Anal. Chem. 1994,66, 2267-2271
I n Vivo Percutaneous Absorptiometry by a Laser Photoacoustic Method Using a Novel Open-Ended Cell Combined with Light Guide Ryulchi Takamoto,t Shinya Yamamoto,t Ryujiro Namba,t Tawku Takamatsu,t Masahiro Matsuoka,t and Tsuguo Sawada'r* Safety & Analytical Research Center, Shiseido Company, Ltd., 1050 Nippa-cho, Kohoku-ku, Yokohama 223, Japan, and Department of Industrhl Chemism, Faculty of Engineering, The UniversHy of Tokyo, 7-3- 1 Hongo, Bunkyo-ku, Tokyo 113, Japan
A laser photoacoustic (PA)method with high sensitivity and accuracy using a novel open-ended PA cell was developed. This PA cell was constructed by processing the end of a light guide as the sample chamber of the PA cell and by attaching an acoustic pipe which was bent optimally to a right angle at one end. This design minimized the dead volume and provided more measurement flexibility. The PA cell design is based on the principle of a resonance permitting a smaller gas volume in the PA cell than that of Helmholtz resonance. As a result, the new PA cell sensitivity was about 10 times that of a conventional PA cell. The sensitivity and reproducibility for in situ PA measurement of an indomethacin (IDM) ointment introduced in a poly(ethy1eneglycol) vehicle were excellent over 10repeated measurements. The signal-to-noise ratio was 46, and the coefficient of variation was not more than 3%for an amount of 2 pg/5 mm i.d. Cood linearity was obtained for amounts of 2-120 pg/5 mm i.d. and the correlation coefficient was not less than 0.99. In vivo percutaneous absorptiometry was attempted using 1%IDM ointment. The diffusion coefficient of IDM through the skin measured by this PA method was 6.7 X 1od cm*/min. This is of the same order of magnitude as results obtained by the conventional in vitro method using a longitudinal diffusion cell. These results confirmed the effectiveness of this simple newly developed in vivo percutaneous absorptiometry technique, compared with conventionalmetbods such as radioisotopes. Evaluation of the percutaneous absorption of a substance
is very important in development of endemic liniments. Various percutaneous absorptiometry methods have been reported'-5 including diffusion cell methods and radioisotopes (RI) , These cannot be used for humans in vivo because skin is either excised or a radioactive compound is used. Therefore, many investigations have used animal substitutes. However, we need information about percutaneous absorption in humans in vivo. Effective human in vivo measurement using the t Shiscido Co., Ltd. 8 University of Tokyo. (1) Bronaugh, R. L.;Stewart, R. F.J. Pharm. Sci. 1986, 75, 109&1097. (2) Southwell, D.; Barry, B. W.; Woodford, R. Int. J. Pharm. 1984,18,299-309. (3) Tojo, K.;Ghannam, M. M.;Sun, Y.;Chien, Y . W.J. Controlled Rclcosc
1985,1,
197-203.
(4) Gummcr,C. L.;Hinz, R. S.;Maibach, H. 1. Int. J. Pharm. 1987,10,101-104. ( 5 ) Guy, R.H.; Carlstrom, E. M.;Bucks, D. A. W.;Hinz, R. S.;Maibach, H. I. 1. Pharm. Sci. 1986, 75, 968-972. QQQ3-27QQl94IQ3662267~Q4.5QlQ @ 1994 Amerlcan Chemical Society
stripping method has been reported by Rougier et al.,6p7 but this method damages the skin and causes measurement problems. There are few methods of measuring percutaneous absorption in humans in vivo reliably and easily. The important conditions for human in vivo measurement are safety, accuracy, spee, and simplicity. Several investigationshave reported the photoacoustic (PA) method an effective The most remarkable point is that the reliability of an open-ended PA cell is influenced by either the surface potential of an in vivo object such as skin or by environmental noise determined by the measurement of signal-to-noise ratios (S/N). Additionally, the PA cell operability must be considered. Several open-ended PA cells have been reportedI2-I4 for obtaining better S/N values. We have reported the development of percutaneous absorptiometry using the laser PA method and its usefulness for in vitro and in vivo measurements. This system was combined with a longitudinal diffusion cell used generally for in vitro percutaneous absorptiometry to substantiate its applicability to in vitro measurement.l5 It was actually applied to human in vivo percutaneous absorptiometryI6 to show the potential compared to conventional PA methods.I2-l4 This PA system used a laser as the light source, and the laser beam was guided to the open-ended PA cell by an optical fiber. This arrangement minimized the noise level and the detected PA signal had a high S/N. We were able to evaluate the percutaneous absorption of subject drugs by measuring the change in the PAsignal with timecorresponding to the amount of drug remaining on the skin. However, when this PA method was tried with many substances, it was apparent that a more sensitive PA device (6) Rougier, A.; Dupub D.; Lot@ C.; Rogua, R.;Schacfcr, H. J. Invcst.Dcrmatol.
I. Aich. Dcrmaiol. Res. 1986, 278, 46-5-469. (8) Roscncwaig, A. Clin. Chcm. 1982,28(9). 1878-1881. (9) Roscncwaig, A.; Pines, E. Bfochfm.Btophys. Acta 1977, 493, 10-23. (IO) Campbell, S.D.; Yee. S.S.;Afromowitz, M. A. 1. Biocng. 1977,1,185-188. ( 1 1) Campbell, S. D.; Ye,S.S.;Afromowitz, M.A. IEEE Trans. Biomcd. Eng. 1979, BME-26(4), 220-227. (12) Poulet. P.; Chambron, J. J. Photoacoust. 1983, 1. 329-346. (13) Kolmcl, K;Sennhenn, B.;Gicsc, K.J. Soc. Cmmcr. Chem. 1986,37,375-385. (14) Gicsc, K.;Nicolaus, A.; Sennhenn, B.; Kolmel, K. Can. J. Phys. 1986.64,
1139-1142.
(IS) Takamoto, R.; Namba, R.; Nakata, 0.;Sawada, T. Anal. Chcm. 1990,62, 674-677 (16) Takamoto. R.; Namba, R.;Matsuoka, M.; Sawada, T. Anal. Chcm. 1992,64, 266 1-2663.
Analjlticel Chemisby, Vol. 66, No. 14, Ju& 15, 1994 2267
was required. This was necessary because we used a light source oscillating in the ultraviolet (UV) wavelength region because many drugs generally absorb light at wavelengths of 200-300 nm. In this case, the most significant problem is related to the safety of skin irradiated with UV light. Use of visible laser light causes no problem because the maximum permissible exposure (MPE) values for human skin are clearly known. However, the MPE values for human skin irradiated by UV light wavelengths (less than 290 nm) are not clearly known because these wavelengths do not reach the earth's surface and experimental data have rarely been reported. Accordingly, the intensity of the light applied to the skin sample must be as low as possible. Under this condition, we must be able to detect a small change in the PA signal when the substance penetrates the skin. We tried to develop an open-ended PA cell with a higher sensitivity than that of the conventionalPA ce11.16 We know that reducing the total gas volume of the PA cell produces higher sensitivity, within limits.17 We designed a PA cell on this basis by determining parameters such as the sample chamber, the microphone chamber, and length and crosssectional area of the acoustic pipe. However, designs based on the conventional open-ended PA cel11*-J4J6increased the total gas volume more than necessary, causing a drop in sensitivity. We needed a design that did not increase the gas volume and minimized the dead volume. In addition, most conventional open-ended PA cells are based on the Helmholtz resonance theory1*to amplify the PA signal. The Helmholtz resonance frequency,f ~is,given by
where c is the speed of sound in the coupling gas, VSCand VMC are the volumes of the sample and microphone chambers, respectively, and d is the diameter of the sample/microphone acoustic coupling tube of length, L, connecting VSCand VMC. The Helmholtz resonance theory requires a certain volume in both the sample and microphone chambers. This means that minimizing the total gas volume in the PA cell has limits. VSC is required to transform heat from the sample into sound, but VMCincreases the total gas volume of the PA cell as a result. Four points occurred in developing a new open-ended PA cell: (1) use resonance theory for causing resonance with no VMC;(2) minimize the influenceof skin surface potential and environmental noise (less than 100 Hz); (3) minimize the dead volume of the PA cell; (4) miniaturize the PA cell for easy measurement. We developed a novel open-ended PA cell on the basis of these concepts. A bundle-type guide was used to guide the UV light from the light source to the PA cell to provide more flexibility during measurement. To minimize the dead volume of the cell, the end of the light guide was used as a cell by attaching an acoustic pipe to the end of the light guide. This PA cell is hardly affected by light scattering in the sample chamber. This design was expected to provide better sensitivity and more accurate measurements than obtained with a conventional PA ce11.16 In vivo absorptiometry was attempted using (17) Aamodt, L. C.; Murphy, J. C.; Parker, J. G. J. Appl. Phys. 1976,47,64-68. (18) Thompson, M. M.; Palmer, R. A. Anal. Chem. 1988,60, 1027-1032.
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Mici?ophone
I l l l l l~
T
SP
i
Acoustic (i. d=O.
(5cm)
Figure 1. Diagram of a novel open-ended PA cell combined with the light guide.
NONLINEAR OPTICAL CRYSTAL
I
I
I
FREQUENCY CONTROLLER
I
I
OPENENDED PA CELL
7.
.:$.
WAVELENGTHS: 240-260 nm (DISCONTINUOUS) (DISCONI'I NUOUS) LIGHT POWER:lOmW (257.2 nm)
Figure 2. Diagram of laser PA system using the open-ended open-ei7ded cell.
a 1% indomethacin (IDM) ointment applied to a hairless mouse. The propriety and effectiveness of this PA method was investigated by comparing the results obtained with this PA method with those from the diffusion cell method for typical percutaneous absorptiometry. EXPERIMENTAL SECTION Figure 1 shows the construction of the novel open-ended PA cell combined with light guide. A bundle-type guide (Asahi Glass Corp., 5 mm diameter X 1 m length), which can transmit more than 85% of the UV light, was used to provide more measurement flexibility and to minimize the dead volume of the cell. The open-ended PA cell uses the end of the light guide as the sample chamber (stainless steel) by attaching an acoustic pipe (@-brass)bent at a right angle inside the end of the light guide. A microphone (Primo, MP- 10) was threaded to the acoustic pipe to control its length and to eliminate the volume of the microphone chamber. The volume and width of the air layer in the sample chamber are 0.04 cm3 and 0.2 cm, respectively. This volume is about 40% of that of the conventional PA ce11.I6 Figure 2 shows an in vivo percutaneous absorptiometrysystem using the open-ended PA cell combined with the light guide. UV light with a CW of 257 nm was used as the light source and was obtained by oscillating a multiple Ar+ laser beam (Spectra Physics, Model-2020) at 5 W and using a P-BaB204 (BBO) crystal wavelength conversion unit (Ascal Corp., UVA-4), which can be microadjusted optically. The UV laser beam was modulated at 2.2 kHz (the resonance frequency of the PA cell) using a light chopper (NF Corp.). The optical path was very easily adjusted because the PA cell was combined with the light guide. The output intensity of the light source was set to 3 mW by the light guide. The PA signal detected by the PA cell was analyzed using a lock-in amplifier (NF Corp, 5610A), recorded using a chart recorder (Rika Denki Kogyo, NP-0393) and calculated using a computer (Nippon Denki Corp., VX).
Indomethacin (C19H&lN04, Wako Pure Chemical, reagent class; for biochemistry), which absorbs light at a wavelength of 257 nm, was used as the model sample. The IDM ointment was prepared using poly(ethy1ene glycol) (PEG) as the vehicle, and the mixture was heated (80 "C). A hairless mouse was used as the subject animal. First, the sensitivity of this new PA cell was optimized. In vivo percutaneous absorptiometry was then applied to the hairless mouse.
RESULTS AND DISCUSSION Resonance Theory of a New PA Cell and Determination of Total Length of Acoustic Pipe. We attempted to clarify the resonance mechanism of this PA cell designed to minimize total cell volume. The total length of the acoustic pipe is L (SP + MP), and the coordinates x are given along the axis of the acoustic pipe. When the sample chamber-acoustic pipe interface is defined as x = 0, the sound field in this PA cell is equivalent to that of a diaphragm vibrating at UOsin(or)at x = 0 and the point (x = L) is closed by the microphone, where UO is the initial particle velocity, o is the angular frequency, and t is time. Since the wavelength of the sound generated in the PA cell is much longer than the inner diameter of the acoustic pipe, only a plane wave progressing along the axis of the acoustic pipe occurs in the air of the PA cell. This sound field is analyzed below. Since the sound is reflected by the microphone, the stationary wave (the sound pressure amplitude (P,) of the reflective sound is equal to that (Pi) of the incident sound) is generated in the acoustic pipe. In addition, at the point of x = L (Pr is in phase of Pi), the sound pressure amplitude (P) of the stationary wave becomes maximum (2Pi) and the particle velocity becomes minimum (0). Accordingly, the distribution P(t,x)of the sound pressure of the stationary wave in the acoustic pipe is expressed as P(r,x) = 2Pi cos[(kx - (cpoi - 'por)/2] sin[wt + (vN + 'por)/2]
(2)
where k is the phase constant, cpoi is the initial phase of the incident sound, and cpor is the initial phase of the reflective sound. Since P is equal to 2Pi at x = L, we can write 'poi
- 'par = 2kL - 4 n ~( n = 0, 1,2, ...)
(3)
Equation 2 then becomes P(r,x) = 2Pi cos[k(L - x)] sin(ot
+ 'poi - kL)
(4)
Bythesame treatment, thedistribution U(t,x)ofthe particle velocity of the stationary wave in the acoustic pipe is given by U(t,x) = (2Pi/pc)sin[k(L - x)] cos(wt + pN- kL)
(5)
where pc is the specificacoustic resistance. Since eq 4 becomes UOsin(or) at x = 0, it can be shown that
Uosin(ot) = (2Pi/p,) sin(kL) cos(wt + 'poi - kL)
(6)
e : THEORETICAL VALUE
t]: EXPERIMEWAL VALUE
Natural Number (m-1 Fundamental Resonance Frequency)
Flgure 3. Theoretical treatment of the resonance frequency caused by changing the total length of the acoustic pipe.
The conditions for forming eq 6 are given by 490i
= kL
+ (2n + l)T/2
( n = 1,2,3, ...)
2Pi/(p,) = V,/[sin(kL) sin(2n + 1 ) ~ / 2 ]
(7) (8)
Consequently, the distribution of the sound pressure and the particle velocity are expressed as P(t,x) = pcUo/[sin(kL)] cos[k(L- x)] cos(wt + T )
(9)
U(t,x) = Vo/[sin(kL)] sin[k(L - x)] sin(wt)
(10)
As obtained from eqs 9 and 10, the conditions for creating resonance in the PA cell are given by kL = m r L = mX/2 where m is the natural number and h is the wavelength of the sound generated in the PA cell. Therefore, the resonance wavelength (XR) and the resonance frequency ( f ~ are ) determined by
XR
2L/m
fR = m(V,/2L)
(13) (14)
where V, is the sound velocity of about 340 m/s at room temperature(l5 "C). Them = 1 resonanceisthefundamental one. From eqs 13 and 14, it is clear that this resonance type is equal to the open-pipe resonance when a sound pressure was given at x = 0. We examined this resonance theory. As shown in Figure 3, the resonance frequencies measured when the total length of the acoustic pipe was changed were in good agreement with those calculated from the resonance theory (eq 14). Consequently, it is obvious that the resonance frequency of this new PA cell is determined by the total length of the acoustic pipe. The total length of the acoustic pipe connecting the sample chamber and the microphone is set to 8 cm, giving a resonance of about 2 kHz, which is hardly affected by skin pulsation and environmental noise. In addition, the resonance can be adjusted by changing the length of the acoustic pipe. Ana&tical Chemktty, Vol. 66, No. 14, July 15, 1994
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Modulation Frequency [kHz] Flgure 4. Comparison of the PA signal amplitude with change In the length of SP and MP (SP MP = 8 cm).
3
Conventional PA c e l l 3
Modulation Frequency [Mz]
Flgure 5. Comparison of the sensitivity of the new PA cell and the conventional PA cell utilizing the Helmholtz resonance theory.
+
Determinationof the Bend Point of an Acoustic Pipe. The acoustic pipe attachment to the PA cell was optimized. We investigated the frequency characteristics of the PA signal amplitude by changing the bend point (SP) of the acoustic pipe so that the total length was 8 cm at the right angle. This bending angle test was carried out only at two points with angles of both 0’ (straight) and 90°, with no other angles between 0’ and 90°, because we judged that the sound behaves similarly to a straight line when the bend angle is smaller and closer to a right angle when the bend angle is bigger. Figure 4 shows the results. The resonance frequency was kept constant at about 2.2 kHz; the PA signal amplitude at the resonance frequency was largest when SP was equal to 5 cm. From this result, we decided that the acoustic pipe should be bent at a right angle with sides of 5 (SP) and 3 cm (MP) in length. Thechanges in the PA signal amplitude accompanying changes in the length of SP seem to be caused by a very complex interaction such as the relationship between the wavelength (16 cm at room temperature, obtained from eq 13) at a frequency of 2.2 kHz and the sound reflection from the bend of the acoustic pipe. This result gave a structure for a PA cell producing a resonance effect much greater than the PA cell (SP = 8), previously reported.’6 The total length of the acoustic pipe can be changed by adding a threaded pipe 3 cm long (MP) to the acoustic pipe. This is effective for depthprofiling analysis performed by changing the measurement frequency. When the length of MP is changed, the changes in the resonance frequency conform to eq 14. The lower the used frequency, the bigger the amplitudes of both the PA signal and noise. When considering that an S/N of more than 5 was obtained for in vivo skin measurement, resonance frequency adjustment is effective over the range of about 8005000 Hz. This method could be used very effectively in depthprofiling analysis. Sensitivity and Reproducibilityof a Novel Open-Ended PA Cellcombined with a Light Guide. The sensitivity of the newly developed PA cell was studied by comparing it with the conventional and Helmholtz resonance type PA ce11.16 Black rubber was used as the PA measurement sample. The Ar+ laser beam (wavelength 488 nm) used as the light source was modulated with the light chopper at 2.2 kHz. The output intensity of the light source was adjusted to 3 mW. The characteristics of the PA signal of the new PA cell based on a specific measurement frequency were compared with those of the Helmholtz resonance PA cell. As shown in Figure 5 , two resonance peaks were found at about 2.2 and 4.0 kHz. 2270
AnalyticalChemistry, Vol. 66, No. 14, July 15, 1994
The intensities of these peaks were about 7 and 26 times, respectively, larger than that for the conventional PA cell. In the new PA cell, the peak intensity of the harmonics generated at about 4.0 kHz is very close to that of the fundamental vibration generated at 2.2 kHz. This suggests that the resonance mechanism of this new PA cell is different from that of Helmholtz resonance and that this resonance is better suited to surface analysis than Helmholtz resonance. In addition, the difference between each resonance curve is based on the different resonance types. The Q value (sharpness of resonance curve) obtained from this PA cell is sharper than that of the conventional PA cell (Helmholtz resonance type). From these results, the sensitivity of the new PA cell is approximately 10 times greater than that of the conventional PA cell. The detection accuracy of PA measurement of IDM was investigated using a UV laser PA system. IDM ointments of concentrations of 0.2%and 1.O% were applied to quartz plates at thicknesses of 100 mm and were used as samples for determining the PA measurement reproducibility. Excellent sensitivity and reproducibility were obtained over 10 repeated measurements; the S/N was 46 and the coefficient of variation (CV) was not more than 3%. Calibrationcurves wereobtained for PA measurements under the same conditions. The PA signal of the novel open-ended PA cell had excellent linearity, with a correlation coefficient of 0.996 for the IDM range (2-24 pg/5 mm i.d.) applied to the skin at in vivo measurement. In addition, a good linear correlation coefficient of 0.99 was obtained for IDM amounts (2-120 p g / 5 mm i.d.) extending the measurement range. The novel open-ended PA cell is effective for in vivo percutaneous absorptiometry using this PA measurement system. When the S/N obtained from IDM ointment on the quartz plate is compared with that of the same sample on in vivo skin, the former is lower than the latter. This is because the gas seal of the PA cell on quartz is worse than on skin because the quartz plate is very hard and inelastic. Hence, although factors such as the skin surface potential or environmental noise decrease the S/N in measurement of in vivo skin, this PA cell is hardly affected. This means that the S/N and accuracy of in vivo skin measurement is better than that of the quartz plate. Consequently, the open-ended PA cell combined with the light guide is extremely useful for in vivo percutaneous absorptiometry. Measurementof In Vivo PercutaneousAbsorption. A series of in vivo percutaneous absorptiometries using the new openended PA cell was performed using 1 % IDM ointment applied to a hairless mouse. Measurements were made every 5 min
1 0
min.
II
Time
Flgure 8. Raw data of the in vivo measurement of percutaneous absorption In the hairless mouse. n=5
9 70 -
I
T
I
I
1% IDM Ointment
where L is the width of the membrane and D is the diffusion coefficient of the substance in a given medium. This principle was applied to the results in Figure 6. A time constant of 607 min was obtained with a correlation coefficient of 0.948: the diffusion coefficient was calculated to be 6.7X 10-6 cm/min for a skin thickness of 0.1 cm. This value is of the same order (3.5 X 10-6cm2/min) as that obtained by the conventional in vitro method using the longitudinal diffusion cell. These results confirmed the effectiveness of this newly developed in vivo percutaneous absorptiometry technique, which is simpler than other methods such as RI. Additionally, this UV laser PA method using a novel open-ended cell may be applicable to in vivo human percutaneous absorptiometry, because we know from experience that the S/N of in vivo human skin is about 5 times better than that of in vivo animal skin. Humans can consciously control their skin pulsation but animalscannot, soa PA signal with a larger S/Nis required for animals. The better performance of the developed PA system is clearly demonstrated by the results of PA measurement for a hairless mouse in vivo.
-i k Vehlcle
lo 00
10
20
30
Time
40
50
CONCLUSIONS
60
[min.]
Ftgurr 7. PA slgnal versus time when measuring hairless m o w in vivo percutaneous absorption.
to avoid accelerated percutaneous absorption as a result of heating by the UV laser beam. The measurement time of 15 s, or half the best measuring time yet, was reduced by the improved S/N. This was very effective in minimizing damage to in vivo skin as much possible. The obtained S/N was about 40 (Figure 6). Figure 7 shows the results of five repeats. When the PA signals obtained by applying only the PEG vehicle were compared with those of the 1% IDM ointment measured under the same conditions, the former was nearly constant with time, while the latter indicated a 5% reduction in the initial PA signal after 60 min. It was confirmed that the reduction of the PA signal is not due to chemical changes in or destruction of the IDM by irradiation. The change in the PA signal is thought to reflect the percutaneous absorption of IDM. We then tried to obtain the diffusion coefficient of IDM across the skin of a hairless mouse in vivo by observing the time constant of the decrease in the PA signal." Since the decrease in the concentration of the IDM was essentially exponential, the time constant was obtained by plotting logarithmicPA signals with time. In addition, from the theory of diffusion of substances across membranes of finite width, it was found that the time constant, T , of such processes is expressed as
A novel in vivo percutaneous absorptiometry system using an open-ended PA cell combined with a light guide was developed. The sensitivity of this system was nearly 1 order of magnitude better than that of the conventional open-ended PA ce11.I6 A 1% IDM ointment was applied to hairless mice, and the in vivo percutaneous absorption was measured. A reasonable diffusion coefficient was obtained from the PA signal related to percutaneous absorption of IDM. This result confirmed the feasibility of in vivo percutaneous absorptiometry using this PA method. Application to in vivo human skin must be safe, the total
UV energy received at the skin was less than 40 mJ, and no ill effects were observed on the skin of hairless mice. Applying this energy to human skin in vivo seems to be safe but final confirmation is required. Experiments to confirm safety for human skin are under way. This newly developed open-ended PA cell has other advantages. Depthwise analysis is possible by changing the length of the acoustic pipe. In addition, the open-ended PA cell can be used as a closed cell by mounting a sample holder made of quartz on the end of the light guide (the sample chamber of the open-ended cell). Consequently, this PA system will be easier to use with higher sensitivity and greater accuracy. Received for review July 21, 1993. Accepted April 20, 1994.e
Abstract published in Aduance ACS Absrracrs, June 1, 1994.
Analytical Chemistry, Vol. 66, No. 14, July 15, 1994
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