Use of Time Resolution To Eliminate Bilirubin Interference in the

Use of Time Resolution To Eliminate Bilirubin Interference in the Determination of Fluorescein. Frank V. Bright, George H. Vickers, and Gary M. Hieftj...
0 downloads 0 Views 365KB Size
1225

Anal. Chem. 1986, 58, 1225-1227 Campana, J. E.; Barlak, T. M.; Colton, R. J.; DeCorpo, J. J.; Wyatt, J. R.; Dunlap, B. 1. Phys. Rev. Len. 1981, 47, 1046-1049. Ens, W.; Beavis, R.; Standing, K. G. Phys. Rev. Lett. 1983, 50, 27-29. Baldwin, M. A,; Proctor, C. J.; Amster, 1. J.; McLafferty, F. W. I n t . J. Mass Spectrom. Ion Processes 1984, 54,97-107. Wlttmaack, K. Phys, Len. A 1979, 69A, 322-325. Castro, M. E.; Russel, D. H. Anal. Chem. 1985, 57,2290-2293. Colton, R . J.; Campana, J. E.; Barlak, T. M.; DeCorpo, J. J.; Wyatt, J. R. Rev. Sci. Instrum. 1980, 51, 1685-1689. McLafferty, F. W.; Todd, P. J.; McGllvery, D. C.; Baldwin, M. A. J. Am. Chem. SOC. 1980, 102,3360-3363. Campana, J. E.; Green, B. N. J. Am. Chem. SOC. 1984, 106, 531-535. Forsberg, L. S.; Dell, A,; Walton, D. J.; Ballou, C. E. J. B o / . Chem. 1982, 257,3555-3563. Katakuse, I.; Nakabushi, H.; Ichihara, T.; Fujita, Y.; Matsuo, T.; Sakurai, T.; Matsuda, H. Mass Spectrosc. 1985, 33, 145-147. Thum, F.; Hofer, W. 0. Surf. Sci. 1978, 90, 331-338. Beuhler, R. J.; Friedman, L. I n t . J. Mass Spectrom. I o n Phys. 197?, 23, 81-97. Staudenmaier, G.; Hofer, W. 0.; Llebl, H. Int. J. Mass Spectrom. I o n PhyS. 1976, 1 1 , 103-112.

(23) Beuhler, R. J ; Friedman, L. Nucl. Instrum. Methods 1980, 170, 309-31 5. (24) Sundqvist, B.; Hedin, A.; Hakansson, P.; Kamensky, 1.; Saiehpour, M.; Sawe, G. I n t . J. Mass Spectrom Ion Processes 1985, 65,69-89. (25) Wang, G. H.; Aberth, W.; Falick, A. M. I n t . J. Mass Spectrom. Ion Processes, in press. (26) Schueler, 6.; Beavis, R.;Ens, W.; Main, 0. E.; Standing, K. G. Surf. Sci. 1985, 160, 571-586. (27) Morita, Y.; Aberth, W.; Burlingame, A. L. Presented at the 32nd Annual Conference on Mass Spectrometer and Allied Topics, San Antonio, TX, May 1984. (28) McEwen, C. N. Anal. Chem. 1983, 55,967-968. (29) Barofsky. D. F.; Giessmann, U.; Bell, A. E.; Swanson, L. W. Anal. Chem. 1983, 55, 1318-1323. (30) McEwen, C. N.; Hass, R. J. Anal. Chem. 1985, 57,892-894. '

RECEIVED for review August 5, 1985. Resubmitted January 21, 1986. Accepted January 21, 1986. This work was supported by the National Institute of General Medical Science under Grant GM 32315.

Use of Time Resolution To Eliminate Bilirubin Interference in the Determination of Fluorescein Frank V. Bright, George H. Vickers, and Gary M. Hieftje*

Department of Chemistry, Indiana University, Bloomington, Indiana 47405

Timeresolved fluorescence spectrometry Is used to eliminate blllrubln Interference In the fluorometrlc datermlnatlon of fluorescein. Desplte complete spectral overlap of the excltatlon and emlsslon of these two gpecles, a difference In fluorescence llfetlme provldes the selectivity parameter used In this method. Results for the determlnatlon of 0.300 nM fluoresceln In the presence of up to 25.00 FM blllrubln (1:8.3 X IO4) using a sampling oscilloscope and an optical delay llne show excellent accuracy (

Figure 3. Instrument response (A) and fluorescence decay curves for bilirubin (B) and fluorescein (C) in 30 p M HSA. The slight negative excursion of the fluorescence decay curve is due to ringing within our system ( 74 - 76).

in the cross-correlation operation, the laser beam is sent to a corner-cube reflector that can be translated along the beam direction on a 2-m-long optical rail. The corner-cube reflector folds L the beam back upon itself with a horizontal displacement of approximately 4 cm; a roundtrip through the entire delay line results in an effective delay of 12 ns. The laser beam then excites L fluorescence or is scattered by the sample, and the resulting signal A S is directed through the monochromator and detected by the PMT. I IcCwUrLQ I E The output from the PMT is directed to the double-balanced R microwave mixer where it is cross-correlated with the reference pulse from the fast photodiode to yield the fluorescence decay curve (19-21). To enhance the signal-to-noiseratio, the laser beam is chopped at a low frequency after the fast photodiode but before the sample cell. This frequency is monitored by a photodiode placed after the sample cell. The reference signal from the photodiode and the output from the mixer are combined in a lock-in amplifier to minimize noise generated in the detection system. Reagents a n d Materials. All solutions were prepared in Figure 2. Schematic diagram of the optical-delay-line-basedtimedistilled deionized water. A 30 pM (0.10 M phosphate buffered, resolution instrument constructed for the determination of fluorescein. pH 7.50) human serum albumin stock solution (HSA, Sigma) was See text for details. used for all sample preparations. Fluorescein was purchased as the disodium salt (Sigma), and a 1.00 mM stock solution was a mode-locker frequency of 40.9800 MHz. Attempts at higher prepared by dissolving the appropriate amount of fluorescein in power operation led to unstable pulse generation. The resulting 100 mL of buffer followed by sonication for 30 min. A 1.00 mM fluorescence emission is collected by a double monochromator bilirubin (Sigma) stock solution was prepared by weighing the (Model 1680 Spectramate, Spex, Inc.). The spectral band-pass appropriate amount of bilirubin and dissolving it in the minimum for all results presented below is 9.0 nm; the emission is monitored volume of 1.0 N NaOH followed by dilution to 10 mL with 1.00 at 520.0 nm. Detection of the resulting fluorescence is achieved mM buffered HSA. It is important to note that the bilirubin will with an RCA 31024 photomultiplier tube (PMT) operated at -3500 rapidly photochemically degrade to biliverdin if it is not in an V dc. The output of the PMT is sent to an oscilloscope (Model approximately equimolar HSA solution. All fluorescence mea7844 mainframe, Model S4 sampling head, and Model 7 S l l surements were carried out in disposable polystyrene cuvettes sampling unit, Tektronix, Inc.), triggered at approximately 12-ns (Fisher) each of which contained 3.000 mL of solution. intervals by the mode-locker drive unit. The oscilloscoperecords Operation. A series of 10 fluorescein standards are employed the instrument response function for the system or the fluoresto generate a calibration curve over the 0.100-10.00 nM range. cence response of the sample, depending on whether a scattering The position of observation on the decay curve is then set either solution or a sample is placed in the sample cell holder, respecby the computer for the oscilloscope or manually for the delay tively. The output of the oscilloscope is sent to the lock-in amline. For all results presented here the delay was positioned at plifier (Model 5101, EG&G Princeton Applied Research), which 6.00 ns. The standards are then run sequentially and the intensity is referenced to the mechanical chopping frequency. The lock-in at the 6.00 ns delay plotted against fluorescein concentration. In amplifier reduces the noise introduced by the sampling head of an analogous fashion a series of fluorescein/bilirubin mixtures the oscilloscope (17). The output of the lock-in amplifier is then are then run under identical conditions, and the resulting sent to a MINC Model 11/23 digital computer that controls the fluorescence signal at the 6.00-11s delay is used to determine the scan rate of the oscilloscope time base and thereby allows indifluorescein concentration from the previously prepared calibration vidual points on the fluorescence decay curve to be monitored curve. Each measurement required 30 s, and each was the average independently. of 100 samplings. Time-ResolvedFluorescence Using an Optical Delay Line and Double-Balanced Microwave Mixer. Figure 2 shows the design R E S U L T S AND D I S C U S S I O N of the instrument used here that employs an optical delay line Figure 3 shows the impulse response function (A) and the and double-balanced microwave mixer (Model ZFM-4, Mini fluorescence decay curves for bilirubin (B) and fluorescein (C). Circuits). The design is similar to that described previously (18). At a delay of 6.00 ns the bilirubin signal is nearly zero, whereas In this design the laser beam is sampled by a fast photodiode that of fluorescein is still almost half of its peak value. Twenty (Model 403B, Spectra Physics, Inc.) that is cross-correlated with replicate determinations (oscilloscope) of the fluorescence the fluorescence or scattering signal. To adjust the delay required

f

ANALYTICAL CHEMISTRY, VOL. 58,

fluorescein (scope)n fluorescein (delay)b bilirubin concn, WM av found' R S D ~ ,av found' RSDd 1.00 3.00 5.00 7.00 9.00 11.00 15.00 20.00 25.00

0.284 0.310 0.297 0.304 0.301 0.296 0.306 0.307 0.302 0.310

2.97 3.81 3.46 3.04 4.07 3.09 3.24 3.71 3.69 4.17

0.299 0.301 0.304 0.300 0.302 0.303 0.299 0.304 0.307 0.305

MAY 1986

1227

nificant that the cost of the delay line approach is much lower than that of the sampling oscilloscope design.

T a b l e I. R e s u l t s f o r t h e D e t e r m i n a t i o n of 0.300 nM F l u o r e s c e i n a s a F u n c t i o n o f A d d e d Bilirubin

0.00

NO. 6 ,

0.96 1.13 1.23 1.26 1.14 1.18 1.26 1.43 2.31 1.81

Values obtained by u s i n g t h e 6.00-11s oscilloscope position. Values o b t a i n e d by using t h e 6.00-11s o p t i c a l delay position. 'Average o f 10 replicates, nM. dRelative standard deviation f o r 10 redicates, %.

lifetimes yielded 3.61 f 0.46 ns for fluorescein and 0.21 f 0.14 ns for bilirubin, in good agreement with literature values (13, 22,23). The fluorescence lifetimes were determined by using the phase-plane method (24-26). Of course, absolute fluorescence lifetimes are not significant here, since it is the difference in fluorescence lifetimes that provides selectivity; as long as the difference remains constant, reliable quantitative information can be acquired (9). Table I shows the results for 10 replicate determinations of 0.300 nM fluorescein in the presence of various concentrations of bilirubin using both instrumental designs. Importantly, both approaches give excellent accuracy (error < 4 % ) and precision, although the delay line approach offers a precision (relative standard deviation