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RESEARCH ARTICLE
Monitoring Lidocaine Single-Crystal Dissolution by
Ultraviolet Imaging
JESPER ØSTERGAARD, FENGBIN YE, JUKKA RANTANEN, ANAN YAGHMUR, SUSAN WENG LARSEN,
CLAUS LARSEN, HENRIK JENSEN
Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen,
Universitetsparken 2, DK-2100 Copenhagen, Denmark
Received 25 November 2010; revised 13 January 2011; accepted 9 February 2011
Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22532
ABSTRACT: Dissolution critically affects the bioavailability of Biopharmaceutics Classification System class 2 compounds. When unexpected dissolution behaviour occurs, detailed studies
using high information content technologies are warranted. In the present study, an evaluation of real-time ultraviolet (UV) imaging for conducting single-crystal dissolution studies was
performed. Using lidocaine as a model compound, the aim was to develop a setup capable of
monitoring and quantifying the dissolution of lidocaine into a phosphate buffer, pH 7.4, under stagnant conditions. A single crystal of lidocaine was placed in the quartz dissolution cell
and UV imaging was performed at 254 nm. Spatially and temporally resolved mapping of lidocaine concentration during the dissolution process was achieved from the recorded images. UV
imaging facilitated the monitoring of lidocaine concentrations in the dissolution media adjacent
to the single crystals. The concentration maps revealed the effects of natural convection due
to density gradients on the dissolution process of lidocaine. UV imaging has great potential
for in vitro drug dissolution testing. © 2011 Wiley-Liss, Inc. and the American Pharmacists
Association J Pharm Sci
Keywords: crystals; dissolution; dissolution rate; imaging methods; natural convection;
UV/Vis spectroscopy; UV imaging
INTRODUCTION
Dissolution is frequently the limiting or ratecontrolling step in drug absorption of poorly watersoluble drugs.1–4 The commonly used in vitro dissolution methods including rotating disc, USP (United
States Pharmacopeia) paddle and basket require relatively large amounts of drug substance and/or dissolution media. A current trend in drug development
is to explore the physical properties of active compounds using high-throughput technologies and to
use the identified solid forms (salt, polymorphic, solvate, co-crystal and amorphous) as a central part of
product development.5 Thus, micro-dissolution techniques requiring only a few milligrams of material,
which may be of particular interest in early drug development, have been developed to overcome limitaCorrespondence to: Jesper Østergaard (Telephone: +4535336138; Fax: +45-35336030; E-mail: [email protected])
This article contains supplementary material available from
the authors upon request or via the Internet at http://
www.wileylibrary.com.
Journal of Pharmaceutical Sciences
© 2011 Wiley-Liss, Inc. and the American Pharmacists Association
tions related to dissolution testing.6–8 In the abovementioned methods, the concentration of dissolved
substance is measured as a function of time in the
bulk release media. Alternatively, for example, in the
case of unexpected dissolution behaviour, more detailed information might be gained by monitoring the
dissolution process immediately next to the surface
of a small amount of the dissolving material. Imaging techniques capable of providing spectrally, spatially and temporally resolved information are emerging tools in more detailed studies of drug dissolution
due to their potentially high information content.9–15
Ultraviolet (UV) imaging technology, which has recently become commercially available, may constitute
an alternative and complementary technology to, for
instance, Fourier transform infrared and magnetic
resonance imaging. UV imaging has the ability to
generate visual images from simultaneous spectroscopic, spatial and time data. With UV imaging, it
is possible to measure the intensity of light in the
UV range passing through an area of a quartz tube
as a function of position and time.16 Thus, UV imaging may facilitate quantification of drug substances
JOURNAL OF PHARMACEUTICAL SCIENCES
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ØSTERGAARD ET AL.
in solution immediately adjacent to the solid material and recording of concentration maps, spatially
and temporarily resolved. Detection in the UV range
is suitable for most drug substances; UV imaging will
therefore be a widely applicable format for conducting advanced drug dissolution studies in drug development. For instance, monitoring dissolution at the
aqueous–solid interface of a crystal may provide information on the rate-limiting steps of the dissolution process.17 It has been shown that detailed insights into the dissolution processes, for example,
face-specific/dependent dissolution,18–21 may be attained from dissolution studies performed on single crystals. Such studies are, however, at present
technically challenging. In addition, measurements of
concentration gradients in the vicinity of the solid
surface may lead to knowledge on the relative importance of diffusion and convective currents to dissolution rates. To this end, the influence of natural
convection caused by density gradients on drug dissolution has been demonstrated.22–24 Furthermore, dissolution studies conducted under conditions with little or no convective currents due to flow or agitation
may also be of relevance, for example, crystal suspensions intended for parenteral administration, that is,
subcutaneous, intra-muscular or intra-articular administration routes.
The objective of the present investigation was to
perform an evaluation of the applicability of a commercially available UV imaging detector for conducting dissolution studies. Using lidocaine as a model
compound, the aim was to develop a simple setup for
measuring the dissolution of lidocaine from a single
crystal into aqueous buffer. Dissolution of lidocaine
into 0.067 M phosphate buffer (pH 7.40) was selected
as an initial model system for UV imaging due to
previous experiences related to intra-articular drug
delivery,25 the high aqueous solubility and associated
rapid dissolution allowing short experimental times.
MATERIALS AND METHODS
Chemicals and Reagents
Lidocaine (Ph Eur (European Pharmacopoeia) 6th
ed.) was obtained from Unikem, Copenhagen,
Denmark. Lidocaine single crystals for the dissolution experiments were obtained from the recrystallisation of lidocaine in n-hexane.26 Both the starting
material and recrystallised lidocaine were identified
to be monoclinic (P21 /c) lidocaine, using X-ray powder diffractometry.27 Sodium dihydrogenphosphate
monohydrate was obtained from Merck (Darmstadt,
Germany). The dissolution medium used was 0.067 M
sodium phosphate, pH 7.40. Purified water from a
Milli-Q deionisation unit (Millipore, Bedford, Massachusetts) was used throughout the study.
JOURNAL OF PHARMACEUTICAL SCIENCES
Figure 1. Scanning electron micrographs of lidocaine
crystals.
UV Imaging Setup
UV imaging was performed using an Actipix SDI300
dissolution imaging system (Paraytec Ltd., York,
United Kingdom) with Actipix flow-through type dissolution cartridges (supporting information Fig. 1). A
syringe pump was used for the infusion of the dissolution medium and the lidocaine standard solutions.
The total detection area of the UV imaging system
is 9 × 7 mm2 (1280 × 1024 pixels); however, the selected imaging area was 9 × 5.5 mm2 . The pixels (7 ×
7 :m2 ) were binned 4 × 4. The light source is a pulsed
Xe lamp, and imaging was performed at 254 nm. The
quartz dissolution cell [7.5 × 3.0 × 63 mm3 (height ×
width × length)] contained approximately 0.56 mL of
dissolution media with the flow-through inserts in
place. Images were recorded (2.3–2.6 images per second) and analysed using Actipix D100 software version 1.3 (Paraytec Ltd. York, United Kingdom). Pixel
intensities were converted into absorbance using the
Actipix software,16 allowing the concentration of lidocaine within the imaging area as a function of position
and time to be determined by the use of a calibration
curve. The procedures for constructing calibration
curves were similar to those reported previously.16
DOI 10.1002/jps
MONITORING LIDOCAINE SINGLE-CRYSTAL DISSOLUTION BY UV IMAGING
Dissolution Studies
The procedure for UV imaging was as follows: dark
images (lamp turned off; 10 s) and reference images
(10 s) were recorded with the dissolution cell filled
with the 0.067 M sodium phosphate buffer (pH 7.40).
After 60 s, data collection was paused and the lidocaine single crystal was mounted in the dissolution
cell. Then, data collection was resumed and the dissolution cell with the crystal in place was filled with
the dissolution buffer delivered by the syringe pump
at a flow rate of 0.5 mL/min. The flow was arrested
upon complete filling of the cell, whereas recording
of UV images was continued for 5–15 min. The single
crystals were oriented differently (horizontally or vertically) by turning the entire UV imaging sensor head
because the crystals could only be mounted in one defined manner inside the dissolution cell. Experiments
were conducted at ambient temperature (19◦ C–23◦ C).
Calibration curves were constructed by flowing lidocaine standard solutions (concentration range: 1.0 ×
10−5 –1.0 × 10−2 M) through the dissolution cell at a
flow rate of 1 mL/min.
The amount of lidocaine dissolved was determined
by averaging absorbance values over the imaging
area. From the increase in average absorbance, the
molar absorptivity and the volume element (area
× light path), the cumulated amount of lidocaine
dissolved was calculated. In the calculation of the
amount of dissolved lidocaine, it is a premise that
the UV absorbance for each pixel read is within the
linear range (Beer’s law is valid) and that lidocaine
does not diffuse out of the imaged volume element.
Scanning Electron Microscopy
The lidocaine crystal samples were analysed using
a scanning electron microscope (JSM 5200; JEOL,
Tokyo, Japan). An acceleration voltage of 10 kV and
a working distance of 20 mm were used. The samples were mounted on a double-faced adhesive tape
and sputter coated for 120 s, with a thin gold–palladium layer in an Auto sputter coater (E5200; Bio-Rad,
Watford, England).
RESULTS AND DISCUSSION
Figure 1 shows scanning electron micrographs of
the lidocaine crystals. Needle-shaped single lidocaine
crystals, 2–3.5 mm in length, were placed in the insert
and mounted in the dissolution cell for UV imaging
(supporting information Fig. 1). The lidocaine crystals
were oriented horizontally or vertically in the dissolution cell (0.56 mL) and surrounded completely by
dissolution medium except at the base. Upon insertion of the lidocaine crystal, the dissolution cell was
filled (0.5 mL/min) with 0.067 M phosphate buffer, pH
7.4, at ambient temperature (19◦ C–23◦ C). The flow
DOI 10.1002/jps
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was stopped while recording the UV images. Figure
2 shows (in false colour) dissolution of lidocaine into
the cell volume in the form of a UV absorbance map
obtained at 254 nm. Figure 2a shows the cell immediately after filling with phosphate buffer. The lidocaine
crystal positioned vertically is apparent as the light
yellow zone. In Figures 2a–2c, the dissolution of lidocaine is apparent as an increase in absorbance (increasing the intensity of the yellow and red colour).
Figure 2 shows that the increase in absorbance is
most predominant in the lower part of the cell. Finally, dissolved lidocaine was removed by applying a
flow of buffer (0.5 mL/min), leaving only the signal
due to the presence of the crystal (Fig. 2d). A video
clip of the lidocaine dissolution process can be found
in the supporting information. The obtained UV observations reveal that the maximum absorbance is
not detected at the position of the lidocaine crystal
as might have been expected. This is due to the fact
that light coming from the fibre optics is striking the
sensor surface behind the crystal. The lidocaine crystal is positioned at approximately 3 mm in front of
the sensor chip. The left-to-right linear arrangement
of the optical fibres allows light, emitted at different
angles, to reach the sensor chip behind the crystal.
Figure 3 depicts a UV image from the dissolution experiment (after 5 min) in which the lidocaine crystal
was oriented horizontally. From inspection of Figures
2 and 3, it is readily apparent that the dissolved lidocaine distributes primarily to the lower part of the
dissolution cell. A small edge effect is observed in Figures 2 and 3 (at the base of the crystal). This is due to
mounting of the crystal that was performed by simply
inserting the crystal into a small hole drilled in the
support. The relatively large increase in absorbance
at the base is due to the lidocaine dissolving in the
(excess) buffer found in this hole when this space was
not fully taken up by the crystal. Additional singlecrystal dissolution experiments with the crystal and
cell oriented differently confirmed that the highest absorbance (concentration) always occurred in the lower
part of the cell. Diffusion of dissolved lidocaine away
from the crystal surface is expected to provide a symmetrical concentration distribution around the crystal in the stagnant liquid. However, convection due
to density gradients may, in addition to diffusion, affect the concentration distribution upon dissolution
of lidocaine from the crystal. The importance of natural convection and density gradients on dissolution
behaviour22–24 can, thus, be spatially imaged in real
time using the present technology. Using a densitometer, the density of the pure phosphate buffer and
a saturated solution of lidocaine in phosphate buffer
at 25◦ C was determined to be 1.0055 and 1.0062 g/
cm3 (Standard deviations were <0.0001 g/cm3 ;
n = 3), respectively.
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ØSTERGAARD ET AL.
Figure 2. Ultraviolet (UV) imaging of lidocaine dissolution at 254 nm from single crystal
positioned vertically in 0.067 M phosphate buffer (pH 7.4). UV images (absorbance) obtained
during (a) immediately upon filling of the cell (t = 0 min), (b) 2 min upon arresting flow, (c) 5 min
upon arresting flow and (d) during flushing the cell with phosphate buffer.
Figure 3. Ultraviolet (UV) imaging of lidocaine dissolution at 254 nm from single crystal positioned horizontally in
0.067 M phosphate buffer (pH 7.4). UV image (absorbance)
obtained 5 min after initiation of the dissolution experiment
(stagnant conditions).
JOURNAL OF PHARMACEUTICAL SCIENCES
Figure 4 shows isoabsorbance contours for a selected image during single-crystal dissolution which
was readily converted into isoconcentration profiles
using the molar absorptivity g254 of 436 M−1 cm−1 determined from the calibration curve. Construction of
the calibration curve providing the molar absorptivity was performed as described previously.16 The conversion of light intensity into absorbance is shown
in supporting information, Figures 2 and 3, for a
lidocaine standard solution, indicating the satisfactory performance of the UV imaging system. It has
to be realised that the concentrations represented by
the isoabsorbance contours correspond to values averaged over the light path (3 mm) and thus, most likely,
deviate significantly from local concentrations that
will be encountered inside the cell. In comparison, the
solubility of lidocaine in 0.067 M phosphate buffer (pH
7.4) has previously been determined to be 44.5 mM at
37◦ C.25
From the increase in absorbance with time, the
amount of lidocaine dissolved from the crystal as a
function of time can be determined when the imaging
area, length of the light path and the molar absorptivity are known. Because attempts to control the dimensions of the crystals investigated were not made,
crystal-to-crystal repeatable dissolution profiles
DOI 10.1002/jps
MONITORING LIDOCAINE SINGLE-CRYSTAL DISSOLUTION BY UV IMAGING
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Figure 4. Isoabsorbance (isoconcentration) profiles obtained by UV imaging of lidocaine single
crystal positioned vertically in 0.067 M phosphate buffer (pH 7.4). UV images (absorbance)
obtained 4 min after initiation of the dissolution experiment (stagnant conditions). The numbers
represent the concentrations in millimolar.
cannot be expected. For the crystal shown in Figure 2,
two dissolution–time profiles are depicted in Figure
5. Consecutive dissolution experiments with the selected crystal were made under stagnant conditions.
Between each experiment, the cell was flushed with
phosphate buffer. The dissolution rate, indicated from
the slope of the profiles, decreased slightly from experiment to experiment. Linear regression analysis provided slope (dissolution rates) of 18.8 and 15.4 nmol/
min for the first and second dissolution profile, respectively. For comparison, the relative standard deviation for the molar absorptivity was 2.1% (n = 7),
indicating that the observed change in slope for the
dissolution profiles is significant. This appears reasonable because the crystal surface area available for
dissolution is smaller in the second dissolution experiment as compared with the first profile. For the
vertically oriented crystal, approximately 23 :g of lidocaine was dissolved after 6 min under the natural
convection conditions prevailing. Prolonged quantitative dissolution experiments were not possible for lidocaine under the stagnant conditions applied due to
deviation from Beer’s law. The images obtained for lidocaine indicate that UV imaging will also be suitable
for less soluble compounds. The present setup allowed
for quantitative measurements of the amount of drug
dissolved in the aqueous medium. Refinements have
to be made with respect to controlling crystal size and
dimensions, crystal positioning and hydrodynamics
in order to take full advantage of the opportunities
that UV imaging offers for single-crystal dissolution
studies. Work along these lines is in progress in our
laboratory. The setup with natural convection only
may be of interest in relation to, for instance, in situ
DOI 10.1002/jps
Figure 5. Lidocaine dissolution profiles obtained in
0.067 M phosphate buffer (pH 7.4) for the crystal shown in
Figure 2 under stagnant conditions. Two consecutive profiles obtained with the same crystal oriented vertically.
suspension forming systems, stents and implants for
parenteral administration.
CONCLUSION
The current report constitutes the first attempt to apply UV imaging for monitoring single-crystal dissolution of drug substances. The UV imaging detector facilitates monitoring of drug substance concentrations
in dissolution media immediately adjacent to a single
crystal of lidocaine in a spatially and temporally resolved format. The ability to attain two-dimensional
images of the dissolution process is likely to
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ØSTERGAARD ET AL.
provide new detailed insights into dissolution processes. For instance, it is immensely difficult to study
natural convection phenomena due to local density
gradients using current dissolution models. Here,
under stagnant conditions, the importance of density gradients was revealed for lidocaine dissolution.
Proof-of-concept was attained and with further refinement, UV imaging has the potential to become an important tool in drug dissolution testing.
ACKNOWLEDGMENTS
This work was supported by the Danish Medical Research Council. Dorthe Kyed Ørbæk is acknowledged
for scanning electron microscopy measurements.
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