Post on 10-Mar-2020
Accepted Manuscript
Title: Time domain NMR as a new process monitoring methodfor characterization of pharmaceutical hydrates
Author: <ce:author id="aut0005"author-id="S0731708516306136-d90313097e751e8ca954b39aa4a141ba"> Stefanie UlrikeSchumacher<ce:author id="aut0010"author-id="S0731708516306136-a239962de7c93803e9c66811af57ca0f"> BennoRothenhausler<ce:author id="aut0015"author-id="S0731708516306136-cceba6d4f33e4bf74d4862cb769401fc"> AlfWillmann<ce:author id="aut0020"author-id="S0731708516306136-de7bda8ebb9e44599a9c93bf17af9a00"> JurgenThun<ce:author id="aut0025"author-id="S0731708516306136-cd5c9f1278a46779586d18575cb901c9"> ReginaMoog<ce:author id="aut0030"author-id="S0731708516306136-9e6d563f118c3caac046a296b76bb43e"> MartinKuentz
PII: S0731-7085(16)30613-6DOI: http://dx.doi.org/doi:10.1016/j.jpba.2017.01.017Reference: PBA 11024
To appear in: Journal of Pharmaceutical and Biomedical Analysis
Received date: 13-9-2016Revised date: 13-12-2016Accepted date: 7-1-2017
Please cite this article as: Stefanie Ulrike Schumacher, Benno Rothenhausler, AlfWillmann, Jurgen Thun, Regina Moog, Martin Kuentz, Time domain NMR as a newprocess monitoring method for characterization of pharmaceutical hydrates, Journal ofPharmaceutical and Biomedical Analysis http://dx.doi.org/10.1016/j.jpba.2017.01.017
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Key Findings
1H time domain NMR (TD-NMR) provides valuable information on molecular water
mobility in hydrates
Methods of spin echo and inversion recovery were suitable to quantify the model hydrates
TD-NMR is a non-destructive method with high potential for process analytics of hydrates
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Time Domain NMR as a new process monitoring method for
characterization of pharmaceutical hydrates
Stefanie Ulrike Schumacher 1, Benno Rothenhäusler
2, Alf Willmann
2, Jürgen Thun
3, Regina
Moog2, Martin Kuentz
1
1University of Applied Sciences and Arts Northwestern Switzerland, Institute of
Pharmaceutical Technology, Gründenstrasse 40, 4132 Muttenz, Switzerland
2Roche Pharma Research and Early Development, Therapeutic Modalities, Roche Innovation
Center Basel, F. Hoffmann-La Roche Ltd, Grenzacherstrasse 124, 4070 Basel, Switzerland
3Roche Pharma Technical Development Actives, Materials Science, Hoffmann-La Roche Ltd,
Grenzacherstrasse 124, 4070 Basel, Switzerland
Address correspondence to
Prof. Dr. Martin Kuentz
University of Applied Sciences and Arts Northwestern Switzerland
Institute of Pharmaceutical Technology
Gründenstrasse 40
4132 Muttenz / Switzerland
T +41 61 467 46 88 / F +41 61 467 47 01
martin.kuentz@fhnw.ch
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Abstract
Hydrates are of great pharmaceutical relevance and even though they have been characterized
thoroughly by various analytical techniques, there is barely literature available on molecular
mobility of the hydrate water studied by NMR relaxation in the time domain. The aim of this
work was to examine the possibility of differentiating hydration states of drugs by 1H time
domain NMR (TD-NMR) regarding spin-spin and spin-lattice relaxation times (T2 and T1)
using benchtop equipment. Caffeine and theophylline were selected as model compounds and
binary mixtures of hydrate to anhydrate were analyzed for each drug using a spin echo and
inversion recovery pulse sequence. It was possible to extract a signal that was specific for the
water in the hydrates so that differentiation from anhydrous solid forms was enabled.
Excellent calibrations were obtained for quantitative analysis of hydrate/anhydrate mixtures
and predicted water contents were in good agreement with water amounts determined in
desiccator sorption experiments. TD-NMR was therefore found to be a suitable new technique
to characterize pharmaceutical hydrates in a non-invasive and hence sample-sparing manner.
Quantification of the hydrate content in pharmaceutical mixtures appears highly attractive for
product development and process monitoring. TD-NMR provides here a valuable and
complementary technique to established process analytics, such as for example Raman
spectroscopy.
Keywords: 1H time domain NMR; hydrates, solid state characterization; T2-relaxation; T1-
relaxation
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1. Introduction
Approximately one third of all active pharmaceutical ingredients (API) are capable of forming
hydrates [1], which indicates their importance in pharmaceutics. Usually, hydrate formation
occurs during crystallization as the final step of chemical manufacturing. It is then often a
challenging task for preformulation and galenics to maintain a defined state of hydration
during downstream processing and drug product manufacturing. Furthermore, hydrate
formation is critical for the selection of the most suitable physical form using a salt and
polymorph screening approach [2, 3]. There is a variety of hydrate types known like for
example: stoichiometric or non-stoichiometric hydrates, channel hydrates, isolated site
hydrates, ion coordinated hydrates or clathrate hydrates [4, 5].
Since only limited drug substance is usually available during early pharmaceutical
development, there is a need for sample-sparing or even non-destructive analytical techniques.
Such methods are also crucial from an industrial perspective in the framework of process
analytical technology (PAT) to assure product quality already during processing. Therefore,
1H time domain NMR (TD-NMR) is an attractive analytical technique as it allows for non-
invasive monitoring of hydrated APIs. Measurements through sealed, non-magnetic vials are
possible and environmental factors, like humidity and temperature, can be closely controlled.
This is especially important for pharmaceutical hydrates, as their stability and transformation
kinetics require distinct conditions. Benchtop TD-NMR may also be easily integrated into a
production process, and the samples drawn can be used for further analytics.
Pharmaceutical hydrates have been thoroughly characterized by various analytical techniques,
including for example X-ray powder diffractometry (XRPD), differential scanning
calorimetry (DSC), thermogravimetric analysis (TGA), Raman spectroscopy, gravimetric
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water vapor sorption (GVS), inverse gas chromatography (iGC), scanning electron
microscopy (SEM), time-resolved synchrotron X-ray diffraction and coherent anti-Stokes
Raman scattering (CARS) microscopy [4, 6-8]. This work used also thermoanalytical and
water sorption methods but the main focus is on 1H time domain nuclear magnetic resonance
(TD-NMR). Yoshioka et al. [9] pioneered in NMR relaxation studies of pharmaceutical
hydrates and their work on samples of a single solid state provides a basis for new research on
mixtures of solid states regarding process analytics. Like TD-NMR, Raman spectroscopy can
also be used for in process control, but it is often not non-destructive and may show limited
differentiation of hydrate states with some compounds. For most applications in the solid state
there is typically a multivariate data analysis required. Therefore, the possibilities of TD-
NMR as an additional or complementary in process control-technique should be evaluated.
T1 and T2 relaxation times of 1H nuclei are typically used to discriminate different types of
water binding. Exponential models of T1 and T2 processes are often employed as a
convention to discriminate for example between loosely bound and strongly bound hydration
water. The TD-NMR work of Yoshioka focused on molecular mobility of hydrate water in
several pharmaceutical hydrates being mentioned in the Japanese pharmacopoeia. In this
study, T1 and T2 relaxation times were interpreted in terms of water mobility and results were
correlated with ease of evaporation as determined by DSC and GVS. A correlation was found
for some of these hydrates, while this was not the case for all compounds. The authors argued,
that correlation failed either, if the T2 relaxation time (of the water 1H-protons) was not
sufficiently different from the relaxation time of 1H-protons that are part of the API or, if the
number ratio of water 1H-protons to API
1H-protons in the samples was not sufficiently large
to sensitively reflected the T1 relaxation time in the total signal.
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During NMR relaxation, the 1H nuclei in a sample exchange energy with each other and with
their surroundings. Relevant interactions in a pharmaceutical hydrate may include the
following: lattice-lattice, water-water and lattice-water [4]. Ahlqvist and Taylor [10]
investigated the interactions between hydrate water and the crystal lattice in detail for the
structurally similar channel hydrates caffeine and theophylline. They used a H/D exchange
protocol in combination with Raman spectroscopy for the assessment of water mobility and
discussed the results with respect to channel structure, channel dimension and molecular
interaction strength. The H/D exchange rate and thus, the water mobility was found
considerably higher in caffeine than in theophylline, although the strength of the hydrogen
bonds between water and drug molecules are similar for both compounds. They argued that
the larger lattice channel of caffeine hydrate allows for higher diffusional water mobility,
whereas the narrow channel of theophylline leads to steric hindering of water molecules and
thus to reduced water mobility and, in addition, to a likely change of the exchange mechanism
from diffusion to 1H-proton transfer as well. Fig. 1 depicts the crystal structure of the hydrates
and provides a comparison of the relative channel dimensions (based on contact surface)
between caffeine and theophylline (Mercury CFS 3.8, CCDC, Cambridge, UK). Ahlqvist and
Taylor [10] used a van der Waals representation of the hydrate lattice and argued, that the
overall size of the channels (caffeine: 5.2 x 5.2 Å; theophylline: 3.6 x 3.3 Å) should be
sufficiently large to allow water molecules to enter the crystal lattice. The estimated size
values may, however, depend on the exact details of the chosen model. Even small
disturbances in the crystal lattice may lead to variations in the estimated channel size, which
might be of significance with respect to water mobility.
A primary goal of the present work was to extract the TD-NMR signal of the hydration water
alone and thereby to practically eliminate the contribution of the API crystal lattice with
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respect to both, T1 as well as T2-relaxation. Another goal was to quantitatively determine the
amount of hydrate water in binary mixtures of hydrates to anhydrates, which is important for
stability testing and the potential use of TD-NMR in a PAT framework. For means of data
comparison, TGA, GVS, XRPD, and Raman spectroscopy are used for characterization as
well.
To select model hydrates with interesting water properties, the two channel hydrates
theophylline and caffeine were used. While theophylline forms a stoichiometric monohydrate,
caffeine comes as 4/5-hydrate [11, 12] and since these drugs have already been studied in the
literature regarding hydration/dehydration behavior, a good data basis was given [12-20] for
the current evaluation of TD-NMR methods.
2. Materials and methods
2.1. Materials
Anhydrous caffeine and theophylline were purchased from Sigma-Aldrich (Buchs,
Switzerland). Hydrate forms of both compounds were prepared as previously described
[10,16]. The weight loss as determined by TGA was 6.2% for caffeine hydrate (theoretical
value for a 4/5 hydrate is 6.9%) and 8.6% for theophylline hydrate (theoretical value is 9.1%
for a 1:1 monohydrate). Powder samples of caffeine hydrate and theophylline hydrate were
delumped by sieving (mesh size 0.5mm) and stored at 75% relative humidity (RH) at RT (25
°C). Partially dehydrated forms of both compounds were prepared by equilibrating the
hydrate forms for up to 40 days in desiccators at various RH% (10, 20, 40, 60, 80, 90).
Humidity levels were maintained by means of water-glycerol-mixtures [21] and monitored
with a digital hygrometer (Testo 608-H2, Testo AG, Lenzkirch, Germany). Equilibration
kinetics of the samples was measured by weighing at periodic time intervals.
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Fully dehydrated forms of the hydrated compounds were prepared by drying over molecular
sieves as confirmed by TGA. The time needed for re-establishment of equilibrium in the
vapor space of the desiccators as well as the time needed for re-equilibration of the samples
(after opening/closing of the lid) was determined in advance and considered during
subsequent sample handling (data not shown).
Powder mixtures containing predetermined amounts of hydrate water were prepared by
mixing the hydrate with the respective anhydrous form of either theophylline or caffeine at
weight ratios of 0/100, 25/75, 50/50, 75/25 and 100/0 (% w/w) using a Turbula shaker-mixer
(Bachofen AG, Switzerland).
Precisely weighed aliquots of each mixture (caffeine: 1.40 g, Theophylline: 1.60 g) were
dispensed into glass vials (14 x 45 mm, type G085, Infochroma AG, Goldau, Switzerland)
and slightly compressed to a filling height of 2.5 cm. Vials were sealed with Parafilm M
(Bemis, Neenah, WI, USA) and inserted into standard Minispec sample tubes (mq-TUB18,
Bruker BioSpin AG, Rheinstetten, Germany) for TD-NMR measurements.
2.2. Methods
2.2.1. Basic solid-state characterization
The anhydrous forms of caffeine and theophylline as well as the hydrate and the dried hydrate
(dehydrate) forms, were analyzed by TGA, XRPD, Raman spectroscopy, GVS and particle
sizing in addition to the TD-NMR measurements.
TGA was performed on a TGA/SDTA851e device (Mettler-Toledo Greifensee, Switzerland).
Raman spectra were recorded in the backscattering modus using a Raman RXN1 analyzer
(Kaiser Optical Systems, Inc., Ann Arbor, USA) equipped with a charge-coupled device
(CCD camera) and a diode laser operating at a wavelength of 785 nm. Measurements were
based on a laser power of 400 mW and background Rayleigh scattering was removed by a
holographic filter during spectra acquisition. The fully hydrated forms were analyzed by
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gravimetric vapor sorption (GVS) using an automated, dynamic sorption analyzer (DVS1,
Surface Measurement Systems, London, UK) at 25°C. We selected an industrial desorption
protocol between 90% and 0 % RH in steps of 10% RH. The relative humidity was decreased
to the next lower level as soon as either one of two stop criteria was met: Change in sample
mass ≤0.002 %/min (5 min average) or measuring time ≥ 1500 minutes at a particular
humidity level. Due to the dynamic measuring protocol, as determined by the selected stop
criteria, the samples may not have reached full desorption equilibrium at each humidity level.
X-ray powder diffraction patterns were obtained in the reflection mode using a Siemens/
Bruker D5000 X-ray Powder Diffraction System (Bruker AXS GmbH, Karlsruhe, Germany).
Finally, particle size distribution (PSD) of the powder samples was determined by static
image analysis (Morphologi®
G3, Malvern, UK)
2.2.2. Time Domain 1H NMR experiments
Low field pulsed TD-NMR measurements were performed on a benchtop Minispec®
A
instrument, (Bruker BioSpin GmbH, Rheinstetten, Germany) equipped with a ratio type probe
head. We worked with an over-filled coil (sample height 2.5 cm) at constant coil temperature
of 30°C. The instrument provides a variety of pre-programmed relaxation time analysis
methods. T2 relaxation processes were analyzed with the Hahn spin echo (T2-SE) method
(Minispec application “t2_se_mb”) and T1 relaxation processes were analyzed using the
inversion recovery (T1-IR) method (Minispec application “t1_ir_mb”). All method
parameters for data acquisition were optimized using the fully hydrated forms of each
compound in order to maximize the dynamic signal range. The application parameters
remained unchanged during subsequent analysis of the various solid forms of a particular
compound.
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For application T2-SE, the first 90°-180° pulse separation was set to 0.02 ms, final pulse
separation was 0.4 ms, and the recycle delay was 3000 ms. For application T1-IR, the first
90°-180° pulse separation was set to 0.03 ms. The last pulse separation and the recycle delay
was either set to 1800 ms and 2800 ms (caffeine) or to 8500 ms and 8000 ms (theophylline)
TD-NMR raw data were exported to Microsoft-Excel and normalized with respect to sample
mass and instrument gain. The signal contribution Mw(t) of the hydrate water alone was
calculated by subtracting the signal of the anhydrous form from the respective signal of the
fully or partially hydrated forms. Data were analyzed by a least square exponential fitting
procedure using either Equation 1 (T1 relaxation) or Equation 2 (T2 relaxation):
( ) ⌊∑ ( ) (
( ))⌋ (1)
( ) ⌊∑ ( )
( )⌋ (2)
where Mw1(t) and Mw2(t) are the calculated TD-NMR signal amplitudes at time t and A1 and
A2 are amplitudes which depend on the amount of hydrate water in the sample. We used
either a mono-exponential (i=1) or bi-exponential (i=2) model for simultaneous fitting of a set
of samples presenting varying amounts of hydrate water and we treated the respective
relaxation times T1 and T2 as shared parameters.
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3. Results and Discussion
3.1. Raman spectroscopy and x-ray powder diffraction
Raman spectroscopy and x-ray powder diffraction were used for a first solid state analysis of
the samples. Fig. 2 shows Raman spectra of the model compounds as anhydrate, hydrate, and
dehydrate (i.e. dried hydrate). The spectra of caffeine exhibited some changes among both
forms but peak shifts were generally quite subtle and for example found around 1450-1500
cm-1
or 480-490 cm-1
. Moreover, caffeine hydrate revealed additional peaks at 880-900 cm-1
and in the range of 280-300 cm-1
. Theophylline showed, by contrast, several pronounced
spectral changes that were for the anhydrous form particularly observed at 100-220 cm-1
, 520-
600 cm-1
, 1320-1360 cm
-1 and 1600-1750 cm
-1. Water in the solid state appeared to more
dominantly affect molecular vibrations in the case of theophylline. Already the results of the
lower frequency range below 600 cm-1
support this view and this part of the Raman spectrum
holds for collective motions of the drugs in the crystal [22]. Such molecular vibrations are
likely influenced by water molecules located directly on the surface of channel pores. This
population of water molecules is expected to dominate the overall water content of
theophylline due to the narrow size of the channels (Fig. 1) [10]. This is different from the
channel geometry of caffeine that can also host water in the center of the channels and
therefore may only have limited effects on molecular vibrations. The supramolecular structure
of the model drugs can to some extent explain why different hydration states were more
dominantly seen in Raman spectra of theophylline as compared to caffeine. This observation
emphasizes the need for complementary methods to vibrational spectroscopy in solid state
analysis.
The different solid forms were also analyzed by means of x-ray powder diffraction (XRPD)
(Fig. 3). Caffeine anhydrate scattered most dominantly at a 2 angle of 11.5 -12° and 26-27°
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in line with the literature [23] and clear differences were noted in the diffraction pattern of the
hydrate (Fig. 3A). The other model drug theophylline typically comes as anhydrous form I
and diffraction patterns (Fig. 3B) were different form that of the monohydrate as it was
expected from the literature [11, 14]. Such clear difference in XRPD patterns do not have to
occur when comparing anhydrous with hydrate drug forms. There is also the known scenario
that dehydration may result in an unaltered crystal lattice and accordingly, hydration state
would barely affect scattering of x-rays [23]. Accordingly, solid state characterization
typically relies on different methods and hence also process analytics would have to offer
alternative methods to cope with all kinds of APIs.
3.2. Time Domain 1H NMR experiments
Initial experiments were about the identification of suitable pulse sequences for 1H TD-NMR
method development. Previous applications of this technique in pharmaceutics were typically
analyzing soft matter [24] for which the Carr-Purcell-Meiboom-Gill sequence is a common
method for T2 determination. As expected, first experiments with the model hydrates did not
provide the needed sensitivity in the millisecond range that enables characterization of 1H
protons in the solid state. More promising were the spin echo methods for spin-spin relaxation
(T2-SE) and an inversion recovery method for spin-lattice relaxation (T1-IR). The latter
method has been used earlier to study pharmaceutical hard capsules and it was feasible to
separate water fractions according to their interaction with the shell material [25]. In the
current work, both TD-NMR methods had to be adapted for the given sample hydrates and
final parameter settings are detailed in the methods section.
Fig. 4 depicts the results of spin-spin relaxation measurement (T2-SE) for caffeine. The decay
of the signal amplitude for caffeine hydrate clearly differed from that observed for the water-
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free forms. By contrast, anhydrous or dehydrated caffeine were not showing relevant
differences given that error bars were mostly overlapping in the spin-spin relaxation curves.
This result was encouraging in that a specific water signal can be obtained by subtracting the
amplitude decay of the anhydrous form from that of the corresponding hydrate. Moreover,
calibration of the TD-NMR method for water content would enable analysis of binary
mixtures containing different blends of anhydrous and fully hydrated form. Fig. 5 depicts the
calculated signal amplitude of the hydrate water in case of caffeine using the T2-SE data.
Open signals represent the contribution of hydrate water in the mixtures with varying amounts
(%, w/w) of the fully hydrated form, whereas the lines represent calculated NMR signals
Mw2(t). A good bi-exponential fit was obtained using the T2 spin-spin relaxation model where
A2(1) and A2(2) were allowed to vary with water content and T2(1) and T2(2) are shared T2
relaxation time constants. The fitted amplitudes and relaxation times are listed in Table 1
together with the results for theophylline. Similar as in the case of caffeine, it was also for
theophylline possible to extract a water signal by using the spin-echo method and very good
calibrations evidenced (analogues to Fig. 5). However, Table 1 lists for theophylline only a
single T2 relaxation time due to a mono-exponential fit that was employed. This simplified
model described experimental data in an adequate way. A single relaxation time could also be
applied for caffeine but the signal contribution of the T2(2) component was higher than for
theophylline so that mono-exponential fit would mean an evident loss in fitting adequacy.
The use of a bi-exponential T2 process in case of caffeine seems also reasonable from a
structural perspective because of the channel geometry [10].Comparatively broad channels of
caffeine, as depicted in Fig. 1A, would result in different types of hydrate water regarding
mobility. Thus, tightly bound water at the inner channel surface can be imagined as distinct
from water molecules that have more rotational or translational degrees of freedom because
they are located toward the channel center. In contrast to caffeine, a differentiation of water
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populations is not straightforward for theophylline due to the very narrow channels (Fig. 1B)
so the assumption of a single T2(1) relaxation time appeared to be reasonable. An additional
T2(2) relaxation time was, however, beneficial to describe the caffeine data in line with the
view that an additional, more loosely bound water population can be assumed here.
Different water populations in terms of mobility would also mean that ease of evaporation is
expected to be different. Already the pioneer work by Yoshioka et al. [9] looked at the ease of
water evaporation by a comparison with TD-NMR results. Current findings may encourage
more research in this direction but the main scope of our work was a process analytical
application for which a T2-SE method proved to be suitable.
An inversion recovery sequence was used in addition to determine the spin-lattice relaxation
signal in terms of T1. Drug hydrates were again compared to the anhydrates as well as
dehydrated forms. The signal amplitudes of hydrates were substantially different from those
of the water-free solids and Fig. 6 displays this time theophylline as example. Amplitudes of
the anhydrate and the dehydrated form were for most of the kinetics showing an overlap of
error bars. To estimate pure hydrate contribution in spin-lattice relaxation, the signal of
anhydrous form was again subtracted from that of the hydrate. This was repeated for different
defined mixtures of anhydrate and hydrate to obtain a calibration that is displayed for
theophylline by Fig. 7. The open symbols report the spin-lattice relaxation of the hydrate
water and the lines represent the calculated signals Mw1(t). Similar to the previously obtained
T2-results, theophylline data were again adequately fitted with a mono-exponential equation,
whereas a bi-exponential resulted in an excellent model for the T1-IR results of caffeine
(Table 2).
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In summary, TD-NMR results suggest that the T2-SE and T1-IR method were both providing
a separation of the water signal of the model hydrates. The signal amplitudes increased
linearly with the amount of hydrate water in blends with predetermined water content. The
obtained calibrations were promising in view of non-invasive measurements or process
analytical use of the benchtop NMR.
3.3. Comparison of TD-NMR with water sorption methods
NMR results were also interpreted with respect to findings of sorption methods. Water
sorption and desorption experiments are helpful to characterize the type of hydrates and data
have been used earlier for thermodynamic modeling [26]. Interesting is further the kinetics of
water sorption and desorption, which has practical consequences for drug handling and
processing. Water sorption/desorption experiments were based on gravimetric testing of
samples equilibrated in desiccators as well as on automated gravimetric vapor sorption
(GVS). The latter method is mostly employed to dynamically change from one relative
humidity (RH) condition to another by using a stop criterion for sufficiently small weight
losses. In case of rather slowly equilibrating samples, it is likely that a typical GVS protocol
may not truly reach equilibrium. Incubation in desiccators can better reflect long-term
behavior of slowly equilibrating samples but the downsides range from higher material and
time consumption to the fact that gravimetric sample measurement is outside of desiccators
and hence outside of the controlled environment.
Thermogravimetric analysis of the prepared caffeine 4/5 hydrate (stored at 75 % RH) showed
a weight loss of 6.2%, which compares to the expected value of 6.9%. Therefore, our storage
conditions at 75% RH were obviously not entirely sufficient to preserve a fully hydrated state
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of caffeine. In line with this, Fig. 8A shows that samples of caffeine hydrate stored at 80% or
90% RH slightly increased water content over time. The extent of water uptake suggests that
the hydrate samples used for equilibrium experiments using desiccators might already have
lost some of their hydrate water compared to the sample that was used for thermogravimetric
analysis.
Fig. 8A shows also that some conditions require quite long equilibration times, which was
also seen with theophylline hydrate (Fig. 8B) in line with a previous study [27]. The two
compounds differed in stability. So was, for example, 60% RH not a stable condition for the
caffeine hydrate in contrast to theophylline hydrate that exhibited pronounced dehydration
only at 40% RH and below. Both channel hydrates change water content within a
comparatively narrow humidity range. It is therefore important to know this critical RH value
of onset of dehydration in order to maintain stability of hydrates throughout the entire chain
of the drug development process from preformulation to solid form development, packaging,
and storage. This requires also that suitable process analytical technologies and control
mechanisms are established. This critical humidity range for stability may also be estimated
based on other thermoanalytical data [28]. Such an approach, however, requires that
equilibrium has been reached in the samples. Care is particularly needed when critical RH
values are measured by automated, dynamic GVS. Both of our model hydrates had
dehydration conditions that were shifted to lower RH values in dynamic GVS experiments so
that hydrates appeared to be stable within a broader humidity range than suggested by
equilibrium experiments. The dynamic GVS desorption curve of caffeine showed a marked
change of mass only below 30% RH while for theophylline, such threshold was only below
20% RH (data not shown). Despite the lack of equilibration, dynamic GVS data have their
merits for practical sample handling that takes place on a comparatively short time scale e.g.
during pharmaceutical manufacturing. However, when dehydration or hydrate formation is to
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be estimated for storage or any other long-term duration then only an equilibrium analysis is
meaningful. We estimated the initial desorption rates of the hydrates from the linear range of
mass change (from the experiments in desiccators), which is displayed for both compounds in
Fig. 9. Negative sorption rates indicate dehydration and absent sorption marks equilibrium
conditions. In fact, a plot of sorption (or desorption) rate against relative humidity is a good
way to use the zero sorption line for estimation of critical RH values regarding a particular
hydration state.
The values of ±0.002 %/min were marked in Fig. 9 because these rate limits were used as stop
criterion in our dynamic RH scans of GVS. This typical limit of an industrial protocol has
been found suitable with respect to balancing equilibration duration at a given condition
against total experiment time. Thus, excessively long data acquisition runs would become
impractical for experimental throughput in an industrial environment. Fig. 9 is helpful to
visualize those RH ranges where dynamic GVS experiments may not have reached
equilibrium.
Apart from the kinetic sorption rates, it was also interesting to consider the final water content
(at different RH after 39 days storage) for a comparison of the different methods. This value
allowed in particular correlating the TD-NMR results with data of the sorption experiments.
Due to the given distribution of the data points, i.e. given heteroscedasticity, we selected a
Spearman rank correlation. This correlation coefficient was determined as between the T2- SE
TD-NMR and equilibrium sorption method: r=0.71 (p=0.11), while the corresponding other
T1-IR TD-NMR method provided r=0.89 (p< 0.05). A comparison of both TD-NMR methods
with the dynamic GVS resulted in lower and non-significant correlations. The correlation
coefficients were certainly affected by the number of RH conditions and the use of non-
parametric statistics comes with comparatively lower discrimination power. More data are
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likely to increase correlation coefficients but current results suggest that TD-NMR appears to
primarily correlate with the equilibrium method regarding final water content.
4. Conclusion
Characterization of pharmaceutical hydrates by means of 1H TD-NMR was found to be
possible by using either a spin echo method for determination of T2-relaxation or an inverse
recovery method to study T1-relaxation behavior. Furthermore, the relaxation of the hydration
water alone was obtained by subtraction of the relaxation anhydrate curve from the relaxation
curve of the hydrate. The good calibrations of hydrate/anhydrate mixtures are highly attractive
for quality monitoring in development or at a later stage. Since the TD-NMR is non-
destructive, it is particularly attractive to analyze samples in early development where drug
availability is limited. A direct measurement through glass vials is further advantageous to
have defined environmental RH conditions. Benchtop TD-NMR holds much promise as PAT
method and may serve as an attractive complementary method to Raman spectroscopy that
provides entirely different molecular information about the solid-state of a drug than NMR in
the time domain.
Acknowledgment
The authors would like to thank Bruker BioSpin AG for support and especially Dr. Jörg
Müller is acknowledged for scientific comments on the TD-NMR method.
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References
[1] D. P. Parikh. Handbook of Pharmaceutical Granulation Technology. Drugs and the
Pharmaceutical Sciences, Informa Healthcare UK, Vol. 198, (2009) p. 481.
[2] D. Giron, Ch. Goldbronn, M. Mutz, S. Pfeffer, Ph. Piechon, Ph. Schwab. Solid State
Characterizations of Pharmaceutical Hydrates J. Therm. Analysis and Calorimetry. 68
(2002) 453–465
[3] S. R. Vippagunta, H. G. Brittain, D. J. Grant. Crystalline solids Adv. Drug Deliv. Rev.
48, 1 (2001) 3–26.
[4] R. K. Khankari, D. J. W. Grant. Pharmaceutical hydrates. Thermochim. Acta. 248
(1995) 61–79.
[5] www.hydrateweb.org, accessed 12th July 2016.
[6] M. Yamauchi, E. H. Lee, A. Otte, S. R. Byrn, M. T. Carvajal. Contrasting the Surface
and Bulk Properties of Anhydrate and Dehydrated Hydrate Materials. Crystal Growth
& Design. 11 (2011) 69 –698.
[7] J.P. Boetker, J. Rantanen, L. Arnfast, M. Doreth, D. Raijada, K. Loebmann, C.
Madsen, J. Khan, T. Rades, A. Müllertz, A. Hawley, D. Thomas, B. Boyd. Anhydrate
to hydrate solid-state transformations of carbamazepine and nitrofurantoin in
biorelevant media studied in situ using time-resolved synchrotron X-ray diffraction.
Eur. J Pharm. Biopharm. 100 (2016) 119-127.
Downloaded from http://iranpaper.irhttp://www.itrans24.com/landing1.html
20
[8] A. Fussell, E. Garbacik, H. Offerhaus, P. Kleinbudde, C. Strachan. In situ dissolution
analysis using coherent anti-Stokes Raman scattering (CARS) and hyperspectral
CARS microscopy. Eur. J. Pharm. Biopharm. 85 (2013) 1141–1147.
[9] S. Yoshioka, Y. Aso, T. Osako, T. Kawanishi. Wide-Ranging Molecular Mobilities of
Water in Active Pharmaceutical Ingredient (API) Hydrates as Determined by NMR
Relaxation Times. J. Pharm. Sci. 97 (2008) 4258–4268.
[10] M.U. A. Ahlqvist, L.S. Taylor. Water dynamics in channel hydrates investigated using
H/D exchange. Int. J. Pharm. 241 (2002) 253–261.
[11] M.D. Ticehurst, R.A. Storey, C. Watt. Application of slurry bridging experiments at
controlled water activities to predict the solid-state conversion between anhydrous and
hydrated forms using theophylline as a model drug. Int. J. Pharm. 247 (2002) 1–10.
[12] U.J. Griesser, A. Burger. The effect of water vapor pressure on desolvation kinetics of
caffeine 4/5-hydrate. Int. J. Pharm. 120 (1995) 83–93.
[13] S. Airaksinen, M. Karjalainen, E.Räsänen, J. Rantanen, J. Yliruusi. Comparison of the
effects of two drying methods on polymorphism of theophylline. Int. J. Pharm. 276
(2004) 129–141.
[14] S. Airaksinen, P. Luukkonen, A. Jorgensen, M. Karjalainen, J. Rantanen, J. Yliruusi.
Effects of Excipients on Hydrate Formation in Wet Masses Containing Theophylline.
J. Pharm. Sci. 92 (2003) 516–528.
Downloaded from http://iranpaper.irhttp://www.itrans24.com/landing1.html
21
[15] M. De Matas, H. G. M. Edwards, E. E. Lawson, L. Shields, P. York. FT-Raman
spectroscopic investigation of a pseudopolymorphic transition in caffeine hydrate. J.
Mol. Structure. 440 (1998) 97–104.
[16] P.L. Gould, J. R. Howard, G.A. Oldershaw. The effect of hydrate formation on the
solubility of theophylline in binary aqueous cosolvent systems. Int. J. Pharm. 51
(1989) 195–202.
[17] A. Hédoux, L. Paccou, P. Derollez, Y. Guinet. Dehydration mechanism of caffeine
hydrate and structural description of driven metastable anhydrates analyzed by micro
Raman spectroscopy. Int. J. Pharm. 486 (2015) 331–338.
[18] E. Suzuki, K.-I. Shirotani, Y. Tsuda, K. Sekiguchi. Water Content and Dehydration
Behavior of Crystalline Caffeine Hydrate. Chem. Pharm. Bull. 33, 11 (1985) 5028–
5035.
[19] E. Suzuki, K. Shimomura, K. Sekiguchi. Thermochemical Sturdy of Theophylline and
Its Hydrate. Chem. Pharm. Bull. 37, 2 (1989) 493–497.
[20] J.S. Tantry, J. Tank, R. Suryanarayanan. Processing-Induced Phase Transitions of
Theophylline – Implications on the Dissolution of Theophylline Tablets. J. Pharm.
Sci., 96 (2007) 1434–1444.
Downloaded from http://iranpaper.irhttp://www.itrans24.com/landing1.html
22
[21] Ch. F. Forney, D.G. Brandl. Control of Humidity in Small Controlled-environment
Chambers using Glycerol-Water Solutions. Technol. & Product Reports, Hort
Technology (1992) 52–54.
[22] A. Hédoux, A.A. Decroix,, Y. Guinet,, L. Paccou,, P. Derollez,, and M. Descamps.
Low- and High-Frequency Raman Investigation on Caffeine: Polymorphism, Disorder
and Phase Transformation. Phys. Chem. B. (2011) 5746-5733.
[23] N.V. Phadnis, R.K. Cavatur,, R. Suryanarayanan. Identification of drugs in
pharmaceutical dosage forms by X-ray powder diffractometry. J. Pharm. Biomed.
Anal. 15 (1997) 929-943.
[24] H. Metz, K. Mäder, Benchtop-NMR and MRI- A new analytical tool in drug delivery
research. Int. J. Pharm. 364 (2008) 170-175.
[25] M. Kuentz, B. Rothenhäusler,, D. Röthlisberger. Time Domain 1H NMR as a New
Method to Monitor Softening of Gelatin and HPMC Capsule Shells. Drug Develop.
Ind. Pharm. 32 (2006) 1165–1173.
[26] J.R. Authelin. Thermodynamics of non-stoichiometric pharmaceutical hydrates. Int. J.
Pharm. 303 (2005) 37-53.
[27] A.K. Salameh, L.S. Taylor. Physical Stability of Crystal Hydrates and their
Anhydrates in the Presence of Excipients. J. Pharm. Sci. 95, 2, (2006) 446-460.
Downloaded from http://iranpaper.irhttp://www.itrans24.com/landing1.html
23
[28] C.H. Gu,D.J.W. Grant. Estimating the Relative Stability of Polymorphs and Hydrates
from Heats of Solution and Solubility Data. J. Pharm. Sci. 90, 9 (2001) 1277-1287.
Downloaded from http://iranpaper.irhttp://www.itrans24.com/landing1.html
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Figure captions
Fig. 1. A) Hydrate crystal structure of caffeine (CSD: CAFINE01) and B) theophylline (CSD:
THEOPH04). Water was removed for clarity of presentation and the channels are depicted as
contact surfaces with probe of 1.0 Å. Pores are larger in the hydrate of caffeine compared
with theophylline.
Fig. 2. Raman spectra (overview) of the different solid state forms in the case of A) caffeine
and B) theophylline.
Fig. 3. A) XRPD-pattern of caffeine anhydrate (red), caffeine hydrate (blue) and dried
caffeine hydrate (i.e. dehydrate)(green). B) XRPD-pattern of theophylline anhydrate (red),
theophylline hydrate (blue) and theophylline dehydrate (green).
Fig. 4. Results of TD-NMR for caffeine using T2 spin echo relaxation. A water signal of the
hydrate can be clearly differentiated from the sample amplitudes of the water-free forms.
Standard deviations (SD) are shown for every third value for clarity of presentation.
Fig. 5. Calculated TD-NMR amplitude of the water signal (arbitrary units, a.u.) vs. time in the
case of caffeine (T2-SE calibration). Different blends of the hydrate with anhydrous form
were analyzed. The fitted amplitudes display suitable linearity (R2=1.00) with the content of
hydrate water (%, w/w). Details are given in the text.
Fig. 6. Results of TD-NMR for theophylline using T1 inverse recovery relaxation. A water
signal of the hydrate can be clearly differenciated from the sample amplitudes of the water-
free forms.
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Fig. 7. Calculated TD-NMR amplitude of the water signal (arbitrary units, a.u.) vs. time in the
case of theophylline (T1-IR calibration). Different blends of the hydrate with anhydrous form
were analyzed. The fitted amplitude display suitable linearity (R2=0.998) with the content of
hydrate water (%, w/w). Details are given in the text.
Fig. 8. Saturation kinetics of A) freshly prepared caffeine hydrate and B) theophylline
hydrate, respectively. Samples were stored in desiccators at room temperature and a given
relative humidity (RH).
Fig. 9. Summary of the sorption kinetics for theophylline hydrate (blue diamonds) and
caffeine hydrate (brown squares). Gray lines mark absent sorption (dotted line) as well as the
limits of what can be detected by a typical industrial protocol of dynamic vapor sorption
(dashed lines).
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Table 1 – Fitted amplitudes and relaxation times obtained by the T2 spin-echo relaxation method.
Details are given in the text.
Caffeine hydrate
100% 75% 50% 25%
A2(1) 427.53 321.35 214.33 107.48
T2(1) (ms)
0.028
A2(2)
2.87 2.64 2.64 1.74
T2(2) (ms) 0.137
Theophylline hydrate
100% 75% 50% 25%
A2(1) 431.81 316.9 165.54 65.1
T2(1) (ms) 0.0238
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Table 2 - Fitted amplitudes and relaxation times obtained by the T1 inverse-recovery method. Details
are given in the text.
Caffeine hydrate
100% 75% 50% 25%
A1(1) 18.53 14.00 8.38 3.69
T1(1) (ms)
366.48
A1(2)
5.33 4.49 3.99 2.66
T1(2) (ms) 111.73
Theophylline hydrate
100% 75% 50% 25%
A1(1) 19.09 15.44 8.74 3.92
T1(1) (ms) 1749.15
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