IOURNALOF

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PHARMACEUTICAL
AND BIOMEDICAL
ANALYSIS
Journal of Pharmaceutical and Biomedical Analysis
ELSEVIER 13 (1995) 1235 1241

Automated analytical systems for drug development studies

3. Multivessel dissolution testing system based on
microdialysis sampling*
Ketan P. Shah 1, Ming Chang, Christopher M. Riley *
Department of Pharmaceutical Chemistry, Center for BioAnalytical Research, University g[" Kansas, 3006 Malott Hall,
Lawrence, KS 66045, USA
Received for review 13 December 1994; revised manuscript received 20 February 1995

Abstract

An automated system consisting of a six-vessel dissolution apparatus, microdialysis sampling, STT E6

multiposition switching valve and a liquid chromatograph was assembled to measure dissolution profiles of
immediate and sustained-release tablets. A DL-5 microdialysis loop probe (BAS, Inc.) was immersed in each
dissolution vessel and perfused with a suitable medium for sampling. The dialystate from each vessel was
injected sequentially onto an on-line liquid chromatography (LC) system for automated analysis. The STT
E6 multiposition switching valve was used to sample up to six vessels simultaneously. After addressing issues
related to sample carry-over and between-probe variability, the automated system was used in a reproducible
manner (RSD < 3%) to measure the dissolution of immediate-release acetaminophen tablets and Accutrim ~
(containing 75 mg phenylpropanolamine HC1) 16 h Precision Release T M tablets. An uneven injection time
sequence was used to monitor three acetaminophen tablets per dissolution run using the automated system
and each vessel was sampled about every 6.5 min. However, with Accutrim * 16 h Precision Release T M
tablets, a longer sampling interval (10 min) was used, the six tablets could be tested in each dissolution run.
The dissolution profiles of acetaminophen and Accutrim ® tablets measured using the automated multivessel
dissolution system compared well with manual and automated single-vessel dissolution systems.

Keywords: Automation; Dissolution testing; Flow-injection analysis; Immediate release; Liquid chromatog-
raphy; Microdialysis sampling; Multivessel; Sustained release

1. Introduction samples for analysis. Previously, a microdialy-

sis sampling system and an on-line liquid chro-
Dissolution testing is used as a routine C o m - m a t o g r a p h were interfaced with a single-vessel
pendial test to m o n i t o r the lot-to-lot variability dissolution apparatus to a u t o m a t e the dissolu-
o f solid dosage forms. The testing procedure is tion testing o f tablets [1]. The a u t o m a t e d sys-
laborious and generates a large n u m b e r o f tem was kept simple during the initial
developmental stages by studying only one
tablet per dissolution run. The effect o f critical
:~ Presented at the Ninth Annual Meeting and Exposition factors, such as system calibration, linearity
of the American Association of Pharmaceutical Scientists and microdialysis drug recovery, on the perfor-
(Analysis and Pharmaceutical Quality Section), San Diego,
CA, 6-10 November 1994. mance o f the a u t o m a t e d system was studied.
* Corresponding author. This paper describes the development o f a
t Present address: Global Pharmaceutical Corp., Philadel- m o r e complicated system designed to sample
phia, PA 19061, USA. up to six dissolution vessels simultaneously.

0731-7085/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved

SSD1 0731-7085(95)01529-9
1236 K.P. Shah et al. / J. Pharm. Biomed. Anal. 13 (1995) 1235 1241

Flushing syringe STT 6portswitching valve

H ~ " " ''%°

"-

O 0 " I.oo.,
1 2 3 ..o*:o° ! " probes I I
~o I
.c o°n

6 5 4 ]

Six vessel dissolution apparatus ~

I I

Fig. 1. Schematicdiagram of the automatedmultivesseldissolutiontesting system.

The advantages of microdialysis sampling to 2.2. Equipment

automate sampling and analyses have been de-
scribed before [1,2]. Essentially, microdialysis The automated multivessel dissolution test-
sampling provides an easy and inexpensive tool ing system (Fig. 1) consisted of a dissolution
for automation. It is a continuous sampling apparatus, a microdialysis sampling system, a
process that does not involve any volume multiposition switching valve, and a liquid
change and allows on-line buffering of the sam- chromatograph. A Hanson Research Corpora-
ples to make them compatible with silica-based tion (Chatsworth, CA) Model SR2 six-vessel
liquid chromatography (LC) column packing dissolution apparatus with temperature-con-
materials. The microdialysis drug recovery can trolled water bath was used in the dissolution
be modified by changing the perfusion rate or experiments. The sampling system consisted of
membrane surface area to avoid an additional DL-5 microdialysis loop probes (5 cm mem-
dilution step for high-dose drug products. The brane length) obtained from BAS, Inc. (West
main limitations associated with microdialysis Lafayette, IN), and a Harvard Apparatus
sampling are the adsorption of drugs onto the (South Natick, MA) Model 55-4140 dual sy-
dialysis membrane and/or connecting tubing
ringe pump for perfusion of the probes. The
and the between-probe variability that necessi-
dialysate was passed through a 2 lal injection
tates calibration of individual probes.
loop mounted on a Valco Instruments Co.
(Houston, TX) Model EF 60 on-line injector.
A multiposition switching valve Model STT E6
2. Experimental
from Valco Instruments Co. was interfaced
2.1. Materials between the dissolution apparatus and the on-
line injector for the simultaneous sampling of
Acetaminophen and phenylpropanolamine six vessels. The LC system consisted of a Beck-
hydrochloride (PPA) were purchased from man Instruments Inc. (Fullerton, CA) Model
Sigma Chemical Co. (St. Louis, MO). Mono- 110A pump, System Gold ® programmable UV
basic and dibasic sodium phosphate and detector Module 166, and an IBM PS/2 Model
methanol were obtained from Fisher Scientific 56SX system controller installed with System
Co. (Fair Lawn, N J). Acetaminophen tablets Gold ® software. An ODS Hypersil column
(325 mg) and Accutrim® 16 h Precision Re- (3 Ixm, 30 mm x 4.6 mm i.d.) was purchased
lease T M tablets (containing 75 mg total PPA, from Keystone (Bellefonte, PA).
20 mg bolus and 55 mg controlled-release core)
were obtained from a local pharmacy. Water 2.3. Dissolution of tablets
processed through the Milli-Q water system
(Waters Corp., Bedford, MA) was used in all Dissolution testing of immediate release ac-
the experiments. etaminophen tablets and Accutrim® 16 h Preci-
 K.P. Shah et al./ J. Pharm. Biomed. Anal. 13 (1995) 1235 1241 1237

sion Release T M tablets was performed accord- dissolution system. Similar perfusion rate and
ing to USP XXII specifications, using the pad- injection intervals were used with the auto-
dle method. In an attempt to demonstrate two mated single-vessel dissolution system, but only
different analytical techniques, the ac- one tablet per dissolution run was studied and
etaminophen was determined by on-line LC the dissolution vessel was sampled every
and the phenylpropanolamine was determined 10min. Dissolution profiles of Accutrim '"~
by flow-injection analysis (FIA). tablets measured using various systems were
compared to the previously reported dissolu-
Acetaminophen tablets tion profile [3].
The dissolution of acetaminophen tablets
was studied in 900 ml of water at 37 °C and
50 rpm. A DL-5 microdialysis loop probe was
suspended in each dissolution vessel and per- 3. Results and discussion
fused continuously with water at a rate of
50 ~tl min-~. The dialysate was collected in a
2 ~tl on-line injection loop and injections were 3.1. Principle of operation
made every 96 s, followed by a blank injection
for 36 s. The latter injection was necessary to The STT E6 switching valve has two sets of
flush the system and eliminate carryover. Only inlets and outlets and six pairs of ports for
three tablets could be studied per dissolution sampling up to six dissolution vessels simulta-
run using the STT E6 valve owing to the rapid neously (see Fig. 1). The syringes mounted on
sampling intervals necessary for the adequate the dual syringe pump were filled with the
description of the dissolution profile of ac- perfusion medium and connected to the two
etaminophen which was released rapidly from inlets of the STT E6 valve. A DL-5 microdialy-
the tablets. Each vessel was sampled once every sis probe was suspended in each dissolution
6.5min. The samples were analyzed on-line vessel and both ends of the probe were con-
using a fast LC column with UV detection at nected to a pair of ports on the STT E6 valve.
280 nm. The mobile phase used to elute ac- At any instance, two probes were perfused ---
etaminophen consisted of methanol-phosphate one probe was flushed to waste and the other
buffer (50 mM, pH 6.2) (15:85, v/v) at a flow connected to the injector. An uneven injection
rate of 2 ml rain ~. The dissolution profiles time sequence was built into the detector
measured using the automated multivessel dis- method to sample and analyze the dissolution
solution testing system were compared to man- medium in an automated fashion. At the end
ual and automated single-vessel dissolution of the injection interval, the STT E6 valve,
testing systems. rotated counter-clockwise, and an on-line injec-
tor, which was an integral part of the LC
Accutrim ~ 16h Precision Release T M tablets system, were switched simultaneously through
The dissolution of Accutrim ~ 16 h Precision a relay on the system controller. The on-line
Release T M tablets was studied in 1 1 water at injector made an injection into the LC column,
37 °C and 100 rpm. Dissolution profiles were whereas the STT E6 valve moved to the next
measured by the automated multivessel disso- position. Fig. 1 shows port # 4 in the flush
lution system described here, by the single-ves- mode (dashed line) and port # 3 in the sample
sel dissolution system described in a previous mode (solid line). Once an injection had been
paper [1], and by manual sampling. Six tablets made and the STT E6 valve moved counter-
could be monitored per dissolution run using clockwise, port # 5 would be in the flush mode
the automated multivessel dissolution system. and port # 4 in the sample mode. The sam-
A DL-5 loop probe was suspended in each pling sequence was designed so that the stag-
dissolution vessel and perfused with water at nant probe was flushed prior to the actual
10 ~tl min J. The perfusate was collected in a sampling from that probe. The flush interval
2 ~tl injection loop and analyzed on-line using was designed to allow the previously stagnant
FIA with water at 1 ml min-~ as a carrier and probe to reach a steady-state condition neces-
UV detection at 215 nm. The six dissolution sary for reproducible sampling. Once the injec-
vessels were sampled sequentially every 10 min. tion sequence was initiated, the data and
Therefore, each dissolution vessel was sampled chromatograms were recorded and stored auto-
every hour using the automated multivessel matically in the system controller.
1238 K.P. Shah et al./ J. Pharm. Biomed. Anal. 13 (1995) 1235-1241

3.2. S y s t e m characterization 1°°t 8

Analyte carry-over studies 7

Carry-over experiments were conducted by g
placing an acetaminophen solution
o 8Q
(350 pg ml t) in dissolution vessel # 1. A DL-5
5
loop probe was suspended in the vessel for
microdialysis sampling and both ends of the
probe were connected to port # 1 on the STT
E6 valve. The remaining five ports were linked i i
0 3
with connecting tubing to pump the perfusion 0 20 40 60 80 100 2u
Flow rate (pLJmin)
medium directly through the valve. Analyte
carry-over from the previous injection through Fig. 2. Effectof perfusion medium flow rate on the relative
the STT E6 valve was studied as a function of ([]) and absolute (D) recoveries of acetaminophen
perfusion rate and injection interval. Table 1 through a DL-5 loop probe. The concentration of ac-
summarizes the results of the carry-over study. etaminophen was approximately 350 lagml ~ in water at
37 °C. The perfusion medium was water.
The analyte carry-over was significant at lower
perfusion rates and reduced at higher perfusion
rates. Less than 1% carry-over was observed at on-line injector, the total dead volume of the
a perfusion rate of 1 0 0 p l m i n ] and a l m i n system was calculated to be about 45 lal or less.
injection interval. The sample carry-over also A volume of perfusate that is twice the dead
decreased with increase in injection interval. At volume must be passed through the system to
10 rain injection interval and 10 lal m i n ] per- ensure acceptable precision in the measure-
fusion rate, the sample carry-over was below ments [1,2]. If the volume of perfusate pumped
1%. through the system is lower than the system
Carry-over of sample may have occurred as dead volume, mixing of the samples could oc-
a result of the common passages of dissolution cur. As seen from Table 1, doubling the perfu-
samples through the STT E6 valve, connecting sion rate from 30 lal m i n ] did not reduce the
tubing and/or the on-line injector. Technical sample carry-over by half. At higher perfusion
information on an STT E6 multiposition rates, the dialysis membrane of the DL-5 loop
switching valve provided by the manufacturer probe showed significant ultrafiltration, result-
confirmed that the two sets of inlet/outlet lines ing in leakage of the dialysate to the dissolu-
are separated in such a way that no sample tion vessel. At a perfusion rate of 60 lal m i n -
mixing could have occurred between the ports. through the probe, only 45 lal of the dialysate
However, each inlet/outlet line had a dead was collected per minute at the other end. This
volume of up to 30pl. Port # 1 used in the reflected in a reduced absolute recovery of the
carry-over study had the largest dead volume. drug at higher perfusion rates, as shown in Fig.
Accounting for the extra-valve dead volume 2. Higher perfusion rates were also detrimental
arising from the connecting tubing and the to the physical integrity of the dialysis mem-
brane. By choosing a larger sampling interval,
lower perfusion rates can be used with negligi-
Table l ble carry-over effect. Larger sampling intervals
Effect of perfusion rate and injection interval on the sam- are ideal for dissolution testing of sustained-re-
ple carry-over from the STT E6 multiposition switching
valve lease formulations, but may not be adequate
for immediate release dosage forms, if a disso-
Perfusion rate Injection interval Sample carry-over (%) lution profile is desired.
(pl rain - 1) (min)

20 1 30 Probe-to-probe variations
30 l 21 Multivessel sampling using the STT E6 valve
30 2 l0 required use of one microdialysis probe per
30 3 5.7 vessel. Inter-probe variations were determined
60 1 14 using three DL-5 loop probes. The probes were
75 1 9.0
100 1 <1 immer-sed in an acetaminophen solution
10 10 <1 (350 lag ml -~) at 37 °C and stirred at 50 rpm to
simulate the conditions of the dissolution ex-
 K.P. Shah et al./ J. Pharm. Biomed. Anal. 13 (1995) 1235-1241 1239

periments. Each probe was perfused with wa- probe, a slight different curve was obtained
ter at 50 lal min-1 and the dialysate was ana- with each probe. The range of the slopes was
lyzed to determine the drug recovery through approximately 15%, consistent with the inter-
the probe. The range for the recoveries of the probe variability described in the previous
three probes was approximately 15%. There- section. The linear calibration curves sug-
fore, it was necessary to calibrate each probe gested that the microdialysis recovery did not
individually. change with drug concentration in the range
The automated multivessel dissolution sys- studied.
tem was calibrated as follows. Before the dis- Similarly, the calibration curves of phenyl-
solution study, the drug recovery from each propanolamine were linear in the concentra-
microdialysis probe at the condition of the tion range of 10-100 lag m l - l . However,
experiment was determined using a standard below 10 lag ml ~ the response was not linear
solution. Next, dissolution studies were con- with concentration, presumably owing to ad-
ducted on a required number of tablets. The sorption of the drug onto the connecting tub-
experiment was concluded by repeating the ing and/or the dialysis membrane. The
same drug recovery experiment. Average peak average microdialysis recovery of drug from
heights of the standard solution before and aqueous solutions at 37 °C and 10 lal min ~ 1
after the dissolution experiments were used to perfusion rate was about 45% over the linear
quantify the results of the dissolution study range of the assay (10 100lagml l).
using a single-point calibration method. The repeatability of the multivessel auto-
mated system was assessed by injecting each
L#tearity and repeatability standard solution in triplicate during the cali-
The linearity of the integrated sampling
bration study. The RSD was less than 3%
and chromatographic system for ac-
for all the standards measured by manual
etaminophen was determined using three DL-
and microdialysis sampling, except for the
5 microdialysis loop probes and a STT E6
100lalml -~ standard measured by probe 2
multiposition valve. Standard solutions of ac-
where the RSD was 4.3%. In general, the
etaminophen in the concentration range 2 5 -
RSD value measured at each concentration
500 lag ml -~ were placed in the dissolution
by microdialysis sampling was lower than
vessel at 37 °C and stirred at 50 rpm (i.e. the
that by manual sampling.
conditions of the dissolution study). A probe
was immersed in each dissolution vessel and
perfused with water at 50 lal min -1 for on-
3.3. Dissolution of acetaminophen tablets
line sampling. The calibration curves of ac-
The dissolution profiles of acetaminophen
etaminophen were linear in the concentration
range studied, as shown in Fig. 3. Since mi- tablets obtained with an automated multives-
crodialysis recovery varied from probe to sel dissolution testing system compared well
with profiles measured by manual and auto-
mated single-vessel dissolution systems [1], as
4
shown in Fig. 4. The sampling interval with
the automated multivessel dissolution system
was limited by the dead volume of the STT
E6 switching valve. An uneven injection se-
2
quence consisting of a 96 s sampling mode
alternated by a 36 s flush mode was used to
avoid carry-over of the sample from the pre-
a. 1
vious injection. The flush time of 36 s was
determined by the retention time of ac-
0 etaminophen on the LC column. With the
0 100 200 300 400 500 600
present configuration, only three tablets per
Concentration (~g/mL)
dissolution run could be studied and each
Fig. 3. Calibration curves of acetaminophen determined by vessel was sampled about every 6.5min.
three different DL-5 loop probes simultaneously using the
Faster sampling rates could be achieved if a
automated multivessel dissolution system. Study condi-
tions: 37 °C and 50 rpm; perfusion rate, 50 ~tl rain-l; perfu- switching valve with a lower dead volume
sion medium, water. were available.
1240 K.P. Shah et al. / J. Pharm. Biomed. Anal. 13 (1995) 1235-1241

120 dissolution profiles of Accutrim® 16 h Precision

ReleaseT M tablets measured by different sys-
11111
i tems. Similar drug release profiles were ob-
o .L
tained by all the systems and the results
compared well with the previously reported
eo release profiles [3]. The percentage of drug
c
e
o 4o released was within the allowable Compendial
a. limits for once-a-day tablets [4], i.e. between 15
2o and 45% released in 3 h, between 40 and 70%
o
o . i , t . t . i . i released in 6 h, and not less than 70% in 12 h.
0 5 10 15 20 25
Larger sampling intervals were possible for the
Time (min)
measurement of the dissolution profile of a
Fig. 4. Dissolution profiles of acetaminophen tablets in slow-releasing tablet. Therefore, dissolution of
water determined by an automated multivessel dissolution six tablets could be studied simultaneously us-
system and compared to automated single-vessel and man-
ual dissolution systems. Study conditions: same as in Fig. ing the multivessel dissolution system. In this
3. ©, Single vessel microdialysis sampling; rT, multivessel case, the dissolution vessels were sampled se-
microdialysis sampling; @, manual sampling. Data from quentially and the same dissolution vessel was
Ref. ll]. sampled every hour.

3.4. Dissolution o f Accutrim ® 16h Precision

Release rM tablets 4. Conclusions

Dissolution profiles of phenylpropanolamine After resolution of the carry-over problem,

from Accutrim~ tablets were measured using the six-port STT E6 switching valve was used
the automated multivessel and single-vessel dis- in a reproducible manner to follow the dissolu-
solution systems based on microdialysis sam- tion of up to six tablets simultaneously. The
pling, and a manual sampling system. The microdialysis probes were permanently
automated systems were calibrated by a single- mounted on the switching valve and the probes
point calibration method using a 75 ~tgm1-1 were suspended in the dissolution vessel at the
standard solution of phenylpropanolamine. time of the study. The automated multivessel
The percentage of drug released was calculated dissolution system could be switched to a sin-
based on the labeled amount of 75 mg PPA per gle-vessel or manual system by simply remov-
tablet. Because of the non-linearity of the assay ing the multiposition valve mounted with
at less than l0 ~tg ml ~, results obtained below probes. Except for cleaning of the dissolution
that value were not reported. Fig. 5 shows the vessels and filling the dissolution medium, the
system was completely automated. The multi-
IO0 position switching valve, on-line injection valve
9O @ and detector were controlled through a com-
8o
puter such that once the testing was initiated,
o°~ samples were removed and analyzed automati-
70
cally and the data was stored in the computer.
~ 6o The multiposition switching valve used in this
-- 50 study could be expanded to 16 ports to study
i° 3O
the dissolution of up to 16 tablets simulta-
neously. However, a valve with a lower dead
20
A
i i i i i
volume must be designed to overcome the
5 10 15 20 25 carry-over problems observed in this study.
Time (Hours)

Fig. 5. Dissolution profiles of phenylpropanolamine from

Accutrim tablets determined by automated multivessel
TM References
and single-vessel dissolution testing systems and a manual
sampling system. Study conditions: 37 °C and 100rpm; [1] K.P. Shah, M. Chang and C.M. Riley, J. Pharm.
perfusion rate, 10 tll min-*; perfusion medium, water. @, Biomed. Anal., 12 (1994) 1519-1527.
Manual sampling; A, single-vessel microdialysis sampling; [2] K.P. Shah, J. Zhou, R. Lee, R.L. Schowen, R. Els-
&, multivessel microdialysis sampling; [], reference data bernd, J.M. Ault, J.F. Stobaugh, M. Slavik and C.M.
from Ref. [3]. Riley, J. Pharm. Biomed. Anal., 12(8) (1994) 993-1001.
 K.P. Shah et al. / J. Pharm. Biomed. Anal. 13 (1995) 1235 1241 1241

[3] H.M. Abdou, in A. Gennaro, B. Migdalof, G.L. Has- 1989, p. 232.

sert and T. Medwick (Eds.), Dissolution, Bioavailabil- [4] The United States Pharmacopeia, XXI1 revision, Mack
ity and Bioequivalence, Mack Publishing Co., Easton, Publishing Co., Easton, 1990.