A Minicomputer-Automated Array Spectrometer for

CLIN. CHEM. 22/9, 1472-1482 (1976)

A Minicomputer-Automated Array Spectrometer for Liquid-Chromatographic Detection of Metabolites Raymond E. Dessy,1 Warren D. Reynolds,2’3 Wayne G. Nunn,” Christopher A. Titus,1 and Gregory F. Moler2 A third-9eneration multiwavelength array spectrometer was developed as a detector for the high-resolution liquid-chromatographic characterization of metabolites. Components include a PDP-8/e minicomputer, matched pair of linear photodiode arrays, holographically-ruled gratings, fiber optics, flow cells, and high intensity xenon light source. The wavelength range is 256 nm differential with 1-nm resolution and can be adjusted from 200 to 800 nm. The system is capable of storing 20 spectra per second (200-456 nm) in a dual-beam mode. Special features include minicomputer-driven signal enhancement via integration as a function of signal strength. The display output includes presentation of the total absorption chromatogram vs. elution time in both real and post-run time as well as selectable single absorption band vs. elution time (post-run time). Application of this dedicated system is illustrated by the separation and characterization of the metabolites of a carcinogen, 4-ethylsulfonyl-1-naphthalenesulfonamide. AddItional metabolites

Keyphrases: data processing

in urine #{149} toxicology #{149} inherited disorders

#{149} screening #{149} ultraviolet spectrometly

#{149}

During the past few years, considerable effort has been expended on developing and improving ultraviolet detectors for high-speed liquid-chromatographic separations of biochemically important components (1-14). Generally, these detectors can be divided into classes

according to their end-use: monitoring and identification (4). For general monitoring purposes, most liquid chromatographs make use of a single-wavelength (element) detector at 254 or 280 nm (12). Other discrete element monitoring has been used, depending upon the specific component being sought (8). For qualitative identification needs, full ultraviolet scanning on each component in the liquid-chromatographic effluent yields additional spectroscopic information. ‘Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, Va. 24061. 2Fl and Drug Administration, National Center for Toxicological Research,

Jefferson, Ark. 72079. to whom reprint requests should be made. Received April 5, 1976; accepted July 3, 1976.

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Although many varieties of rapid scanning spectrometer systems are available, the first -generation units relied on moving mechanical parts to scan the spectrum (12, 14). The second-generation instruments incorporated vidicon tubes, which eliminated the construction and maintenance problems associated with rotating mirrors or vibrating galvanometers (17, 18). But the vidicons suffer from substantial coherent pattern noise and limited integration time because of high. dark current and memory effects (15, 16). The recent availability of inexpensive solid-state linear photodiode arrays as simultaneous wavelength detectors has enabled the development of third -generation spectrometers for use in liquid chromatography.4 The abUity to record total ultraviolet spectra instantaneously is accompanied by several advantages beyond simple component identification. These advantages become apparent by inspection of both time domain and spectral domain (12-20). In the time domain case, the simultaneous detection of all dispersed radiation (n spectral or spatial resolved elements) reduces the observation time by a factor of n in the case of measurements limited by the signal-to-noise ratio and improves this ratio by a factor of for a fixed observation time as in the case of fast liquid-chromatographic peaks (19, 20). However, further improvementa in the signal/noise ratio based on the Hadamard or Fourier methods are limited in the case of ultraviolet spectroscopy, because the noise is statistical and source dependent (15, 19). In the spectral domain, detection is improved by virtue of having access to information on the total ultraviolet spectrum. By summing the output intensity over all discrete 1-nm channels from 200-350 nm as a single output intensity, an advantage is gained over select single channel (254 nm) detectability.4 This increase in detectability varies from component to com4Dessy,

photodiode

R., Nunn, W. C., Reynolds, W. D., and Titus, C. A., Linear

array spectrometers as detector systems in automated liquid chromatographs. 27th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 1976, paper 375.

ponent but depends on the ratio of the integrated area under the spectral curve (200-350 nm) to that at the selected single channel (250 nm): 35O

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In most cases, this can produce a five- to 500-fold increase in sensitivity of detection. For high-speed liquid-chromatographic separations of biological samples, for which sample size may be limited or trace-level components (hormones) may be present in complex physiological mixtures (urine), this improved sensitivity would be an advantage (21). Along with the expanding use and development of pesticides, herbicides, food additives, plasticizers, and other environmental chemicals, there has been a growing concern for possible untoward effects of these chemicals in man. Long-term, low-dose feeding effects and related mechanistic studies of a few of these agents and model compounds are under investigation at this Center.2 Identification and monitoring of these agents and their metabolites can be highly useful in studies of an agent’s activity, toxicity, and mechanism of action. High-resolution anion-exchange liquid chromatography is an excellent analytical tool for studies of this kind because both unconjugated and conjugated metabolites can be separated for structural determination in a single sample without the need for hydrolysis or extensive sample pre-treatment. Anion-exchange has been used to separate diphenylhydantoin and its metabolites, the glucuronide and sulfate conjugates of p-nitrophenol, and p-hydroxyacetanilide metabolites as well as acetaminophen and its metabolites in human urine (22-24). Two recent books summarize the application of ionexchange liquid-chromatographic methods to problems in biochemistry and biology (25, 26). This system is composed of a minicomputer plus linear photodiode array spectrometer, for use as a detector for high-resolution ion-exchange liquid chromatography. We illustrate its application by describing the separation and characterization of conjugates of 4-ethylsulfonyl-1-naphthalenesulfonamide in urine from BALB/C mice..

Instrument General From experience with a prototype system consisting of a Reticon detector, light source, optics, and microprocessor, which was previously constructed by this group,4 we decided on the requirements for this type of dedicated system as follows: 1. The liquid chromatographic detector system would need to include a dual-beam spectrometer to accommodate gradient elution. 2. The system must be capable of taking about 25 spectra per second to accommodate signal enhancement techniques (if required).

3. The spectral range must be 200-450 nm. 4. The system must be capable of operation in the absorption mode for ease of computer data handling. 5. The ultraviolet source and electronics must be stable over long periods, because a typical liquid chromatographic run with an ion-exchange column might require 80 hours. 6. Because of the large volume of data (2.5 million data words/run) expected, all meaningful raw spectral data must be stored on disk for limited post-run appraisal and then transferred to industry-compatible tape for storage or transfer to the central computing facility, where it would be subjected to various in-depth data-reduction programs. 7. The output format should include total integrated absorption at all measured wavelengths vs. elution time (real-time liquid chromatogram) and selectable singleS band wavelengths vs. elution time as well as individual spectra. 8. For data handling and reduction, a minicomputer with 12K core and a teletype, disk, scope, magnetic tape, and plotter devices will be required. These were the ideal criteria, which might not all necessarily be met, or need to be met in ultimate use. The exploration stage would involve a period when it was not known which wavelengths (200-450 nm) were of most importance to the metabolite characterization research. It might be necessary to rescreen previous runs many times to finally focus on the observations that would be used in component identification or for routine monitoring. The present automated liquid chromatography/ultraviolet spectrometry system consists of several major units, including the high-resolution liquid chromatograph with associated electro-mechanical and electronic controls, a 12-bit computer unit based around a PDP-8/e CPU with 12K of memory and with four interactive storage and display devices, and a spectrometer unit consisting of a xenon light source, fiber optics, and a pair of holographically ruled gratings along with two cooled (-30 #{176}C) photodiode arrays. Each of these major units is discussed in the following sections. Liquid Chromatograph A high-resolution liquid chromatograph unit utilizing the 150-200 cm ion-exchange column similar to that developed by Scott and co-workers was fabricated, with some additions and changes (27). A pH-gradient section was added that develops a descending hyperbolic gradient in pH (8 4.40) during the first 5 h of the 60-h run. The system is gradient-programmable in five separate inter-dependent functions. The five functions control the two separate gradient development cycles and a final wash cycle as well as start and stand-by modes. The pH and buffer gradients are developed by use of two solution reservoirs, an initial mixing vessel solution, and a wash reservoir. The mixing vessel starting solution contains 0.1 mol/liter buffer (ammonium acetate/acetic acid) adjusted to pH 9 while the other two source reservoirs contain “acid solution” (pH -

CLINICAL CHEMISTRY,

Vol. 22, No. 9, 1976

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of high pressure anion-exchange liquid

chromatograph

4.0, 3 mol/liter buffer) and “buffer solution” (pH 4.40, 6 mol/liter), respectively. The mixing vessel (stirred) is fed from the three reservoirs through three separate solenoid valves and a mixing-vessel feeder pump (two pre-set speeds). A sequence of mixing steps is pre-determined by the operation of the electronic counter/ timers and sequential pump speed control. Any combination of curve/linear gradients can be set by this operation. For example, a descending pH gradient can be superimposed on a slowly increasing linear buffer gradient. A flow diagram of the liquid chromatograph unit is shown in Figure 1. All materials contacting the sample and solutions are Grade 316 stainless steel, glass, or Teflon. Several columns (3 mm o.d. X 75, 100, and 150 cm) have been used for the determination of the optimum separation of components in the urine of BALB-C mice. Various anion-exchange resins and particle sizes, from several manufacturers, have been used. Aminex A-27 (BioRad), average particle diameter 13.5 m, was used, with fair resolution but with a high pressure drop (34.5 mPa for 8 mol/liter buffer). Another Aminex resin (1X8L: 9160) of smaller size (5-8 iim, according to our measurements) was used in a 100-cm column with excellent resolution, but the pressure drop was excessive (-42.8 mPa) at 70 iil/min. A nominal 8 ± 1 m resin (which we measured as 5.2-6.0 zm) in a 100-cm column gave nearly identical resolution; this resin was from Durrum Instrument Corp., Palo Alto, Calif. 94303. However, the Aminex A-28 (5 tm, 150-cm column) appeared to be the best compromise between flow-rate (elution time), pressure drop, and resolution of the many urinary components from the conjugates of the model compound, 4-ethylsulfonyl-naphthalene-1 -sulfonamide. Figure 2 exemplifies the separation of both the conjugates of 14C-labeled 4-ethylsulfonyl-1naphthalenesulfonamide and urinary components. 1474

CLINICAL CHEMISTRY, Vol. 22, No. 9, 1976

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Reticon Spectrometer A high-intensity 150 W xenon arc lamp and h’ousing with regulated power supply was utilized for the ultraviolet source (Model 7301, Oriel Corp., Stamford, Conn. 06902). A fairly smooth continuum output develops from 190 to 750 nm, as seen in the typical spectral output curve (Figure 3). The source size is 0.5 mm X 2.2 mm; the image size at the focal point (76 mm from housing) is 0.9 mm X 4.0 mm. Flow-cell. The micro flow-cells, one each for the reference and sample beams, were fabricated in the usual Z configuration. The dimensions chosen depended both on the size of the available light beam from the fiber optics and the optimum absorption lightpath/volume ratio (1 cm/31 tl) for chromatograph band-width (21). These flow-cells were made from stainless steel, with quartz windows and Teflon gaskets. Grating. The necessity of attaining sub-nanogram sensitivity required a system with best available optics. We used a high-quality holographically-ruled concave grating (600 lines/mm, Model 3B; J-Y Optical Systems, Metuchen, N. J. 08840), which minimized problems with spherical aberration as well as keeping reflection and transmission losses to a minimum (17). Optical path and sensitivity. A metal housing with the interior painted a nonglossy black, which was also light-tight and purgeable with nitrogen, was fabricated for the optics system. All external entrances and exits were constructed so as to minimize light leakage. An optical configuration based on a Rowland circle (F/3.0, 200 mm) as shown in Figure 4 was placed inside this housing. Transmission and reflection losses were kept Light

source.

to a minimum by using a grating and fiber-optic bundles. The fiber-optics bundle further reduced the optical alignment problem and allowed the system to run for long periods without recalibration. The overall system involved an F/3.0 rather than the earlier prototype of F/4.5.4 Design and optimized performance calculations, which are summarized in Appendixes 1,11 and III, were completed on the optical system from the light source to signal read-out. The maximum dynamic range of a pixel5 in the linear array was calculated to be 40 000/1 (Appendix I). Theoretical design calculations of source strength and sensitivity indicate that with a liquidchromatographic effluent sample containing guanosine = 11 050) and an estimated peak width of 3 mm (31-zl cell volume), as little as 1 ng can be detected (Appendixes I and II). Reticon detector. The silicon photodiode

linear array detector uses a reverse biased p-n junction diode as the photosensitiveelement. These linear arrays are available in several pixels per array sizes and pixel areas. Currently, arrays can be obtained with 128 to 1024 pixels per array with pixel areas of either 1 X 1 mil or 1 X 17 mil. These linear arrays are light sensitive from below 200 to above 900 nm and are most efficient in the 600-820 nm region, as shown in the photon efficiency curve (Figure 5). The quantum efficiency at each wavelength is corrected to a maximum value of 1.0 by a computer assembly language sub-routine before the data are stored. Because the wavelength region of interest for the present instrument is 200-400 nm, a 256-linear array was selected that would enable 1-nm resolution (1 mil centers) and a 256-nm range (200-456 nm). However, this range can be adjusted to any 256-nm band in the 200-900 nm region. Two matched arrays, which are individually housed with Pelletier cooling plates (-30 #{176}C), were selected based on their identical energy response (Model RL256 EC/17; Reticon Corp., Sunnyvale, Calif. 94086). The cooled detectors decreased electronic noise originating from room-temperature by a factor of 20 (15). A 256-linear photodiode array with a printedcircuit background is illustrated in Figure 6 and a schematic of the linear array is shown in Figure 7. The photodiode array operates as follows: A pixel isa single photodiode

and, in the present

design,

it receives

the light from a single one nm band. CLINICAL CHEMISTRY,

Vol. 22, No. 9, 1976

1475

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1. As light strikes the surface of the detector, electron hole-pairs are created, and charge migration occurs toward the capacitor element (Figure 7). 2. As the light continues to strike the detector surface, this action leads to a gradual discharge of the capacitor. 3. Over the integration time, the charge on the capacitor drops an amount equivalent to the amount of integrated light falling on th&detector. 4. When the photodiode (pixel) is “read,” a multiplexer attaches the detector to a voltage source that charges the capacitor back up to a standard potential. 5. A signal, corresponding to the current-flow necessary to recharge the voltage source, is sent down a wire (video-line).

6. The multiplexer then causes the voltage source to be switched to the next photodiode in the detector, and the process is repeated. Solid-state “read-out”.

linear

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(Reticon)

In the fast scanning environment of high resolution liquid-chromatographic operation, continuous monitoring of the photodiode array is unnecessary. 1000 spectra per second provides more data than can be either stored or handled by a dedicated minicomputer. In practice, it is normal to initiate data collection only periodically. The data-collection process begins by clearing all photodiodes of previous stored charge. During a dead period, when the detector is not being 1476

CLINICAL CHEMISTRY,

Vol. 22, No. 9, 1976

used, the light striking the detector’s surface rapidly leads to saturation of all photodiodes. (See Appendix I for “saturation time”). Before spectral information is gathered, the array must be “dummy read” by applying a read pulse that starts the drive electronics, and all photodiodes in the array are recharged back to their starting charge points. The jth photodiode in the array is read-out at a different time than the zth photodiode. However, the time interval between the reading out of the jth photodiode in successive scans is the same for successively read zth photodiodes. Because cycling the photodiodes is fast compared to the liquid-chromatographic effluent concentration changes being observed, a truly simultaneous multiwavelength detector is involved. Consequently, each individual photodiode is continually “on” except for a very short “blink” out of each cycle and can be “on” for as much as 95% of the time. If one views the single video line output of the device, a series of pulses appear, each corresponding to the amount of light striking a photodiode in the array (Figure 8). These arrays operate with the multiplexer switching between photodiodes at a clock rate of 1 kHz to slightly above 1 MHz. A complete reading of the array (256 photodiodes) can be as fast as 12 ms, as slow as 0.25 s. The integrate time can be altered between the limits of the array “shift out” rate (20 kHz) and the time when the dark-current noise becomes a serious problem. This places an upper limit of 80 spectra/s to a lower limit of 1 spectra/bOO s (4 s/pixel) for the cooled array. For optimum integration time, a rate of 20 spectra/s was chosen. The serial pulse train arising from the common video line of the detector is passed through an amplifier, integrate, sample, and hold system with high rejection of periodic noise. Most of the electronic noise in the detector arises from switching transients induced at times shown in Figures 8 and 9. Each pulse is integrated over most of its width, which leads to a reduction in noise by the analog integration operation. A schematic of the detector and amplifiers for both “real time” recording output (strip-chart) and minicomputer data acquisition (absorbance, transmittance) is shown in Figure 10. The recording output is a sum of the absorbance signal over all measured wavelengths (200-450 nm) for a given set of “scans”.

Fig. 9. Typical switchingtransientdevelopedduringa single pixel read-out

Fig.

11. Mini-computer configuration with I/O devices

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Computer General. The basic computer configuration used to acquire, process, and display the formatted spectral data is based around a 12-bit minicomputer (PDP 8/e; Digital Equipment Corp., Maynard, Mass. 01754) with 12K words of core memory. Additional bus-bar hardware components include a 10 bit A/D convertor (AD8A), real-time clock (DK8E-P), 10-Hz dayclock, D/A convertor (VC-8E point plot) and hardware multiply/divide processor (KE8E). The storage devices include two 3.2-million data word disk and controller units (RK8E) and a nine-track magnetic tape (TU-lO). All the preceding is from Digital Equipment Corp. The input/output devices include a multispeed alphaI numeric terminal (LA-36, Digital Equipment Corp.), incremental plotter (Cal-Comp No. 565; California Computer Products, Anaheim, Calif. 92801) with interface (XY8E; Digital Equipment Corp.) and display oscifioscope (No. 5051; Tekronix Corp., Beaverton, Ore. 97005). Figure 11 shows a configuration diagram of the computer system. Operation. The central processor unit (CPU) uses a 10-bit A/D convertor to change the incoming analog signal from the detector to digital, which is stored in a 4K data word input buffer in the CPU. The data input rate is controlled by two separate clocks. The first real-time clock is used to control the integration or exposure time of the linear array. The second clock is used as a day clock; it is started when a liquid chromatographic run is started, read each time a spectrum is

taken, and allows data to be taken only when there is a signal above the baseline. If the response in the reference beam falls below a pre-set level, the integration time is increased in binary progression. This operation corresponds to slit opening on a regular spectrometer, but without loss in resolution. The integration time (a two-bit number) and the output of the A/D (a 10-bit number) are ANDED to provide a 12-bit data point for each photodiode (pixel) in the array yielding a dynamic range of 1000:1. The three data modes (absorbance, transmittance, or integral) are carried by the analog multiplexer that acts as an auxiliary to the A/D convertor. The data are collected in ping pong double buffers. As each buffer is filled, it is transferred to the disks by using assembler drivers that are interrupt oriented. By using this type of rotating buffer for data storage, the possibility of missing incoming data is small. The data are stored on disk in single-word-binary formatted files. The analog amplifiers perform all the correction functions except the one for responsivity of the linear array (see Figure 5). This calibration function is accomplished in the computer by binary adding the appropriate correction factor stored in memory for each wavelength as a log. The addition function is much faster than other math operations since data throughput is at a premium for the computer signal enhancement technique.. Software. A series of software routines were written in various levels of computer languages (assembler, BASIC, and FORTRAN) to acquire data, transfer to mag-tape, and to format and display the data. After the liquid-chromatographic run is over, User Service Routines and assembly coding is used to rewrite the directory area of the Operating System (OS-8) so that access to the data may be had by using conventional keyboard or User Service Routine assembler calls. An overview schematic of the software support is shown in Figure 12. A flow-chart for the shortest of the 10 software programs is shown in Figure 13. I/O display. During the 60-h liquid-chromatographic run, a large amount of data (approximately 2.5 X 106 data words) will be acquired that requires display in standard spectral and chromatographic format. During a liquid-chromatographic run, the Total Absorbance Chromatogram (TAC) is plotted out on a Y/t plotter CLINICAL CHEMISTRY,

Vol. 22, No. 9, 1976

1477

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associated with the system. This is the normal liquidchromatographic plot of response vs. time that is typical of single-wavelength monitors. The post-run operations include the following: 1. Plotting time vs. absorbance at any single wavelength, continuous band of wavelengths, or selected group of wavelengths. A digital incremental plotter is used to provide the resolution and labeling required for documentation purposes. 2. Listing every chromatographic peak and its retention time on the system terminal so that the technician can determine the elution time of a component for preparative purposes. 3. Present an oscilloscopic display of any given spectrum. 4. Dumping of the data to industry-compatible tape for storage, or transfer to a larger computer for full spectral display purposes, or library search and matching of cataloged spectra.

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Application Pathological

CUTION (CHAIN)

Effects (Mice)

4-Ethylsulfonylnaphthalene1-sulfonamide has several effects on the mouse bladder epithelium (C57 female, 12 weeks). A single oral dose (5-160 mg/kg 6ody wt) induces epithelial Iyperplasia of the bladder in mice. Administration of 100 mg/kg of diet for eight weeks produces a greater degree of epithelial hyperplasia and, frequently, bladder tumors at 30 weeks (30-32). The compound initially causes a violent proliferative epithelial response, which later settles into a chronic phase in which cell tumors vary from animal to animal and site to site within the same bladder. The sulfonamide group appears to be essential to the activity of the molecule (31). Activity was maximum when the sulfonamide group was attached to an aromatic system (benzene or naphthalene) containing an alkylsulfonyl or sulfonamide group (31). These implications of structure/activity relationship have a bearing on the non-nutritive artificial sweeteners (30). [‘4C] 4-Ethylsulfonyl-1-naphthalenesulfonamide metabolites The metabolism phthalenesulfonamide 1478

of [‘4C] 4-ethylsulfonyl1-nain mice strains is not fully

CLINICAL CHEMISTRY,

Vol. 22, No. 9, 1976

Fig. 13. Software

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known. In the current series of studies, three female BALB/C mice (20 weeks old) were each fed 1.05 iCi of 14C-labeled 4-ethylsulfonyl-1-naphthalenesulfonamide (sp. acty., 11 Ci/mol). A 24-h urine was collected in special metabolism cages. The pooled urine was filtered through a Millipore filter (0.2 tin av pore size) before chromatography. Twenty-five microliters of the ifitered urine was injected onto the 3 mm>< 150 cm Aminex A-28 anion-exchange column and eluted with both the pH and buffer gradients during the 55-h period. The separation was conducted under the following conditions: 0.1 ml/min flow-rate, 70 #{176}C (isothermal), hyperbolic descending pH gradient (8 4.40) during the first 5 h, ascending buffer gradients 0.1 to 6 mol/liter (aminonium acetate/acetic acid) during a 0-30 h period, holding 6 mol/liter buffer constant during the remaining elution period (hours 30 to 55). Individual 14C-contaming fractions were counted by a Nuclear-Chicago, Mark II liquid scintillation instrument equipped with a Model PDS/3 data reduction system. The 1-ml frac-

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tions were added to a vial containing 12 ml of PCS solubilizer (Amersham-Searle Corp., Arlington Heights, Ill. 60005) and counted for 20 mm. The count-rate data (dpm) vs. retention time for each lO-min fraction having significant radioactivity (dpm >50) was plotted as shown in Figure 2 (cross-hatch). The radioactive unmetabolized [‘4C]4-ethylsulfonyl1-naphthalenesulfonamide and its individual radioactive metabolites are shown at retention times of: 26.5, 31.0, 35.5, 43.3, and 53.0 h. These radioactive peaks account for 96% of the total measured radioactivity in the 25-id urine sample. Liquid Chromatography/Ultraviolet Spectrometry Display Work

is continuing on improving the computerplotter display of both the Total Absorbance Chromatogram and the Absorbance Band Chromatogram. A recent example of the spectral output for both [14C]4-ethylsulfonyl-1 -naphthalenesulfonamide (retention time, 31.0 h) and a suspected sulfonate conjugate (retention time, 43.3 h) is shown in Figures 14 and 15, respectively. driven

Discussion Our interest in the computerized liquid chromatography/ultraviolet spectrometry system is twofold. First, the generation of a data base of ultraviolet spectral data

on urinary components (human and selected animal) that are associated with normal and pathological states, with subsequent computer retrieval and matching, is required for profiling of various inborn errors of metabolism. The acquisition of this data bank would aid immensely in the selection of ultraviolet spectral “windows” or binary descriptors for use in developing profiles of urinary components in specific human pathological states, by computer pattern-recognition techniques. The second purpose is the general use of the system for rapid ultraviolet data characterization of toxic chemical agents and their metabolites in urine. As shown by the current system, computer data manipulation can enhance sensitivity of detection of components and can help in the chromatographic resolution of compounds. Liquid chromatography has been hindered by the lack of a universal detector, and, consequently, no equivalent for the gas chromatograph/mass spectrometer system has yet been described. In the present work, we have begun to demonstrate the utility of a computerized liquid chromatography/ultraviolet spectrometry system for rapidly acquiring ultraviolet data and identifying various toxic chemical agents and their metabolites. Computer retrieval and display of selected ultraviolet spectra upon completion of a liquid-chromatographic run saves many hours of tedious collection of many hundreds of fractions and transfer to an ultraviolet spectrometer. Because one can re-run the entire liquid chromatogram at a wavelength specific for a chemical toxicant or its metabolites, they can be rapidly located in the chromatogram for further spectroscopic study. At this stage in the development, we have not “fine tuned” the system for maximum detectability. However, the spectrometer system has demonstrated increased sensitivity over single-band monitoring by virtue of its computerized signal-integration routine. Enhanced sensitivity will enable several improvements in liquid column chromatography. Although the capacities of conventional resins are several hundred fold greater than those of pellicular resins, the improved sensitivity will permit smaller sample loadings along with increased resolution and so will permit use of pellicular resins for identifying and screening for urinary metabolites. Future data manipulations for enhanced spectral presentation will include: (a) use of ratios at two or more selected wavelength for determination of chromatographic peak homogeneity, (b) spectral baseline or background subtraction technique by computer routine, (c) peak deconvolution for improved resolution, and (d) on-line quantitative analysis.

The mini.computerized spectrometer unit was developed jointly by the Instrumentation and Computer Interfacing Group, Depart. ment of Chemistry, Virginia Polytechnic Institute and State Uni. versity, and the National Center for Toxicological Research under Contract No. 222.75-2047(c). Mouse urines containing (14CJ4-ethylsulfonyl-1-naphthalenesulfonamide and metabolites were kindly supplied by Dr. James Stanley, Molecular Biology Division, National Center for Toxicological Research. CLINICAL CHEMISTRY,

Vol. 22, No. 9, 1976

1479

References

spectrometer for ultraviolet Chem. 47, 25 (1975).

1. Callmer, K., and Nilsson, 0., Modification of a Varian liquid chromatography u.v. detector for high sensitivity. Chromatographia 6,517(1973). 2. Morris, C. J. 0. R, Chromatographic detectors. Lab. Pract. 23,513 (1974). 3. Veening, H., Recent developments in instrumentation for liquid chromatography. J. Chem. Educ. 50, A481 (1973). 4. Krejci, M., Experimental comparison of some detectors used in high performance liquid chromatography. Chem. Li8ty 57, 843 (1973). 5. Watson, E. S., Spectrophotometer for simultaneous absorption measurements of two substances at two wavelengths. Ger. Offen. 2, 258, 208, 7 June (1973). 6. Pellizzari, E. 0., and Sparacino, C. M., Scanning fluorescence spectrometry combined with u.v. detection of high pressure liquid chromatographic effluents. Anal. Chem. 45, 378 (1973). 7. Baker, 0. R., Williams, R. C., and Steichen, J. C., A comparison of photometric detectors for high speed liquid chromatography. J.

18. Pardue, Applications

Chromatogr. Sci.

12, 499 (1974).

8. Sonnenschein, A., Theory variable wavelength detectors Instrum. 12, 123 (1974).

and practice for using continuously vs. other monitoring systems. Anal.

9. Koszewski, J., Bylina, A., Sybilska, Chromatographic spectrophotometric

1,339,475, 5 December

0., and Gravowski, apparatus. Brit.

Z. R., Patent

1973.

10. Steichen, J. C., Dual-purpose absorbance-fluorescence for high pressure liquid chromatography. J. Chromatogr.

detector 104, 39

(1975).

13. Sauer, B., New detector for high pressure liquid chromatography. GITFachz. Lab. 17, 1152 (1973). 14. Bylina, A., Sybilska, D., Grabowski, Z. R., and Koszewski, Rapid scanning spectrophotometry as a new detection system chromatography. J. Chromatogr. 83, 357 (1973).

J., in

15. Talmi, V., Applicability of TV-type multichannel spectroscopy. Anal. Chem. 47, 659A (1975).

to

16. Talmi,

Y., TV-type

multichannel

detectors.

detectors

Anal. Chem. 47, 697A

(1975). 17. Milano, M. J., and Pardue, H. L., Evaluation

of a vidicon

scanning

Appendix I

Em

=

46, 1000

(1.4)

2(0.28) (1o6)

23. Anders, M. W., and Latorre, J. P., High-speed liquid chromatography of glucuronide and sulfate conjugates. J. Chromatogr. 55, 409 (1971). 24. Mrochek, J. E., Katz, S., Cristie, W. H., and Dinsmore, S. R., Acetaminophen metabolism in man as determined by high-resolution liquid chromatography. Clin. Chem. 20, 1086 (1974). 25. Khym, J. X., Analytical and Biology. Prentice-Hall,

Ion Exchange Procedures in Chemistry Inc., Englewood Cliffs, N. J., 1974. 26. Brown, P. R., High Pressure Liquid Chromatography; Biochemical and Biomedical Applications. Academic Press, New York, N. Y., 1973. 27. Scott, C. D., High pressure ion exchange chromatography as applied to the separation of complex biochemical mixtures. Sep. Purif. 3, 263 (1974).

28. Talmi, spectra of Chem. 48, 29. Olson,

Am.

Y., Crosmum, R., and Larson, N. M., Characteristic noise some common analytical spectrometric sources. Anal. 326 (1976). G. G., Applications of an optical multichannel analyzer. Lab., p69 (February 1972).

30. Flaks, A., Hamilton, J. M., and Clayson, D. B., Effect of ammonium chloride on incidence of bladder tumors induced by 4-ethylsulfonylnaphthalene-1.sulfonamide. J. Nat. Cancer Inst. 51, 2007

(1973). 31. Clayson, D. B., Bedford, A. J., and Turner, R., Acute response of the mouse bladder to derivatives of 4-Ethylsulfonylnaphthalene. 1-sulfonamide and bladder carcinogens measured by the uptake of 5.iodo-2’deoxyuridine (1251). Chem.-Biol. Interact. 6, 107 (1973). 32. Levi, P. E., Knowles, J. C., Cowen, D. M., et al., Disorganization of mouse bladder epithelium induced by 2-acetylaminofluorene and 4-ethylsulfonylnaphthalene-1-sulfonamide. J. Nat. Cancer Inst. 46,

337 (1971).

x

1 10)

\/

1 10

and

or, N N

=

=

total photon

(0.114)(1.25

x

-

E1

=

where photons/W.s 1480

hv

=

8 X 1012

ergs/photon

is given by:

CLINICAL CHEMISTRY,

Vol. 22, No. 9, 1976

1018

flux at mirror

photon W’s

(at 250 nm)

(250 nm):

108) 1.42 X 1017 photons

(at 250 nm)

5

The photon mated

E2

1.25 X

=

=

0.114W(250nm)

pa-

22. Anders, M. W., and Latorre, J. P., High-speed ion exchange chromatography of barbiturates, diphenyihydantoin and their bydroxylated metabolites. Anal. Chem. 42, 1430 (1970).

(250) (40.3) =

Anal.

(1974).

21. Karger, B. L., Martin, M., and Guiochon, G., Role of column rameters and injection volume on detection limits in chromatography. Anal. Chem. 46, 1640 (1974).

/

Calculation of photon rate, reference beam rate, saturation rate, dynamic range, and sample beam detectability. light source: from spectral irradiance curve (Figure 3), S1 = 0.28 tW/cm2#{149}nM, and total energy striking source mirror, Em is:

spectrometry.

H. L., McDowell, A. E., Fast, D. M., and Milano, M. J., of a vidicon spectrometer to analytical problems in clinical chemistry. Clin. Chem. 21, 1192 (1975). 19. Marshall, A. G., and Comisarow, M. B., Fourier and Hadamard transform methods in spectroscopy. Anal. Chem. 47, 491A (1975). 20. Plankey, F. W., Glenn, T. H., Hart, P. L., and Winefordner, J. D., Hadamard spectrometer for uv-visible spectrometry. Anal. C/tern.

Methods

11. Dimov, N. P., Interrupted high speed liquid chromatography with simultaneous spectroscopic identification. DokI. BoIg. Akad. Nauk 27, 1407 (1974). 12. Denton, M. S., DeAngelis, T. P., Yacynych, A. M., et al., Oscillating mirror rapid scanning ultraviolet visible spectrometer as a detector for liquid chromatography. Anal. Chem. 48, 20 (1976).

molecular absorption

loss to entrance

of fiber optics is approxi-

by the area ratios of image to fiber end: 0.9 X 1.5 .9 X 4.0

=

0.38

The total photons/s entering the fiber optics, assuming no scattering loss upon entrance:

(by cooling to -30 #{176}C, the D.R. may improve by a factor of 20)

x

beam: from previous calculation, 10 = 1.40 X photon/s @ 250 nm, assume a guanosine concentration at 109 g/25 sl mouse urine (BALB-C); for

N1 =

0.38

=

1.42 X 10’

sample

1016

5.39 X 106 photons/s

The fiber optics are split into 2 beams, or: N2

Assume

=

2.70 X 1016 photons/s

a 50% loss through N3

=

guanosine,

the fiber optics:

X 1016

1.35

t250

LC peak-width

photon/s

These are the number of photons/s at 250 nm presented to each of the reference and sample beams. reference beam: assume 0.6 mol/liter acetic acid/ acetate buffer (pH 4.4) and at 250 nm, the measured absorbance is 0.015 (1 cm pathlength). The photons/ second that exist from the flow cell is: 1 ln (2) 2.3

-

=

A, where lo

N3

=

1.4

=

1016

solving for I

=

0.1 ml/min; VR shape, where w C( V)

=

L (mw = 283) mole ‘ cm 3 mm (est. for 10 gm); flow-rate 0.3 ml; assume 10 g solution

= =

$

w V’r

et

V2

a true gaussian of:

=

peak

(see Appendix

V- V02)/2Q2

II)

V1

yields: w1 = 2.40 X 10b0 g or, the concentration in the light beam is: c1 = 2.68 X 10-8 mol/liter Then: Total A = (A)buffer + (A)guan (at 250 nm) or,

I assume

11 050

=

=

1 ln 2.3

1.3533 X 1016 photon/s

-

exit fiber optics to grating,

50% loss through

()

=0.015+a-b.c

I

1.40 X 1016

where:

or I = 6.72 X 10’s photon/s at 250 nm assume 50% energy transfer through grating vergence loss proportional to area ratios: I

=

(.0137)(6.72

=

9.135 X 10’s photon/s

and di-

X 1015)

=

5.67 X 10

=

5.67 X 10

=

7.09 X 10 photons

X 1.25

i

X 10’s

I

W-s

(N.E.)CH =

correcting for loss through exit fiber-optics and grating divergence loss (.0068): I = 9.192 X 10’s ph/s; the differential signal developed between the reference and

X 106

dldet

(at 250 nm)

given as 6.9 X 10-6

(9.192-9.135)

=

5.7 x lO

i0’

per second

photons/s

photons/s

W. or, Sdet

=

(5.7

1.45 X 10

.0352

=

sample detectors will be the net photons times the photodiode sensitivity:

6.9 X 10-6 X 2.1 x 10

=

lnI

-

2.68 X 10-8 g

=

1.36 X 1016 photons/s

=

=

noise per channel (N.E.): s/cm2 at 30 #{176}C then,6

11 050, c

=

37.1778

saturation exposure (S.E.): given as 0,27 i W.s/cm2 (ref. 34) but, the area of the 250 nm pixel (channel) is: ar 2.11 X 10 cm2 or, (S.E. )CH

a = 1 cm, b substituting gives:

=

X 1.25 x 1018 x 10-6

x

10)(8

x

10_12)(10_7)(106)(12

=

5.2 X 10

X 10)

2.2 X 10 =

1.8 X 10 photons

Sdet

dynamic range: defined as maximum signal at saturation to the noise signal both measured at a fixed pixel, DR -

Appendix II Solution of gaussian curve for concentration of guanosine in 0.030 ml detector cell. See Figure 16 for details and peak measurements.

(SE)CH

=

(NE)cjj

7.09 X 10

1.8

x

10

where, V0

=

4 X 10 at 30 #{176}C

retention Vb

-

V2Robert R. Buss, personal communication, Benicia Ave., Sunnyvale, Calif. 94086. 6

A

Reticon

Corp.,

V0 V1

volume of guanosine = =

.030 ml 0.3 ml

910

w

=

10

g guanosine

CLINICAL CHEMISTRY,

Vol. 22. No. 9, 1976

1481

V2-V1 6 then xb = 6V/(V2 V1) where V = 0.015, w and V2 - V1 given previously Thus A = 2 X i0 IF(6 X .015)/.3 .5 -

-

vi

=

V2

=

Fig. 16. Gaussian peak shape for guanosine

2 X i0

{F(.3)

-

2 X ‘i0

(.6179

-

=

.5)

2.36 X 1010g

Appendix Ill The curve is of the general

let, Va

=

V0

-

r

w

-

form: (V-V0)2

expV for the area: AT Vb

Comparison of a single pixel in the linear photodiode array (512 pixels) to the photomultiplier (ref. 28, 29) For the Reticon:

2r2

/S\

C(V)dv

.IVQVb

=

V0 + V

=

where N because

the area in the cross-hatched

metrical

about V0, we simplify:

A1

The function, F(x) is a cumulative of a uniform normal distribution sively tabulated, or

=

region is sym2 fva

Vb

=

Q0 =

C(V)dv

Te

=

input photons/s (= 10) quantum efficiency at specified nm (.32 at 250nm) integration or dwell time per channel (1 noise (#{235}/frame) ( 770) number of frames ( 1)

=

N1

=

S

by making the transformation, = dv/o

x

(V

=

V0)/c,

then dx

where N

=

2W{F(Xb)

WF(O))

-

-

1482

CLINICAL CHEMISTRY,

NPM

0.51

for the LC curve, o- can be approximated

Vol. 22, No. 9, 1976

sensing

for

NPQPGK NPM

input photons/s ( 10) quantum efficiency (.1 at 250 nm) G = gain ( 106) K = coulombs/esu ( 1.6 X 10’9) = photomultiplier dark current noise (6 1012 A) =

Q,.,=

C( V)dv] {WF(Xb)

=

N1CH

25v0VbC(V)dv=2[f’bC(V)dv_f..,o =2

-4.1:1

For photomultiplier, single wavelength grating (250 nm, 1 nm bandwidth):

WF(x’)

=

3

(770)(1)

/S\

j.V

thenA

(10)(.32)(1)

()CH

-

NN1

s)

probability function and has been exten-

e----dt

F(x)==J

NpQvTe

NicH

as follows:

1S\

-

1\NIPM

-

(104)(.1)(106)(1.6 6

x

1012

x 10_19)

-

-

6

-

261

X