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Design and Characterization of a New Miniaturized Rotating Disk Equipment for In Vitro Dissolution Rate Studies
Design and Characterization of a New Miniaturized Rotating Disk Equipment for In Vitro Dissolution Rate Studies ¨ F,3 WALTER LINDBERG,4 ANITA M. PERSSON,1 KAJSA BAUMANN,2 LARS-OLOF SUNDELO 1 1 ANDERS SOKOLOWSKI, CURT PETTERSSON 1
Division of Analytical Pharmaceutical Chemistry, Uppsala University, BMC, Box 574, SE-751 23 Uppsala, Sweden
2
Department of Chemistry, Go¨teborg University, SE-412 96 Go¨teborg, Sweden
3
Division of Organic Pharmaceutical Chemistry, Uppsala University, BMC, SE-751 23 Uppsala, Sweden
4
Lead Generation Department, AstraZeneca R&D, SE-431 83 Mo¨lndal, Sweden
Received 16 March 2007; revised 17 August 2007; accepted 25 September 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21235
ABSTRACT: A miniaturized apparatus for the determination of the apparent in vitro dissolution rate has been designed, constructed and characterized. The miniaturized apparatus was based on a low volume dissolution cell and a disk in a rotating magnetic bar. The disk tablet is pressed directly into the bar with a press designed and constructed for this purpose. It requires approximately 5 mg of substance. The disk was positioned eccentrically on the bar with an external flow of medium to increase the rate of solvent flow over the disk surface. Six different drug substances were used. The dissolution media were sodium phosphate buffer, pH 7.0, and ammonium acetate buffer, pH 6.8. All quantifications were made by integrating the dissolution cell with high-performance liquid chromatography (HPLC) using diode-array detection (DAD). The obtained results were compared with data from a conventional rotating disk equipment, where the disk was centrically mounted. The dissolution rates at 100 rpm seemed to be on an average of 2–3 times higher for the miniaturized apparatus (RSD 0.2–56%). The preliminary studies of this prototype indicate that the miniaturized rotating disk is a promising design for the qualitative estimation of dissolution rates of substances, for example during screening in early drug discovery. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:3344–3355, 2008
Keywords: dissolution rate; in vitro models; physicochemical properties; analytical chemistry; HPLC (high-performance/pressure liquid chromatography); miniaturization
INTRODUCTION It is important to investigate the physicochemical properties of new drug candidates in order to maximize chances of success in later development. The advances in parallel chemistry, computa-
Correspondence to: Curt Pettersson (Telephone: þ46-018471-4340; Fax: þ46-018-471-4393; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 97, 3344–3355 (2008) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
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tional modeling, and high throughput screening have generated a trend of less soluble lead compounds.1–3 The dissolution rate may be a limiting factor in the administration of an oral dosage form to achieve a desired clinical effect.4 The dissolution rate depends on a combination of factors related to the intrinsic properties of the substance (solubility, crystallinity, amorphism, polymorphism, hydration, solvation, etc.), as well as the physical features of in vitro experimental arrangements (agitation, compression of the substance and the surface structure, size of area,
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speed of rotation, flow pattern over the disk, temperature, etc.).5,6 Apart from this, the chemical properties of the dissolution medium (pH, type of buffer, additional components, etc.) also play a decisive role for the dissolution rate.7–10 Different techniques have been used in dissolution rate studies, for example, USP rotating disk method5 as described by Wood et al.,11 flow-through cell or channel flow methods,12–14 stationary disk with rotating fluid system15 and rotating disk measurements in combination with isothermal miniaturized calorimetry.16 However, all those methods consume a relatively large amount of substance, often more than 100 mg, since many of them are based on standard spectrophotometrical detection. Furthermore, the detection step is often not an online arrangement which should be preferred to reduce the workload and sources of errors. The use of an online high-performance liquid chromatography (HPLC) instrument can provide quantification of low solute concentrations (higher sensitivity) since an enrichment step can be performed on the analytical column.17 The column can also give a separation between the drug of interest and other components present, some of which resulting from more complex dissolution media or impurities. The main goal of the present study was to characterize the newly designed and constructed miniaturized rotating disk equipment for the determination of the apparent in vitro dissolution rate. The miniaturized rotating disk equipment is based on a Plexiglas cell connected to a HPLC system with UV-detection. The disk tablet is pressed directly into a gold plated magnetic bar using a press constructed for this purpose. A continuous flow of dissolution medium is maintained over the disk surface. The scope was not to describe the hydrodynamic conditions in the new dissolution cell in detail, only to compare obtained results to data from conventional rotating disk measurements.5 The test substances were selected from all of the classes in the Biopharmaceutics Classification System (BCS).18
THEORETICAL Factors that are important for the dissolution process were described quantitatively by Noyes and Whitney already in 1897.19 A few years later Bruner and Tolloczko20 and Nernst and Brunner21,22 extended the Noyes–Whitney equation by taking the diffusion process and instruDOI 10.1002/jps
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mental setup into account, which meant a considerable improvement. Essentially this equation has since then been utilized to analyze experimental data from various types of experimental arrangements for the determination of the intrinsic rate of dissolution, k1, which is defined as the amount of substance that goes into solution from unit area in unit time. The determination of k1 by a generalized rotating disk method, taking the liquid flow rate into account and proposing extrapolation procedure to infinite rate of flow, was discussed by Nicklasson et al.23 some 20 years ago. The hydrodynamics in the new flow cell is complex due to a combined effect of flow rate and rotation speed. Especially higher flow rates and rotation speeds are assumed to promote turbulence. A general qualitative equation for the dissolution rate was derived for this cell. Thus the experimentally determined dissolution rate, G, at a given speed of revolution, v, was calculated from
G¼
CV A
(1)
where C is the concentration in the dissolution medium leaving the cell as determined by HPLC from a standard curve, V is the volume flow rate of the dissolution medium through the dissolution cell and A is the area of the drug surface of the disk. Expressing these factors in appropriate units will give the dissolution rate, G, as mg/s/ cm2. This is the mass flux from the disk into the solution. The derivation indicates various possibilities to extrapolate the values of G to infinite speed of revolution, giving the intrinsic dissolution rate (k1 or G1). However, this will not be further discussed in this article.
MATERIALS AND METHODS Chemicals Naproxen and ketoprofen met USP specifications, nortriptyline hydrochloride, and furosemide minimum 98% and carbamazepine 99%, were all from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Ciprofloxacin 98.0% was obtained from Fluka BioChemika, Chemie GmbH (Buchs, Switzerland). Sodium di-hydrogen phosphate monohydrate (NaH2PO4 H2O), pro analysi (p.a.), Acros Organics (Springfield, NJ), di-sodium hydrogen phosphate dihydrate (Na2HPO4 2H2O) p.a. and trifluoroacetic acid 99.0%, both from JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
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Fluka Chemika, Chemie GmbH (Buchs, Switzerland). Ammonium acetate (NH4Ac) p.a., Riedel-de Hae¨ n, Sigma-Aldrich Laborchemikalien GmbH (Seelze, Germany). Acetonitrile, HPLC grade, Fisher Scientific, UK Limited (Leicestershire, UK) and water purified in a Milli-Q1 Academic system (18.2 MV cm/0.22 mm; Millipore, MA). Instrumental For the traditional rotating disk experiments a manual laboratory press from Perkin-Elmer (Waltham, MA), with a stainless steel rotating disk die (Ø 8 mm) similar to that described by Wood et al.11 was used. The dissolution bath was a Dissolutest, Prolabo, France and the thermometer a Testo 110 from Testo, Inc. (Lenzkirch, Germany). pH monitoring was carried out using a pH Meter 744, Metrohm, Schweiz with electrode CMAW711 (Ø 4.5 mm) from Thermo Russell (Auchtermuchty Fife, Scotland). For the new miniaturized equipment the press cylinder (DNC standard) was purchased from FESTO (Hampshire, England). The design and construction was done in-house and is described in greater detail in the next section. A magnetic stirrer with graded stirring speeds was obtained from Heidolph MR 3001K (Steinheim, Germany). The external HPLC-pump, see Figure 4, was a Jasco PU-1585, Jasco, Inc. (Tokyo, Japan). The chromatography in all dissolution rate studies was performed on an Agilent 1100 Series HPLC system with a binary pump, degasser, autosampler and diode-array detector (DAD), Agilent Technologies, Inc. (Palo Alto, CA). A six-position switching valve with a 20 mL stainless steel loop was also purchased from Agilent Technologies, Inc. Data were collected by ChemStation Rev.A.10.02 from Hewlett Packard, Agilent Technologies, Inc. The analytical column was a Zorbax Eclipse XDB-C8 (2.1 mm 50 mm, 5 mm) purchased from Agilent Technologies, Inc. The surface solid-state properties were characterized by Raman spectroscopy with a Labram HR 800 instrument from HORIBA Jobin Yvon Ltd. (Stanmore, Middlesex, UK). Experimental Preparation of the Dissolution Media Sodium phosphate buffer pH 7.0 0.1 (65.5 mM) and ammonium acetate buffer pH 6.8 0.3 (10 mM) with the ionic strengths of 150 and 10 mM respectively were prepared. The pKa-values used JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
for phosphoric acid are 1.89, 6.67, and 11.68 (I ¼ 0.165 M, 25.08C),24 for ammonium 9.20 and for acetic acid 4.53 (both at I ¼ 0.15 M).25 Traditional Rotating Disk Approximately 100 mg of substance was compressed in the die at 195 MPa compression force. The final compression force was chosen by altering the pressure over the disk in values of approximately 195, 585, and 976 MPa. The pressure was maintained for 1 min during disk compression. The substance compact, fixed in the die, was entered into the center of the vessel containing 500 mL of the dissolution medium at room temperature (21.08C 1.58C). The medium was deaireated with helium gas for about 30 min. The die was placed 2.45 cm from the bottom of the vessel and spun at 100 rpm. Samples of 1.0 mL of the medium were withdrawn from time zero (directly after lowering the rotating disk into the vessel) and every minute for the first 10 min followed by every fifth minute until 15 or 30 min had passed, depending on the dissolution rate of the substance tested. The samples were taken midway between the surface and the bottom of the vessel. To avoid problems with adsorption to filters, which had been noticed during initial dissolution studies of the substances, no filtration step was included before injecting the dissolution samples into the HPLC. Samples were not diluted. The pH was constant before and after sampling. Three to six disks for each drug substance and buffer system were evaluated, and the average dissolution rates were calculated together with the relative standard deviations (RSD). Miniaturized Rotating Disk Apparatus The dissolution flow cell chamber was made of two halves of Plexiglas. The inner diameter of the cell was 10.2 mm and the height above the disk surface was 2.4 mm when closed. A gold plated magnetic bar holding the disk was placed at the bottom of the Plexiglas chamber, positioned on a magnetic stirrer, as shown in Figure 1. The Plexiglas chamber held a volume of 300 mL. Inlet and outlet was 1/16 inch capillary tubes made of PEEK in order to fit standard HPLC equipment. The two halves of the Plexiglas flow cell were tightened together by two screws and sealed by an O-ring. The flow from an external HPLC pump was started so that the cell was filled with the deaireated dissolution medium at room temperature (21.08C 1.58C). No air bubbles DOI 10.1002/jps
A NEW MINIATURIZED ROTATING DISK EQUIPMENT
Figure 1. The Plexiglas dissolution flow cell with the gold plated magnetic bar at the bottom of the lower half. The cell is standing on a magnetic stirrer. The two straight arrows indicate the flow direction of the dissolution medium.
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schematically shown in Figure 2. The bar was gold plated in order to avoid corrosion from the dissolution media. The disks had a maximum weight of approximately 5 mg of substance. In order to make the miniaturized disks directly into the magnetic bar, a pneumatic tablet press was designed and constructed. The press is partly depicted in Figure 3. A milled cavity in the piston housing of the press was made at the top, fitting the magnetic bar. Through the housing a piston hole was drilled. This hole was filled with drug substance and then the magnetic bar was fixed in the cavity at the top. The hole of the bar was facing down to the piston entrance while locking the two pieces together by a lid. When making the disks a pressure of 4 bars for approximately 15 s were used, generating a force of approximately 182 MPa. For each substance three to six disks were evaluated. Two to five injections for each disk were made during one run in both buffers, depending on the dissolution rate and rotation
should be trapped in the dissolution cell. If so, the rotation speed was raised to press the bubbles out of the chamber. The flow rate of the medium through the cell was set to 1.0 mL/min. Rotation speeds of 100 and 1000 rpm were tested for quantification. The magnetic bar with the dimensions of 10 mm 4 mm 2.30 mm (Ø 1.5 mm disk) is
Figure 2. A schematic figure of the rotating disk in the eccentric design of the miniaturized equipment, where v is the angular velocity and Ø is the diameter of the pressed substance disk in mm. DOI 10.1002/jps
Figure 3. Schematic figure of the press for the making of miniaturized disks in the gold plated magnetic bar. The lid is pictured at the top of the figure followed by the magnetic bar, the piston housing with the piston inside and then the press cylinder itself. The swing arm located at the right in the figure is placed above the lid to lock the piston housing into position when making the disks. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
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speed. The average dissolution rates together with the RSD were calculated. Flow Rate Evaluation The influence of the flow rate (0.2–1.2 mL/min) through the Plexiglas chamber on the apparent dissolution rate was investigated. The study was performed at constant rotation speeds of 100 and 1000 rpm. Naproxen was used as test substance and the dissolution medium was phosphate buffer at pH 7.0. Characterization of Crystallinity Since the dissolution rate and solubility depend on the surface solid-state properties of a substance, for example the degree of crystallinity, or polymorphic change it was of interest to investigate this by Raman spectroscopy. Raman spectra were obtained for naproxen powder, powder compressed in traditional and miniaturized disk devices as described earlier. A miniaturized disk after exposure to ammonium acetate buffer for approximately 30 min (dried before analysis) was also investigated. The sampling time was 10 points each second, with an aperture of 1000 mm and the laser set at 780 nm. Analyses were carried out at room temperature over the range 300–1800 cm 1. Automatic spike removal was used during the analysis. HPLC Analysis The dissolution chamber for the miniaturized apparatus was connected to the HPLC system as shown in Figure 4. During the dissolution experiments a continuous flow of medium was pumped through the Plexiglas chamber. The first run was always discarded due to substance debris not firmly attached to the magnetic bar after compression. The sampling time of the switching valve was calibrated so that the volume from the dissolution cell was equal to the volume of the standards aspirated by the autosampler. The autosampler in the HPLC system was giving the start signal for the switching valve by injecting 1 mL of the mobile phase. Evaluation of injection volumes was made by comparing the slopes of the calibration curves obtained from the switching valve and the autosampler respectively. The statistical assessment used the 95% confidence level. The temperature of the column compartment was ambient (25–288C) and constant within approximately 28C in one experiment. The injecJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
Figure 4. The instrumental setup for measuring the dissolution rate by the miniaturized apparatus. The sampling loop was positioned in a switching valve.
tion volume was always 20 mL and the mobile phase flow through the column 1.0 mL/min. The light absorption value was always measured at the absorption maximum, lmax, as obtained by the DAD. The HPLC analyses were performed in isocratic mode using three different mobile phase compositions containing acetonitrile (ACN), MilliQ water and trifluoroacetic acid (TFA) as shown in Table 1. A 2 mM potassium nitrate (KNO3) solution vas used as unretained species to determine the dead time, t0. No analysis was longer than 2 min, which gives a total run time of less than 3 min. Standard curves were made from external standards and the concentration range was chosen to fit the anticipated dissolution rate values. No less than six different concentrations were used and the coefficient of determination (R2) was always 0.990 or better for the regression lines.
RESULTS AND DISCUSSION Chromatographic Evaluation To compare the injection volumes of the autosampler with the switching valve setup a comparison of the regression lines was performed. Naproxen, ciprofloxacin, and nortriptyline HCl were test substances. It was found that the closest relationship between the two injection types for 20 mL was achieved if the switching valve was allowed to be open for 0.03 min during the injection, using a flow of 1.0 mL/min through the dissolution cell. The confidence interval DOI 10.1002/jps
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Table 1. Mobile Phase Compositions, Measured Values of lmax, Retention Factors for the Substances, pKa and Intrinsic Solubilities (S0) for the Solutes. Retention Factor, K, calculated From (tR t0)/t0 Substance (BCS Class)
Volume ACN (mL)
Volume Milli-Q Water (mL)
Volume TFA (mL)
lmax (nm)
Retention Factor (k)
pKa
Intrinsic Solubility (mg/mL)
350 350 300 300 300 150
650 650 700 700 700 850
1 1 1 1 1 1
230 257 234 212 240 278
5.3 5.1 3.8 2.7 7.5 3.3
4.1829 3.9829 3.5229 N/A28 10.2131 6.2032; 8.5932
0.01429 0.1229 0.005929 0.2630 0.02731 0.1332
a
Naproxen (2 ) Ketoprofen (2b) Furosemide (4a) Carbamazepine (2a) Nortriptyline HCl (1c) Ciprofloxacin (3a) N/A, not applicable. a From Ref.26 b From Ref.27 c From Ref.28
(a ¼ 0.05) for the slopes of the calibration curves were covering the digit 1 for all substances. Thus no statistical difference between the two types of injection was observed.
Structural Analysis During the optimization of the disk compression force it was seen that some substances generated fragile disks at higher pressures, a phenomenon
Flow and Dissolution Rate The influence of flow rate on the apparent dissolution rate in the miniaturized rotating disk system was investigated. The results of the HPLC determination of concentration, C, as a function of flow _ are shown in Figure 5A. If now C V, _ which rate, V, is proportional to the apparent in vitro dissolution rate (in Eq. 1), is plotted versus V_ as in Figure 5B it is seen that above a certain critical flow rate the apparent dissolution rate becomes constant. This critical flow rate increases with increasing speed of disk revolution, reflecting the increased apparent rate of dissolution. Before reaching the plateau an accumulation of the solute is obtained in the cell and hence no sink condition is obtained. At the plateau sink condition is assumed. Obviously these results indicate that the experiments must be performed so that the minimum flow rate condition is fulfilled. A restriction of the study was that the cell of Plexiglas only could withstand a moderate pressure (approximately 10 bar) since the two halves of the cell were tightened together by two screws and sealed by an O-ring. With the current seal 1.2 mL/min seemed to be an upper limit to accommodate a safety margin for leakage and the risk of breaking the Plexiglas due to raise in pressure. As only one substance was tested in the study of the flow rate, a compromise was made and 1.0 mL/min was used for all substances at both rotation speeds since this is in the upper region of the practical flow rate. DOI 10.1002/jps
Figure 5A & B. The influence on the apparent dissolution rate by varying the flow rate of the dissolution medium through the Plexiglas chamber. Naproxen in phosphate buffer at pH 7.0 using 100 rpm (&) respective 1000 rpm (^) as rotation speed. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
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Figure 6. The Raman spectra of naproxen powder and disks.
that has been reported by others.28 At the optimized compression force no such occurrence was seen for the model substances. The dissolution rates of the test drugs (naproxen, nortriptyline HCl, and ciprofloxacin) did not vary significantly during the study of the three compression forces, cf. Experimental, RSD of an average of 11% (data not shown) was obtained. The difference in dissolution rate versus compression force has been investigated previously.28 No differences in the Raman spectra for naproxen powder, traditional disk, miniaturized disk, or miniaturized disk exposed to ammonium acetate buffer was observed, see Figure 6. Roughly the same compression forces were used in making of the disks (195 MPa respective 182 MPa) and thus it was assumed that differences in the dissolution rates were not originating from any solid state changes when comparing the two apparatuses.
Apparent In Vitro Dissolution Rate and Dissolution Media The apparent dissolution rate, G, was for the traditional rotating disk calculated from the initial slope of a straight line, where the concentration of the dissolved solute (y-axis) plotted against sampling time (x-axis). The line was drawn from 11 to 14 samples withdrawn at different times from the dissolution vessel and analyzed with HPLC. Sink condition was assumed as long as the graph did not deviate from linearity (R2 0.990). For the miniaturized system the dissolution rate was calculated from an analysis according to Eq. (1), using a flow of dissolution medium at 1.0 mL/min. The average apparent in vitro dissolution rate for naproxen, ketoprofen, nortriptyline HCl, furosemide, carbamazepinex, and ciprofloxacin JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
in phosphate buffer of pH 7.0 and ammonium acetate buffer of pH 6.8 are summarized in Tables 2 and 3, respectively. The rotation speed was set to 100 rpm for both apparatuses and also 1000 rpm for the miniaturized equipment. In view of the fact that the miniaturized apparatus has an additional flow over the surface (and can be set to higher rotation speeds), this will naturally generate a different fluid pattern compared to the traditional setup. The eccentric design (and higher rotation speeds) will also affect the thickness of the layer adjacent to the surface since the angular velocity, v, is increased. Hence the increased apparent dissolution rate of the solutes observed in Tables 2 and 3 when using the novel equipment is expected. Some substances may convert from anhydrous form to the hydrate upon exposure to aqueous medium,33,34 but since the same aqueous media were tested for all substances in both apparatuses this eventual effect was assumed to be equivalent. Linear regression analysis with 95% confidence level was used to compare the traditional rotating disk to the miniaturized one for the respective buffer system (Fig. 7). The statistical evaluation established a difference between the two apparatuses since the p-values were lower than 0.05. The p-value was below 0.05 for the ammonium acetate evaluation even though the sample point of nortriptyline HCl, differing from the cluster in Figure 7B, was removed ( p ¼ 0.03, R2 ¼ 0.836, and slope ¼ 0.396). From the results presented in Tables 2 and 3 the following general observations can be made. When performed at the same speed of revolution the miniaturized rotation disk apparatus gives on average 2–3 times higher values for the apparent dissolution rates compared to the traditional rotating disk experiments. The exception is ciprofloxacin. When in the miniaturized disk experiments the speed of revolution is increased from 100 to 1000 rpm the apparent dissolution rate increases consistently with the exception of ciprofloxacin. Increasing the speed of revolution by a factor of ten gives on an average an increase in the apparent dissolution rate of the order of 2.5 for the miniaturized disk experiments. It is important however to establish the critical flow rate for the drug substances at the set rotation speed for accurate comparision (Fig. 5). Ciprofloxacin is the only ampholyte in the group of substances tested here, and this may generate the deviation of the trend observed in Tables 2–4. The large RSD (53% and 56%) of ciprofloxacin may DOI 10.1002/jps
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Table 2. Average Apparent Dissolution Rates, G, Using Sodium Phosphate Buffer at pH 7.0 as Dissolution Medium
Traditional RD, 100 rpm Substance (n ¼ Number of Disks) Naproxen (n ¼ 6) Ketoprofen (n ¼ 3) Furosemide (n ¼ 3) Carbamazepine (n ¼ 3) Nortriptyline HCl (n ¼ 6) Ciprofloxacin (n ¼ 6)
Miniaturized RD, 100 rpm
Miniaturized RD, 1000 rpm
G (mg/s/cm2)
RSD (%)
G (mg/s/cm2)
RSD (%)
G (mg/s/cm2)
RSD (%)
5.4 12.2 8.4 0.35 30.5 0.08
4 3 6 10 21 8
9.8 18.9 17.1 1.1 67.7 0.53
14 6 3 17 10 53
23.2 70.1 41.7 2.4 193.2 0.81
8 5 6 0.2 14 27
RD, rotating disk.
forces to achieve a flat and rigid surface. However, disks of ciprofloxacin and nortriptyline HCl also swelled in the traditional rotating disk apparatus. But due to the centric design without an added flow over the surface, and in addition more substance quantities to withstand the disk erosion, this effect was not assumed as critical as for the miniaturized setup. The presence of irregularities on the surface may result in increased turbulence in the adjacent fluid.35 An increase of the available surface area will also increase the dissolution rate. A more precise and reproducible way of making the disks is probably necessary in order to improve the results. A noteworthy difference in the dissolution rates between the two aqueous buffer systems is found among the substances containing a weak carboxylic acid. It has been shown that the maximum dissolution rate for a solid acid will occur when the pKa for the buffer is two or more pH units above the pKa of the acid,7 in combination with the intrinsic solubility of the solute.
be explained by the fact that the disk swelled in the two buffers. When spun at 100 rpm this swollen disk was not affected as much as at a rotation speed of 1000 rpm. This higher speed caused the crest to be cut off, leaving a smoother surface behind. During 100 rpm however the crest was remained at the surface for a longer time, causing an irreproducible surface area until the crest was totally broken up leaving the smoother surface in contact with dissolution medium. The swelling of the disk surface, was very high for ciprofloxacin in the ammonium acetate buffer. This generated an increased risk of breaking the cell of Plexiglas due to the raise in pressure since the PEEK capillary for the outflow was obstructed. In general it could be noted that very low (ciprofloxacin) and very high (notriptyline HCl) dissolution rates may cause some experimental difficulties, most likely due to problems with the surface of the disk. Differences in physical properties of these compounds may require different compression
Table 3. Average Apparent Dissolution Rates, G, Using Ammonium Acetate Buffer at pH 6.8 as Dissolution Medium Traditional RD, 100 rpm Substance (n ¼ Number of Disks) Naproxen (n ¼ 6) Ketoprofen (n ¼ 3) Furosemide (n ¼ 3) Carbamazepine (n ¼ 3) Nortriptyline HCl (n ¼ 6) Ciprofloxacin (n ¼ 6, 100 rpm/n ¼ 2, 1000 rpm)
Miniaturized RD, 100 rpm
Miniaturized RD, 1000 rpm
G (mg/s/cm2)
RSD (%)
G (mg/s/cm2)
RSD (%)
G (mg/s/cm2)
RSD (%)
0.48 1.4 0.83 0.48 30.0 0.08
3 1 14 21 2 29
1.2 3.9 1.9 1.3 60.3 1.3
16 18 3 18 11 56
2.8 10.4 5.0 3.0 177.5 7.2
12 4 4 15 4 8
RD, rotating disk. DOI 10.1002/jps
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Some important differences between the two aqueous buffer systems are the type of buffer (i.e., type of ions present), the concentration, the buffer capacity at the pH chosen and the ionic strength. The buffer capacity of ammonium acetate at pH 6.8 is very low. A better buffer capacity at this pH is achieved with the phosphate buffer, but since the ammonium acetate buffer was originating from an LC-UV/MS method for solubility measurements this buffer was tested in addition. The use of mass spectrometry detection is desired in future development and for this reason phosphate buffer is not optimal. The presence of diprotic buffers might also increase the dissolution rates of weak acids.9 An increased buffer concentration may as well generate higher rates, depending on the solutes intrinsic solubility.9,36 The dissolving substance is not affecting the pH of the dissolution medium if the concentration of the solute is 100 times lower compared to the buffer capacity (b) of the dissolution medium. This can easily be calculated for the phosphate buffer (b ¼ 0.0328 M at pH 7.0) by using the Henderson–Hasselbalch and buffer equations.37,38 If a strong base, c ¼ 3.28 10 4 M, is added to the buffer the pH will change by 0.01 which is negligible. In this new apparatus the concentrations of the substances during the dissolution rate studies were all fulfilling this criterion, except for nortriptyline HCl in the ammonium acetate buffer. However, since this is a salt form of nortriptyline it is not assumed to affect the pH of the dissolution medium significantly. The intrinsic solubility of a protolytic compound will affect the pH in the adjacent layer of the disk, with high solubility providing the most significant pH change in contrast to a low soluble
Figure 7. The line fit plots from the linear regression analysis of the two apparatuses compared to each other with respective buffer at 100 rpm and 1.0 mL/min for the miniaturized apparatus. The p-values at 95% confidence level were; 1.1 10 4 for the phosphate, A, and 3.8 10 8 for the ammonium acetate, B, using linear regression analysis.
Table 4. Relative Rates of Dissolution, That Is, Apparent Rate of Dissolution (G) in Sodium Phosphate Buffer ¼ Index P (from Tab. 2) Divided by Apparent Rate of Dissolution (G) in Ammonium Acetate Buffer ¼ Index A (from Tab. 3) Traditional RD, Miniaturized RD, Miniaturized RD, 100 rpm 100 rpm 1000 rpm Substance Naproxen Ketoprofen Furosemide Carbamazepine Nortriptyline HCl Ciprofloxacin
G_P/G_A
G_P/G_A
G_P/G_A
Average
11.2 8.7 10.1 0.73 1.0 1.0
8.2 4.8 9.0 0.85 1.1 0.40
8.3 6.7 8.3 0.80 1.1 0.11
9.2 6.7 9.1 0.79 1.1 0.50
RD, rotating disk. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
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substance. If a more concentrated buffer, with the best buffer capacity at the chosen pH, is used as dissolution medium this will have an effect on the boundary conditions. The pH will be held more controlled by this buffer, compared with a buffer at low concentration and poor buffer capacity. As a buffer is changed it is therefore obvious that the apparent dissolution rate will change reflecting the interaction of the dissolving substance with the buffer components. If apparent dissolution rates from the sodium phosphate buffer (Tab. 2) are divided by the corresponding data for the ammonium acetate buffer (Tab. 3) one obtains the values as given in Table 4. The data are consistent, but the value for the ampholyte deviates from the more homogeneous pattern. It is not obvious that the ratios can be assumed to be the same comparing the two apparatuses since the hydrodynamics in the systems are very different, but here it can be implied that the dissolution experiments also could be used to single out substances with deviating properties (physical as well as chemical). This in itself could be valuable in the process of drug design, providing that the surface of the disks are comparable. Table 4 gives fairly precise information about the relative change in dissolution rate when going from one buffer to another. A comparison of the dissolution rate in the buffer systems at the same concentration and ionic strength could probably improve the results. When performing dissolution rate studies it is therefore always necessary to specify all relevant information of the medium that could influence the dissolution rate of solutes. This new apparatus determines the dissolution rate in a faster way than the traditional with a gain in drug substance and dissolution medium. In addition the dissolution rate can be correlated to the solubility of a substance, as described previously.12,39,40 This correlation will be evaluated for this new apparatus in future studies. From all these observations it can be concluded that the experimental setups perform in a very consistent way and that the miniaturized disk method is at least as reliable as the traditional one. All this holds for data from the same buffer.
CONCLUSIONS A new miniaturized apparatus for the determination of the apparent in vitro dissolution rate has DOI 10.1002/jps
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been designed, constructed, and characterized. The apparatus was compared to a traditional rotating disk equipment. It is shown that this new technique gives data consistent with the traditional one and with a precision approximately as good as for the latter. This novel apparatus is mainly intended for use in screening purposes early in drug development. The numerical values of the apparent dissolution rate for the miniaturized setup are by a factor slightly larger than two as compared to the traditional one at the same rotation speed. This can be explained by the more efficient flow of solvent and eccentric design in the novel apparatus. Although the hydrodynamics in the miniaturized system is complex, as it depends on a combined effect of the flow rate and rotation speed, the obtained results are promising as discussed above. However, more studies are needed in order to present a theory to derive an equation that more accurately describes the dissolution rate in the complex flow profile of the miniaturized flow cell. When comparing data from two different buffer systems the relative changes in apparent in vitro dissolution rate as determined by the two techniques agree quite well. Even if the present results may be good enough for relative comparisons during screening, the problem of reproducible disk formation must be addressed in the future. The miniaturized rotating disk arrangement has the great advantage that only minute quantities of solid, about 5 mg, are required and that the dissolution rate, G, can be obtained directly from the results of one analysis. Furthermore, this technique provides a means to determine intrinsic dissolution rates by varying the speed of revolution during an experiment and extrapolating to infinite speed or to correlate the dissolution rate to the solubility more rapidly. Another advantage of the new equipment is that it is simple to use and enables an easy change of the dissolution medium.
ACKNOWLEDGMENTS We would like to thank Anders AS. Karlsson and Anders S. Carlsson at AstraZeneca Mo¨ lndal (Sweden) for providing drug substances, Matti Ahlqvist at AstraZeneca Mo¨ lndal (Sweden) for doing the Raman spectroscopy measurements and Magnus Persson, Structural Biology Laboratories at the Biomedical Centre in Uppsala (Sweden) for help with drawings in the manuscript. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
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REFERENCES 1. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. 2001. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev 46:3–26. 2. McFarland JW, Avdeef A, Berger CM, Raevsky OA. 2001. Estimating the water solubilities of crystalline compounds from their chemical structures alone. J Chem Inf Comput Sci 41:1355–1359. 3. Bergstrom CA, Norinder U, Luthman K, Artursson P. 2002. Experimental and computational screening models for prediction of aqueous drug solubility. Pharm Res 19:182–188. 4. Dressman JB, Reppas C. 2000. In vitro-in vivo correlations for lipophilic, poorly water-soluble drugs. Eur J Pharm Sci 11:S73–S80. 5. USP. 2006. Intrinsic dissolution. US Pharmacopeia National Formulary 2006, 29 (chapter <1087>): 2923–2924. 6. Missel PJ, Stevens LE, Mauger JW. 2004. Reexamination of convective diffusion/drug dissolution in a laminar flow channel: Accurate prediction of dissolution rate. Pharm Res 21:2300– 2306. 7. McNamara DP, Amidon GL. 1988. Reaction plane approach for estimating the effects of buffers on the dissolution rate of acidic drugs. J Pharm Sci 77:511– 517. 8. Ramtoola Z, Corrigan OI. 1989. Influence of the buffering capacity of the medium on the dissolution of drug-excipient mixtures. Drug Dev Ind Pharm 15:2359–2374. 9. Aunins JG, Southard MZ, Myers RA, Himmelstein KJ, Stella VJ. 1985. Dissolution of carboxylic acids. III: The effect of polyionizable buffers. J Pharm Sci 74:1305–1316. 10. Mooney KG, Mintun MA, Himmelstein KJ, Stella VJ. 1981. Dissolution kinetics of carboxylic acids II: Effect of buffers. J Pharm Sci 70:22–32. 11. Wood J, Syarto J, Letterman H. 1965. Improved holder for intrinsic dissolution rate studies. J Pharm Sci 54:1068. 12. Shah AC, Nelson KG. 1975. Evaluation of a convective diffusion drug dissolution rate model. J Pharm Sci 64:1518–1520. 13. Sun W, Larive CK, Southard MZ. 2003. A mechanistic study of danazol dissolution in ionic surfactant solutions. J Pharm Sci 92:424–435. 14. Peltonen L, Liljeroth P, Heikkila T, Kontturi K, Hirvonen J. 2003. Dissolution testing of acetylsalicylic acid by a channel flow method-correlation to USP basket and intrinsic dissolution methods. Eur J Pharm Sci 19:395–401. 15. Khoury N, Mauger JW, Howard S. 1988. Dissolution rate studies from a stationary disk/rotating fluid system. Pharm Res 5:495–500.
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16. Terada K, Kitano H, Yoshihashi Y, Yonemochi E. 2000. Quantitative correlation between initial dissolution rate and heat of solution of drug. Pharm Res 17:920–924. 17. Frei RW, Zech K. 1988. Selective sample handling and detection in high-performance liquid chromatography. J Chrom Libr, Elsevier, Amsterdam 39A: 43–54. 18. Amidon GL, Lennernas H, Shah VP, Crison JR. 1995. A theoretical basis for a biopharmaceutic drug classification: The correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm Res 12:413–420. 19. Noyes AA, Whitney WR. 1897. The rate of solution of solid substances in their own solutions. J Am Chem Soc 19:930–934. 20. Bruner L, Tolloczko S. 1900. On the velocity of solution of solid bodies. Zeit Physikal Chem 35: 283–290. 21. Nernst W. 1904. Theory of reaction velocity in heterogenous systems. Zeit Physikal Chem 47:52– 55. 22. Brunner E. 1904. Reaction velocity in heterogenous systems. Zeit Physikal Chem 47:56–102. 23. Nicklasson M, Brodin A, Sundelo¨ f L-O. 1982. On the determination of true intrinsic rates of dissolution by means of a generalized rotating disk method. Acta Pharm Suec 19:109–118. 24. Sirius, (UK) SAIL. 1994. Sirius Technical Applications Notes (STAN). 1. 25. RefinementPro2, Sirius, (UK) SAIL. 26. Wu CY, Benet LZ. 2005. Predicting drug disposition via application of BCS: Transport/absorption/elimination interplay and development of a biopharmaceutics drug disposition classification system. Pharm Res 22:11–23. 27. Granero GE, Ramachandran C, Amidon GL. 2006. Rapid in vivo dissolution of ketoprofen: Implications on the biopharmaceutics classification system. Pharmazie 61:673–676. 28. Yu LX, Carlin AS, Amidon GL, Hussain AS. 2004. Feasibility studies of utilizing disk intrinsic dissolution rate to classify drugs. Int J Pharm 270:221– 227. 29. Avdeef A, Berger CM, Brownell C. 2000. pH-metric solubility. 2: Correlation between the acid-base titration and the saturation shake-flask solubility-pH methods. Pharm Res 17:85–89. 30. Loftsson T, Hreinsdottir D, Masson M. 2005. Evaluation of cyclodextrin solubilization of drugs. Int J Pharm 302:18–28. 31. Aburub A, Allgeier M, Box K, Comer J, Dage J, Hoaglund-Hyzer C, Kotiyan P, Riebesehl B-U, Sanchez-Felix M, Semones S, Stuart M. 2005. Differences between the kinetic and intrinsic solubility of a series of pharmaceutical compounds generated using a new solubility technique (CheqSol). Poster presented at AAPS Annual Meeting and Exposition, Nashville TN, USA.
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32. Escribano E, Calpena AC, Garrigues TM, Freixas J, Domenech J, Moreno J. 1997. Structure-absorption relationships of a series of 6-fluoroquinolones. Antimicrob Agents Chemother 41:1996–2000. 33. Li X, Zhi F, Hu Y. 2007. Investigation of excipient and processing on solid phase transformation and dissolution of ciprofloxacin.Int JPharm328:177–182. 34. Murphy D, Rodriguez-Cintron F, Langevin B, Kelly RC, Rodriguez-Hornedo N. 2002. Solutionmediated phase transformation of anhydrous to dihydrate carbamazepine and the effect of lattice disorder. Int J Pharm 246:121–134. 35. Levich VG. 1962. Physicochemical hydrodynamics. New Jersey, USA: Prentice-Hall Inc Englewood Cliffs. pp 162–171. 36. Ozturk SS, Palsson BO, Dressman JB. 1988. Dissolution of ionizable drugs in buffered and unbuffered solutions. Pharm Res 5:272–282.
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37. Van Slyke DD. 1922. On the measurement of buffer values and on the relationship of buffer value to the dissociation constant of the buffer and the concentration and reaction of the buffer solution. J Biol Chem 52:525–570. 38. Sille´ n LG, Lange PW, Gabrielson CO. 1948. Fysikalisk-kemiska ra¨ kneuppgifter. Uppsala, Sweden: Almqvist & Wiksells Boktryckeri Aktiebolag. pp 173–174. 39. Hamlin WE, Northam JI, Wagner JG. 1965. Relationship between in vitro dissolution rates and solubilities of numerous compounds representative of various chemical species. J Pharm Sci 54:1651– 1653. 40. Nicklasson M, Brodin A, Nyqvist H. 1981. Studies on the relationship between solubility and intrinsic rate of dissolution as a function of pH. Acta Pharm Suec 18:119–128.
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Division of Analytical Pharmaceutical Chemistry, Uppsala University, BMC, Box 574, SE-751 23 Uppsala, Sweden
2
Department of Chemistry, Go¨teborg University, SE-412 96 Go¨teborg, Sweden
3
Division of Organic Pharmaceutical Chemistry, Uppsala University, BMC, SE-751 23 Uppsala, Sweden
4
Lead Generation Department, AstraZeneca R&D, SE-431 83 Mo¨lndal, Sweden
Received 16 March 2007; revised 17 August 2007; accepted 25 September 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21235
ABSTRACT: A miniaturized apparatus for the determination of the apparent in vitro dissolution rate has been designed, constructed and characterized. The miniaturized apparatus was based on a low volume dissolution cell and a disk in a rotating magnetic bar. The disk tablet is pressed directly into the bar with a press designed and constructed for this purpose. It requires approximately 5 mg of substance. The disk was positioned eccentrically on the bar with an external flow of medium to increase the rate of solvent flow over the disk surface. Six different drug substances were used. The dissolution media were sodium phosphate buffer, pH 7.0, and ammonium acetate buffer, pH 6.8. All quantifications were made by integrating the dissolution cell with high-performance liquid chromatography (HPLC) using diode-array detection (DAD). The obtained results were compared with data from a conventional rotating disk equipment, where the disk was centrically mounted. The dissolution rates at 100 rpm seemed to be on an average of 2–3 times higher for the miniaturized apparatus (RSD 0.2–56%). The preliminary studies of this prototype indicate that the miniaturized rotating disk is a promising design for the qualitative estimation of dissolution rates of substances, for example during screening in early drug discovery. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:3344–3355, 2008
Keywords: dissolution rate; in vitro models; physicochemical properties; analytical chemistry; HPLC (high-performance/pressure liquid chromatography); miniaturization
INTRODUCTION It is important to investigate the physicochemical properties of new drug candidates in order to maximize chances of success in later development. The advances in parallel chemistry, computa-
Correspondence to: Curt Pettersson (Telephone: þ46-018471-4340; Fax: þ46-018-471-4393; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 97, 3344–3355 (2008) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
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tional modeling, and high throughput screening have generated a trend of less soluble lead compounds.1–3 The dissolution rate may be a limiting factor in the administration of an oral dosage form to achieve a desired clinical effect.4 The dissolution rate depends on a combination of factors related to the intrinsic properties of the substance (solubility, crystallinity, amorphism, polymorphism, hydration, solvation, etc.), as well as the physical features of in vitro experimental arrangements (agitation, compression of the substance and the surface structure, size of area,
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speed of rotation, flow pattern over the disk, temperature, etc.).5,6 Apart from this, the chemical properties of the dissolution medium (pH, type of buffer, additional components, etc.) also play a decisive role for the dissolution rate.7–10 Different techniques have been used in dissolution rate studies, for example, USP rotating disk method5 as described by Wood et al.,11 flow-through cell or channel flow methods,12–14 stationary disk with rotating fluid system15 and rotating disk measurements in combination with isothermal miniaturized calorimetry.16 However, all those methods consume a relatively large amount of substance, often more than 100 mg, since many of them are based on standard spectrophotometrical detection. Furthermore, the detection step is often not an online arrangement which should be preferred to reduce the workload and sources of errors. The use of an online high-performance liquid chromatography (HPLC) instrument can provide quantification of low solute concentrations (higher sensitivity) since an enrichment step can be performed on the analytical column.17 The column can also give a separation between the drug of interest and other components present, some of which resulting from more complex dissolution media or impurities. The main goal of the present study was to characterize the newly designed and constructed miniaturized rotating disk equipment for the determination of the apparent in vitro dissolution rate. The miniaturized rotating disk equipment is based on a Plexiglas cell connected to a HPLC system with UV-detection. The disk tablet is pressed directly into a gold plated magnetic bar using a press constructed for this purpose. A continuous flow of dissolution medium is maintained over the disk surface. The scope was not to describe the hydrodynamic conditions in the new dissolution cell in detail, only to compare obtained results to data from conventional rotating disk measurements.5 The test substances were selected from all of the classes in the Biopharmaceutics Classification System (BCS).18
THEORETICAL Factors that are important for the dissolution process were described quantitatively by Noyes and Whitney already in 1897.19 A few years later Bruner and Tolloczko20 and Nernst and Brunner21,22 extended the Noyes–Whitney equation by taking the diffusion process and instruDOI 10.1002/jps
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mental setup into account, which meant a considerable improvement. Essentially this equation has since then been utilized to analyze experimental data from various types of experimental arrangements for the determination of the intrinsic rate of dissolution, k1, which is defined as the amount of substance that goes into solution from unit area in unit time. The determination of k1 by a generalized rotating disk method, taking the liquid flow rate into account and proposing extrapolation procedure to infinite rate of flow, was discussed by Nicklasson et al.23 some 20 years ago. The hydrodynamics in the new flow cell is complex due to a combined effect of flow rate and rotation speed. Especially higher flow rates and rotation speeds are assumed to promote turbulence. A general qualitative equation for the dissolution rate was derived for this cell. Thus the experimentally determined dissolution rate, G, at a given speed of revolution, v, was calculated from
G¼
CV A
(1)
where C is the concentration in the dissolution medium leaving the cell as determined by HPLC from a standard curve, V is the volume flow rate of the dissolution medium through the dissolution cell and A is the area of the drug surface of the disk. Expressing these factors in appropriate units will give the dissolution rate, G, as mg/s/ cm2. This is the mass flux from the disk into the solution. The derivation indicates various possibilities to extrapolate the values of G to infinite speed of revolution, giving the intrinsic dissolution rate (k1 or G1). However, this will not be further discussed in this article.
MATERIALS AND METHODS Chemicals Naproxen and ketoprofen met USP specifications, nortriptyline hydrochloride, and furosemide minimum 98% and carbamazepine 99%, were all from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Ciprofloxacin 98.0% was obtained from Fluka BioChemika, Chemie GmbH (Buchs, Switzerland). Sodium di-hydrogen phosphate monohydrate (NaH2PO4 H2O), pro analysi (p.a.), Acros Organics (Springfield, NJ), di-sodium hydrogen phosphate dihydrate (Na2HPO4 2H2O) p.a. and trifluoroacetic acid 99.0%, both from JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
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Fluka Chemika, Chemie GmbH (Buchs, Switzerland). Ammonium acetate (NH4Ac) p.a., Riedel-de Hae¨ n, Sigma-Aldrich Laborchemikalien GmbH (Seelze, Germany). Acetonitrile, HPLC grade, Fisher Scientific, UK Limited (Leicestershire, UK) and water purified in a Milli-Q1 Academic system (18.2 MV cm/0.22 mm; Millipore, MA). Instrumental For the traditional rotating disk experiments a manual laboratory press from Perkin-Elmer (Waltham, MA), with a stainless steel rotating disk die (Ø 8 mm) similar to that described by Wood et al.11 was used. The dissolution bath was a Dissolutest, Prolabo, France and the thermometer a Testo 110 from Testo, Inc. (Lenzkirch, Germany). pH monitoring was carried out using a pH Meter 744, Metrohm, Schweiz with electrode CMAW711 (Ø 4.5 mm) from Thermo Russell (Auchtermuchty Fife, Scotland). For the new miniaturized equipment the press cylinder (DNC standard) was purchased from FESTO (Hampshire, England). The design and construction was done in-house and is described in greater detail in the next section. A magnetic stirrer with graded stirring speeds was obtained from Heidolph MR 3001K (Steinheim, Germany). The external HPLC-pump, see Figure 4, was a Jasco PU-1585, Jasco, Inc. (Tokyo, Japan). The chromatography in all dissolution rate studies was performed on an Agilent 1100 Series HPLC system with a binary pump, degasser, autosampler and diode-array detector (DAD), Agilent Technologies, Inc. (Palo Alto, CA). A six-position switching valve with a 20 mL stainless steel loop was also purchased from Agilent Technologies, Inc. Data were collected by ChemStation Rev.A.10.02 from Hewlett Packard, Agilent Technologies, Inc. The analytical column was a Zorbax Eclipse XDB-C8 (2.1 mm 50 mm, 5 mm) purchased from Agilent Technologies, Inc. The surface solid-state properties were characterized by Raman spectroscopy with a Labram HR 800 instrument from HORIBA Jobin Yvon Ltd. (Stanmore, Middlesex, UK). Experimental Preparation of the Dissolution Media Sodium phosphate buffer pH 7.0 0.1 (65.5 mM) and ammonium acetate buffer pH 6.8 0.3 (10 mM) with the ionic strengths of 150 and 10 mM respectively were prepared. The pKa-values used JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
for phosphoric acid are 1.89, 6.67, and 11.68 (I ¼ 0.165 M, 25.08C),24 for ammonium 9.20 and for acetic acid 4.53 (both at I ¼ 0.15 M).25 Traditional Rotating Disk Approximately 100 mg of substance was compressed in the die at 195 MPa compression force. The final compression force was chosen by altering the pressure over the disk in values of approximately 195, 585, and 976 MPa. The pressure was maintained for 1 min during disk compression. The substance compact, fixed in the die, was entered into the center of the vessel containing 500 mL of the dissolution medium at room temperature (21.08C 1.58C). The medium was deaireated with helium gas for about 30 min. The die was placed 2.45 cm from the bottom of the vessel and spun at 100 rpm. Samples of 1.0 mL of the medium were withdrawn from time zero (directly after lowering the rotating disk into the vessel) and every minute for the first 10 min followed by every fifth minute until 15 or 30 min had passed, depending on the dissolution rate of the substance tested. The samples were taken midway between the surface and the bottom of the vessel. To avoid problems with adsorption to filters, which had been noticed during initial dissolution studies of the substances, no filtration step was included before injecting the dissolution samples into the HPLC. Samples were not diluted. The pH was constant before and after sampling. Three to six disks for each drug substance and buffer system were evaluated, and the average dissolution rates were calculated together with the relative standard deviations (RSD). Miniaturized Rotating Disk Apparatus The dissolution flow cell chamber was made of two halves of Plexiglas. The inner diameter of the cell was 10.2 mm and the height above the disk surface was 2.4 mm when closed. A gold plated magnetic bar holding the disk was placed at the bottom of the Plexiglas chamber, positioned on a magnetic stirrer, as shown in Figure 1. The Plexiglas chamber held a volume of 300 mL. Inlet and outlet was 1/16 inch capillary tubes made of PEEK in order to fit standard HPLC equipment. The two halves of the Plexiglas flow cell were tightened together by two screws and sealed by an O-ring. The flow from an external HPLC pump was started so that the cell was filled with the deaireated dissolution medium at room temperature (21.08C 1.58C). No air bubbles DOI 10.1002/jps
A NEW MINIATURIZED ROTATING DISK EQUIPMENT
Figure 1. The Plexiglas dissolution flow cell with the gold plated magnetic bar at the bottom of the lower half. The cell is standing on a magnetic stirrer. The two straight arrows indicate the flow direction of the dissolution medium.
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schematically shown in Figure 2. The bar was gold plated in order to avoid corrosion from the dissolution media. The disks had a maximum weight of approximately 5 mg of substance. In order to make the miniaturized disks directly into the magnetic bar, a pneumatic tablet press was designed and constructed. The press is partly depicted in Figure 3. A milled cavity in the piston housing of the press was made at the top, fitting the magnetic bar. Through the housing a piston hole was drilled. This hole was filled with drug substance and then the magnetic bar was fixed in the cavity at the top. The hole of the bar was facing down to the piston entrance while locking the two pieces together by a lid. When making the disks a pressure of 4 bars for approximately 15 s were used, generating a force of approximately 182 MPa. For each substance three to six disks were evaluated. Two to five injections for each disk were made during one run in both buffers, depending on the dissolution rate and rotation
should be trapped in the dissolution cell. If so, the rotation speed was raised to press the bubbles out of the chamber. The flow rate of the medium through the cell was set to 1.0 mL/min. Rotation speeds of 100 and 1000 rpm were tested for quantification. The magnetic bar with the dimensions of 10 mm 4 mm 2.30 mm (Ø 1.5 mm disk) is
Figure 2. A schematic figure of the rotating disk in the eccentric design of the miniaturized equipment, where v is the angular velocity and Ø is the diameter of the pressed substance disk in mm. DOI 10.1002/jps
Figure 3. Schematic figure of the press for the making of miniaturized disks in the gold plated magnetic bar. The lid is pictured at the top of the figure followed by the magnetic bar, the piston housing with the piston inside and then the press cylinder itself. The swing arm located at the right in the figure is placed above the lid to lock the piston housing into position when making the disks. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
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speed. The average dissolution rates together with the RSD were calculated. Flow Rate Evaluation The influence of the flow rate (0.2–1.2 mL/min) through the Plexiglas chamber on the apparent dissolution rate was investigated. The study was performed at constant rotation speeds of 100 and 1000 rpm. Naproxen was used as test substance and the dissolution medium was phosphate buffer at pH 7.0. Characterization of Crystallinity Since the dissolution rate and solubility depend on the surface solid-state properties of a substance, for example the degree of crystallinity, or polymorphic change it was of interest to investigate this by Raman spectroscopy. Raman spectra were obtained for naproxen powder, powder compressed in traditional and miniaturized disk devices as described earlier. A miniaturized disk after exposure to ammonium acetate buffer for approximately 30 min (dried before analysis) was also investigated. The sampling time was 10 points each second, with an aperture of 1000 mm and the laser set at 780 nm. Analyses were carried out at room temperature over the range 300–1800 cm 1. Automatic spike removal was used during the analysis. HPLC Analysis The dissolution chamber for the miniaturized apparatus was connected to the HPLC system as shown in Figure 4. During the dissolution experiments a continuous flow of medium was pumped through the Plexiglas chamber. The first run was always discarded due to substance debris not firmly attached to the magnetic bar after compression. The sampling time of the switching valve was calibrated so that the volume from the dissolution cell was equal to the volume of the standards aspirated by the autosampler. The autosampler in the HPLC system was giving the start signal for the switching valve by injecting 1 mL of the mobile phase. Evaluation of injection volumes was made by comparing the slopes of the calibration curves obtained from the switching valve and the autosampler respectively. The statistical assessment used the 95% confidence level. The temperature of the column compartment was ambient (25–288C) and constant within approximately 28C in one experiment. The injecJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
Figure 4. The instrumental setup for measuring the dissolution rate by the miniaturized apparatus. The sampling loop was positioned in a switching valve.
tion volume was always 20 mL and the mobile phase flow through the column 1.0 mL/min. The light absorption value was always measured at the absorption maximum, lmax, as obtained by the DAD. The HPLC analyses were performed in isocratic mode using three different mobile phase compositions containing acetonitrile (ACN), MilliQ water and trifluoroacetic acid (TFA) as shown in Table 1. A 2 mM potassium nitrate (KNO3) solution vas used as unretained species to determine the dead time, t0. No analysis was longer than 2 min, which gives a total run time of less than 3 min. Standard curves were made from external standards and the concentration range was chosen to fit the anticipated dissolution rate values. No less than six different concentrations were used and the coefficient of determination (R2) was always 0.990 or better for the regression lines.
RESULTS AND DISCUSSION Chromatographic Evaluation To compare the injection volumes of the autosampler with the switching valve setup a comparison of the regression lines was performed. Naproxen, ciprofloxacin, and nortriptyline HCl were test substances. It was found that the closest relationship between the two injection types for 20 mL was achieved if the switching valve was allowed to be open for 0.03 min during the injection, using a flow of 1.0 mL/min through the dissolution cell. The confidence interval DOI 10.1002/jps
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Table 1. Mobile Phase Compositions, Measured Values of lmax, Retention Factors for the Substances, pKa and Intrinsic Solubilities (S0) for the Solutes. Retention Factor, K, calculated From (tR t0)/t0 Substance (BCS Class)
Volume ACN (mL)
Volume Milli-Q Water (mL)
Volume TFA (mL)
lmax (nm)
Retention Factor (k)
pKa
Intrinsic Solubility (mg/mL)
350 350 300 300 300 150
650 650 700 700 700 850
1 1 1 1 1 1
230 257 234 212 240 278
5.3 5.1 3.8 2.7 7.5 3.3
4.1829 3.9829 3.5229 N/A28 10.2131 6.2032; 8.5932
0.01429 0.1229 0.005929 0.2630 0.02731 0.1332
a
Naproxen (2 ) Ketoprofen (2b) Furosemide (4a) Carbamazepine (2a) Nortriptyline HCl (1c) Ciprofloxacin (3a) N/A, not applicable. a From Ref.26 b From Ref.27 c From Ref.28
(a ¼ 0.05) for the slopes of the calibration curves were covering the digit 1 for all substances. Thus no statistical difference between the two types of injection was observed.
Structural Analysis During the optimization of the disk compression force it was seen that some substances generated fragile disks at higher pressures, a phenomenon
Flow and Dissolution Rate The influence of flow rate on the apparent dissolution rate in the miniaturized rotating disk system was investigated. The results of the HPLC determination of concentration, C, as a function of flow _ are shown in Figure 5A. If now C V, _ which rate, V, is proportional to the apparent in vitro dissolution rate (in Eq. 1), is plotted versus V_ as in Figure 5B it is seen that above a certain critical flow rate the apparent dissolution rate becomes constant. This critical flow rate increases with increasing speed of disk revolution, reflecting the increased apparent rate of dissolution. Before reaching the plateau an accumulation of the solute is obtained in the cell and hence no sink condition is obtained. At the plateau sink condition is assumed. Obviously these results indicate that the experiments must be performed so that the minimum flow rate condition is fulfilled. A restriction of the study was that the cell of Plexiglas only could withstand a moderate pressure (approximately 10 bar) since the two halves of the cell were tightened together by two screws and sealed by an O-ring. With the current seal 1.2 mL/min seemed to be an upper limit to accommodate a safety margin for leakage and the risk of breaking the Plexiglas due to raise in pressure. As only one substance was tested in the study of the flow rate, a compromise was made and 1.0 mL/min was used for all substances at both rotation speeds since this is in the upper region of the practical flow rate. DOI 10.1002/jps
Figure 5A & B. The influence on the apparent dissolution rate by varying the flow rate of the dissolution medium through the Plexiglas chamber. Naproxen in phosphate buffer at pH 7.0 using 100 rpm (&) respective 1000 rpm (^) as rotation speed. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
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Figure 6. The Raman spectra of naproxen powder and disks.
that has been reported by others.28 At the optimized compression force no such occurrence was seen for the model substances. The dissolution rates of the test drugs (naproxen, nortriptyline HCl, and ciprofloxacin) did not vary significantly during the study of the three compression forces, cf. Experimental, RSD of an average of 11% (data not shown) was obtained. The difference in dissolution rate versus compression force has been investigated previously.28 No differences in the Raman spectra for naproxen powder, traditional disk, miniaturized disk, or miniaturized disk exposed to ammonium acetate buffer was observed, see Figure 6. Roughly the same compression forces were used in making of the disks (195 MPa respective 182 MPa) and thus it was assumed that differences in the dissolution rates were not originating from any solid state changes when comparing the two apparatuses.
Apparent In Vitro Dissolution Rate and Dissolution Media The apparent dissolution rate, G, was for the traditional rotating disk calculated from the initial slope of a straight line, where the concentration of the dissolved solute (y-axis) plotted against sampling time (x-axis). The line was drawn from 11 to 14 samples withdrawn at different times from the dissolution vessel and analyzed with HPLC. Sink condition was assumed as long as the graph did not deviate from linearity (R2 0.990). For the miniaturized system the dissolution rate was calculated from an analysis according to Eq. (1), using a flow of dissolution medium at 1.0 mL/min. The average apparent in vitro dissolution rate for naproxen, ketoprofen, nortriptyline HCl, furosemide, carbamazepinex, and ciprofloxacin JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
in phosphate buffer of pH 7.0 and ammonium acetate buffer of pH 6.8 are summarized in Tables 2 and 3, respectively. The rotation speed was set to 100 rpm for both apparatuses and also 1000 rpm for the miniaturized equipment. In view of the fact that the miniaturized apparatus has an additional flow over the surface (and can be set to higher rotation speeds), this will naturally generate a different fluid pattern compared to the traditional setup. The eccentric design (and higher rotation speeds) will also affect the thickness of the layer adjacent to the surface since the angular velocity, v, is increased. Hence the increased apparent dissolution rate of the solutes observed in Tables 2 and 3 when using the novel equipment is expected. Some substances may convert from anhydrous form to the hydrate upon exposure to aqueous medium,33,34 but since the same aqueous media were tested for all substances in both apparatuses this eventual effect was assumed to be equivalent. Linear regression analysis with 95% confidence level was used to compare the traditional rotating disk to the miniaturized one for the respective buffer system (Fig. 7). The statistical evaluation established a difference between the two apparatuses since the p-values were lower than 0.05. The p-value was below 0.05 for the ammonium acetate evaluation even though the sample point of nortriptyline HCl, differing from the cluster in Figure 7B, was removed ( p ¼ 0.03, R2 ¼ 0.836, and slope ¼ 0.396). From the results presented in Tables 2 and 3 the following general observations can be made. When performed at the same speed of revolution the miniaturized rotation disk apparatus gives on average 2–3 times higher values for the apparent dissolution rates compared to the traditional rotating disk experiments. The exception is ciprofloxacin. When in the miniaturized disk experiments the speed of revolution is increased from 100 to 1000 rpm the apparent dissolution rate increases consistently with the exception of ciprofloxacin. Increasing the speed of revolution by a factor of ten gives on an average an increase in the apparent dissolution rate of the order of 2.5 for the miniaturized disk experiments. It is important however to establish the critical flow rate for the drug substances at the set rotation speed for accurate comparision (Fig. 5). Ciprofloxacin is the only ampholyte in the group of substances tested here, and this may generate the deviation of the trend observed in Tables 2–4. The large RSD (53% and 56%) of ciprofloxacin may DOI 10.1002/jps
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Table 2. Average Apparent Dissolution Rates, G, Using Sodium Phosphate Buffer at pH 7.0 as Dissolution Medium
Traditional RD, 100 rpm Substance (n ¼ Number of Disks) Naproxen (n ¼ 6) Ketoprofen (n ¼ 3) Furosemide (n ¼ 3) Carbamazepine (n ¼ 3) Nortriptyline HCl (n ¼ 6) Ciprofloxacin (n ¼ 6)
Miniaturized RD, 100 rpm
Miniaturized RD, 1000 rpm
G (mg/s/cm2)
RSD (%)
G (mg/s/cm2)
RSD (%)
G (mg/s/cm2)
RSD (%)
5.4 12.2 8.4 0.35 30.5 0.08
4 3 6 10 21 8
9.8 18.9 17.1 1.1 67.7 0.53
14 6 3 17 10 53
23.2 70.1 41.7 2.4 193.2 0.81
8 5 6 0.2 14 27
RD, rotating disk.
forces to achieve a flat and rigid surface. However, disks of ciprofloxacin and nortriptyline HCl also swelled in the traditional rotating disk apparatus. But due to the centric design without an added flow over the surface, and in addition more substance quantities to withstand the disk erosion, this effect was not assumed as critical as for the miniaturized setup. The presence of irregularities on the surface may result in increased turbulence in the adjacent fluid.35 An increase of the available surface area will also increase the dissolution rate. A more precise and reproducible way of making the disks is probably necessary in order to improve the results. A noteworthy difference in the dissolution rates between the two aqueous buffer systems is found among the substances containing a weak carboxylic acid. It has been shown that the maximum dissolution rate for a solid acid will occur when the pKa for the buffer is two or more pH units above the pKa of the acid,7 in combination with the intrinsic solubility of the solute.
be explained by the fact that the disk swelled in the two buffers. When spun at 100 rpm this swollen disk was not affected as much as at a rotation speed of 1000 rpm. This higher speed caused the crest to be cut off, leaving a smoother surface behind. During 100 rpm however the crest was remained at the surface for a longer time, causing an irreproducible surface area until the crest was totally broken up leaving the smoother surface in contact with dissolution medium. The swelling of the disk surface, was very high for ciprofloxacin in the ammonium acetate buffer. This generated an increased risk of breaking the cell of Plexiglas due to the raise in pressure since the PEEK capillary for the outflow was obstructed. In general it could be noted that very low (ciprofloxacin) and very high (notriptyline HCl) dissolution rates may cause some experimental difficulties, most likely due to problems with the surface of the disk. Differences in physical properties of these compounds may require different compression
Table 3. Average Apparent Dissolution Rates, G, Using Ammonium Acetate Buffer at pH 6.8 as Dissolution Medium Traditional RD, 100 rpm Substance (n ¼ Number of Disks) Naproxen (n ¼ 6) Ketoprofen (n ¼ 3) Furosemide (n ¼ 3) Carbamazepine (n ¼ 3) Nortriptyline HCl (n ¼ 6) Ciprofloxacin (n ¼ 6, 100 rpm/n ¼ 2, 1000 rpm)
Miniaturized RD, 100 rpm
Miniaturized RD, 1000 rpm
G (mg/s/cm2)
RSD (%)
G (mg/s/cm2)
RSD (%)
G (mg/s/cm2)
RSD (%)
0.48 1.4 0.83 0.48 30.0 0.08
3 1 14 21 2 29
1.2 3.9 1.9 1.3 60.3 1.3
16 18 3 18 11 56
2.8 10.4 5.0 3.0 177.5 7.2
12 4 4 15 4 8
RD, rotating disk. DOI 10.1002/jps
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Some important differences between the two aqueous buffer systems are the type of buffer (i.e., type of ions present), the concentration, the buffer capacity at the pH chosen and the ionic strength. The buffer capacity of ammonium acetate at pH 6.8 is very low. A better buffer capacity at this pH is achieved with the phosphate buffer, but since the ammonium acetate buffer was originating from an LC-UV/MS method for solubility measurements this buffer was tested in addition. The use of mass spectrometry detection is desired in future development and for this reason phosphate buffer is not optimal. The presence of diprotic buffers might also increase the dissolution rates of weak acids.9 An increased buffer concentration may as well generate higher rates, depending on the solutes intrinsic solubility.9,36 The dissolving substance is not affecting the pH of the dissolution medium if the concentration of the solute is 100 times lower compared to the buffer capacity (b) of the dissolution medium. This can easily be calculated for the phosphate buffer (b ¼ 0.0328 M at pH 7.0) by using the Henderson–Hasselbalch and buffer equations.37,38 If a strong base, c ¼ 3.28 10 4 M, is added to the buffer the pH will change by 0.01 which is negligible. In this new apparatus the concentrations of the substances during the dissolution rate studies were all fulfilling this criterion, except for nortriptyline HCl in the ammonium acetate buffer. However, since this is a salt form of nortriptyline it is not assumed to affect the pH of the dissolution medium significantly. The intrinsic solubility of a protolytic compound will affect the pH in the adjacent layer of the disk, with high solubility providing the most significant pH change in contrast to a low soluble
Figure 7. The line fit plots from the linear regression analysis of the two apparatuses compared to each other with respective buffer at 100 rpm and 1.0 mL/min for the miniaturized apparatus. The p-values at 95% confidence level were; 1.1 10 4 for the phosphate, A, and 3.8 10 8 for the ammonium acetate, B, using linear regression analysis.
Table 4. Relative Rates of Dissolution, That Is, Apparent Rate of Dissolution (G) in Sodium Phosphate Buffer ¼ Index P (from Tab. 2) Divided by Apparent Rate of Dissolution (G) in Ammonium Acetate Buffer ¼ Index A (from Tab. 3) Traditional RD, Miniaturized RD, Miniaturized RD, 100 rpm 100 rpm 1000 rpm Substance Naproxen Ketoprofen Furosemide Carbamazepine Nortriptyline HCl Ciprofloxacin
G_P/G_A
G_P/G_A
G_P/G_A
Average
11.2 8.7 10.1 0.73 1.0 1.0
8.2 4.8 9.0 0.85 1.1 0.40
8.3 6.7 8.3 0.80 1.1 0.11
9.2 6.7 9.1 0.79 1.1 0.50
RD, rotating disk. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
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substance. If a more concentrated buffer, with the best buffer capacity at the chosen pH, is used as dissolution medium this will have an effect on the boundary conditions. The pH will be held more controlled by this buffer, compared with a buffer at low concentration and poor buffer capacity. As a buffer is changed it is therefore obvious that the apparent dissolution rate will change reflecting the interaction of the dissolving substance with the buffer components. If apparent dissolution rates from the sodium phosphate buffer (Tab. 2) are divided by the corresponding data for the ammonium acetate buffer (Tab. 3) one obtains the values as given in Table 4. The data are consistent, but the value for the ampholyte deviates from the more homogeneous pattern. It is not obvious that the ratios can be assumed to be the same comparing the two apparatuses since the hydrodynamics in the systems are very different, but here it can be implied that the dissolution experiments also could be used to single out substances with deviating properties (physical as well as chemical). This in itself could be valuable in the process of drug design, providing that the surface of the disks are comparable. Table 4 gives fairly precise information about the relative change in dissolution rate when going from one buffer to another. A comparison of the dissolution rate in the buffer systems at the same concentration and ionic strength could probably improve the results. When performing dissolution rate studies it is therefore always necessary to specify all relevant information of the medium that could influence the dissolution rate of solutes. This new apparatus determines the dissolution rate in a faster way than the traditional with a gain in drug substance and dissolution medium. In addition the dissolution rate can be correlated to the solubility of a substance, as described previously.12,39,40 This correlation will be evaluated for this new apparatus in future studies. From all these observations it can be concluded that the experimental setups perform in a very consistent way and that the miniaturized disk method is at least as reliable as the traditional one. All this holds for data from the same buffer.
CONCLUSIONS A new miniaturized apparatus for the determination of the apparent in vitro dissolution rate has DOI 10.1002/jps
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been designed, constructed, and characterized. The apparatus was compared to a traditional rotating disk equipment. It is shown that this new technique gives data consistent with the traditional one and with a precision approximately as good as for the latter. This novel apparatus is mainly intended for use in screening purposes early in drug development. The numerical values of the apparent dissolution rate for the miniaturized setup are by a factor slightly larger than two as compared to the traditional one at the same rotation speed. This can be explained by the more efficient flow of solvent and eccentric design in the novel apparatus. Although the hydrodynamics in the miniaturized system is complex, as it depends on a combined effect of the flow rate and rotation speed, the obtained results are promising as discussed above. However, more studies are needed in order to present a theory to derive an equation that more accurately describes the dissolution rate in the complex flow profile of the miniaturized flow cell. When comparing data from two different buffer systems the relative changes in apparent in vitro dissolution rate as determined by the two techniques agree quite well. Even if the present results may be good enough for relative comparisons during screening, the problem of reproducible disk formation must be addressed in the future. The miniaturized rotating disk arrangement has the great advantage that only minute quantities of solid, about 5 mg, are required and that the dissolution rate, G, can be obtained directly from the results of one analysis. Furthermore, this technique provides a means to determine intrinsic dissolution rates by varying the speed of revolution during an experiment and extrapolating to infinite speed or to correlate the dissolution rate to the solubility more rapidly. Another advantage of the new equipment is that it is simple to use and enables an easy change of the dissolution medium.
ACKNOWLEDGMENTS We would like to thank Anders AS. Karlsson and Anders S. Carlsson at AstraZeneca Mo¨ lndal (Sweden) for providing drug substances, Matti Ahlqvist at AstraZeneca Mo¨ lndal (Sweden) for doing the Raman spectroscopy measurements and Magnus Persson, Structural Biology Laboratories at the Biomedical Centre in Uppsala (Sweden) for help with drawings in the manuscript. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008
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