Microwave Assisted Polymorph Selection in Pharmaceutical Drugs

A Major Qualifying Project Report:

Submitted to the Faculty

of

WORCESTER POLYTECHNIC INSTITUTE

In partial fulfillment of the requirements for the

Degree of Bachelor of Science

by

Sofia M. Kniazeva

Date: April 24, 2008

Approved:

Professor Venkat R. Thalladi, Advisor

Department of Chemistry and Biochemistry

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Table of Contents

Abstract ............................................................................................................................... 3

1 Background and Scope .................................................................................................... 4

1.1 Polymorphism and Pseudopolymorphism ................................................................ 4

1.2 Microwave Heating................................................................................................... 5

1.3 Compounds Examined.............................................................................................. 7

2 Experimental .................................................................................................................... 8

2.1.1 Caffeine Recrystallization from Wet Methanol..................................................... 8

2.1.2 Caffeine Recrystallization from Aqueous Solution ............................................... 8

2.1.3 Theophylline Recrystallization from Aqueous Solution........................................ 8

2.2 Method Development for Microwave Dehydration.................................................. 9

2.3 Differential Scanning Calorimetry............................................................................ 9

2.4 Powder X-ray Diffraction ......................................................................................... 9

2.5 Control experiments................................................................................................ 10

3 Results and Discussion ................................................................................................. 11

3.1 Recrystallization ..................................................................................................... 11

3.2 Microwave Assisted Dehydration........................................................................... 12

3.3 Control experiments................................................................................................ 18

4 Conclusion .................................................................................................................... 23

5 References...................................................................................................................... 25

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ABSTRACT

Microwave heating is rapid, efficient, selective and volumetric. We explore the

application of these unique properties in the dehydration and subsequent polymorph

selection of active pharmaceutical ingredients (APIs). Traditional heating methods, such

as hot plates, oil baths, and ovens are often lengthy in time and require the application of

heat to the entire material. Microwave heating, on the other hand, takes advantage of the

affinity and selectivity of the microwaves to the water molecules present in hydrated

crystalline structures, therefore allowing for unique dehydration paths. This project

explores the outcomes of using microwave heating to dehydrate caffeine – an API

commonly used in the pharmaceutical industry.

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1 BACKGROUND AND SCOPE

1.1 Polymorphism and Pseudopolymorphism

Polymorphs, molecular solids exhibiting different packing arrangements (Figure 1), pose

a daunting challenge to the pharmaceutical industry.1 Polymorphs of the same compound

exhibit different physical and chemical properties such as bioavailability, dissolution rate,

melting point, density, and compressibility.2 It is therefore imperative to isolate the

appropriate form of a potential drug compound to ensure that the optimum formulation is

released to patients. To meet this end, pharmaceutical companies rigorously screen drug

candidates for polymorphism using a high-throughput approach where conditions such as

concentration, temperature, pressure and cooling rates are varied in an empirical fashion.3

Despite this rigorous approach, there is no guarantee that all polymorphs of a given

compound will be observed.

Figure 1. Crystal structures4 of monoclinic acetaminophen (a) and orthorhombic acetaminophen (b). Notice the presence of slip planes in (b). Inset are the predicted crystal morphologies using Bravais Friedel Donnay Harker theory (BFDH).

An additional type of polymorphism is known as pseudopolymorphism.5 Water or solvent

molecules may be periodically included in the lattice with the drug compound (Figure 2).

These two pseudopolymorphs are called hydrates and solvates respectively.

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Figure 2. Crystal structure of theophylline hydrate.6 The water molecules serve as an intermolecular glue between layers of theophylline.

A number of recent papers have focused on the dehydration (or desolvation) processes of

these types of compounds in an attempt to isolate novel anhydrous polymorphs.7

Theophylline monohydrate is an example of a hydrate compound that dehydrates to a

metastable polymorphic modification. To initiate the dehydration process, the solid

theophylline was either heated in an oven or stored in vacuo.

1.2 Microwave Heating

Molecules possessing a permanent dipole moment will rotate in the presence of an

oscillating electric field; therefore, when polar molecules are exposed to radiation in the

microwave regime (2.45 GHz), they attempt to align themselves with the changing field.

Since the frequency is slightly too fast for the molecules to keep pace with the field and

they begin to rotate out of phase, and as a result, random collisions that occur between

molecules cause the medium to heat up rapidly (Figure 3). 8

6

Figure 3. Schematic illustrating dipolar polarization. Molecules possessing a permanent dipole moment rotate out of phase with the oscillating electric filed component of microwave radiation.

There are a number of significant advantages associated with microwave assisted

dehydration. Conventional dehydration methods (such as oven dehydration) supply heat

to the entire solid. By heating the entire mass it is possible to induce phase

transformations that may lead to an undesired modification. In the case of microwave

heating, the water is selectively heated allowing dehydration to occur at temperatures

significantly less than those reported for traditional methods.

The rapid nature of microwave heating also presents an advantage. Conventional heating

methods can take upwards of 24 hours to fully dehydrate the sample, whereas the

microwave approach can dehydrate compounds in less than ten minutes. This paper

focuses on the dehydration of active pharmaceutical hydrates using microwave

irradiation. Experiments were performed on channel and stoichiometric hydrates in order

to elucidate any structural dependence on the efficiency of microwave dehydration.

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1.3 Compounds Examined

We examined the effect of microwave assisted dehydration on caffeine and theophylline.

Caffeine is a xanthine derivative used to treat drowsiness. It is known to crystallize in two

anhydrous polymorphs, α and β (Figure 4), as well as a non-stoichiometric channel

hydrate (Figure 6).9 The α modification is metastable at room temperature and is

obtained by heating the β form at 150° C for a period of three days.

Figure 4. Powder X-ray diffractograms of the anhydrous forms of caffeine. The α modification (blue) displays a single peak at 27° 2θ. The β form (red) displays a doublet at 27° 2θ.

Theophylline, a widely prescribed bronchodilator, possesses two anhydrous polymorphs,

a monohydrate, and a metastable anhydrous form obtained through dehydration of the

monohydrate.10 Of the two stable anhydrous forms, one is stable at room temperature

while the other is only obtained by recrystallization at elevated temperatures.

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2 EXPERIMENTAL

2.1 Method Development for Recrystallization

Different methods were applied in order to determine the most efficient way of producing

the hydrated form of caffeine and theophylline via recrystallization of commercially

available anhydrates. Powder X-ray diffractograms of the recrystallization product were

recorded for each method and compared to the literature diffractograms.

2.1.1 Caffeine Recrystallization from Wet Methanol

Three Erlenmeyer flasks were filled with 5mL of methanol and 5%, 10% and 15% of

water (respectively). Each solution was placed on low heat and 50 mg of caffeine was

added to each flask, the solution was stirred until the entire compound was dissolved. The

flasks were then taken off the heat and, while still hot, each of the solutions was

distributed among 5 vials (approximately 1 mL in each). The vials were covered with

perforated aluminum foils and left undisturbed for a period of a few days until

recrystallization was observed. PXRD was taken to identify the crystals.

2.1.2 Caffeine Recrystallization from Aqueous Solution

Approximately 900 mg of commercial caffeine was massed and added to an Erlenmeyer

flask containing 5 mL of water (Caffeine’s solubility: 180 mg/mL at 80 °C). The solution

was heated until all solid was dissolved and boiled further in order to dissolve any tiny

aggregates that were still present. The solution was hot filtered into two vials, covered

and allowed to slowly evaporate until crystals appeared in two to five days.

2.1.3 Theophylline Recrystallization from Aqueous Solution

40 mL of water was poured into an Erlenmeyer flask and heated to about 85 °C. 280 mg

of commercial theophylline was then added and stirred until all the solid was observed to

dissolve. The solution was hot filtered into eight vials, covered and allowed to slowly

evaporate until crystals were grown.

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2.2 Method Development for Microwave Dehydration

Theophylline and caffeine hydrates were placed in a glass PXRD sample holder and

microwaved in a domestic microwave oven (Chefmate 2.45 GHz) for different time

intervals. The samples were removed from the oven and immediately characterized using

PXRD and differential scanning calorimetry (DSC).

It was unclear if dehydration occurred because of microwave heating of the sample or

convenctional heating of the sample holder. To resolve this issue, we used a Teflon based

sample holder which was essentially microwave transparent. A PXRD diffractogram of

the bare Teflon plate was recorded to identify Teflon related peaks.

Validation experiments were done for the caffeine samples to try and replicate results, as

well as further microwave experiments where microwave time intervals were varied from

two to ten minutes, consecutively. At this point vacuum drying was introduced as part of

the procedure (after recrystallization/before microwave dehydration) in an attempt to

minimize the effect of the convective heating that might occur when excess water is

present and heated by microwave irradiation.

2.3 Differential Scanning Calorimetry

Measurements were carried out with DSC-2920 (TA Instruments) in hermetically sealed

and crimped aluminum pans. Samples were subjected to heating in the range 30-300 °C

at a rate of 10 °C per minute. The monohydrate showed a distinct endotherm near 40 °C

corresponding to the loss of water; the anhydrous form showed no such endotherm.

Thermal gravimetric analysis (TGA-2950, TA Instruments) of the monohydrate showed

that the endotherm at 40 °C in DSC corresponds to complete loss of water.

2.4 Powder X-ray Diffraction

Data were collected on a Bruker D8 Advance diffractometer. The instrument was

equipped with a vertical goniometer and a scintillation counter as a detector and applied

Bragg-Brentano geometry for data collection. X-rays were generated at a power setting of

10

40 kV and 40 mA. If the crystals were larger they were pulverized using a mortar and a

pestle prior to diffraction analysis. We also subjected the smaller crystals of monohydrate

to diffraction without grinding; there was no significant change in the relative intensities

of the diffraction peaks. Samples were transferred to a glass sample holder that had

loading dimensions 1.6 cm × 2 cm and exposed to X-rays over the 2θ range 5-50° in

0.05° steps and at a scan rate of 2° per minute.

2.5 Control experiments

Experiments were designed to estimate the temperature reached in the microwave during

the heating intervals. An empty glass vial was microwaved for two minutes and a thermal

sticker was placed on it immediately after to determine glass temperature. Following that,

different organic solids with known melting points were exposed to microwaves for five

minutes both in a glass vial and on a Teflon plate. Any phase transformations, from solid

to liquid, were observed and recorded as an indicator of the temperature.

In order to ensure that the temperature reached in the microwave was not high enough to

induce a polymorphic transformation on its own, samples of commercial caffeine were

microwaved for periods of five and eight minutes, after which PXRD and DSC were used

to evaluate any polymorphic changes that may have occurred. One of the samples was

microwaved with a drop of water prior to DSC analysis, to eliminate the effect of residual

water while exposed to microwave irradiation.

Caffeine hydrates were also dehydrated using conventional methods (oven and oil bath)

to demonstrate that dehydration occurring at temperatures lower than 140 °C could not

induce a phase transformation to the α form. In the first experiment, caffeine hydrates

were placed in a 60 °C oven for a period of one week, after which PXRD was taken to

evaluate the results. In the second experiment, the hydrate was placed in an oil bath, at a

constant temperature of 110°C for one day. Subsequently, PXRD was used to determine

dehydration effects.

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3 RESULTS AND DISCUSSION

3.1 Recrystallization

The recrystallization of commercial caffeine from 5%, 10% and 15% wet methanol

solutions over a period of three to five days resulted in the formation of a white

polycrystalline substance. PXRD analysis identified the material as the β modification of

anhydrous caffeine (Figure 5).

Figure 5. PXRD diffractogram of the product obtained by recrystallization from 5% wet methanol solution. All three methanol solutions (5%, 10% and 15%) produced almost superimposable PXRD patterns. The pattern exhibits the characteristic anhydrous peak at 11.8° 2θ and a doublet at 27° 2θ which are characteristic of the β modification.

Recrystallization was repeated from saturated aqueous solutions (literature values

indicate the solubility of caffeine in water at 80 °C to be around 180 mg – this estimate

was used to determine the amount of caffeine that was needed to make a saturated

solution).9 This recrystallization technique resulted in the formation of white, needle-like

crystals, observed to form almost immediately after the hot, saturated solution was

distributed among the glass vials. PXRD patterns taken after recrystallization, shown in

Figure 6, identified the product as caffeine hydrate.

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Figure 6. PXRD pattern of caffeine hydrate obtained by recrystallization from saturated aqueous solution.

The recrystallization of theophylline from saturated aqueous solutions was also successful,

and produced white, needle-like crystals over a period of three days. The crystals were

determined to be the hydrated form of the compound through PXRD (Figure 7).

3.2 Microwave Assisted Dehydration

The recrystallized theophylline hydrate was microwaved continuously for a period of

nine consecutive minutes, and immediately analyzed by PXRD. Diffractograms of the

theophylline hydrate, the commercially available theophylline and the microwaved

sample were compared in order to determine whether dehydration occurred (Figure 7).

Figure 7. PXRD pattern of commercial theophylline (navy); theophylline hydrate obtained by recrystallization from saturated aqueous solution (red), and theophylline hydrate after nine minutes of microwave exposure (light blue).

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We noted that dehydration of theophylline in a microwave oven generated mixtures of

metastable and stable modifications. This is very similar to results reported in the

literature concerning conventional approaches to dehydration. During conventional

dehydration processes (oven, in vacuo) mixtures of the stable and metastable forms are

obtained. We observed that the metastable modification fully converted to the stable

modification after approximately eight hours at room temperature. This is most likely due

to the presence of quantities of the stable modification that facilitated the phase transition

to the stable anhydrous form. In addition, we noticed that theophylline hydrate slowly

converted to the metastable form while exposed to the atmospheric conditions of our

laboratory. The low humidity level present in this region during the winter months most

likely helped dehydrate the compound. For these reasons we focused our efforts on the

dehydration of caffeine.

Initially, caffeine hydrate was microwaved for five minute time intervals as a starting

point. After each run, the sample was immediately analyzed using PXRD. Figure 8

illustrates the transition from caffeine hydrate to anhydrous caffeine. After exposure to

microwave irradiation, the characteristic hydrate peak at 10.7° 2θ completely disappeared

while the anhydrous peak at 11.8° 2θ significantly increased in intensity. More

importantly, a singlet at 27° 2θ appeared indicating that the hydrate had dehydrated to the

metastable modification. Validation experiments were performed to verify the

reproducibility of the results. In all subsequent experiments, similar results were obtained

as illustrated in Figure 9.

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Figure 8. PXRD patterns of caffeine hydrate before microwave dehydration (navy); caffeine hydrate after five minutes in the microwave (light blue) and after ten minutes in the microwave (red).

Figure 9. PXRD patterns of caffeine hydrate before microwave dehydration (navy) and caffeine hydrate after ten minutes in the microwave (red).

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Figure 9(a). PXRD pattern of validation experiment (Figure 9) at the θ=24-30º range. The single peak at 26.6 °2θ is characteristic of the metastable polymorph of caffeine. It is easily distinguishable from the double peak of the hydrate at the same 2θ value.

We hypothesized that residual water present on the surface of the crystals was heating up

during the microwave dehydration process and as a result, causing the observed product

to form due to convective heating. To prevent this effect, the caffeine hydrate crystals

were vacuum filtered after isolation to aid in the removal of residual water. Microwave

dehydration experiments performed on the vacuum filtered hydrate exhibited complete

dehydration in two minutes (Figure 10) compared to the ten minutes required to

dehydrate the moist caffeine hydrate. We surmise that the residual water may be limiting

the amount of microwave irradiation that reaches the channels present in the hydrate.

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Figure 10. PXRD pattern of caffeine hydrate (navy); caffeine hydrate after two minutes in the microwave (red) and α modification of anhydrous caffeine. The disappearance of the characteristic hydrate peak (at 10.7º 2θ), and appearance of the characteristic anhydrous peak (11.8º 2θ) verify dehydration. The singlet at 26.6º 2θ observed on the microwaved sample’s PXRD corresponds to the singlet characteristic of the α modification.

DSC thermograms were recorded to corroborate the PXRD data. In addition, this method

allowed quantification of the relative amount of the α form present after microwave

exposure. Studies performed on caffeine11 indicate the following enthalpic data for the β

modification: ∆fusHm = 19.86 kJ/mol and ∆transHm = 3.43 kJ/mol; therefore, starting with

100% of the β-form would result in a ratio of 5.58 (∆fusHm vs ∆transHm). As illustrated in

Figure 11, we obtained the following data from the commercial caffeine sample: ∆fusHm

= 14.67 kJ/mol and ∆transHm = 2.284 kJ/mol. The calculated ratio, 6.42, is within range of

the literature data, confirming that the sample is in the β form. If less β form was present

the ratio between the two areas would increase. This methodology was used to quantify

the relative amount of the α form present after microwave irradiation.

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Figure 11. DSC thermogram for commercial caffeine with no microwave heating.

The ratio obtained from the heats of fusion and phase transformation of a caffeine sample

dehydrated in the microwave, Figure 12, confirms the presence of the α polymorph. Its

value, 13.78, is almost double that of the standard caffeine sample, indicating that as

much as 45% of the dehydrated caffeine is in the metastable form.

Figure 12. DSC thermogram for caffeine hydrate microwaved for five minutes.

1.999 J/g : 27.55 J/g

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1.999 J/g : 27.55 J/g1.999 J/g : 27.55 J/g

40 90 140 190 24040 90 140 190 240

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3.3 Control experiments

A series of control experiments were performed in order to determine whether the

dehydration results were not due to convective heating and were a result of microwave

assisted dehydration.

If the temperature in the microwave exceeds 140ºC, it can induce a phase transformation

from the β anhydrous caffeine to the α form, thereby dismissing the effectiveness of

microwave dehydration.

In order to estimate the microwave temperature range, various organic solids with known

melting points were heated on Teflon and glass plate sample holders in the microwave

and any phase transformations were recorded in Table 1 below.

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Table 1. Compounds used to determine the maximum temperature reached inside the microwave.

Compound Melting Point Heating Vessel Time Observation

Decanodioic acid 131ºC Glass vial 5min Did not melt

Diphenylamine 52ºC Glass vial 5min Melted

2-fluorocarboxaldehyde 85ºC Glass vial 5min Melted

Terephtalodehyde 114ºC Glass vial 5min Melted

Diphenylamine 52ºC Teflon plate 5min Melted

2-fluorocarboxaldehyde 85ºC Teflon plate 5min Did not melt

From the two Teflon plate runs of diphenylamine and 2-fluorocaroxaldehyde it was

concluded that the temperature conducted through the Teflon plate as a result of

microwave irradiation was no greater than 85ºC, and therefore was not sufficient to

induce phase transformation between the two anhydrous polymorphs. This was further

validated by exposing a sample of the anhydrous β modification used as the starting

material for all microwave experiments to microwave irradiation for a period of eight

minutes. As demonstrated in the PXRD pattern below (Figure 13), the sample did not

undergo a phase transformation, therefore verifying that the temperature reached in the

microwave was not a factor.

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Figure 13. PXRD of commercial caffeine after eight minutes in the microwave. The doublet at 27º 2θ confirms the presence of the stable caffeine polymorph.

DSC analysis provided evidence that microwave irradiation of commercial caffeine did

not induce a phase transformation. Calculations of the ∆fusHm to ∆transHm ratios for two

different commercial caffeine samples microwaved for five minutes, one dry and one

with a drop of water, resulted in values that were within the range of a standard

commercial caffeine sample (5.53 and 6.01 to 6.42, respectively), as demonstrated in

Figures 14 and 15 below.

Figure 14. DSC of commercial caffeine after five minutes in the microwave.

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8.608 J/g : 47.63 J/g

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8.608 J/g : 47.63 J/g

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Figure 15. DSC of moist commercial caffeine after five minutes in the microwave

Additionally, conventional dehydration (in an oven and oil bath) was performed on

caffeine hydrate to verify that prolonged exposure to temperatures slightly below the

phase transformation temperature would not induce a transition from β to α. Caffeine

hydrate held at 60° C for one week (Figure 16) as well as the sample held at 110° C for

one day (Figure 17) displayed no phase transformations.

Figure 16. PXRD of caffeine hydrate after oven dehydration (one week, 60ºC). The doublet observed at 27º 2θ on the oven dehydrated sample pattern (light blue) matches the doublet observed on the commercial caffeine pattern (navy), and is unlike the singlet observed on the pattern of the α form (red) and the pattern of the sample that was dehydrated by the microwave (violet).

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10.15 J/g : 61.02 J/g

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10.15 J/g : 61.02 J/g

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Figure 17 – PXRD of caffeine hydrate after oven dehydration (one day, 110ºC). The doublet observed at 27º 2θ on the oil bath dehydrated sample pattern (light blue) matches the doublet observed on the commercial caffeine pattern (navy), and is unlike the singlet observed on the pattern of the α form (red) and the pattern of the sample that was dehydrated by the microwave (violet).

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4 CONCLUSION

This work demonstrates that microwave assisted dehydration leads to the formation of the

metastable polymorph of caffeine at significantly lower temperatures than reported using

conventional means. It was further proven through control experiments that this unique

result is due to the effect of the microwaves on the hydrated caffeine sample and is not a

result of convective heating. One explanation for this outcome could be the interaction of

the microwaves with the water molecules present in channel hydrates, such as caffeine.

The water molecules may attempt to align with the oscillating electric field and leave the

lattice through the channels. Conventional heating techniques like oil baths and ovens

disperse heat through the hydrate, affecting both the water molecules and the caffeine

itself. Microwaves selectively target water molecules, as illustrated in Figure 18 below,

leaving the rest of the compound unaffected.

Figure 18. Proposed method of microwave dehydration of pharmaceutical hydrates, such as caffeine.

The ability to dehydrate compounds with microwave irradiation may permit access to

new metastable polymorphs of pharmaceutical interest. The rapid and selective nature of

dehydration using microwaves proves to be an efficient route to generation of the

API layer

API layer

API layer

API layer

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metastable modification of caffeine. Future work will focus on elucidating the

mechanism responsible for these results.

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5 REFERENCES

1. S. R. Byrn, R. R. Pfeiffer, J. G. Stowell, Solid-State Chemistry of Drugs, 2nd ed., SSCI, West Lafayette, 1999; Polymorphism in Pharmaceutical Solids (Ed.: H. G. Brittain), Marcel Dekker, New York, 1999; J. Bernstein, Polymorphism in Molecular Crystals, Oxford University Press, New York, 2002; Polymorphism in the Pharmaceutical Industry (Ed.: R. Hilfiker), Wiley-VCH, Weinheim, 2006.

2. S. R. Chemburkar, J. Bauer, K. Deming, H. Spiwek, K. Patel, J.Morris, R. Henry, S. Spanton, W. Dziki, W. Porter, J. Quick, P.Bauer, J. Donaubauer, B. A. Narayanan, M. Soldani,D. Riley, K. McFarland, Org. Process Res. Dev. 2000, 4, 413 – 417.

3. Recently, this classical approach has been automated into a highthroughput method. See, for example: S. L. Morissette, S. Soukasene, D. Levinson, M. J. Cima, O. Almarsson, Proc. Natl. Acad. Sci. USA 2003, 100, 2180 – 2184.

4. Structures were obtained from the Cambridge Crystallographic Database. Monoclinic structure from Refcode HXACAN09; orthorhombic structure from Refcode HXACAN08.

5. G. R. Desiraju, CrystEngComm 2003, 5, 466 – 467; K. R. Seddon, Cryst. Growth Des. 2004, 4, 1087; G. R. Desiraju, Cryst. Growth Des. 2004, 4, 1089 – 1090; J.Bernstein, Cryst. Growth Des. 2005, 5, 1661 – 1662; A. Nangia, Cryst. Growth Des. 2006, 6, 2–4.

6. Structure was obtained from the Cambridge Crystallographic Database. Monohydrate structure from Refcode THEOPH.

7. Amado, Ana M.; Nolasco, Mariela M.; Ribeiro-Claro, Paulo J. A. Probing pseudopolymorphic transitions in pharmaceutical solids using Raman spectroscopy: hydration and dehydration of theophylline. Journal of Pharmaceutical Sciences 2007, 96(5), 1366-1379.

8. Santagada V; Perissutti E; Caliendo G The application of microwave irradiation as new convenient synthetic procedure in drug discovery. Current medicinal chemistry 2002, 9(13), 1251-83; Mavandadi, Farah; Pilotti, Aake. The impact of microwave - assisted organic synthesis in drug discovery. Drug Discovery Today 2006, 11(3 & 4), 165-174; Kremsner, Jennifer M.; Stadler, Alexander; Kappe, C. Oliver. The scale-up of microwave - assisted organic synthesis. Topics in Current Chemistry 2006, 266 Microwave Methods in Organic Synthesis, 233-278.

9. Pirttimaki, Jukka; Laine, Ensio; Ketolainen, Jarkko; Paronen, Petteri. Effects of grinding and compression on crystal structure of anhydrous caffeine. International Journal of Pharmaceutics 1993, 95(1-3), 93-9; Manduva, Radhesh; Kett, Vicky L.; Banks, Simon R.; Wood, John; Reading, Mike; Craig, Duncan Q. M. Calorimetric and spatial characterization of polymorphic transitions in caffeine using quasi-isothermal MTDSC and localized thermomechanical analysis. Journal of Pharmaceutical Sciences 2007, 97(3), 1285-1300.

10. Airaksinen, Sari; Karjalainen, Milja; Raesaenen, Eetu; Rantanen, Jukka; Yliruusi, Jouko. Comparison of the effects of two drying methods on polymorphism of theophylline. International Journal of Pharmaceutics 2004, 276(1-2), 129-141;

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Legendre, Bernard; Randzio, Stanislaw L. Transitiometric analysis of solid II/solid I transition in anhydrous theophylline. International Journal of Pharmaceutics 2007, 343(1-2), 41-47.

11. Bothe, H.; Cammenga, H. K. Composition, properties, stability and thermal dehydration of crystalline caffeine hydrate. Thermochimica Acta 1980, 40(1), 29-39; Griesser, U. J.; Burger, A. The effect of water vapor pressure on desolvation kinetics of caffeine 4/5- hydrate. International Journal of Pharmaceutics 1995, 120(1), 83-93.