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Anal. Chem. 1985, 57, 1408-1411
Evaluation of Robot Automated Drug Dissolution Measurements Andrew N. Papas,* Maurice
Y. Alpert, Salvatore M. Marchese, a n d J a m e s W.Fitzgerald
U.S. Food and Drug Administration, Winchester Engineering and Analytical Center, 109 Holton Street, Winchester, Massachusetts 01890 Michael F. Delaney
Department of Chemistry, Boston University, Boston, Massachusetts 02215
A controlled study was conducted to evaluate the feaslblllty of robotlc automatlon of routlne regulatory drug dlssolutlon measurements. A commercially available laboratory robot system automated a number of dlssolutlon analyses while concurrent manually acquired samples were analyzed by the same analytlcal method. Five welltharacterlzed drug tablet types were employed, lncludlng the two current “Unlted States Pharmacopeia” (USP) Method I I calibrators, These tablet types spanned a range of dlslntegratlonand dlssolutlon rates. I n all, over 150 tablets were analyzed. The results were subjected to a statlstlcal analysls of varlance (ANOVA). ARer some small sources of systematic blas were eliminated, the results obtained by the robot were statlstlcally lndistlngulshable from the manual results. Addltlonally, the overall ranges of total drug dissolved were comparable to those obtalned from a dlssolutlon Interlaboratory collaborative study and to range llmlts for the USP calibrator tablets.
Dissolution measurements of pharmaceutical formulations are routinely performed both as a quality assurance check on the final product and as a convenient surrogate for evaluating the in vivo bioavailability of the drug (I). Due to the inherent variability in dissolution characteristics between individual tablets, these measurements require careful attention to detail for both the apparatus and the experimental procedure (2, 3). Recent developments in extended- and delayed-release dosage forms ( 4 ) ,employing, for example, sintered polymers (5)or transdermal handages (6),are necessitating multipoint dissolution measurements be conducted over increasing time periods. The trend in current pharmaceutical technology is to attempt to achieve a more constant physiological drug concentration, resulting in longer dosing intervals, which therefore requires multiple dissolution measurements to demonstrate proper formulation. Manual drug dissolution measurements are time-consuming and tedious. Attempts to automate the process often result in systems which do not completely agree with manual values-this is undesirable in both regulatory and quality control environments (7).This laboratory conducted a detailed, yet time-restricted, evaluation on the feasibility of using a commercially available laboratory robot system for automated dissolution analyses. It was felt that a robot using standard laboratory devices could be both time- and costeffective, yet should provide results in close agreement with manual determinations. In this study concurrent manual and robot sampling resulted in simultaneous analysis using tablets displaying a range of disintegration-dissolution characteristics. The quantitative results were subjected to a statistical analysis designed to detect any systematic bias of differences in precision. The accuracy of both was determined by comparing the ranges obtained to those obtained from a dissolution
Table I. Components of the Laboratory Robot System 1 2
3 4 5 6 7 8
9 10
11 12
system controller robot arm general purpose hand microliter syringe hand pill pusher rod hand kettle sipper hand fraction collector turntable master laboratory station (3 computer-controlledsyringes and valves) power and event controller test tube racks filter tip rack and verifiction switch fixed sample sipper station
13 nrinter . . . . . . . interlaboratory collaborative study for three table types and to range limits for the two current USP calibrators. EXPERIMENTAL SECTION Laboratory Robot. A 2100 Zymate laboratory robot system (Zymark Corp., Hopkington, MA) was used with a number of standard options as shown in Table I. Two nonstandard manipulators were provided by the manufacturer. The first was used for pushing (or pulling) tablets into the dissolution kettles from the rim of the kettle. This “hand” consisted of a long (-45 cm) horizontal rod with a flat “snowplow” at the end. The other nonstandard “hand” used for taking filtered aliquots from the kettles consisted of a horizontal rod with a vertical stainless steel tube at the end. The lower end of the tube was designed to hold, by friction fit, a standard pipet filter tip. To the upper end of the tube was connected coiled Teflon tubing which connected to a solenoid valve and motor-driven 10-mL syringe in the 2510 Master Laboratory Station (Zymark). Dissolution Apparatus. A six-kettle dissolution apparatus (Hanson Research Corp., Northridge, CA) with synchronized mechanically rotated spindles was used for all measurements, The paddles were rotated at either 50 or 100 rpm as dictated by the method. A Circulating System-200 (GCA/Precision Scientific) temperature control and pump system was used to maintain the dissolution kettles at 37.0 f 0.5 “C. The apparatus was evaluated and adjusted, if necessary, to meet all USP specifications (leveled kettles; centered nonwabbling shafts; and all other spatial requirements). Similarly, the robot was programmed to sample each kettle at a time and location in accordance with USP requirements. Spectrometers. Static absorbance measurements were made at a wavelength appropriate to each drug with a Cary 118 spectrophotometer (Varian Instrument Group, Palo Alto, CA). Flow-cell absorbance measurements were made with a Model DBGT spectrophotometer (Beckman Instruments, Inc., Fullerton, CA) using a 1 cm path length, 1 mL volume flow cell (Pyrocell Manufacturing Co., Westwood, NJ). Tablet Samples and Dissolution Conditions. Five tablet types were studied. The first two, prednisone and salicylic acid, are USP calibrator tablets which are used to demonstrate that the apparatus and the chemistry are capable of performing acceptable dissolution measurements. Limits on the percent dissolved under the specified test conditions are distribut,ed by USP with each lot of calibrator tablets.
This article not subject to U.S. Copyright. Published 1985 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 7, JUNE 1985
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Table 11. Formulation, Dissolution Medium, and Quantitation Wavelength for each Tablet Type drug
formulation
source
dissolution medium
prednisone salicylic acid hydrocortisone acetaminophen L-dopa
50 mg tablet 300 mg tablet 20 mg tablet
USP dissolution calibrator; lot G USP dissolution calibrator; lot H
HzO
242
0.05 M, pH 7.46 phosphate buffer
a a a
HzO 0.05 M, pH 5.8 phosphate buffer 0.1 M HC1
249
a
325 mg tablet 500 mg tablet
A,,
nm 296 248 280
paddle rpm 50 50 50 50 100
U.S.F.D.A. Philadelphia District Office Inter-Laboratory Collaborative Study.
Three types of commercially available tablets were also studied, hydrocortisone, acetaminophen, and L-dopa. These samples were obtained from the FDA Philadelphia District Office and were used in an interlaboratory dissolution collaborative study (12). The tablet’s declared formulation, dissolution medium, and wavelength for quantitation are reported in Table 11. Data Analysis. All raw experimental data were entered into the RS/1software package (Bolt Beranek and Newman Software Products, Cambridge, MA) running on a VAX-11/750 computer (Digital Equipment Corp., Marlboro, MA). RS/1 was used for all statistical calculations (descriptive statistics, normality tests, accuracy test, precision test, and one-way analysis of variance) as well as the graphical presentation of results. For each drug investigated the raw data of one run consists of absorbance measurements (manual vs. robot) for each kettle. The data from five runs for each sample type, totally 30 tablets, were evaluated together and tested for differences in magnitude and dispersion (accuracy and precision) between robot and manual sampling. The difference values (robot - manual) were first tested under the null hypothesis of a normal distribution using the Wilk-Shapiro normality test. In most cases the hypothesis of a normal distribution was accepted at the 95% confidence level. A paired t test was used to examine the results for each drug to see if the robot and manual procedures yielded results which were statistically different. In addition, any drug which failed the normdity test was also examined using a nonparametric paired rank-sign test. Both of the statistical procedures take advantage of the fact that robot and manual samples were acquired simultaneously for each tablet, which should eliminate the effect of differences between tablets. Since the tablets are not identical, the only way to obtain replication would be to have two robots and two chemists simultaneously sampling each kettle, but this was a practical impossibility. If each run was treated as a replicate determination, it was possible to use one-way analysis of variance (ANOVA) to test for between-kettles bias. Similarly, by consideration of the values obtained in each kettle as replicates, it was possible to use ANOVA to detect between-run bias.
RESULTS AND DISCUSSION Operation of the Robot. One chemist was designated as the robot operator. He received 1day of on-site instructions from the manufacturer, which included physical arrangement of the robot arm, its hands, and other peripherals. Within 2 days, he completed programming the robot to conduct dissolution measurements, and the evaluation runs began. The entire study was conducted over a 3-week period. An important feature which facilitated expedient program development is the layered architecture of the robot’s interpreted command language. At the top-most layer, the program reads much like a manual laboratory procedure: put.tablets.into.kettles sample.aliquots dilute.samples feed.and.read.spectrophotometer print.results Each underlying layer contains a more detailed set of operations similar to procedure declarations in PASCAL. The bottom layer of the program contains symbolic references to absolute robot coordinates. These robot-positioning coordinates we established during the set-up phase using the robot’s remote-control module. This allows the chemist to describe
Table 111. Robot Drug Dissolution Procedure 1 pick up “pusher” hand and wait for the operator’s “begin”
signal 2 at timed intervals (e.$., 3 min apart) push each of the six
tablets into a kettle 3 move six test tubes from the rack to the carousel 4 wait for the dissolution time to expire (30 min) 5 at the proper time take a filtered aliquot from each kettle. a. place a filter tip on the “sipper”hand, and verify its presence b. remove a filtered aliquot (20 mL) c. dispense the aliquot into a test tube d. flush the “sipper” line with air e. discard the filter tip 6 dilute each of the six aliquots a. using the syringe hand, take a measured volume (e.g., 4 mL) from the sample test tube b. dispense the aliquot into a clean test tube on turntable c. using the second motor-driven 10-mL syringe, dispense a measured volume of diluent (16 mL) into aliquot 7 measure the absorbance of each diluted aliquot a. bring the tube up to the remote nozzle with robot hand b. mix the sample by bubbling air through the nozzle cannula c. suck the sample into the spectrophotometer flow cell using the third motor driven 5-mL syringe d. make 10 absorbance readings and compute the average e. pump the sample back into the test tube f. put the test tube back into the rack locations to the robot using sensible names (e.g., above. the.test.tube.rack) rather than by using numerical coordinates. This tremendously reduces the time and effort needed to program the analysis procedure. A summary of the dissolution program is given in Table 111. Evaluation of the Robot’s Precision. Apart from the dissolution procedure itself, the precision of the robot in quantitatively manipulating solutions and measuring absorbances employing a spectrophotometer with a flow cell was evaluated in three experiments. In the first study, as aliquot of distilled water was dispensed and the amount transferred was quantitated by weight. Six aliquots of 4 mL each, dispensed with a 10-mL motor driven-syringe averaged 3.998 g with an RSD of 0.044%. Six aliquots of 4 X 1mL each, dispensed with a 1OOO-hL precision syringe averaged 4.049 g with a relative standard deviation of 0.164%. In the second experiment, a stock prednisone solution was diluted (1:2) and then pumped to the flow cell spectrophotometer. The analog signal was converted by the A/D converter and then the values were printed out. This was repeated 23 times and resulted in a relative standard deviation of 0.806 % . In the third experiment, 24 prednisone solutions, spanning a range of concentrations with absorbances between 0.2 and 1.6, were measured by both the flow-cell spectrometer and a Cary 118 UV/VIS spectrophotometer instrument. Excellent agreement was observed between the instrument; the regression line was linear (correlati9n coefficient = 0.991) with an intercept indistinguishable from zero at the 95% confidence level. One measurement appeared to be an outlier, giving a
ANALYTICAL CHEMISTRY, VOL. 57, NO. 7, JUNE 1985
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Table IV. Summary of the Dissolution Results and a Comparison with the Interlaboratory Study drug
n
prednisone salicyclic acid hydrocortisone acetaminophen L-dopa
30 30 30 30 24
manual mean, %
RSD
mean, %
robot RSD
av 6-tablet RSD robot Cs"
1.52 1.65 2.55 3.84 4.08
44.28 16.69 97.43 96.58 96.58
1.68 1.61 2.55 3.82 3.97
2.09 2.86 2.68
43.69 16.22 97.29 96.98 94.67
3.10 2.61 2.02
"Average RSD for a six-tablet run from each of the laboratories participating in the collaborative study. Prednisone Dissolution 650~
Levodopa D i s s o l u t i o n 1050~
/
1
/
0
8,
0
4 500
500
600
550
I
650
800
E50
900
950
io00
1050
Manual
Manual
-
Experimental
Experimentel -0.910888*X
8
ts9.49349
Robot vs. manual results for 30 prednisone tablets (0) and the regression line. Figure 1.
high value on the flow-cell instrument. This may have been caused by an air bubble in the cell-a problem virtually eliminated by selecting a suitable aliquot volume and pumping flow rate. From these three experiments it was concluded that the accuracy and precision of the dissolution would dot be adversely constrained by the robot's ability to dispense solutions and measure absorbances. It was therefore decided to allow the robot to conduct all dilution and quantitation steps. Dissolution Runs. Five sets (in one case, these were only enough tablets to allow four runs), six kettles per set, were conducted for each of the five tablet types using USP Method I1 (paddles) (8). A summary of the manual and robot dissolution results is presented in Table IV, and plots of the individual tablet results for two of the drugs are shown in Figures 1 and 2. There is typically a significant amount of scatter in these plots which is indicative of differences in the tablets. The first table type studied was the USP Calibrator Prednisone current lot G (Figure 1). These tablets, which have been studied extensively by others (9-11), represent the most demanding challenge. They disintegrate rapidly yet the drug dissolves slowly, yielding particles that swirl around inside the dissolution kettle. We therefore studied this calibrator at the slower 50 rpm paddle speed. It is the most studied tablet because of its extreme sensitivity to dissolution parameter changes. A paired t test on the 30 individual tablet results (manual vs. robot) indicated that there was small systematic bias yielding robot results consistently higher than the manual values. This bias is reflected in the average of the prednisone dissolution, results being about 0.6% higher for the robot, as compared to the manual results (based on percent of declared). Further consideration indicated three possible causes of this bias: (1)the porosity of the filter tip was larger than that used manually; (2) the robot sampling time was slightly later than the manual sampling time (This was caused by the robot needing more time to take a sample aliquot than the manual
0 744306NX + 2 5 i , 8 1 7 3 a kettle 1
__ ~
Robot vs. manual results for 24 L-dopa tablets (0)and the regression line. The tablets labeled (W) are from kettle 1 and were biased high by the robot due to a run-to-run carry over.
Flgure 2.
Table V. F Tests for Each Tablet TypesR drug
no. of tablets
prednisone salicylic acid hydrocortisone acetaminophen L-dopa
30 30 30 30 24
standard deviation manual robot 1.52 1.65 2.55 3.84 4.08
1.68 1.61 2.55 3.82 3.97
F 1.22 1.04 1.00 1.01 1.06
"All precision differences are insignificant a t the 95% confidence level. procedure); (3) the robot may have been sampling deeper in the kettle. Each area was addressed: 8-pm filters replaced 0.45-pm filters used in the manual sampling. The program was modified, and careful attention was paid to the sampling site. Once these biases were removed, two more prednisone runs conducted gave results with no significant systematic bias. Overall, every individual result was well within the acceptable range for the USP prednisone calibrator tablets (33-51%). The other USP calibrator tablet, salicylic acid current lot H, was studied next. These nondisintegrating tablets remain intact, and only 13-23% dissolves in 30 min a t 50 rpm. For five runs, the robot results averaged about 0.5% higher than the manual values. Presumably this small systematic bias would have been eliminated in the same way as it was for prednisone, but time prevented repeating these measurements. For hydrocortisone and acetaminophen, no systematic bias was found at the 95% confidence level. However for L-dopa, a small systematic bias was noted. Kettle 1 robot results in each run were noticeably higher than the kettle 1 manual values, Figure 2). This was caused presumably by a run-to-run carry-over and could be easily eliminated by including a thorough probe cleaning a t the conclusion of a run. When the values for kettle 1 were excluded, the differences between
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Anal, Chem. 1985, 57, 1411-1415
the robot and the manual procedure were statistically insignificant. Precision. By use of an F test on the dissolution results, there is no significant difference in precision between the robot and the manual procedure (Table V). This satisfactory observation is somewhat misleading. One would have predicted that the robot, with more control over sampling timing and position, would yield better precision. However, since the precision may possibly be limited by the differences in dissolution rates between the tablets, the more controlled sampling of the robot would not yield more precise results. In order to more completely evaluate the precision of robotic drug dissolution, we obtained the results of an interlaboratory dissolution collaborative study conducted by the FDA Philadelphia District Office involving 10 FDA field laboratories. This study included three of the tablet types used in the Philadelphia study: hydrocortisone, acetaminophen, and levodopa. A comparison of these results is shown in the last two columns of Table 111. To compare precisions, average six-tablet relative standard deviations were calculated for the robot and for the laboratories in the collaborative study. By use of an F test, it is seen that the robot's precision is not significantly different from that obtained in the other laboratories.
CONCLUSIONS Two major concerns were addressed in this time-limited feasability study. The first focused on the accuracy and precision of robot automated drug dissolution. We have shown that the robot automated analysis and all its associated quantitative steps were comparable to those of manual analyses. Although we evaluated USP Method 11drugs, we anticipate similar findings with USP Method I (basket) drugs. The second concern was that of time savings which ultimately translates to cost savings. As time of analysis was
secondary in this pilot study, the programmed procedures were not optimized. However, given that the equipment set-up time (excluding the robot programming time) was roughly equivalent, the analyst was freed up approximately two-thirds of the total analytical time required for dissolution. This was realized only after the entire dissolution analysis was automated: dropping in tablets, sampling, diluting, and quantitating. This time savings will be substantial when multipoint, time-profile analyses are automated by robotics; a project we hope to undertake in the near future.
Registry No. Prednisone, 53-03-2;salicylic acid, 69-72-7; hydrocortisone, 50-23-7;acetaminophen, 103-90-2; L-dopa, 59-92-7. LITERATURE CITED Knapp, G. Pharm. Techno/. 1977, 7 (4), 12. Cox, D. C.; Douglas, C. C.; Furman, W. B.; Kichhoefer, R. D.; Myrlck, J. W.; Wells, C. E. Pharm. Techno/. 1978, 2 (4), 40. Hanson, W. A. "Handbook of Dissolution Testing"; Pharmaceutical Technology Publicatbns: Springfield, OR, 1982. Leeson, L. J.; Peterson, R. V.; Robinson, J. R. Pharm. Forum 1984, 10, 4103. Cohen, J.; Slegel, R. A.; Langer, R. J . Pharm. Sci. 1984, 73, 1034. Chien, Y. W. J . Pharm. Sci. 1984, 73, 1064. Wells, C. E. J . Pharm. Sci. 1981, 70, 232. "The United States Pharmacopeia" 20th rev.; U S . Pharmacopeia1 Convention, Inc.: Rockville. MD, 1980; p 959. Cox, D. C.; Furman, W. B.; Thornton, L. K.; Moore, T. W., Jefferson, E. H. J . Pharm. Sci. 1983, 72, 910. Cox, D. C.; Furman, W. B. J . Pharm. Sci. 1984, 73, 670. Cox, D. C.; Furman, W. 0. J . Pharm. Sci. 1984, 73, 1125. Gurbarg. M., private communications no. 127, FDA Philadelphia District Laboratory, Philadelphia, PA.
RECEIVED for review December 17,1984. Accepted February 19,1985. Reference to any commercial materials, equipment, or process does not in any way constitute approval, endorsement, or recommendation by the Food and Drug Administration.
Spectrophotometric Determination of Mercury in Zinc Blende and Pharmaceutical Preparations with 1-Salicylidene-5-(2-pyridylmethylidene)isothiocarbonohydrazide Daniel Rosales* and Jose L. Giimez Ariza
Department of Analytical Chemistry, Faculty of Chemistry, University of Sevilla, 41012 Sevilla, Spain
A slmple, rapid, selectlve, and sensitive method for the spectrophotometric determlnatlon of mercury has been developed based upon the formation of l-sallcyildene-5-( 2pyridyhethylldene)lsothlocarbonohydrazlde-Hg( I I ) complex. A yellow color Is formed at pH 4.7 EDTA-acetate buffer in a medium contalnlng 40 % (v/v) dlmethyHormamide and measured at 400 nm. The molar absorptivity is 6.4 X lo4 L mol-' cm-l and the relatlve standard deviation 1.3% (0.75 ppm mercury). The effect of interferences was studied. The method has been applled to the determlnatlon of mercury In rlnc blende (relative standard devlatlon 5 % ) and In 15 pharmaceutlcal preparatlons prlor to destructlon of the organlc matter by uslng a "0,-KMnO, mixture (relative standard devlations 1-6 % ).
Mercury is one of the most toxic elements known to man. 0003-2700/85/0357-1411$01.50/0
The governmental agencies of many countries have set the maximum tolerated amount of this element in foods between 0.05 and 0.5 mg kg-' ( I ) , so a routine method of mercury determination must be simple, reliable, sensitive, and selective. It can be said that all analytical techniques have been applied to mercury determination, but those normally used are the spectrophotometric, neutron activation analysis, and atomic absorption spectrometry techniques. However, the results obtained by the last two leave much to be desired for the analysis of small amounts of this element (2). Dithiazone has been unquestionably the most widely spectrophotometric reagent used (31, but this highly sensitive reagent requires very careful handling, and the determination of small amounts of mercury involves the reextraction of the reagent in excess (single-color method) (4). During a systematic study on Schiffs bases derived from thiocarbohydrazide (5,6),it was found that the asymmetric compound l-salicylidene-5-(2-pyridylmethylidene)isothio0 1985 Amerlcan Chemical Society
