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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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1

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


2

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

[email protected]


3

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


4

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


5

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.


6

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


7

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.


8

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


9

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.


10

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.


11

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°


12

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-


13

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


14

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).


15

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


16

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


17

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


18

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.


19

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24

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.


25

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).


26

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


27

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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