View - Clinical Chemistry

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

A Minicomputer-Automated 

for Liquid-Chromatographic 

Array Spectrometer 

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 Keyphrases: data processing #{149} screening #{149} 

metabolites in urine #{149} toxicology #{149} ultraviolet spectrometly 

#{149} inherited disorders 

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. 

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 com- 

4Dessy, R., Nunn, W. C., Reynolds, W. D., and Titus, C. A., Linear 

photodiode array spectrometers as detector systems in automated 

liquid chromatographs. 27th Pittsburgh Conference on Analytical 

Chemistry and Applied Spectroscopy, March 1976, paper 375. 

1472 CLINICAL CHEMISTRY, Vol. 22, No. 9, 1976

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): 

F 

35O 

I 

J200 

i25t 

I 

J250 

AdA 

AdA 

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 1473

L 

0 

C.) 

2 

Fig. 1. Flow diagram of high pressure anion-exchange liquid 

chromatograph 

E 

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 (1- 

X8L: 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-1- 

naphthalenesulfonamide and urinary components. 

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Fig. 3. Spectral irradiance curve vs. wavelengths of 150 watt 

xenon source 

DrncT0#{149} 

DIT1CTOI 

(NAHOMITS) 

HOLO0IAPHICALY 

1ULID CONCAVI 

OIATINGI 

Fig. 4. Optical path including liquid chromatographic flow-cells 

for photodiode array spectrometer 

Reticon 

Spectrometer 

Light source. 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 

--- 

WAVII.UIGTH 

Fig. 5. Quantum efficiency response vs. wavelength of the 

photodiode array (Reticon) 

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. 

(nm) 

1 

I 


0 

rrrTTTrnr 

10O. 

TIME m S 

Fig.8. Time base responseof a single pixel (element)to varying 

light intensity (lower). Video line pulse signal to the single pixel 

capacitor (upper) 

Fig. 6. Photodiode linear array (256 pixels) with printed circuit 

background 

LI 

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ELEMENT 1 2 

T 

INDICATE 

SHIFT EEGISTU JO END OF 

SCAN 

. . . S. 

A PHOTODIODE 

E!:.J.) 

4 

VIDEO 

0 COMMON 

1+5 VOLTS) 

Fig. 7. Schematic of photodiode linear array-operational 

view 

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 linear photodiode array (Reticon) 

“read-out”. 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 

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 

Detail from Figure 8 

Fig. 11. Mini-computer configuration with I/O devices 

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 

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Fig. 10. Schematic of Reticon Array Detectors (sample, reference) 

and analog “front-end” for computer input 

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 

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 

AISOSIANCI 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. 

T.ANsMITtAHcI 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 


USIa DIaVICI 

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(HillS) 

INPUT ITS, 11101 IKTUIN, HALT 

DIVICI HANDLII 11101 HAIDW*II PAftull 01 

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PLOT IDA 3) MONITOI PILl IDIOt ON DLI 

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3) 

Fig. 12. Overview of computer system data flow and display 

CONVIIT DATA 

TO 3 WelD 

INTIOII 

)LOOP 2. LOOP I 

INPUT 

IUOO.SV 

FILl 

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. 

Application 

Pathological Effects (Mice) 

4-Ethylsulfonylnaphthalene- 1-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 of [‘4C] 4-ethylsulfonyl- 1-naphthalenesulfonamide 

in mice strains is not fully 

DIGIN 

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Fig. 13. Software program for binary to three word floating point 

convertor 

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- 

(CHAIN) 


IN 

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z 

4 

0 

Ia 

1.0 

0.7 

0.5 

Ia 

4 

I 

0.3 

II 

I I----a 

20 250 300 350 

_a 

WAVELENGTH 

Fig. 14. Ultraviolet spectrum (200-350 nm) of [14C]4-ethylsulfonyl-1-naphthalenesulfonamide, 

from Reticon spectrometer 

IN 

U 

z 

1.0 

(nm) 

‘I S 

I 

I \ 

I 

0.3 s / 

/ 

I 

I 

0.3 I I 

/ 

‘a, I’ 

C II (III I I I II 

200 230 3 

WAyft1GTh 

Fig. 15. Ultraviolet spectrum of [‘4C]4-ethylsulfonyl-1-naphthalenesulfonamide 

metabolite, from Reticon spectrometer 

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-ethylsulfonyl- 1-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 computerdriven 

plotter 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. 

Discussion 

I 

‘I 

A 

‘I.. 

Our interest in the computerized liquid chromatography/ultraviolet 

spectrometry system is twofold. First, 

the generation of a data base of ultraviolet spectral data 

(em) 

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. 


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Appendix 

I 

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: 

Em = (1.4) 2(0.28) (1o6) (250) (40.3) 

and 

= 0.114W(250nm) 

E2 - E1 = hv = 8 X 1012 ergs/photon 

where photons/W.s is given by: 

/ 1 \/ 1 

x 10) 10 

photon 

= 1.25 X 1018 (at 250 nm) 

W’s 

or, N = total photon flux at mirror (250 nm): 

N = (0.114)(1.25 x 108) 

= 1.42 X 1017 photons (at 250 nm) 

5 

The photon loss to entrance of fiber optics is approximated 

by the area ratios of image to fiber end: 

0.9 X 1.5 = 0.38 

.9 X 4.0 


The total photons/s entering the fiber optics, assuming (by cooling to -30 #{176}C, the D.R. may improve 

no scattering loss upon entrance: of 20) 

by a factor 

N1 = 0.38 x 1.42 X 10’ sample beam: from previous calculation, 10 = 1.40 X 

1016 photon/s @ 250 nm, assume a guanosine concen- 

= 5.39 X 106 photons/s tration at 109 g/25 sl mouse urine (BALB-C); for 

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

N2 = 2.70 X 1016 photons/s t250 = 11 050 L (mw = 283) 

mole ‘ cm 

Assume a 50% loss through the fiber optics: LC peak-width = 3 mm (est. for 10 gm); flow-rate = 

N3 = 1.35 X 1016 photon/s 0.1 ml/min; VR = 0.3 ml; assume a true gaussian peak 

shape, where w = 10 g solution of: 

These are the number of photons/s at 250 nm presented 

to each of the reference and sample beams. w $ V2 et V- V02)/2Q2 (see Appendix II) 

reference beam: assume 0.6 mol/liter acetic acid/ C( V) = V’r V1 

acetate buffer (pH 4.4) and at 250 nm, the measured 

absorbance is 0.015 (1 cm pathlength). The photons/ yields: w1 = 2.40 X 10b0 g or, the concentration in the 

second that exist from the flow cell is: light beam is: c1 = 2.68 X 10-8 mol/liter 

Then: 

- 1 

2.3 

ln (2) = A, where lo = N3 = 1.4 1016 Total A = (A)buffer + (A)guan (at 250 nm) 

solving for I or, 

1 

I = 1.3533 X 1016 photon/s - ln 

2.3 () =0.015+a-b.c 

assume 50% loss through exit fiber optics to grating, 

where: 

or 

I = 1.40 X 1016 

I = 6.72 X 10’s photon/s at 250 nm 

assume 50% energy transfer through grating and di- a = 1 cm, b = 11 050, c = 2.68 X 10-8 g 

vergence loss proportional to area ratios: substituting gives: 

I = (.0137)(6.72 X 1015) 37.1778 - lnI = .0352 

= 9.135 X 10’s photon/s I = 1.36 X 1016 photons/s 

saturation exposure (S.E.): given as 0,27 i W.s/cm2 correcting for loss through exit fiber-optics and grating 

(ref. 34) but, the area of the 250 nm pixel (channel) is: divergence loss (.0068): I = 9.192 X 10’s ph/s; the difar 

2.11 X 10 cm2 or, ferential signal developed between the reference and 

(S.E. )CH = 5.67 X 10 i W-s sample detectors will be the net photons per second 

times the photodiode sensitivity: 

= 5.67 X 10 X 1.25 X 10’s X 106 

dldet = (9.192-9.135) i0’ photons/s 

= 7.09 X 10 photons (at 250 nm) 

= 5.7 x lO photons/s 

noise per channel (N.E.): given as 6.9 X 10-6 W. 

s/cm2 at 30 #{176}C then,6 

or, 

Sdet = 

(N.E.)CH = 6.9 X 10-6 X 2.1 x 10 

(5.7 x 10)(8 x 10_12)(10_7)(106)(12 X 10) 

= 1.45 X 10 X 1.25 x 1018 x 10-6 

2.2 X 10 

= 1.8 X 10 photons Sdet = 5.2 X 10 A 

dynamic range: defined as maximum signal at saturation 

to the noise signal both measured at a fixed 

pixel, Appendix II 

DR = 

(SE)CH 

(NE)cjj 

Solution of gaussian curve for concentration of guanosine 

in 0.030 ml detector cell. See Figure 16 for details 

and peak measurements. 

- 7.09 X 10 

1.8 x 10 where, V0 = retention volume of guanosine 

4 X 10 at 30 #{176}C Vb - V0 = .030 ml 

V2- V1 = 0.3 ml 

6 Robert R. Buss, personal communication, Reticon Corp., 910 

Benicia Ave., Sunnyvale, Calif. 94086. w = 10 g guanosine 

CLINICAL CHEMISTRY, Vol. 22. No. 9, 1976 1481

V2-V1 6 

vi 

V2 

Fig. 16. Gaussianpeak shape for guanosine 

The curve is of the general form: 

- w r (V-V0)2 

exp- 2r2 

let, Va = V0 - V for the area: AT = .IVQVb C(V)dv 

Vb = 

V0 + V 

because the area in the cross-hatched region is symmetrical 

about V0, we simplify: A1 = 2 fva Vb C(V)dv 

The function, F(x) is a cumulative probability function 

of a uniform normal distribution and has been extensively 

tabulated, or 

F(x)==J 

e----dt 

by making the transformation, x = (V - V0)/c, then dx 

= dv/o 

j.V = WF(x’) 

thenA 25v0VbC(V)dv=2[f’bC(V)dv_f..,o 

C( V)dv] 

=2 {WF(Xb) - WF(O)) 

= 2W{F(Xb) - 0.51 

for the LC curve, o- can be approximated as follows: 

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 

Appendix 

Ill 

= 2 X i0 {F(.3) - 

= 2 X ‘i0 (.6179 - .5) 

= 2.36 X 1010g 

Comparison of a single pixel in the linear photodiode 

array (512 pixels) to the photomultiplier (ref. 28, 29) 

For the Reticon: 

/S\ 

NicH 

NpQvTe 

NN1 

where N = input photons/s (= 10) 

Q0= quantum efficiency at specified nm (.32 at 

250nm) 

Te = integration or dwell time per channel (1 

s) 

= noise (#{235}/frame) ( 770) 

N1 = number of frames ( 1) 

S (10)(.32)(1) 3 

()CH (770)(1) -4.1:1 

For photomultiplier, single wavelength sensing for 

grating (250 nm, 1 nm bandwidth): 

/S\ 

N1CH 

= NPQPGK 

NPM 

where N = input photons/s ( 10) 

Q,.,= quantum efficiency (.1 at 250 nm) 

G = gain ( 106) 

K = coulombs/esu ( 1.6 X 10’9) 

NPM = photomultiplier dark current noise (6 X 

1012 A) 

1S\ - (104)(.1)(106)(1.6 x 10_19) - - 261 

1\NIPM - 6 x 1012 - 6 -

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