Determination of Tryptophane in Plasma with a Spectrofluorometer System Hans Steinhart * Institute of Nutrition Physiology, Technical University of Munich, D- 8050 Freising- Weihenstephan, West Germany
Jiirgen Sandmann Kontron Technik GmbH, 0-8057 Eching, West Germany
The OMA system of PAR was adapted to the Farrand spectrofluorometer 801 to give rise to a highly sensltive and highly resolvlng spectrofluorometer system. The fluorescence signal Is received by the target of the vldlcon (the receiver of the OMA system). High monochromacy and dlsperslon of the emission spectrum are thus obtalned. I n quantitative fluorometrlc measurements, the sensitivity can be Increased by the multiple-count effect. The slngle measurements are added and the total signal Is either reglstered or stored. Because there are two storages, the background signal can be stored or a background compensated measurlng signal can be obtained. The signal/nolse relation (S/N)Is improved by the multiple-count effect, so that a remarkable Increase in sensltivity is obtained. I n experlments to determlne the free tryptophane content in the plasma of chicks, a detection sensitivity of 0.005 nmol with a standard devlatlon of 4.1 % could be obtained, the monochromacy being high at 5, 2, and 1 nm In the emlsslon part.
Fluorometric determinations gain importance in many analytical methods as compared to spectrophotometric ones, because they result in a better specification of the sample by selection as well by an excitation wavelength as by an emission wavelength. In addition, the fluorometric method delivers a far better sensitivity, because the active emission energy is measured by the fluorometric method, whereas by photometric methods only the energy which has not been absorbed by the sample is detected. The reason for higher sensitivity of fluorometric vs. photometric measurements is the better signal/noise ratio (S/N) of the former; that is, more noise is generated by a photomultiplier in photometric measurement because of the higher light level of the former. It has been the aim of the present development to take full advantage of the high resolution provided by the monochromators and to increase the analytical sensitivity markedly by improvement of the signal/noise rate. It was desirable to simultaneously record both excitation and emission spectra. A Farrand spectrofluorometer 801 was used as basic equipment of the new spectrofluorometer system. ks Farrand spectrofluorometers are very versatile, the set aims could be achieved by a relatively simple adaptation of an optical multichannel analyzer (OMA) system produced by Princeton Applied Research Corporation to the spectrofluorometer.
PRINCIPLE Principle of Function. Figure 1shows the block schematic of the spectrofluorometer system. The energy of a 150-W xenon high pressure lamp reaches the excitation monochromator, which permits an optical resolution of 0.25 nm. The high monochromacy is obtained by the use of suitable entrance and exit slits. The sample is excited to 950
ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977
Figure 1. Schematic of the spectrofluorometer system. M, mirror; G, lattice; L, xenon lamp; S, slit; SMPL, sample; T, target; V, vidicon; VE, vidicon electronics; 0, scope; P, plotter; R, recorder fluorescence by the extremely monochromatic radiation. The fluorescence spectrum emitted by the sample now enters the emission monochromator, which has the same optical characteristics as the excitation monochromator. Other than with the known construction of the fluorometer, the emission monochromator does not need the exit slit because in this construction the emission spectrum is focused on the target of the vidicon (i.e., the receiver of the OMA system). This target, which is used instead of the usual photomultiplier, and which is divided into 500 optoelectrical channels with 128 diodes each on a surface of 12.5 X 10 mm, can conduct the emission spectrum to the control unit as a total optoelectronic signal. In order to measure differences on the basis of electronic storage, samples A and B were measured separately with the same vidicon, both results being fed to separate storages in the control unit. The sensitivity of the measurement can be increased by choosing an addition factor K at the control unit. Each signal recall takes 32 ms and is repeated until the addition factor K is reached. The storage signals A and B can now be recalled alternatively as separate signals or as a difference signal, oscillatorily or digitally. Thus, a background compensated emission spectrum can be represented. The stored signals in a choosen spectrum range can also be conducted to printers, recorders, or other instruments by recalling from the storage of the control unit. Increase of Analytical Sensitivity and Spectral Dispersion of the Emission Spectrum. Before discussing
the characteristics of the vidicon more closely, some details of various receivers should be basically described. The sensitivity of optical receivers (photomultiplier etc.) greatly depends on S/N. Considerations are based on the fact that 14 photons correspond to 1 count. The photomultiplier RCA 4818 used in the Farrand spectrofluorometer 801 is a very sensitive receiver if compared to the usually requested spectral qualities of 150 to 200 dark counts on an average per second. The given figures are average values, because in the production of photomultipliers the single types do not come out identically and therefore show distinct tolerances in the S/N. The vidicon used here has about 200-250 dark counts per second. Therefore the photomultiplier RCA 4818 is more advantageous than the vidicon for simultaneous (real time) measuring. However, it is not necessary to register analogous values if the sample does not change within a short time (a few seconds), that is when there are no kinetic effects. The target of the vidicon consists of 500 channels of 256 diodes each, out of which only 128 diodes are used for measuring. The remaining diodes serve to compensate the dark counts of the vidicon. This is the only means of obtaining the dark count factor of 200-250 counts per second. The increase in sensitivity is caused by the fact that the measurement can be conducted up to several thousand times by introducing a K-factor (i.e. an addition factor); one measurement (real time) takes 32 ms, the total of all the measurements is indicated. The multiple count effect of the system causes the result to be superimposed for a longer term by a statistical medium noise which is very well reproducible. Thus an absolute increase of sensitivity is obtained by improvement of the relative S/N in relation to the K-factor. The S / N improves with the square root of the real time measurements (i.e., the K-factor). The formula for the calculation of the single S / N is
where Np= Number of photons per channel, N , = electronic noise (pre-amplifier), q = quantum yield, T = exposure time, and K = K-factor. Because each channel of the target has about the same sensitivity as any conventional receiver, it is possible to recall each channel like a separate receiver or to plot it oscillatorily. At first, the term of spectral purity is to be discussed. The monochromator consists of the entrance slit, the lattice, and the exit slit. The entrance slit indicates the monochromacy of the monochromator, the lattice the dispersion, and the exit the bandpass. In the described system, the exit slit of the emission monochromator is replaced by the target of the vidicon, each channel of the target corresponds to a single slit of 0.2 nm. The spectral bandpass of a channel depends on the monochromator. The lattice used in these experiments has 14400 lines/inch, which corresponds to a spectral bandpass of 0.2 nm per target channel. If the lattice has 28800 lines/inch, the spectral bandpass is 0.1 nm per target channel. The selection of the lattices depends on two points: a) on the optical dispersion wanted and b) on the spectral range which is to be plotted on the oscillograph. In this system a spectrum of 100 nm can be plotted simultaneously and digitally. With a spectral bandpass of 0.1 nm per target channel, an emission spectrum of 50 nm can be reached. The best dispersion of the lattice with 14400 lines/inch is 0.5 nm and that of the lattice with 28800 lines/inch is 0.25 nm. Therefore in our experiments two target channels have to be joined to receive one dispersion unit. It is possible to recall single channels or the total of several ones, because the control unit of the OMA system can store every spectrum channel separately. If for instance the value
Figure 2. Reaction scheme: tryptophane
-
norharman
with a relative bandpass of 5 nm is to be indicated, 25 target channels have to be added up for a lattice of 14400 lines/inch. The two-channel system allows storing the spectral properties of the solvent on channel A. The emission spectrum of the sample is fed to channel B. Now a background compensated signal is obtained by indication of the difference. As all values are stored in the control unit, it is possible to transfer the complete signal on a X-Y recorder or a printer later on.
EXPERIMENTAL Only 0.01, and 0.02 mL of plasma were available in experiments with two-weeks-old chicks to determine the concentration of free tryptophane. In order to do sufficient parallel experiments and other investigations, the plasma was diluted to 1 mL with a physiological NaCl solution. After deproteinization with TCA each time, 0.1 mL of the diluted plasma was used for the determination. Tryptophane was determined as its derivative norharman, which is synthesized in the presence of formaldehyde and FeC13 The method is first described by Hess and Udenfriend ( I ) and improved by Denckla and Dewey ( 2 ) and Bloxam and Warren (3) (Figure 2). The final volume of the reaction mixture was 5 mL. The standard-curve was made in the range of 0.05 to 1 nmol/mL. As only 0.1 mL of tryptophane solution was taken for the synthesis of norharman, the limit of detection is at 0.005 nmol tryptophane. The measurements were made in a 100-pL quartz continuous flow cuvette. The excitation wavelength was 373 nm, the emission wavelength 452 nm. To diminish stray light, filter 7/54 was put in front of the excitation monochromator, and filter 3/73 in front of the vidicon target. The bandpass of the slits of the excitation monochromator were 10 nm and the bandpass of the slit in front of the emission monochromator was 5 nm. Twenty-five channels were added up in the vidicon. This corresponds to a bandpass of 5 nm. The factor K was 1100. The fluorescence of the reaction mixture without tryptophane was stored, so that the values could be corrected automatically. RESULTS Table I shows the plasma tryptophane values of the chicks fed with various protein carriers when fasted for 16 h and 2 h after feeding. Six measurements were made per animal. Standard deviations were 4.1% a t a maximum. The results corroborate the plasma tryptophane values obtained from slaughtered chicks. But the results also show that the tryptophane concentrations vary considerably concerning the source of protein, and are dependent on the state of feeding. The fluorescence of norharman could easily be measured at bandpasses of 5 nm in the emission part of the fluorometer. Now comparative measurements were made, in which the slits were kept at 10 nm a t the excitation monochromator, but in the emission part the entrance slit was narrowed to 2 or 1nm. Accordingly 10 or 5 channels were added in the vidicon (this corresponds to bandpasses of 2 or 1 nm). To take full advantage of the storages of the control unit, the factor K was increased to 1600 with 10 or 5 channels. The values found with K = 1100 and the bandpass 5 nm were converted into K = 1600. The results are summarized in Table 11, which shows the relative values. ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977
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Table I. Tryptophane Concentration in the Plasma of Chicks with Various Feed Protein Carriers, Fasted and 2 Hours after Feeding Feed A 2 h after feeding Fasted animals Ani- nmol/mL pg/lOO mL nmol/mL pg/100 mL mal plasma plasma plasma plasma
3
40.5 40.5 47.0
1 2 3
79.0 79.5 81.0
1
2
58.0 827.14 58.0 827.14 67.0 959.90 Feed B 1613.44 1623.65 1654.29
1184.55 1184.55 1368.36
120.0
2042.33 2266.99 24 50.80
96.50 90.50 108.00
1970.85 1848.31 2205.72
100.0 111.0
Feed C 47.0 44.0 55.0
1
2 3
959.90 898.63 1123.28
,
I
I
I
I
I
I
I
I
200 2LO 280 320 360 LOO LLO L80 520 560 nm
Table 11. Comparison of Various Bandpasses in the Emission Part Animal
5nm/25 channels
2nm/lO
1nm/5
channels
channels
Fasted animals 1
2 3
2 13.7 214.1 217.6
22.8 21.8 24.2
6.20 6.20 1.83
2 hours after feeding
1
2 3
242.5 246.1 275.6
26.0 27.4 31.5
6.06 6.23 8.52
As can be seen in Table 11,even bandpasses of 2 and 1nm bring about useful results.
DISCUSSION The fluorometer OMA system has considerable advantages as compared to the conventional fluorometers with photomultipliers as detectors. S / N can be very much improved in quantitative measurements by introducing multiple measurements of the samples which are to be determined (factor K ) , so that sensitivity can be increased as compared to the usual quantitative determinations. Denckla and Dewey (2) reached a limit of detection of 0.04 nmol tryptophane in the example of the fluorometrical determination of tryptophane with the derivative norharman. In our experiments with diluted plasma of chicks and with tryptophane solutions, 0.005 nmol could still be determined. Denckla and Dewey (2) used a bandpass of 20 nm in the emission part; in our measurements the bandpass was only 5 nm. Considering the fact that in our experiments the plasma had been diluted by the factor 25, compared to the experiments made by Denckla and Dewey (2), the sensitivity is increased by the factor 25 as compared to the authors cited. Figure 3 shows that the emission spectrum of norharman covers a range of about 120 nm. At the maximum of the fluorescence, the section 5 nm and the section 20 nm can be taken as a rectangle; therefore it is justified to assume a decrease in energy of the factor 4 X 4 = 16 if lowering the bandpass from 20 to 5 nm. In spite of the better monochromacy having the advantage that possible interactions with other substances present in biological material are diminished, a considerable increase in sensitivity concerning the determination of tryptophane is possible. In the extreme case that the bandpass of the entrance slit of the 852
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700
Figure 3. Emission spectrum of norharman synthesized from chick's
plasma emission monochromator is 20 nm and if 100 target channels of the vidicon are added, the limit of detection can still increase by the factor of 4 X 4 = 16, i.e., an overall factor of 16 X 25 = 400. As experiments with the bandpasses 2 and 1 nm show, it is possible to go back to 2 or 1nm and still receive good results. To take advantage of the potential sensitivity of the system, a few points have to be considered as compared to conventional fluorometer systems. The continuous flow cuvette should always be fixed permanently in the cuvette holder. Otherwise the slightest jam of the cuvette will cause disturbances. The cuvette holder should be thermostable because the fluorescence of many compounds depends on temperature. The stability of the lamp energy is essential for the quality of the measurements. Only choice xenon high pressure lamps with the highest stability of energy and excellent energy stabilizers may be used, otherwise the S/N improved by the multiple measurements is disturbed. It is best to use a double beam fluorometer. In quantitative investigations, the bandpass can be of great importance in fluorometric experiments with biological material. There are two ways to determine a substance in a heterogenous solution. Either the substance in question is isolated or there is a method by which the substances are seized selectively. It is advantageous to avoid the processes of isolation and cleaning, because they require a lot of work. In the method described here, interactions of attendant substances with the substance which is to be determined can be neutralized by lowering the slit passes and by the better dispersion of the spectrum thus obtained. Therefore cleaning procedures of certain substances can be omitted wholly or partially. Since the fluorescence measurements can be limited in time, substances which are labile to radiation can be determined more easily in quantity. Dependent on the sensitivity of the sample the measuring time can be shortened to 32 ms. The system is suitable for kinetic investigations of fast reactions, because so far the measuring of changes in the fluorescence of a chemical system has not been possible in 32 ms. Fluorescence spectra of radiation labile substances can be plotted, because it is possible to show fluorescence spectra simultaneously on the screen. Besides, shifts caused by radiation influences can be detected at once and be exploited
for kinetic investigations.
(3) D. L. Bloxam and W. H. Warren, Anal. Biochem., 60, 621 (1974).
LITERATURE CITED (1) S. M.Hess and S.Udenfriend, J. pharmacal. €xp. Ther., 127, 175 (1959). (2) W. D. Denckla and H. K. Dewey, J. Lab. Clin. Med.,69, 160 (1967).
for review September 20! lg7& Accepted February 22, 1977.
Determination of Gentamicin Complex Components in Fermentation Broth by in-situ Fluorimetric Measurements of 4-Chloro-7-nitrobenzo-2-oxa- 1,3-diazole Derivatives Peter Kabasakalian, Sami Kalliney, and Anita W. Magatti” Development Division, Schering Corporation, Bloomfield, New Jersey 07003
A method for quantitating the individual components of gentamicin during the progress of a fermentation is described. Fluorimetric measurements are carried out in situ on the 4-chioro-7-nitrobenzo-2-oxa-l,&diaroie (NBD chloride) derivatives formed after thin-layer chromatography (TLC) of the clarified fermentation broth. This fluorimetric procedure is 800 times as sensitive as its ninhydrin analog. /-Methionine Is shown to be Involved in the methylation of gentamicin C1, to C2 and then to C1.
Gentamicin, a broad-spectrum aminoglycoside antibiotic complex produced by Micromonospora purpurea (1-3), is composed of three components, C1, Ct, and C1, (Figure 1). These differ from each other in the degree of methylation at the 6‘ position. Although the component ratio can be followed 24 during a fermentation by microbiological estimation (4), h is required. The fast, direct densitometric method of Wilson et al. (5), using the ninhydrin chromogenic spray procedure after resolution by thin-layer chromatography (TLC), is not sensitive enough to follow directly the course of a fermentation. Since fluorescence is 10-100 times more sensitive than colorimetric procedures, fluorigenic labeling techniques were investigated. 4-Chloro-7-nitrobenzo-2-oxa-l,3-diazole (NBD chloride), which reacts with primary and secondary amines (6) while yielding a nonfluorescent hydrolysis product, was found to be the fluorigenic reagent of choice.
EXPERIMENTAL Apparatus. A Schoeffel SD 3000 double-beam densitometer equipped with a SDC 300 density computer-recorder (Schoeffel Instruments, Westwood, N.J.) was used in the reflectance (single beam) mode. It contained a high pressure xenon-mercury lamp and a miniature quartz-type monochromator set at 420 nm (excitation). The reflectance mode accessary had a built-in 420-nm sharp cut-off filter and an insertable wedge filter which was set at 530 nm (fluorescence). The inlet and exit beam slits were 1.5 and 1 mm, respectively. The settings in the density computer were positive, ratio, and numerator. Scanning and recorder speeds of 10 cm/min were used. Peak areas were measured with a Disc Integrator, Model 252A, Disc Instruments, Inc., Santa Ana, Calif. Precoated silica gel plates (0.25-mm thickness) on 20 X 20 cm glass purchased from Analtech, Inc., Newark, Del., were scored into 20 parallel lanes with a Schoeffel Scoring Device. The Chromaflex Dipping Tank was obtained from Kontes, Vineland, N. J. Reagents. 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD chloride) was purchased from Aldrich Chemical Co., Milwaukee,
Wis. Gentamicin and sisomicin standards and gentamicin fermentation broths (7) were obtained from Schering Research Division. Procedure. The gentamicin fermentation broth samples were acidified to pH 2 with sulfuric acid, then centrifuged to separate mycelium and particulate matter. The pH of the aqueous solution was adjusted to 12 with sodium hydroxide and appropriate dilutions were made. TLC plates were spotted using Microcaps (Drummond Scientific Co., Broomall, Pa.) to deliver 5 pL of solution. Samples were spotted according to the “data-pair” technique of Frei et al. (8). The plates were developed according to the procedure of Wilson et al. (5), air dried, dipped in methanolic NBD chloride (0.25 mg/mL) for 2 s, heated in an oven at 120 OC for 10 min, cooled, and rechromatographed in methanol in the same direction as the first development. The plates were scanned using excitation and emission wavelengths of 420 and 530 nm, respectively.
RESULTS AND DISCUSSION Sisomicin, a broad-spectrum antibiotic produced by Micromonospora inyoensis, and a 4‘,5‘-dehydro derivative of gentamicin C1, (9), was used as the model for studying the various quantitative TLC schemes since it has only one major component. The slope of the plot of the instrumental response (integrated area) vs. the amount of sisomicin spotted (in area unitslpg sisomicin) under optimum instrumental conditions was used as the criterion of sensitivity. This value for the ninhydrin chromogenic spray procedure (5) was 0.8 area units/kg sisomicin. Spray reagents which did not require heat for fluorigenic labeling were first investigated. The fluorescamine quantitative TLC procedure of Sherma and Touchstone (10) looked promising. Although it was sensitive (27 area unitslpg sisomicin), the fluorescence decayed rapidly despite the use of a triethylamine stabilizing spray. o-Phthalaldehyde (OPT), which is soluble and stable in aqueous buffers, was reported by Benson and Hare (11)to be 5-10 times more sensitive than fluorescamine when reacted at rcmm temperature with primary amines in the presence of 2-mercaptoethanol in aqueous solution. There has been no reported use of OPT for TLC systems. The use of OPT as a fluorigenic spray for the sisomicin TLC system failed to produce any significant fluorescence even though it worked in solution. This approach was abandoned after the reason for its failure could not be ascertained. We then turned to spray reagents which required heat for fluorigenic labeling. The literature (12) indicates a preference for NBD chloride (which reacts only with primary and secondary alkylamines) over dansyl chloride (l-dimethyl-amiANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977
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