Anal. Chem. 1986, 58, 2647-2649
measurement, the shapes of both curves were identical where overlapped. With the multimode spectra the shapes were drastically different in these regions, and splicing was not possible. As a result, overlap was eliminated to facilitate plotting the data. The flaring on the low wavelength end indicates that the square of the average power is under-correcting due to a decrease in the peak-to-average power ratio. Although the high wavelength end of the spectrum does not have the correct shape, the effect is much less severe. This difference in behavior is possibly due to variations in the optical cavity length as the laser is tuned. The bis-MSB corrected spectrum of a-NPO is included in Figure 2 so that a direct comparison to the single-mode data is possible. In this experiment both the signal and the reference were measured simultaneously. The signal was divided by the reference and multiplied by the relative bis-MSB data given in Table I. Again splicing was accomplished by minimizing x2 across the overlap region. The average discrepancy between the two curves was less than 1% . The multimode experiment used a 5050 metallic beam splitter to evenly divide the laser between the sample and the reference. Other arrangements of sample and reference gave less reproducible results, for example, interchanging sample and reference solutions (double beam in time), or refocusing the laser after the sample. This latter arrangement is intrinsically inferior because the sample can change the peakto-average power ratio of the source due to two-photon absorption. Bis-MSB has been shown to be an ideal chemical reference for removing the source dependencies from two-photon spectra. The emission is devoid of any one-photon contributions, generating a response due only to the instantaneous
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power of the optical field. As such, the molecule provides information not available from average power measurements. The spectrally broad absorption provides correction factors that vary slowly as demonstrated by the values in Tables I and 11. Although these studies used half of the laser power to irradiate the reference, this was only done for convenience. Due to the extremely large bis-MSB cross section, it should be possible to use about 80% of the beam for sample excitation. The only nonideal behavior is the discrepancy between the results obtained with linear and circularly polarized radiation. As a result, the linear-to-circular transformation has to be made between the beam splitter and the sample. This requires the assumption that no wavelength-dependent changes in the peak-to-average power ratio are generated by the quarter-wave plate or Fresnel rhomb used to effect the transformation.
LITERATURE CITED (1) Kaiser, W.; Garrett, C. G. fhys. Rev. Lett. 1961, 7 , 229-231. (2) McCiain, W. M. J. Chem. fhys. 1971, 5 5 , 2789-2796. (3) Mieienz, K. D. Optical Radiation Measurements : Measurement of Photoluminescence;Academic Press: New York, 1982; Vol. 3. (4) Johnson, M. C.; Lytle, F. E. J . Appl. fhys. 1980, 51, 2445-2449. (5) Swofford, R.; McCiain, W. M. Chem. fhys. Lett. 1975, 3 4 , 455-460. (6) Cataiano, I. M.; Cingolani, A. Appl. fhys. Lett. 1981, 38, 745-747. (7) Beriman, I. B. Handbook of Nuorescence Spectra of Aromatic Molecules; Academic Press: New York, 1971. (8) Weber, H. P. I€€€ J. Quantum Electron. 1971, E - 7 , 189-195. (9) Johnson, M. C. MS Thesis, Purdue University, 1979.
RECEIVED for review May 29, 1986. Accepted July 14, 1986. This research was supported by National Science Foundation Grant CHE-8320158. S.M.K. acknowledges support of the Purdue Research Foundation through a David Ross Fellowship.
Spectrofluorometric Method for the Determination of A/-Nitroso Compounds Dolores Ruiz Lopez,* Carmen Lopez Martinez, and Rafael Garcia Villanova Departamento de Bromatologia, Toxicologia y Analisis Quimico Aplicado, Facultad de Farmacia, Universidad de Granada, 18001 Granada, S p a i n
A spectrofluorometric method Is proposed for the determination of N-nitroso compounds. This method is based on the reductlon of these compounds wlth the alloy Ni-Ai in an aikailne medium In order to obtain the corresponding secondary amines. The amines that are obtained are then sublected to a firstphase reaction with fiuorescamlne and a secondphase reactlon with L-leucine-L-alanine to obtain the fluorescent product. The process of reducing N-nitroso compounds to amines was carried out using gas chromatography. The relationship between the intensity of fluorescence and the concentration was vaild for quantities of the N-NO group in the range of 0.5-20 nM. The pH plays an essential role in both phases of the reactlon.
The investigation of nitrosamines, especially in food, is a topic of growing importance due to the carcinogenic action of these compounds ( 1 ) . The presence of these N-nitroso
Scheme I
compounds in food is a serious sanitary problem because of their role in the origin of cancers, above all in hepatic cancer. Such potential hazards necessitate the analysis of foods and beverages when nitrate or nitrite ions have been used in their preparation and when processes of fermentation have been applied. The analytical methods for the detection and determination of N-nitroso compounds are numerous (2-4). The majority
0003-2700/86/0358-2647$01.50/0@ 1986 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 13, NOVEMBER 1986 U w
Table 1. Yield in the Reduction of N-Nitroso Compounds to Amines nitrosamine
initial amt! mg
yield, 70
std dev
error over the av, %
NDMA NEMA NDEA NDPA NEBA NDBA NPYR
3.12 2.82 2.82 2.73 2.76 2.73 3.24
89.2 85.0 94.7 93.0 85.6 82.8 76.1
0.748 0.894 0.489 1.166 0.748 1.019 0.489
1.16 1.25 0.53 3.44 1.58 4.19 0.75
P
of these methods are based on gas chromatography with different detectors. The spectrofluorometric method for the global determination of nitrosamines, the method that we propose, is based on the condensation of secondary amines with fluorescamine (5) (the first phase of the reaction). The resulting product is made fluorescent by means of a reaction with the primary amine L-leucine-L-alanine(the second phase), according to the reaction given in Scheme I. The aim of this study is to confirm the suitability of the fluorometric method proposed for the determination of N nitroso compounds. I t permits a global determination of the >N-NO group by the reduction to secondary amines. EXPERIMENTAL SECTION Instrumentation. All measurements were made in a fluorescent spectrophotometer (Perkin-Elmer Model 204), equipped with a Osram X80 xenon lamp (Hitachi Perkin-Elmer trademark). The products of reduction of the nitrosamines to amines were analyzed with a Sigma 3-B gas chromatograph with N/P detector and a Sigma 15 computer-register (both from Perkin-Elmer). A column (2 m X 2 mm i.d.) of Carbowax 20 M at 10% + 2% KOH on Chromosorb W-AW was used under the following conditions: detector and injector temperature, 250 "C; initial temperature of the oven, 80 "C for 1 min; ramp, 20 "C/min; final temperature, 170 "C; and flow rate, 20 mL/min. Reagents. Analytical grade reagents were used: Ni-A1 alloy (FEROSA) N-nitrosodimethylamine (NDMA), N-nitrosoethylmethylamine (NEMA), N-nitrosodiethylamine (NDEA), N nitrosodipropylamine (NDPA), N-nitrosoethylbutylamine (NEBA), N-nitrosopropylbutylamine (NPBA), and N-nitrosopyrrolidine (NPYR)(SigmaChemical). Stock Solutions. The following stock solutions were used: fluorescamine solution (Fluka) 0.02% in acetone (Merck) and M, diethylamine L-leucine-L-alanine (Sigma Chemical) 2 X solution (DEA),dipropylamine (DPA), dibutylamine (DBA),and pyrrolidine (PYR) 5 X M. Buffer solutions were prepared by combining appropiate volumes of 0.1 M KH2P04and 0.1 M NaOH for pH 6.5 and 0.1 M H,B03 and KCl and NaOH for pH 9.
Procedure. Reduction of Nitrosoamines. In a graduated centrifuge tube, 3 pL of each of the above pure nitrosamines is dissolved in 2 mL of ethyl ether, whose concentration is shown in Table I. Two milliliters of 0.5 M KOH solution is added. The organic solvent is evaporated in a 60 "C water bath. Powdered Ni-A1 alloy (0.1 g) is added. After allowing 30 rnin for reduction time, it was centrifuged and 100 pL of the supernatant was taken. This volume was diluted with a borate buffer solution (pH 9) until 10 mL was obtained. This dilution was carried out in order to obtained adequate nitrosamine concentrations (0.5-20 nM/50 pL) for the standard curve. For condensation with fluorescamine, 50 pL of the above solution was taken, to which 100 pL of the borate buffer solution (pH 9) and 100 pL of fluorescamine solution were added. After 1 min of agitation, it was placed in an ice bath for 5 min. Afterward, 1 mL of buffer phosphate solution (pH 6.5) and 0.5 mL of L-leucine-L-alaninesolution were added. This was maintained in a water bath at 70 "C for 15 min. Finally, 1mL of twice-distilled water was added, and fluorescence was measured at 20 "C, 390 nm of excitation and 480 nm of emission. Before the intensity was measured, the zero of the apparatus was adjusted with the blank and the 100 adjusted with 20 nM
9P
0
2
4
6
TIME (rnin. 1
Figure 1. Chromatogram of the process of reduction of NDEA (2.83 mg/2 mL of 0.5 M KOH) by DEA: (a) at 15-min reduction time and (b) at 30-min reduction time.
of DEA, according to the process of condensation with fluorescamine. R E S U L T S AND DISCUSSION Characteristics of the Reaction. The reaction product could be observed when the reaction was monitored by gas chromatography. For this purpose, NDEA (2.82 mg) was reduced under the conditions already described, and 2 KLof the final product of reduction was injected into the gas chromatograph. The DEA peak was identified with a pure solution of this amine. I t can be observed that the reduction of DEA took 30 rnin to complete (Figure 1). The yield in the reduction of nitrosamines to amines was calculated for each of the nitrosamines tested using the measurements of fluorescent intensity (Table I). Intensity of Fluorescence in Relation to Concentration of Amino. The relationship between the intensity of fluorescence and the concentration of the secondary amine was established. Tests were conducted using solutions of DEA between 0.5 and 20 nM after condensation with fluorescamine. In this range of concentration the relation intensity of fluorescence/concentration is a linear function. This same experiment was also conducted using an equimolecular solution of secondary amines (DEA, DPA, DBA, PYR) in the same range of concentration (0.5-20 mM), expressed in amine group. An identical linear function was obtained. Influence of pH i n the Fluorescamine-Secondary Amine Reaction. The effect of p H in the first phase of the reaction f fluorescarnine-secondary amine) as well as in the second phase (product of condensation with L-leucine-L-alanine) was studied. Figure 2 shows the effect of p H in each phase. I t can be seen that the maximum intensity of fluorescence in achieved a t pH 8-9 for the first phase and pH 6.5 for the second phase. In the first phase, the secondary amine condenses with fluorescarnine and opens the lactonic ring, causing the fluorescamine molecule to lose its rigidity and, consequently,
Anal. Chem. 1988, 58, 2649-2653
7 40
Figure 2. Effect of pH on the first and second phases of the fluorescamine-secondary amine reaction.
2849
fluorogenic reagent. If, on the other hand, the pH is slightly higher, the fluorescamine is hydrolyzed and therefore unable to react with the amine. The fluorescamine-secondary amine reaction was found to remain stable for over 24 h. The proposed method does not require the identification of each of the nitrosamines present in a sample (as occurs with the chromatographic method), since the N-nitroso compounds are evaluated as a whole, given their reduction to secondary amines. The sensitivity of this method is also considerable, and the minimum detectable level for this procedure is 10 nM of N-nitroso compounds/mL. Registry No. Ni-AI alloy, 11114-68-4;NDMA, 62-75-9; NEMA, 10595-95-6;NDEA, 55-18-5;NDPA, 621-64-7; NEBA, 4549-44-4; NPBA, 25413-64-3;NPYR, 930-55-2;fluorescamine,38183-12-9; L-leucine-L-alanine,7298-84-2.
LITERATURE CITED its innate fluorescence. In the second phase, the lactonic ring closes up when the previous product reacts with a primary amine (L-Leu-L-Ala). The pH plays a vital role in these two phases of reaction. If the pH is low, the amines could be charged by the protons, making a reaction with fluorescamine impossible. Moreover, an increased acidity could provoke the precipitation of the
(1) Magee, P.; Barnes, J. 8 . J. Cancer 1958, 70, 114-122. (2) Cutaia, A. J. J. Assoc. Off. Anal. Chem. 1982, 65(3), 584-587. (3) Sen, N. P.; Seaman, S.; Karpinsky, K. Assoc. Off. Anal. Chem. 1984, 67(2), 232-235. (4) Samueisson, R. Anal. Chim. Acta 1979, 708, 213-219. (5) Nakamura, H.; Tamura, 2. Anal. Chem. 1980, 52, 2087-2092.
RECEIVED for review May 2, 1986. Accepted June 27, 1986.
Determination of Protein in Human Serum by High-Performance Liquid Chromatography with Semiconductor Laser Fluorometric Detection Kouji Sauda, Totaro Imasaka, and Nobuhiko Ishibashi* Faculty of Engineering, Kyushu University, Hakozaki, Fukuoka 812, Japan
Indocyanlne green (ICG) was found to become fluorescent when bound with proteln. Then, protein in human serum was labeled with ICG,and the complex was separated by a gel flitration column. The eluted sample was detected by a fluorometrlc system uslng a semiconductor laser (780 nm, 15 mW) as an exciting source. a,-Lipoprotein and yglobulln were preferentlally comblned wlth I C 0 and gave large peaks In the chromatogram, though the albumin content was more than 10 tlmes larger than those compounds. The detectlon limit was 1.3 pmoi for albumln, which was -1-2 orders of magnitude better than the value obtained by conventlonal spectrophotometric and fluorometric detectors.
Laser fluorometry has been used as a sensitive detector for high-performance liquid chromatography (HPLC) because of its good beam coherence and large photon flux. Various lasers such as a continuous wave (CW) argon ion laser and a pulsed nitrogen laser pumped dye laser are used as a light source for the fluorometric detector in HPLC. Diebold and Zare constructed the windowless flow cell, which was designed to reject background light scatter from the cell windows, and they demonstrated ultratrace analysis of aflatoxins by using a helium-cadmium laser (325 nm) (I). Other researchers have also shown advantages of the laser fluorometric detector and have demonstrated many analytical applications (2-9), but, the lasers have large dimensions and are expensive. Moreover, the lasers have some difficulties in their operation and 0003-2700/86/0358-2649$01.50/0
maintenance. As such, the laser fluorometric HPLC detector has not been practical for use in the commercial instrument. Recently, a semiconductor laser has been developed for the application to a videodisc system, since it is very small apd less expensive. The continuous wave semiconductor laser is currently used as a light source for photoacoustic spectrometry (lo), conventional absorption spectrometry (11-13), fluorometry (14), thermal lens spectrometry (151, and heterodyne spectrometry (16). On the other hand, a picosecond and highly repetitive pulsed semiconductor laser is advantageous for measurements of the fluorescence lifetime by a time-correlated photon counting system (17). Among these spectrometric methods, semiconductor laser fluorometry is most sensitive, allowing sample detection at M levels when a 3-mW laser is used. It is emphasized that blank fluorescence from a solvent is completely negligible in near-infrared fluorometry. However, the wavelength of the semiconductor laser commercially available is limited to 750-1300 nm, and therefore, it is useful only in a few analytical applications (14). Some polymethine dyes are known to have absorption bands at around 600-900 nm. They have large molar absorptivities (>l00OOO) and are strongly fluorescent, and as such, they have been used as laser dyes. Therefore, they have been detected at ultratrace levels by semiconductor laser fluorometry (14, 17,18). However, most samples such as biochemical substances have neither an absorption band nor fluorescence emission in this wavelength region. For determination of such substances, the sample molecule should be labeled with a 0 1986 American Chemical Society
