Anal. Chem. 1995, 67, 2858-2863
UV/Visible Spectral Dissolution Monitoring by in Situ Fiber-optic Probes J. H. Cho,t P. J. Gemperline,t A. Salt,* and D. S. Walker*l* Department of Chemistv, East Carolina University, Greenville, North Carolina 27858, and Burroughs Wellcome Co., Analytical Development Laboratories, P.0. Box 1887, Greenville, North Carolina 27835
A seven-channelfiber-optic W/visible spectrograph has been developed for in situ dissolution testing of pharmaceutical products. The system employs a spectrograph and a two-dimensional charge-coupled device for detection. This arrangement permits simultaneous monitoring of six dissolution vessels and a seventh reference vessel. Wavelength calibration algorithms were developed to ensure that spectra recorded on each of the seven probes are mutually compatible. Standard reference materials and placebo ingredients were used to create multicomponent calibration models. Multivariate calibration techniques were developed in an effort to produce rugged analytical methods. Four products with single active ingredients were studied. We also conducted a series of studies to determine the reproducibility,both day-to-day and probe-to probe, and linearity and lack of interference from excipients for the assays developed using the new instrument. It was determined that in situ dissolution testing is feasible for the formulations studied. The application of this technology is attractive because it saves time and labor and reduces the need for analytical solvents. Dissolution testing is a requirement for the regulatory approval of solid dosage form pharmaceutical products. Without automation, it is a labor-intensive process. Traditionally, automating the process has focused on removing sample aliquots for analysis at allotted times. This has been accomplished using automated liquid samplers and laboratory robotics. We have developed a system that eliminates the need for sample removal. By placing fiber-opticprobes directly into the dissolution vessels, W/visible spectra of the absorbing components are monitored in real-time. The application of fiber optics to pharmaceutical applications has been limited up to this time. Brown and En1 studied the dissolution of Sudafed Plus (tradename of Burroughs Wellcome Co.) by W/vis spectroscopy using a fiber-optic apparatus. The results indicate that despite poor optical efficiency, 2-5%, they could successfully track the dissolution of a sample directly in a dissolution bath over a 20 min period without diluting or filtering the sample. It has also been shown by Josefson et al.2that sample turbidity in an optical W/vis assay is not problematic if appropriate calibration sets are available for the sample. The group employed partial least-squares methods (PIS) to correct for the disturbance caused by sample turbidity in felodipine tablets. ~~
A
~~~~
~~
East Carolina University.
* Burroughs Wellcome Co. (1) Brown, C.W.; Lin, J. Appl. Spectrosc. 1993,47, 615. (2)Josefson, M.; Johansson, E.; Tortensson, A. Anal. Chem. 1988,60,26662671.
2858 Analyfical Chemistry, Vol. 67, No. 17, September 7, 1995
The multichannel, multicomponent W/vis fiber-optic spectrograph described in this paper utilizes commercially available technologies that have experienced a large growth in their fields of application. This type of technology, remote monitoring with fiber optics, is not completely new, as it has been used to monitor fuel types in the petroleum indu~try.~Other workers have described the measurement of W/vis absorption with fiber-opticbased instrumentation. Ren and Synovec described a single-fiber instrument for multiwavelength absorbance measurements! A fiber-optic photometer for monitoring groundwater and soil contamination and also for wastewater monitoring has also been d e ~ r i b e d The . ~ development and application of fiber-optic sensors were recently reviewed by Angel et aL6 Our system is unique in that it can simultaneously collect W/ vis spectra (200-450 nm) from seven fiber channels with no moving parts, with low noise (1 electron pixel-' h-l), and at a faster rate than is possible with commercially available systems. Under appropriate conditions, spectra recorded on any of the seven fiber channels can be used for calibration of other fiber channels. Major advantages of this technology over existing commercial dissolution systems include reducing the complexity of existing W/vis dissolution assays and reducing the cost of dissolution measurements through reduction of filter and glassware use, human effort, and solvents used. Four formulations were chosen for the study, which will be referred to as products A, B, C, and D, so as not to reveal proprietary information of the Burroughs Wellcome Co. The weight of active ingredients in these products covered a wide range, from about 30 mg/tablet to '750 mg/tablet. These products were chosen because each formulation contains a single active component that can be quantitated by W absorbance measurements, the active components span a relatively wide range of W absorptivities, the four products span a wide ratio (w/w) of active to excipient ingredients, and all four are high-volume products in terms of the number of dissolution tests performed. EXPERIMENTALSECTION Instrument. The UV/vis fiber-optic spectrograph was assembled from commercially available technologies. The basic spectroscopic setup is shown in Figure 1. A deuterium lamp (Oriel, Stratford, CT) fitted with f/1.8 optics was coupled to the frontal portion of an incoherent fiber bundle (PolymicroTechnologies, Phoenix, AZ) consisting of seven fibers. The distal end of (3) Adar, F.; Grayzel, R; Purcell, F. Remote Raman Analysis for Process Monitoring Applications. Technical note, SPEX Industries: Edison, NJ, 1993. (4)Ren, C. N.; Synovec, R E. A n d . Chem. 1990,62, 558-564. (5) Mainer, S . M.; Kautsandreas. J. D.; Eccles, L. ASTM Spec. Tech Publ. 1988, 963,370-380. (6)Angel, S. M.; Kulp, T. J.; Myrick, M. L.; Langry, K. C. Chem. Sens. Technol. 1991,3,163-183.
0003-2700/95/0367-2858$9.00/0 0 1995 American Chemical Society
Deuterium
lamp
a
1-s7
Dissoulution
bath 7-,1
Spectrograph
fiber
bundle Cooled CCD io mk
Control compute,
Controller
Figure 1. Optical layout for the in situ fiber-optic UVhris spectrograph. Only four fiber connections are shown for clarity.
Figure 2 Diagram of UVhris fiber probes. Table 1. Dissolution Parameters and Conditions Used in the Feasibility Study
paddle
the bundle was split into individual fibers, which were used as independentsources for each dissolution bath location. Each fiber in the fiber bundle was terminated with an SMAconnector Fiber Inshument Sales, Oriskany. NY) and attached to a switchboard to allow for easy probe changes and calibration. The detection section used a coherent fiber bundle at the aforementioned switchboard, with the frontal end coming from the sample and the distal end at the detector. The distal end holds the fibers stacked on top of each other, forming a vertical line (see Figure 1). This fiber bundle line was matched through a fused silica lens to the entrance slit of an imaging spectrograph with a fixed grating monochromator (Instruments SA/Spex, Edison, NJ). The collected light is separated in wavelength on the horizontal axis while maintaining the vertical separation of the individual fibers. ?he dispersed light from each fiber is imaged onto the focal plane of a liquid nitrogencooled charge coupled device (CCD) with an &e area of 1152 horizontal pixels x 298 vertical pixels (Instruments SA/Spex). Data acquisition was accomplished by use of a 486DX2/66 computer running DOS 6.2 and Windows 3.1 (Microsoh Inc., Redmond, WA). A memory resident driver provided by Inshuments SA/Spex controlled the CCD camera. A Matlab m e Mathworks, Inc.. Natick, MA) function was milten to interface with the driver. To initiate data acquisition, the Matlab function sends a signal to the memory resident driver which specifies the integration time, horizontal binning, and vertical binning to be used. ?he driver returns the acquired image data to the Matlab function, which then packs it into a Matlab data matrix for storage or postprocessing. AU postprocessing of image data was performed using Matlab programs mitten in our laboratoly. A Matlab program with peakfinding routines was used to automaticaliy determine the position of fiber channels in the CCD image and to extract the raw intensity profiles. A Matlab program for automatic wavelength calibration was also mitten. Fmt the seven receiving fibers were illuminated with a mercury emission lamp, and the resulting CCD image was recorded. This image was postprocessed to automatically determine the pixel positions of the Hg emission lines at 435.83,404.66, 365.48,313.16, and 253.70 nm. Least-squares fitting was used to independentiy calibrate each fiber channel. Interpola!ion was then used to estimate spectral intensities at 1 nm intends. Absorbance
measurement times after 15 min
rotation speed
first 15 min
formulation
medium
products 4 B. and C product D
water
50 rpm
every 1min
water
100 rpm
every 1min
every 5 min until 45 min
every 5 min
until 45 min
spectra of samples were obtained by taking the appropriate log ratios of wavelength calibrated blank and sample intensity profiles. Using this strategy, absorption spdm recorded using the inshument gave excellent matches from fiber channel to fiber channel. The reproducibility of the measurements was sufficiently good that we were able to use calibration data sets recorded from one probe to estimate composition of "unknowns" recorded on other probes. We investigated two probe designs, a reflection probe and a transmission probe F i r e 2). All probe body parts were made with 316 stainless steel. The reilection probe held the fibers side by-side and viewed the sample twice by reflecting the source light onto the collection fiber by a polished stainless steel reflector. ?he path length could be adjusted by moving the reflector closer to or farther from the fibers. A sapphire window was used to seal the fibers from the sample solution. ?he transmission probe used a fused silica prism to redirect the incoming light 180".?he path length was adjusted by changing the distance between the face of the prism and the illumination fiber. All source and detection fibers were terminated using SMA connectors. A dissolution system Wankel Industries Inc., Edison, NJ) complying with the USP Apparatus 2 specification @addle method) was used in all tests. ?he temperature of the water bath was maintained at 37.5 "C (within M.5 "C) for all dissolution tests. Table 1 lists the other dissolution conditions specified for each of the above formulations. Note that the measurement times are the times at which in situ measurements were made in this study. These are not the sampling times used in conventional dissolution testing of the above formulations. Materials. All formulationsand standard reference materials were supplied by Burroughs Wellcome Co. (Research Triangle Park, NC) and were used without further purification. Burroughs Wellcome Co. also supplied placebo materials to match the four products studied. Some pertinent information about the products Anawical Chemistry. Vol. 67, No. 17, September
1, 1995
2859
Table 2. Formulations and Their Compositions Used in the Feasibility Study' av tablet % active path length wavelength PCR product wt, mg (w/w) used, mm range, nm factors
A B C D
1067.5 483.3 639.5 176.5
74.9 15.5 15.6 17.0
1.5 5.0 3.0 15.0
295 300 250-340 230-360
b b 2 2
max abs (1 tablet/ 900 mL)
0.277 (295 nm) 0.325 (300 nm) 0.915 (250nm) 0.070 (257 nm)
PCR calibrations were used for products C and D. Single wavelength calibration models were used for productsA and B. * Single wavelength.
q 2 120
c) v)
5 n 110
0
0 X
I
X
Table 3. Composition of Calibration Standards, Expressed as Percent of Labeled Strength.
full simplified calibm stds calibrn stds active active std ingr t!xcipients std ingr 1 2 3 4 5 6 7 8 9 10 11
80 80 120 120 100 100 100 70 130 100 100
80 120 80 120 100 100 100 100 100 70 130
1 2 3 4 5
25 50 75 100 120
subset validn stds active std ingr excipients 1 2 3 4 5
90 90 120 120 100
90 120 90 120 100
The full set of calibration standards was used for products B, C, and D. The simplified calibration standards were used for product A. (I
m
70
60
80 100 120 Placebo, %Labeled strength
140
Figure 3. Experimental design (two-factor, three-level central composite factorial design) used for the calibration set (0)and the validation set ( x ) .
is listed in Table 2 to illustrate the useful range of absorbance signals that can be accommodated by our fiber-optic instrument. A wide range of absorption signals was accommodated by the use of our variable path length probes. Concentrated stock solutions of all active ingredients were prepared by accurately weighing the standard materials to give a concentration of -200% of the labeled strength for the formulated product, or the equivalent of dissolving two tablets in 900 mL of water. Concentrated stock solutions of the excipients were prepared from the corresponding placebos to give a concentration of excipients equivalent to dissolving 10 active tablets in 900 mL of water. In order to prepare these standards, placebo tablets were ground with an agate mortar and pestle. The desired amount of powdered material was then accurately weighed into a volumetric flask and diluted to the mark with the appropriate medium. The excipient stock solutions were thoroughly mixed immediately prior to withdrawing aliquots for dilution to ensure that insoluble particulate matter was evenly dispersed in the flask. To facilitate calibration, linearity testing, and noninterference testing, two-factor, three-level central composite factorial designs were used to prepare mixed working standards of the active ingredient plus excipients for the products in Table 2. The twofactor, three-level central composite is shown in Figure 3. At the center of the design (100%active ingredient and 100%placebo), three replicates were used. Assay concentration ranges were designed to provide greatest precision at 100%dissolution, where regulatory assays are normally performed. Mixed calibration standards and validation standards were prepared by dilution of the appropriate stock solutions to give the 2860 Analytical Chemisfry, Vol. 67, No. 17,September 1, 1995
desired percentages of labeled concentrations shown in Table 3. In order to obtain accurate calibration results, it was necessary to include excipients in calibration standards for products B, C, and D (see table footnote for details). Accurate calibration results were obtained for product A without including excipients in the calibration standards. Mixed validation standards were used to establish the linearity of each method and noninterference of excipients. For products A, C, and D, separate preparations of the full calibration standards 1-11 were used for validation (see Table 3). For product B, the subset validation standards were used for validation. Procedures. Spectra of blank solutions, calibration mixtures, and validation mixtures were measured off-line with the in situ dissolution spectrograph by dipping the fiber-optic probes into 50 mL beakers filled with the appropriate solutions. Flasks containing calibration and validation mixtures with excipients were thoroughly mixed prior to dispensing aliquots into the 50 mL beakers to disperse particulate matter. These mixtures were cloudy in appearance, and visible evidence of settling could be observed when the mixture was left stand for a few minutes. Spectra of these mixtures were acquired immediately after the aliquots were dispensed; if the mixtures were left standing, they were stirred immediately prior to measurement of the spectra to ensure that particulate matter was dispersed throughout the beaker as evenly as possible. Dissolution profiles of single tablets were obtained under the conditions described in Table 2. The dissolution medium (water in these experiments) was degassed immediately prior to filling the vessels by vacuum filtration through a 0.45 pm nylon filter. The dissolution vessels were filled with the appropriate dissolution medium and allowed to equilibrate to the proper temperature. Single tablets were manually dropped into the vessels at the same moment data acquisition was initiated. principal component regression (PCR) calibration models were used for products C and D. The optimum numbers of factors for PCR models were selected to give minimum prediction error for validation data sets. A single-wavelength calibration models was used for product A and B. The single-wavelength models were chosen because the excipients showed no interference at the selected wavelengths and the performance of these models was as good as that of the best PCR models. Small baseline offsets
Table 4. ReproduclbllltyStudy Using Calibration and Valldatlon Mixtures To Match Product A Tablets'
deviations (actual-estimated). % labeled strength probgto-probe, same day day-to-day,probe 1 actual concn, % labeled probe probe probe probe Nov Nov Nov 3 4 7 strength 1 2 3 4 ~~
~~
~
80.05 80.05 120.07 120.07 100.06 100.06 100.06 70.04 130.07 100.06 100.06
av bias std dev overall bias overall std dev
1.58 0.97 0.77 1.62 0.86 1.39 0.56 -0.44 -1.79 0.40 -1.26 -2.00 2.51 2.26 0.92 1.33 2.88 1.72 1.37 1.08 0.30 2.60 3.45 1.82 -0.70 -2.43 -2.35 1.86 1.51 0.71 0.13 1.09 0.16 1.43 1.06
0.72 1.65
0.11 1.46
0.47 0.15 -1.93 -0.88 3.11 0.41 0.73 0.91 -1.41 0.51 -0.04 0.18 1.33 0.61 1.45
1.28 -0.56 -1.80 -1.70 -0.19 0.61 1.23 2.84 -1.24 1.74 0.75
Table 5. Reproducibility Study Using Calibration and Validatlon Standards To Match Product D Tablets'
deviations (actual-estimated), % labeled s t r e n d Nov 15 probe 1 actual concn, Nov 11 %labeled probe probe probe probe Nov Nov Nov 2 3 2 3 strength 14 15 16
2.99 2.86 -1.68 -0.02 0.23 2.11 -1.25 -0.04 -5.14 -1.61 -2.82
0.97 1.41 -3.25 -2.13 -0.01 0.67 1.41 1.46 -0.66 -0.21 1.16
80.0 80.0 120.0 120.0 100.0 100.0 100.0 70.0 130.0 100.0 100.0
0.96 -1.45 2.64 -0.34 -0.89 -0.03 -0.05 -2.84 0.44 1.54 -1.62
0.42 2.32 0.31 0.47 -0.28 0.14 0.08 0.39 0.37 -0.39 -0.74 2.88 -0.12 1.52 2.57 0.58 -0.48 -0.66 -1.02 1.51 1.87 -2.12 0.22 3.33 2.23 3.84 -0.40 -0.91 1.90 2.02 4.03 -2.21 -0.12
2.91 2.67 1.47 0.70 1.48 0.78 1.98 -0.66 0.75 1.25 0.97 -0.65 1.59 0.25 2.22 1.52 1.66 0.77 2.88 1.34 1.06 0.53
0.27 -0.40 1.46 2.21
0.07 1.53 -0.02 1.86
av
-0.15
-0.61
1.61
Tne table shows deviations from expected values for test mixtures when individual probes are recalibrated each day. The variation from standard-to-standardis not significantly different from the variation from probe-to-probe or day-to-day as determined by one-way analysis of variance (not shown) at the 90% confidence level.
were corrected in the product A single-wavelength calibration models by subtracting background absorbance signals at 340 nm. The active ingredient and excipients in product A did not exhibit significant absorption bands at this wavelength. RESULTS AND DISCUSSION Probe Design. Both probe designs, the reflectance and transmission types (see Figure 2), proved to be suitable for collecting spectra. However, the reflectanceprobe suffered from low light throughput and required a signiscantly longer integration time, on the order of 40 s. This was considered unacceptable when compared with the transmission probe, which required much shorter integration times, on the order of a few seconds. The long integration times were found to be related to the fabrication of the reflectance probe bodies, which had the reflector off-axiswith the fibers. Transmission probes proved to be quite rugged and reliable and were used in all of the studies reported. Bias and Reproducibility. The bias, short-term reproducibility within days, and reproducibility withii probes were studied by measuring a set of calibration and validation standards repeatedly with each probe over a period of several days. The resulting calibration data were used to estimate the composition of the validation standards by matching calibration and validation data sets from the same probe and the same day. Table 4 shows the results of the study using standards for product A. One-way analysis of variance (ANOVA, table not shown) was used to study probe-to-probe and day-todayvariation. The differences were not significant at the 90% confidence level. As can be seen from the table, the bias for different probes and different days was smaller than the overall standard deviation. This indicatesthat an attempt to make a bias correction would introduce more error than it would remove. The overall standard deviation was about 44.5% of labeled strength between probes and f2.M of labeled strength between days. Similar results were obtained for other products. This level of reproducibility was judged to be satisfactory in this application.
0.61
2.14
0.89
2.47 1.63 -1.31 0.52 0.67 -2.18 -0.37 0.09 -0.26 1.83 1.12 0.38
The table shows deviations from expected values for validation standards. The calibration data set from probe 1acquired on Nov 11 was used to estimate the composition of all validation samples. The variation from standard-to-standardis not significantly different from the variation from probe-to-probewhen calibration data from the same day are used. The variation from day-today is marginally signiticant (see text) when calibration data from different days are used. The long-term reproducibility of fiber-optic dissolution assays was tested by pairing calibration data and validation data from different days or different probes. Table 5 shows the bias and reproducibility using a calibration data set for product D obtained using probe 1on Nov 11to estimate the composition of validation samples acquired with different probes or on different days. ANOVA (not shown) was used to determine the statistical significance of differencesfrom probe-to-probe and day-today. In the case of the data acquired on Nov 11,there was no significant difference bias between probes, even though the calibration set acquired using probe 1was used to estimate measurements made with probes 2 and 3. In the case of the data acquired on Nov 15, there was a slight difference between probes. Specifically, the bias for probe 3 was marginally signiticant, e.g., it was significant at the 95%confldence level but not at the 99%confldence level. Similar results were obtained with the other products studied. Iinearity and Noninterference. Response surface modeling of the validation data from central composite experimental designs was used to demonstrate linearity of assays and noninterference of excipients. In this kind of analysis, linear and quadratic models were computed from the estimated compositions of validation samples. Models used for linearity testing: linear model quadratic model
+ a,x, 2 jJ = a, + a,x, + up, jJ = a,
(1)
(2) In the above equations,9 is the estimated composition of the active ingredient, X I is the actual composition of active ingredient, and xz is the actual composition of placebo. The coefficients uj are determined by least-squares methods. In eq 1, an ideal linear calibration would have an intercept equal to 0 (a0 = 0) and a slope equal to 1 (a1 = 1). An R e s t was then performed to see if the quadratic model was significantly better than the linear one. A positive outcome for this test indicated that the method under investigation was linear, e.g., the coefficient a2 was not significantly different from 0. Analytical Chemistty, Vol. 67, No. 17, September 1, 1995
2861
Table 7. Linearity and Noninterference Test Results Using Calibration and Validation Standards To Match Product A Tablets.
Table 6. Results Used for Linearity and Noninterference Testing U8ing Callbration and Validation Standards To Match Product A Tablets.
active concn 80.05 80.05 120.07 120.07 100.06 100.06 100.06 70.04 130.07 100.06 100.06 av bias
excipient concn 80.00 120.00 80.00 120.00 100.00 100.00 100.00 100.00 100.00 70.00 130.00
skew overall bias overall skew a
estimated concn, active ingredient probe probe probe probe 1
2
3
4
81.62 81.66 120.63 120.47 102.57 102.94 101.43 72.64 129.37 101.92 101.15 1.43 0.958
81.01 80.90 119.63 118.81 102.32 101.78 101.14 73.49 127.65 101.57 100.21 0.71 0.928
80.82 81.44 118.28 118.07 100.97 101.39 100.36 71.86 127.72 100.77 100.19 0.11 0.928
80.51 80.19 118.14 119.19 103.17 100.47 100.79 70.95 128.67 100.56 100.01 0.18 0.960 0.61 0.943
All values expressed as % labeled strength.
Models used for noninterference testing: linear model interaction model
(3)
jJ= a0 01x1
+
+
probe
SSE
df
1
5.301
9
2
9.261
9
3
4.112
9
4
12.220
9
SSE
quadratic 3.435 interaction 5.100 quadratic 7.502 interaction 8.097 quadratic 2.235 interaction 3.915 9.096 quadratic interaction 11.750
df
Fcalc
8 7 8 7 8 7 8 7
4.347 0.138 1.876 0.503 6.719 0.176 2.747 0.140
Fcit
5.320 4.740 5.320 4.740 5.320 4.740 5.320 4.740
Columns labeled SSE represent the sum-squared error for the corresponding model. Linear models for each probe are compared to the interaction models and quadratic models. Interaction or noninterference is indicated by a large reduction in SSE (see text). All four probes respond linearly without interference from excipients. Similar results were obtained for other formulations. df, degrees of freedom.
m\\
0.5
To test for noninterference, a linear model with an extra coefficient for interaction between excipients and the active ingredient was compared in an F-test with a simple linear model. When the model with interaction is significantly better than the simple linear model, interference between the excipients and the active ingredient is indicated for the method under investigation. A positive outcome for this test indicated a lack of interference, e.g., the coefficients a2 and a3 were simultaneously not significantly different from 0.
full models
linear models
'
0.4
-.I
580
\
285
\\Y
290
295 3W 305 Wavelength (nm)
310
315
320
Figure 4. Absorbance spectra of product A tablets acquired during dissolution testing.
+
jJ = a, alxl a,p2 agx,x, (4) Table 6 shows the results obtained from validation standards for product A. The average bias and skew for each probe are shown. Skew is defined as the slope obtained from the leastsquares fit of eq 1, and bias is the average difference between the estimated and actual concentrations. The overall bias for the four probes was 0.61%labeled strength, and the overall skew was 0.943. Similar results were obtained for other produces. This level of error was deemed acceptable in this application. The results show in Table 6 were used for linearity and noninterference testing. The estimated concentrations for the active ingredient were fit to the linear, quadratic, and interaction models described above. The results of the statistical tests are shown in Table 7. The columns labeled SSE represent the sum-squared errors for the respective models. The linear models for each probe are compared to the interaction and quadratic models. A large decrease in SSE for the quadratic or interferencemodels indicates non-linearity or interference, respectively. The decrease is determined to be significant when Fcalc> Fcit.Critical values of F are reported in Table 7 at the 95% confidence level. The response of probe 3 is slightly curved. All of the other probes exhibit linear response to the active ingredient in product A. All probes show freedom from interference for the excipients of product A. Tablet Dissolution Profiles. Initially,we experienced a great deal of difficulty obtaining useful in situ dissolution spectra from 2862 Analytical Chemistty, Vol. 67,No. 17, September 1, 1995
I I:/ 01
0
I I 5
10
15
20
25
30 Time Elapsed (min.)
35
40
45
50
Figure 5. Dissolution profiles of three product A tablets. Dissolution test conditions: 900 mL of water, 50 rpm, 37.5 f 0.5 "C.Spectra were acquired at 1 min intervals for the first 15 min and at 5 min intervals thereafter.
the dissolution vessels because bubbles tended to form on the optical surfaces of the probes, causing light to be scattered away from the desired beam path. We found that virtually all of these problems could be avoided by degassing the dissolution medium. Spectra and dissolution profiles of products A, C, and D are shown in Figures 4-9 , respectively. Each tablet showed a slightly different dissolution pattern due to the chaotic nature of tablet disintegration. The initial appearance of active ingredient in solution always coincided with the initial stages of tablet disintegration. Rapid increases in the concentrations of the active
1
0.8
15
o.6
60-
2 0.4
8 40-
0.2
0 230
20
240
250
260
270
280
290
300
310
320
0 0
330
Wavelength (m)
Figure 6. Absorbance spectra of product C tablets acquired during
dissolution testing. loo 80 -
40t
0.2r
3
I
1
I
0.15
e
SI 9
0.1
0.05
0'
220
230
240
250
260
270
280
290
300
310
-
I 320
Wavelength (m)
Figure 8. Absorbance spectra of product D tablets acquired during dissolution testing.
ingredients were observed for tablets that disintegrated quickly. In the case of product C, there was a long disintegration period of -7 min, followed by a rapid increase in the amount of dissolved active ingredient. Compared to the other products studied, product D had the longest dissolution time. CONCLUSIONS The fiber-optic dissolution system showed excellent reproducibility when individual probes were recalibrated each day. For single component formulas with excipients that do not interfere with the assay, recalibration is easily accomplished with a single component standard. If individual probes are not recalibrated every day, the reproducibility from day to day is slightly worse. This means the analytical results may be influenced by uncontrolled conditions, such as light loss due to differences in the
5
10
15
20 25 30 Time Elapsed (min)
35
40
45
Figure 9. Dissolution profiles of three product D tablets. Dissolution test conditions: 900 mL of water, 100 rpm, 37.5 0.5 "C.Spectra were acquired at 1 min intervals for the first 15 min and at 5 min intervals thereafter.
*
radius of fiber bends from day to day, slight changes in probe path length from day to day, or the degree of cleanliness of fiber and prism surfaces exposed to the dissolution medium. Unreproducible light loss due to fiber bending can be minimized by precision alignment of probe fibers in connectors and maintenance of fairly straight fiber cable runs. Precision alignment of fibers in connectors ensures that light is launched into the fiber such that most of the intensity is carried by the low-order transmission modes. These modes are less susceptible to bending losses than high-order transmission modes. Noninterference of excipients was easy to demonstrate for product A, which has low excipient weights. Single-wavelength calibrations using standards of pure active ingredients were adequate for this product. In order to obtain non-interfering assays for products B, C, and D, it was necessary to include excipients in mixed calibration standards and to use multivariate calibration. It is suspected that the dyes used in these formulationsinterfere in the UV wavelength regions used for these assays. The rapid in situ data collection capability of the fiber-optic spectrograph made it possible to characterize the shape of dissolution curves easily compared to other timeconsuming methods of analysis, like HPLC or conventional UV/visible spectrophotometry, where small aliquots must be withdrawn, filtered, diluted, and processed. We recognize that the placement of probes can affect the hydrodynamics of the dissolution medium and therefore can affect the dissolutionkinetics. We are currently working to miniaturize our prototype probes to minimize these effects. It should be pointed out that there are many other issues to be resolved before this measurement technique can be used in a regulated laboratory. The application of the system to other products will be investigated for both development and multicomponent formulations. Currently, the most significantvalue of this technology is in the study and development of pharmaceutical products with controlled dissolution. ACKNOWLEDGMENT This research was supported jointly by the Burroughs Wellcome Co. and by Zymark Corp. Received for review March 15, 1995. Accepted June 5,
1995.B AC950258L @
Abstract published in Advance ACS Abstracts, July 15, 1995.
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