100-
>
60-
> 0
,
W ti
ar
4c-
0
0
L
-
5
L 10
15
ACID CONCENTRATIONS,
20
-25
%(V./V'
Figure 5. Comparison of acid interference on arsenic (As") recovery when 2 pellets of NaBH, were used. (A)H2S04.( 0 )NH03, (0) HC104 in 15 mL of 20 % (v/v) HCI solution. (A)H2S04,(e)HN03, (H) HCIO, in 30 mL of 2 0 % (v/v) HCI solution
T h e KI pretreatment did not compensate for the acid interference. Up to 5 mL of 20% (w/v) K I solution added to the acid solution did not improve the peak height. Peak area was not measured because of multiple peaks. The effect of the oxidation state of arsenic in the solution on arsenic measurement is shown in Table 111. The solution containing 1 Fg of As" produced a smaller peak than that containing As3+. However, prior treatment of the solution containing 1 pg of As5+ with 1 mL of 20% (w/v) K I increased the peak height to that of As3+. When two pellets of N&H4 were added and the gas collection method was used, the two arsenic species (As3+,As") produced similar peak heights. This indicates that prereduction of As5+to As3+by KI is not necessary when the above method is used. Peak area was not affected by the oxidation state of arsenic in the solution. A similar observation has been reported by others ( 4 ) . Biological samples were analyzed by the gas collection method using 2 pellets of NaBH, with the effect of acid interference on recovery of arsenic in mind. Over 95% recovery of added arsenic was found in all samples when the amount of residual acids was carefully controlled prior to NaBH, addition (Table IV).
CONCLUSIONS The experiments performed allow the following conclusions. (I). Calibration curves for As5+, obtained using the continuous flow method, were more linear than those constructed using the gas collection method. (2). When peak height was used to calculate arsenic, all three acids (H2S04,"OB, HC10,) interfered with arsenic recovery at higher concentrations. The pattern was found not to be dependent on the total amount of acid present, b u t rather on its concentration. Peak area remained relatively constant irrespective of acid concentrations in the solution u p to 24% (v/v). An attempt to overcome this acid interference by K I addition was not successful. (3). T h e two arsenic species (As3+,As5+) produced similar peak areas while peak heights from As5+were lower than those of As3+. Pretreatment of As5+ solution with 1 mL of 20% (w/v) K I solution compensated for this variance. (4). By controlling the amount of residual acid prior to NaBH4 addition, according to the above findings over 95% recovery of added arsenic (As"+)was achieved in all biological samples. In summary, the overall conclusion to be made is that residual acids in the samples do interfere with arsine generation. Steps to reduce the amount of residual acid in samples should be taken, or the measurement of peak area can be employed to correct for interferences posed. ACKNOWLEDGMENT The authors thank Richard Harnish of UCLA for his technical assistance. LITERATURE CITED (1) (2) (3) (4) (5) (6)
F. J. Fernandez, At. Absorppr. News/., 12 (4) 93 (1973). A. E. Smith, Analyst (London), 100, 300 (1975). T. Matura and G. Sudoh, Anal. Chim. Acta, 7 7 , 37 (1975). J. A. Fiorino. J. W. Jones, and S. G. Capar, Anal. Chem., 48, 120 (1976). F. D. Pierce and H. R. Brown, Anal. Chem., 48, 693 (1976). F. A . Cotton and G. Wilkinson. Adv. Inorg. Chem., 572 (1966).
RECEIVED for review March 14, 1977. Accepted July 22, 1977. This investigation was supported by research grant R803798-01, U. S. Environmental Protection Agency.
Isotopic Assay of Nanomole Amounts of Nitrogen- 15 Labeled Amino Acids by Collision-Induced Dissociation Mass Spectrometry James H. McReynolds' and Michael Anbar' Mass Spectrometry Research Center, Stanford Research Institute, Menlo Park, California 94025
A method of measuring the a-"N content of amino acids by field ionization and collision-induced dissociation mass spectrometry is described. Molecular ions produced by field ionization are subjected to collisional dissociation to produce nitrogen-containing fragments that can be used to measure the I5Ncontent of 0.15 atom % enrichment on 50 nmol of an amino acid with an accuracy and precislon of 10% of the absolute I5N content. P r e s e n t address, D e p a r t m e n t of B i o p h y s i c a l Sciences, State U n i v e r s i t y of New Y o r k at Buffalo, 4234 R i d g e L e a Road, A m h e r s t ,
N.Y. 14226. 1832
ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
A number of different methods have been developed for the analysis of lSN in metabolites. The traditional methods involve the careful separation of the compounds of interest from other nitrogen-containing materials and Kjeldahl digestion to convert the sample nitrogen to ammonia and a final oxidation of the NH3 to N1 with hypohromide. T h e I5N content of the nitrogen may be determined by using either precision isotope ratio mass spectrometry or emission spectrometry ( 1 , 2 ) . These methods, besides being unable to determine the number or location of the "N atoms in the original molecule, require several milligrams of sample to obtain the amount of pure nitrogen needed for an accurate
analysis (3-5). T h e use of computer control and ion counting techniques has reduced t h e sample requirements for isotope ratio analysis allowing a precision of 0.5 per mil standard deviation with g of carbon as C 0 2 ,while 'jN has been analyzed with 10% accuracy as NH4+using isobutane chemical ionization mass spectrometry (CIMS) with samples as small as 0.25 pmol (6, 7 ) . For measurements t h a t d o not require high precision, it is also possible t o measure the 'jN content by direct recording of t h e ratio of a suitable molecular or fragment ion from the undegraded sample molecule. Biemann determined t h e 15N content of a mixture of amino acid ethyl esters using this technique; however, the measurable enrichments were limited t o about 0.3% (8). Using both t h e direct ratio recording technique and a dual collector system, Waller and his coworkers were able t o measure 15N enrichment in ricinine (9). T h i s type of measurement is sensitive t o interference from fragment ions with t h e nominal mass of either nitrogen containing species. Ion-molecule reactions in t h e source created additional variance, thus requiring measurements at a constant source pressure. Another approach involves t h e measurement of t h e isotopic multiplets at a single nominal mass on a high resolution instrument. A precision of 0.5% for creatinine at natural abundance was reported for this technique using a few micrograms of sample (10). Isotopic abundance can also be measured by ion kinetic energy spectrometry (IKES), b u t t h e potential of this technique has not been as thoroughly explored as those described above ( I I , 1 2 ) . In this paper we show the potential usefulness of this technique by demonstrating t h e isotopic assay of '"N in two amino acids, alanine and leucine, in quantities of 50 nmol using field ionization-collision induced dissociation mass spectrometry (FI-CID-MS). An interference-free determination is achieved by monitoring a simple secondary ion (NH,') formed by CID from a protonated molecular ion formed by FI. EXPERIMENTAL Apparatus. All spectra were obtained with a FI-CID-MS system that has been described previously (13). For recording ratios of CID fragment ions produced from different molecular ions, a special accelerating voltage switching circuit and two 4096-channel multichannel analyzers (MCA) (Ortec Model 6240) were used. The switching circuit consists of two digital clocks with independently preset periods ranging from 0.01 to 10.0 s. Upon receiving a manual start pulse, timer A triggers the first MCA and gates the multiplier output pulses into this analyzer. The analyzer sweep drives the electrostatic analyzer (ESA) voltage to sweep a selected fragment ion mass range. At the end of its preset period, timer A starts timer B, triggers an analog signal used to program the ion accelerating voltage, starts the second MCA, and gates the multiplier into the second MCA. The ESA is swept through the same mass range to record the fragment ions produced from a different primary ion beam. The start pulses for each timer pass through adjustable delays to allow settling of the ion acceleration voltage between switching. Upon receipt of a stop pulse, the system runs until timer B completes its cycle. The programming voltage is applied through an operational amplifier with adjustable gain to the accelerating voltage supply (Spellman Model RHR15PN30/RVC). The detaih of the circuit are described elsewhere in greater detail (14). Reagents. All unlabeled amino acids were obtained from Nutritional Biochemicals Corporation. Samples of a-I5N labeled glycine, alanine, and leucine were obtained from Prochem Ltd., Maplewood, N.J. FIMS of Amino Acids. Glycine, alanine, valine, leucine, and isoleucine were analyzed by evaporation from a solid probe at temperatures ranging from 50-120 "C. The temperature of the multipoint FI source was maintained between 150 and 200 "C. All samples were analyzed with a nominal point potential of +7.5 kV and a counterelectrode potential of +6 kV.
C H I - COOH hV2
Y
,
I
30
coed
" Z
44
I
",c o o i kH2
72
I
I ' 118
Flgure 1. Field ionization spectra of free amino acids. (a) Glycine, (b) Alanine, (c) Valine, (d) Leucine. F I Spectrum of isoleucine was identical to that of leucine
FI-CID Analysis of Amino Acids. The molecular ions produced by FI were subjected to CID using hydrogen collision gas a t a collision cell pressure of approximately 5 x Torr, resulting in a pressure of 1 X Torr in the analyzer. CID spectra were recorded by integrating repeated ESA sweeps (10 s) on the MCA over a period of appxoximately 5 min. Preparation of Calibration Curves. The percentage of "N in the labeled alanine and leucine was established as 94.3% and 98.270, respectively, from the peak area ratios of the molecular ion region of the FI spectra. In calcullating the "N content, the peak ratios were corrected for the ratios of M. M + 1, M + 2, etc. observed for the unlabeled standard. Stock solutions of labeled and unlabeled standards were made up in water to contain 50 nmol in a 10-pL aliquot ( - 5 mM). The FI mass spectra indicated that the amino acid standards were not chemically pure, so the relative concentration ratios of the hbeled to unlabeled stock solutions were determined mass spectrometricly. The 15N stock solution was diluted with the unlabeled stock solution to give a series of standards ranging from 0.16 to 2.6 atom 90 enrichment for alanine and from 0.20 to 6.76 atom 90 for leucine. Procedure for 15N/''N Isotope Ratio Determinations. Using the standard solutions, 10-pL aliquots containing approximately 50 nmol of either alanine or leucine were dried on the probe sample holders. Samples were evaporated into the source under the same conditions used for obtaining F I and FI-CID spectra. The magnet was tuned to the molecular ion of the amino acid with the analog output of the peak switching circuit off. The analog output signal was manually turned on and its level adjusted to focus the MH + 1 ion on the collision cell slit. The sweep width and offset controls of the ESA control were set to cover the voltage range corresponding to a mass range of 16-22 amu. In order to enhance the counting statistics, the timers and MCAs were set to provide a 1- or 2-s '3weep time for MH and a 10-s sweep time for M H + 1. Following the above tuning procedure, the collision gas was admitted and the automatic peak switching circuit was started to record the selected collisional fragments from MH and MH + 1 alternately until the samples were exhausted. RESULTS AND DISCUSSION FI Spectra of Free Amino Acids. T o establish t h e fundamental information for this work, we recorded t h e FI mass spectra of a number of free amino acids as shown in Figure 1. All of t h e amino acids studied gave a significant ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
1833
~
L GLYCINE MW
L - L E U C I N E MW 131 FI-CID
75
I "qk
CH2-COOH
7c
lOOX
"2 30
-,
I
i 28
A
I 1
1
1
I
I
0
.
1
1 50
n e 5"
-7
I
L ISOLEUCINE MW 131 F I C I D
I
1
+* I
CCZH
r 132
L ALAb4,NcEDMW 89
I
18
I!
28
At- ...AI
Y
50
100
50
re
Flgure 3. Field ionization-collisional dissociation spectra of leucine and isoleucine
100
me
Figure 2. Field ionization-collision induced dissociation spectra of glycine, alanine, and valine
yield of the protonated molecular ions (MH+) with the amine fragment (R-CH-NH2+) present a t 1 0 4 0 % of it. For all of the amino acids studied, the nonprotonated molecular ion was less t h a n 1% of the intensity of the protonated form. T h e yield of the amine fragment relative to MH' was observed to increase with higher source temperatures or increasing field. FI-CID S p e c t r a of Free Amino Acids. The CID spectra of the above amino acids were recorded according to the procedures described in the Experimental section and elsewhere (13). The FI-CID spectra are shown in Figures 2 and 3. As noted previously, the CID spectra are qualitatively similar to the E1 spectra of the same materials (15, 16). In particular, note that leucine and isoleucine are distinguishable by the CID spectra. In the case of isoleucine, m / z 29,41, 57, 69, and 74 are elevated relative to leucine, which produces a prominent m / z 44 in the low mass region. These same features are also present in the E1 spectra of the ethyl esters of these compounds (17). In all of the above CID spectra, there is a fragment at m / z 18 which may be due to either NH,+ or HzOf. Due to the 1834
ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
I
~
Figure 4. CID spectra of glycine, m l e 18 region (a) Fragments from m l e 76 (M 1) of 14N glycine, (b) Fragments from m l e 76 (MH) of 15N glycine
+
ubiquitous background of H 2 0 in all mass spectrometric systems, the m / z 18 ion is generally not meaningful. In the CID case, however, the m / z 18 can only arise from the primary ion being examined. To further elucidate the composition of the m / z fragment ions, the CID spectra were recorded on the M H 1 ions of alanine and valine (carrying natural abundances of 13C,D, and 15N). These spectra showed as additional peak a t m / z 19,
+
I
Table-I. Calibration Curves from Alanine and Leucine Observed :ratio Expected No. of (%), mean ratio, Detns ( + std dev) 1 5 1141, ~ Alanine
'I
0.382 0.563 0.746 1.05 1.69 2.98
II
I
0.45 0.56 0.73 1.09 1.67 3.04
(0.02) (0.02) (0.02)
5 2
(0.01) (0.01) (0.40)
3 2
2 3
Leucine 0.37 ( 0 . 0 3 ) 0 . 6 1 (0.08) 0.78 (0.03) 1.17 (0.3.4) 1.97 (0.01) 3.67 (0.34) 6 . 8 1 (0.09)
0.382 0.601 1
I
*c
1
I ' 20
m e
1
0.800 1.20
I
1
, .5
20
2.02 3.69 7.21
n e
Figure 5. Integrated CID spectra from alanine containing 2.6 atom % I5N. (a) Fragments from MH, 1 slsweep, (b) Fragments from MH 1 , 10 s/sweep
3 3 3
5 3
3 3
+
indicating that a significant portion of the m / z 18 was due to NH4+. This was verified by the analysis of -95% a-"N glycine, alanine, leucine, and lysine. Figure 4 shows this region of the FI-CID spectrum of m / z 76 of 14N-glycine and "Nglycine. D e t e r m i n a t i o n of the "N C o n t e n t of S t a n d a r d Solutions. The peak switching circuit and MCA were used as described in the Experimental section to obtain the ratios of m / t 19 from M H + 1 vs. m / z 18 from MH. Using 50 nmol of the amino acids, the counts were integrated for approximately 15 min during the sample evaporation. From the integrated spectra, the counts a t m / z 18 and 19 from M H and m / z 18, 19, and 20 from M H + 1 were recorded. T h e lsN/14N ratio was computed from the observed ion counts in the individual amu windows by the following formula:
In this equation, Z, and i, represent the ion counts a t fragment mass j produced from M H and the isotopic M H + 1, respectively. T h e times t M H and t M H + l are the dwell times on the two molecular ions. T h e correction, &, is a measure of the randomly scattered ion background. In addition to this background, some of the counts a t ili, may be due to H30+and to the tail of the larger adjacent peak, i18.Peak overlap is due to the broadening of the CID peaks by the conversion of internal bond energy into kinetic energy of the fragments (18). T h e second term of the numerator of the above equation corrects the i19counts for both of these contributions. T h e ratio computed also includes a minor contribution from 14NH3D+of about 0.06%, but since it is constant at all levels of 15N enrichment, a correction can easily be made. T h e CID technique avoids any sporadic background interference, since only specific fragments originating from specific parent ions are monitored, e.g., in the case of leucine only fragments of m / z 19 which originate from parent ions of 132 amu could interfere with the assay of 15NH4+from 15N leucine. T h e corrected counts a t m / z i19 in a typical sample at natural abundance ranged from 200 to 300 while 50 000 to 7 5 000 counts were recorded for Z18. T h e total background corrections were typically 100 to 200 counts a t iI9for such a sample. Figure 5 shows the ESA scans recorded for an alanine sample containing 2.6% cr-'jN. Table I summarizes the results obtained from the dilution curves on alanine and leucine. The expected isotope ratios were
Table 11. Regression Analysis of Calibration Curve Data Slope,
i
dev
std
Intercept, + std dev
r
0.055 (0.04) Cl.033 (0.06)
0.989 0.988
Alanine Weighted Unweighted
0.982 ( 0 . 0 4 ) 1.001(0.04)
Weighted Unweighted
0.962 (0.018) 0.014 (0.026) 0.996 0.949 (0.01) 0.044 (0.043) 0.998
Leucine
calculated from the known dilutions a n d the isotopic abundances of the labeled and unlabeled standards according to the method reviewed by Schoeller (19). The observed ratios were fitted to the theoretical ratios by linear regression analysis. The analysis was performed on bloth the unweighted data and on the data weighted by the inverse of the theoretical ratios, which is equivalent to a weighting according to the inverse of the variance predicted by counting statistics (19). T h e regression parameters of both curves are given in Table
11. The precision and accuracy of this method are within 10% of the isotope ratio down to the natural abundance level. The lowest levels determined in the dilution curve (0.15 atom YO enrichment) could still be measured on the level of individual amino acids in plasma using 1-mL sample sizes. This method should, therefore, be applicable to the study of amino acid metabolism and protein turnover rates. P r o j e c t e d I m p r o v e m e n t s i n t h e Methodology. T h e methodology described in this paper may be improved in four directions: (1)the efficiency of generation of the primary ions; (2) the efficiency of transmittance of the mass selected primary ion to the collision chamber; (3) the efficiency of producing the desired secondary ions; and (4) the resolution and abundance sensitivity of the CID spectrum. T h e overall primary ion formation and transmission efficiency achieved in this study is about 1015 ions/mol, giving about 5 x 10' primary ions of the major isotopic species and about 2 x I O 5 "N amino acid primary ions at natural abundance. Following collision, we obtain 0.1 to 1% of secondary ions of the desired composition (e.g., 15NH4)which limits our sensitivity and precision to 50 nmol and lo%, respectively. T h e relatively low efficiency of transmittance of FI generated ions is due to their inherently large angular divergence, which overshadows their reasonably high ionization yield (20). This limitation can be minimized by enhanced acceleration of the ions emerging from the FI source (13). It may be estimated that ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
1835
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. Bkhem., 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) a n d 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 a n d 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
