Sci. Instrum ., submitted. (15) F. W. McLafferty, P. F. Bente, R. Kornfeld. S.C. Tsai, and I. Howe, J . Am. Chem. SOC.,95, 2120 (1973). (16) K. R. Jennings, Int. J . Mass Specfrom. Ion Phys., 1 , 227 (1968). (17) K. Biemann in “Mass Spectrometry, Organic Chemical Applications”, McGraw-Hill, New York, N.Y., 1962, p 263. (18) J. H. Beynon and R. G. Cooks, J . Phys. E., 7, 10 (1974). (19) D. A. Schoeller. Biomed. Mass Spectrom., 3, 265 (1976). (20) H. L. Brown, R. H. Cross, and M. Anbar. Int. J . Mass Specfrom. Ion Phys., 23, 63 (1977).
RECEIVED for review April 20, 1977. Accepted June 30, 1977. This work was supported in part by the National Institute of General Medical Sciences Contract GM 21835,and by NCI Grant No. 5 R O l CA 13312-05.
Short Excitation Wavelength Fluorometric Detection in High-pressure Liquid Chromatography of Indole Peptide, Naphthyl, and Phenol Compounds G. J. Kroi,” C. A. Mannan, R. E. Pickering, D. V. Amato, and B. T. Kho Analytical Research and Development, Ayerst Laboratories, Rouses Point, New York 72979
A. Sonnenschein Schoeffel Instrument Corporation, Westwood, New Jersey 07675
A direct and relatively sensitlve (picogram range) fluorometrlc detection technique for Indole decapeptide lutelnizlng hormone (LH), a naphthyl adrenergic blocklng agent (propranolol), and phenol (estrogen) compounds separated by high pressure llquki chromatography (HPLC) was Investigated. The fluorometric detection involved a deuterium llght source and excitatlon Wavelengths below 250 nm. The observed detection limits were more sensitlve by a factor of ten than those obtained with excitation wavelengths above 260 nm. The observed sensltivlty gain was deduced from the UV absorptlon spectra which are slmilar to corrected fluorescence excltatlon spectra.
T h e inherent sensitivity and specificity of fluorometric detection was exploited in a number of liquid chromatographic procedures. However, much of the previous work involved chromatography of fluorescent derivatives such as 5-dimethylamino-1-naphthalenesulfonyl chloride (Dansyl-C1) and 4-phenylspiro[furan-2(3H),l’-phthalan]-3,3’-dione (Fluorescamine) and detection a t relatively long (above 260 nm) 1836
ANALYTICAL CHEMISTRY, VOL. 4 9 , NO. 12, OCTOBER 1 9 7 7
excitation wavelengths (1-4). Although derivatization was often used to enhance detection sensitivity, it can complicate the analytical procedure. Furthermore, this complication is not always necessary since there are compounds (e.g. indoles, phenols, and naphthyls) which yield relatively high fluorometric intensity in the underivatized state, provided that they are excited with a light source which generates sufficient energy a t excitation wavelengths below 250 nm. Recent availability of a commercial liquid chromatography spectrofluorometric detector with a deuterium light source prompted us to investigate detection of several naturally fluorescent compounds and fluorescent derivatives a t excitation wavelengths below 250 nm. Our objective was to simplify and/or enhance sensitivity and specificity of fluorometric detection in liquid chromatography. Fluorometric detection of underivatized compounds is more direct and reduces the chances of interference from other compounds which may also react with the same derivatizing reagent (e.g. Dansyl-Cl). Our approach was based on the information obtained from the corrected excitation or UV absorption spectra. (In dilute
solutions, the two spectra are similar if instrumental artifacts are removed ( 5 , 6).) Since the corrected excitation and UV absorption spectra often have higher maxima a t wavelengths below 250 nm, it was conceivable that one might observe higher fluorometric emission intensity a t shorter excitation wavelengths. T h e potential of short excitation wavelength fluorometric detection is illustrated in this study by several compounds of pharmaceutical interest. Although the selected compounds fluoresce a t excitation wavelengths above 260 nm, excitation wavelengths below 250 nm yielded considerably more sensitive detection limits provided that a suitable light source (e.g., Deuterium lamp) is used. EXPERIMENTAL Materials. All compounds chromatographed in this study were obtained from internal (Ayerst Laboratories Inc., Rouses Point, N.Y.) sources. Dansyl-C1 reagent was purchased from Regis Chemical Company, Morton Grove, Ill. Spectro-grade (distilled in glass) solvents were purchased from Burdick and Jackson Laboratories, Muskegon, Mich. E. Merck LiChrosorb chromatographic supports were obtained from Brinkmann Instruments, Westbury, N.Y. Spherisorb ODS ( 5 pm) support was purchased from Spectra-Physics, Santa Clara, Calif. All chromatographic columns were packed in an acetone slurry with a Haskel (Haskel Engineering and Supply Co., Burbank, Calif.) constant pressure pump (part of DuPont Model 820 Liquid Chromatograph). A 50-mL stainless steel reservoir was connected in series with the chromatographic column and was used to contain the acetone slurry prior to column packing. The 4.0-mm i.d. chromatographic columns were purchased from Alltech Associates, Arlington Heights, Ill., while the 3.2-mm i.d. columns were purchased from Altex Scientific Inc., Berkeley, Calif. Equipment. A Model SF 770 Variable Wavelength UV Liquid Chromatography detector and a Model FS970 Liquid Chromatography Fluorescence detector were obtained from Schoeffel Instrument Corp., Westwood, N.J. The KV 370, and 550 cut-off filters were obtained from Schott-Optical Glass Inc., Duryea, Pa. A Model 740 constant flow rate pump was purchased from Spectra-Physics Inc., Santa Clara, Calif. A Model 314 constant flow rate screw driven syringe pump was purchased from Instrumentation Specialties Co., Lincoln, Neb. A Model 282, 10-in. strip chart recorder was purchased from Linear Instruments Corp., Irvine, Calif. Procedure. The estrogen compounds were chromatographed on LiChrosorb SI 60 (5 pm) 25 cm X 3.2 mm (i.d.1and LiChrosorb NHz (10pm) 25 cm X 3.2 mm (i.d.) columns connected in series and eluted with pentane-methanol (94:6 v / v ) solvent at 1.0 mL/min flow rate. The eluate was monitored by the UV and fluorescence detectors also connected in series. The UV detection wavelength was 220 nm. The fluorescence excitation wavelengths were 210 and 290 nm while the emission was limited by the KV-370 cut-off filter. The Dansyl derivative of a proline-leucine-glycine tripeptide was chromatographed on LiChrosorb SI 60 ( 5 pm) 25 cm X 3.2 mm (id.) column. The column was eluted with pentane-methanol (88:12 v/v) solvent a t 1 mL/min flow rate. The eluate was monitored by the fluorescence detector at 218, 250, and 340 nm excitation wavelengths; emission was limited by KV 370, and 550 cut-off filters. The underivatized indole decapeptide compound was chromatographed on Spherisorb ODS support packed in a 15 cm X 4.0 mm column. The column was eluted with 0.05 M pH 6.5 phosphate buffer-methanol-acetonitrile (6:3:1 v/v) solvent at 0.6 mL/min flow rate. The eluate was monitored by the UV detector at 220 nm, and by the fluorescence detector set at 220 nm excitation with KV-370 filtered emission. The propranolol and its 4-hydroxy analogue were chromatographed on Spherisorb ODS support packed in a 15 cm x 4.0 mm (i.d.1 column. The column was eluted with 0.02 M pH 6.0 phosphate buffer-methanol-acetonitrile (6:3:1 v/v) solvent at 1.2 mL/min flow rate. The eluate was monitored by the UV detector at 220 nm and by the fluorescence detector adjusted to either 210 or 280 nm excitation wavelength with KV-370 filter on the emission side.
E l u t i o n T i m e [nitn)
Figure 1. HPLC separation of phenolic estrogens on coupled in series LiChrosorb SI 60-LiChrosorb NH, columns; fluorometric vs. UV absorbance detection. Fluorometric (210 rim, excitation) and UV absorbance (220 nm) detectors are coupled in series
RESULTS AND DISCUSSION Since the detection limits of a chromatographic system depend not only on the detector but also on the chromatographic efficiency, a relatively efficient chromatographic system yielding about 8000 theoretical plates was developed for the separation of estrogens. Based on a previous study a low viscosity, pentane base chromatographic solvent was used to increase chromatographic efficiency. This system was also sufficiently selective t o resolve within 30 min the 17a-0 isomers of estrogen diols as well as estrone-equilin and estradiol-dihydroequilin pairs. Coupling of the LiChrosorb SI 60 support column to the LiChrosorb NH2 column was based on the observation that LiChrosorb SI 60 column alone yielded good resolution between a-p pairs but poor resolution of the estrone-equilin pair. In contrast, LiChrosorb NH2 column gave good resolution between estrone and equilin but poor resolution between 0-0 pairs. Addition of an amine to the chromatographic solvent used with LiChrosorb SI 60 column gave a resolution similar t o that obtained with LiChrosorb N H 2 column eluted without amine in the chromatographic solvent. Figure 1 illustrates the separation achieved with the LiChrosorb SI 60-LiChrosorb N H 2 columns coupled in series and contrasts the UV and fluorometric detection sensitivity; the spectrofluorometric and UV detectors were also coupled in series. Note that since equilenin estrogens have two conjugated aromatic rings which yield higher relative fluorometric responses than the estrogens with only one aromatic ring, the amount of equilenins injected on column is relatively small. Thus the chromatogram in Figure 1 was obtained with a mixture of estrogens containing 17 rig of estrone (ES),9 ng of equilin (EQ), about 0.3 ng of equilenin (EQN), 33 ng of 17a-estradiol (WED),17@-estradiol(@-ED),17a-dihydroequilin (a-DHEQ) and 170-dihydroequilin (P.DHEQ), and about 0.2 ng of 17a-dihydroequilenin (a-DHEQN) and 170-dihydroequilenin (0-DHEQN). A similar Chromatogram was obtained with the spectrofluorometric detector adjusted t o a 290-nm excitation wavelength; however, compared t o 210-nm excitation, detection a t 290-nm excitation was found t o be 6-9 times less sensitive for estrone, equilin, estradiols, and di-
(a,
ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
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I
1
I
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E l u t i o n Time (min)
Flgure 2. HPLC separation of estrone (Es) and equilenin (Eqn) on coupled in series LiChrosorb SI 60-LiChrosorb NH, columns: fluorometric detection limits at 210 nm excitation. On-column injection: estrone 0.4 ng, equilenin 12 pg
L
i L I 200
300
400
W a v e l e n g t h [nm)
Figure 3. UV absorbance spectrum of Dansyl tripeptide (prolineleucine-glycine) derivative
hydroequilins, and 13 times less sensitive for equilenin and dihydroequilenin compounds. Since the chromatograms illustrated in Figure l were obtained with the UV and fluorometric detectors coupled in series, the amounts of estrogen compounds injected on column are close to the detection limit of the UV detector, but above the detection limit of the fluorometric detector. Figure 2 illustrates separation and detection of E S and EQN in amounts close to their fluorometric detection limits at 210-nm excitation wavelength. At a signal to noise ratio 2:1, the apparent detection limit of ES is about 0.1 ng while EQN could be detected at low (4-2) pg level. Although the above UV detection limits were 100 to 200 times less sensitive than fluorescence a t 210-nm excitation wavelength, they were nevertheless in the range of reported fluorometric detection limits of Dansyl estrogen and amino acid derivatives ( I , 8). The apparent low sensitivity of the reported fluorometric detection limits of Dansyl derivatives may be attributed to less efficient detectors and to the fact that the commonly used excitation and emission wavelengths (about 340 and 520 nm, respectively) with Dansyl derivatives do not yield the most sensitive detection limits possible. According to the UV absorption spectrum (which is similar to the corrected fluorescence excitation spectrum ( 5 , 6),the Dansyl derivative has excitation maxima a t 340, 250, and 218 nm (See Figure 3); the commonly used excitation wavelength is 340 nm which corresponds to the lowest peak on the UV absorption spectrum (or corrected fluorescence excitation spectrum). This observation was confirmed by the relative size of H P L C peaks observed with a Dansyl tripeptide derivative monitored at 218, 250, and 340-nm excitation wavelengths; at 218 nm, the detection limit was 15 pg (Figure 4, a , b, c , e ) . The above fluorometric sensitivity gain at short excitation wavelengths was also observed in HPLC analysis of a large molecular weight LH-FSH releasing decapeptide hormone (9) 1838
ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
E l u t i o n Time (min)
Figure 4. HPLC chromatograms of Dansyl tripeptide derivative: UV absorbance vs. fluorometric detection at different excitation wavelengths. ( a ) and (e), fluorescence at 218 nm excitation: ( b ) , fluorescence at 250 nm excitation: (c), fluorescence at 340 nm excitation; ( d )and (0, UV absorbance at 218 nm; detectors are coupled in series. On column injection: ( a ) - ( d ) = 300 ng, ( e ) - ( f ) = 30 pg
which contains one tryptophan moiety. Although the uncorrected fluorescence excitation spectrum of the above underivatized peptide scanned with a xenon light source spectrofluorometer suggested a 283-nm excitation wavelength, the UV absorbance spectrum of the same peptide had considerably higher absorbance a t 220 nm than at 283 nm which implies significant sensitivity gain a t 220-nm excitation wavelength. The 220-nm excitation maximum yielded about a 3.4 times more sensitive detection limit than the 283-nm excitation maximum (See Figure 5 a , and b). At 220-nm excitation wavelength, the HPLC detection limit of the tryptophan containing decapeptide was found to be about 0.3 ng (See Figure 5d) which corresponds to 0.25 pm since the molecular weight of the decapeptide is about 1200. The UV detection limit was about 5 times less sensitive (Figure 5 e ) . The fluorometric detection limit observed in this study of a tryptophan (indole) peptide may be compared to previously reported detection limits of fluorescamine amino acid derivatives ( 2 )and fluorometric detection of indole compounds ( I O ) . Both previously reported studies involved an excitation wavelength above 260 nm and achieved 5-15 ng or 25-75 pm detection limits. As shown above, the detection limit observed with a tryptophan containing decapeptide at 220-nm excitation is about 0.3 ng, which is equivalent t o 0.25 pm. A significant short excitation wavelength sensitivity gain was also observed with propranolol, an adrenergic blocking agent, and its 4-hydroxy analogue. Comparison of Figure 6 and illustrates the relative intensity of the fluorometric signals
0 E U v)
E
-a !A
t
al
.-
-mal
a
I /
L
0
c 1
I
0
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I
io
I 20
Elution Time (min)
Figure 7. HPLC separation and detection of propranolol and 4hydroxypropranolol. Top chromatogram: fluorometric detection at 210-nm excitation, bottom chromatogram: IJV absorbance at 220 nm. On-column injection: 0.4 ng of each compound
analogue at 210-nm excitation is about 40 pg. The reported secondary amine Dansyl derivative detection limits were in the 1-10 ng range (3). i
I
7.5
7.5
E l u t i o n T i m e (rnin)
Figure 5. HPLC chromatograms of LH-FSH releasing indole decapeptde hormone: UV absorbance vs. fluorometric detection at different excitation wavelengths. ( a ) and ( d ) , fluorescence at 220 nm excitation; ( b ) , fluorescence at 283 nm excitation; (c)and ( e ) ,UV absorbance at 220 nm. On column injection: ( a ) - ( c ) = 39 ng, ( d ) and ( e ) = 1.6 ng
tI
n
/I
al
.-c
-m
0
K
CONCLUSIONS The results presented in this study indicate that phenolic estrogens, tryptophan (indole) peptides, and a naphthyl adrenergic blocking agent (propranolol) yielded about 10 times more sensitive detection limits with excitation wavelengths below 250 nm than with excitation wavelengths above 260 nm. A similar gain in sensitivity was observed with the Dansyl derivative of a tripeptide. The observed gain in the sensitivity of fluorometric detection was deduced from the UV absorption spectra which are similar t o corrected fluorescence excitation spectra. However, in order to take advantage of this sensitivity gain, a fluorometer with a light source which generates sufficient energy a t short wavelengths (such as a Deuterium lamp) is necessary. Of course some compounds such as polyaromatics, which have their major UV absorbance and corrected fluorescence excitation maxima above 250 nm, will not yield the sensitivity gains observed in this study with excitation wavelengths below 250 nm. ACKNOWLEDGMENT The authors thank R. D. Daley for hlelpful discussions and review of the manuscript and C. E. Olrzech for preparation of Dansyl derivative of tripeptide. LITERATURE CITED
0
10
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E l u t i o n Time (min)
Figure 6.
HPLC separation and detection of propranolol and 4hydroxypropranolol. Top chromatogram: fluorometric detection at 280 nm excitation, bottom chromatogram: UV absorbance at 220 nm, on-column injection: 10 ng of each compound
observed with 280-nm and 210-nm excitation wavelengths and the UV absorbance signal at 220 nm. T h e above comparison indicates that the fluorometric response of propranolol and its 4-hydroxy analogue with 210-nm excitation is about 10 times more sensitive than with 280-nm excitation, and about 20 times more sensitive than UV absorbance a t 220 nm. The apparent detection limit for propranolol and its 4-hydroxy
(1) R. W. Frei and J. F. Lawrence J . Chromatogr., 83. 321 (1973). (2) S.Udenfriend, S.Stein, P. Bohlen, W. Dairlman, W. Leimgruber, and M. Weigele, Science, 178, 871 (1972). (3) R. W. Frei, W. Santi, and M. Thomas, J . Chromatogr., 116,365 (1976). (4) R. W. Frei and W. Santi, Fresenius' Z . Anal. Chem., 277, 303 (1975). (5) G. G. Guilbault in T. S. West, Ed., "MTP International Review of Science, Analytical Chemistry, Part 1. Volume 12,"Butterworths, London 1973, Chap. 7, p 219. (6) D. A. Terhaar and T. J. Porro, Paper No. ,483,Pittsburgh Conference, Cleveland, Ohio, February 28-March 4, 1'377. (7) G. J. Krol, C. A. Mannan, F. 0. Gemmill, Jr , G. E. Hicks, and 6.T. Kho, J . Chromatogr.. 74, 43 (1972). (8) L. P. Penzer and G. W. Ortel, J . Chromalog., 51, 325 (1970). (9) Y. Baba, H. Matsuo, and A. V. Schalb, B b c k m . Biophys. Res. Commun., 44, 459 (1971). (IO) A. P. Graffeo and B. L. Karger, Clin. Che,m. (Winston-Salem, N . C . ) , 22, 164 (1976).
RECEIVED for review May 23, 1977. Accepted July 27, 1977. ANALYTICAL CHEMISTRY, VOL. 49,NO.
12,OCTOBER 1977
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