Establishing a new method to evaluate the recrystallization of nano

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Establishing a new method to evaluate the recrystallization of nano-gram quantities of paracetamol printed as a microarray using ink-jet printing Mohammed S.N. Algahtani, Martyn C. Davies, Morgan R. Alexander, and Jonathan C Burley Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b01121 • Publication Date (Web): 06 Dec 2018 Downloaded from http://pubs.acs.org on December 10, 2018

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Crystal Growth & Design

Establishing a new method to evaluate the recrystallization of nanogram quantities of paracetamol printed as a microarray using ink-jet printing

Mohammed S N Algahtani1, Martyn C Davies2, Morgan R Alexander2, Jonathan C Burley2

1 Pharmaceutical Department, School of Pharmacy, Najran University, Najran, Saudi Arabia

2 Laboratory of Biophysics and Surface Analysis, School of Pharmacy, University of Nottingham, Nottingham, NG7 2RD, United Kingdom

Abstract In pharmaceutical pre-formulation it is important to be able to screen a drug compound for possible solid forms (amorphous, crystal, polymorphs, salts etc) prior to scale-up of manufacture for formulation, and as this screening is undertaken prior to scale-up, there is often only a small amount of drug material available. Minimising the amount of sample required for these solid form investigations is therefore of paramount importance. Typical industrial work-flows require hundreds of milligrams of compound, while a small number of research papers have suggested that new approaches based on conventional inkjet printing may allow only a few milligrams to be used, but even these small quantities of the sample may not be available in early-stage drug development. Herein we report an approach based on pico-litre inkjet printing. Employing paracetamol as a model compound, we illustrate how a basic solid-form screen may be run using only nano-grams of material (around six orders of magnitude less material than previously reported approaches). For the first time, we were able to monitor the recrystallization of nanogram amorphous paracetamol to meta-stable crystalline forms II and III. Cross-polarised microscopy and Raman spectroscopy were employed to monitor the recrystallization of the

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paracetamol microarray. We suggest that further development of this concept may allow preformulation to occur far earlier in drug development than is currently the case, and also a more extensive parameter space to be explored using this new approach in a micro-array format.

Introduction At an early stage of medicine development, the lead optimization phase is important in order to decide as early as possible the optimum physical form and formulation, and to reduce product iteration during the discovery and the development phases 1. This includes the pure drug (amorphous, crystalline and any polymorphs) as well as multi-component forms (salts, cocrystals, solvates, etc) with a view to understanding the formulation options for the solid API. Great efforts have been focused in creating high throughput techniques that use the minimum amount of a new drug candidate, which is typically available in very limited quantities, and screen for solid forms by applying variables of compositions and processing in parallel to understand the effect of these variables on the solid-state outcomes2–4. The fully high throughput (HT) crystallization systems that have been designed and are employed in industry consist of robotic arms for dispensing and handling, controlled by experimental software, such as CrystalMax technology5. These HT systems are essentially miniaturised versions of lab-scale or production scale systems and can run a screen using 20-100 mg of API; they are widely used in the pharmaceutical industry. Inkjet printing technologies were utilized as a dispensing method to study the physical properties of APIs in small scale. For example, Buanz et al., were able to rapidly prepare pharmaceutical cocrystals using a desktop thermal inkjet printer6. Also, the same group used a piezoelectric system inkjet printer to deposit picoliter droplets of glycine onto a glass and aluminium substrate. A highly metastable β glycine form was produced on all the surfaces used7. Also, inkjet printing technology has been explored by a very small number of researchers for HT solid-form screening. Initial work in the field was undertaken by the group of Kazarian, who printed a limited number of drug: polymer formulations onto a focal plane array ATR-IR system, which allowed rapid analysis of the formulations under a range of conditions including applied humidity8. Later, the group of Bradley used an elegant polymer microarray to provide a wide range of substrates for


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the solid-form screening of different drugs through polymer-induced hetero-nucleation. A polymer micro-array was first inkjet-printed, and then drugs were inkjet-printed directly on top of the polymer spots, using the same solvent as used to dispense the polymer. This allowed screening using as little as 9 μg of drug per printed spot (i.e. ca. 2.5 mg for a 256-spot screen)9. The group of Matzger moved away from inkjet printing to contact-based printing, which allowed them to dramatically increase the number of samples to 1536, with samples being confined in a custom-fabricated well-plate, and less than 1mg of drug sample being required 3. The Bradley approach employed the same solvent for both polymer deposition and subsequent drug deposition, which produces the significant risk of re-dissolved polymer affecting the crystallisation of the drug; the Matzger approach employed photo-polymerisation of monomers which avoided this; however their use of a pin-based contact method for printing reduces flexibility and control for the amount of drug in each printed micro-dot. While the innovative approaches described above have demonstrated that a significant reduction in drug sample required for solid form screening is achievable, they also open up a number of questions. None of the studies outlined above addressed the physical stability of the printed drugs as a function of time. Stability is a key parameter in the formulation, affecting (for example) shelf-life of the medicine, requirements for cold-chain distribution, etc. Also, while the approaches above demonstrated an excellent level of miniaturisation, it is not clear at the present time where the limits of sample reduction might lead. Therefore in the current work, we investigate whether the inkjet approach of Bradley and Kazarian is amenable to HT stability screening while employing simpler substrates for printing which are not expected to re-dissolve into the printed drug solution. We also investigate whether a significant reduction in sample quantity is possible by using ultra-low volume pico-litre printing in this context for the first time. For the micro-array approach, due to the small quantities of material in each printed spot, traditional analytical techniques such as x-ray powder diffraction (XRPD) can be challenging at best, and we, therefore, employ both cross-polarised optical microscopy and Raman microscopy



as assays (following Bradley et al. and Matzger et al. for the latter). Both are well-suited to microscopic amounts of sample, and both distinguish rapidly and clearly between amorphous, and crystalline material9,10 Raman microscopy can detect chemical decomposition via the midwavenumber frequency molecular vibrations in the 400-4000 cm-1 range and can also reliably distinguish between solid forms (e.g. amorphous vs crystalline, different polymorphs etc), especially if the low-wavenumber frequency range 10-400 cm-1 is employed10. This lowwavenumber Raman region has not been applied to the analysis of printed micro-arrays to date, and our work therefore also addresses its utility in this respect for the first time. As a model compound, we employ paracetamol, also known as acetaminophen for which several solid forms (amorphous and three polymorphs) have been reported. For the polymorphs, solution crystallisations typically yield form I (which is used in medicines), form II is typically produced by crystallisation from the solid amorphous state from an uncovered sample but has been reported from solution crystallisations3,11,12, form III has mainly been reported to occur when crystallisation from the solid amorphous state takes place under confined conditions, e.g. under a microscope cover slip13, or in a solid-state NMR sample holder14 or in capillaries for powder X-ray microdiffraction (PXRD) analysis15. We note that paracetamol was also used as a model compound by Matzger et al., who identified forms I and II from their high-throughput polymer-induced hetero-nucleation screen which was outlined above3.

Experimental Details Laboratory grade paracetamol was purchased from Sigma-Aldrich and used as supplied; differential scanning calorimetry (DSC) and XRPD data indicated that the as-received form was the common and stable crystalline form I as expected. Magnesium chloride hexahydrate (MgCl2 6H2O) was also purchased from Sigma-Aldrich. Ultrapure water was freshly obtained from a water purification system (ELGA, USF, High Wycombe, Bucks, UK) with a resistivity 18.2 MΩ·cm at 25 °C. As substrates, initially polystyrene (hydrophobic) slides were employed, with a view to printing compact well-formed microdots. These slides were simply constructed from a transparent PS petri dish (Corning, Sigma Aldrich). Gold coated glass slides were subsequently


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employed as described in the text below; these were made by a filament evaporation technique in the University of Nottingham engineering labs. Sample Preparation and Printing A 6 mg/ml solution of paracetamol was prepared in ultrapure water (the aqueous solubility of paracetamol under the conditions employed is expected to be ca. 17 mg/ml16, i.e. our feed-stock solution was well below the saturation solubility). The paracetamol microarray was constructed via an ink-jet printer (Scienion S5 SciFlex Arrayer) equipped with a piezoelectric dispenser. The dispenser was programmed to aspirate the paracetamol sample from the well plate and print 10, 50, 100, and 200 drops of paracetamol in triplicate in a predefined area on the polystyrene slide (Figure 1 A). The micro-dots were separated by 1000 µm, and each droplet ejected from the dispenser had a volume of 245 pico-litres, which was obtained by adjusting the nozzle voltage to 100 V for 55 µs. Volumes of samples dispensed, therefore, ranged from 2.45 to 49 nano-litres, while the amount of paracetamol per printed micro-dot was therefore 14, 73, 147 and 294 nanograms, respectively. The process was programmed to wash the dispenser between each sample spotting using ultrapure water. A second micro-array was printed and stored as outlined above, but using a gold-coated glass substrate rather than polystyrene.

After the droplets had been totally evaporated by leaving the slide on the printer (in less than 1 minute), the slide was placed under an optical microscope and was stored in a closed small chamber. This chamber provides an atmosphere of specific humidity by using of the saturated salt bath. The chamber was constructed from two Petri dishes stacked and sealed above each other. A saturated salt solution of MgCl2 6H2O was placed in the bottom petri dish and two windows were opened between the Petri dishes to allow the humidity circulation as depicted in Figure 1 B. The humidity chamber controlled the humidity to 33% relative humidity17. The slide was stored at room temperature, which in the laboratory is automatically controlled at 23ºC. Each paracetamol micro-dot image was recorded separately under the optical microscope. Raman single point measurements and Raman surface mapping were obtained from the micro-



dots of interest (Figure 1 C and D). The Raman measurement of a recrystallized micro-dot was taken on the day of crystallization, as detected by cross-polarised optical microscopy and visual inspection of bright-field images.

Figure 1 Depiction of the experimental design. (A) Paracetamol microarray was fabricated using inkjet printing technology. (B) The paracetamol microarray slide was stored in humidity chamber that controlled by humidity salt. The two red rectangles are small windows to allow the humidity circulation. (C) The paracetamol recrystallization was monitored under the optical microscope using the Bright-field and cross-polarised mode. (D) Raman single-point measurements and Raman surface mapping were used to assess the crystallization.

The changes in appearance and potential recrystallisation of the printed micro-dots was monitored using a bright-field and cross-polarised microscope for 13 days. The paracetamol microarray was monitored under the microscope every day for any changes, and at day 13 the slide was taken to the Raman spectroscopy for further analysis. Single point measurements and Raman surface mapping were also employed to obtain spectroscopic data from the micro-dots of interest. To obtain paracetamol reference spectra for the form I paracetamol, A Raman spectrum was simply collected of the as-received material, which was shown by XRPD to be form I. For form II and III the established literature protocol was followed13. A sample of crystalline form I (as-


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received paracetamol) was melted between a glass microscope slide and a cover slip at 180 ⁰C and then cooled to room temperature at 30 ⁰C/min to produce amorphous paracetamol. This was then heated until crystals appeared (ca. 70 ⁰C) and then cooled. Spectra were collected from a thin layer of crystals around the edge of the sample, and from spherulites from the centre of the sample, which provided the reference spectra of forms II and III respectively. Optical Microscope Monitoring Bright-field and cross-polarised images were obtained using Prior Lux Pol™ polarising microscope (Prior Lux Pol™, Prior Scientific, Cambridge, UK). Sample focusing of this instrument is achieved by using focusing eyepieces and free objectives, which range from 4× to 40× as required. A CCD camera was mounted above the microscope in order to record the microscopic images and was connected to a computer with Q-capture™ software provided by Qimaging®. This software is able to control the brightness and the light exposure time, plus other important features that enhance the image quality. Bright-field and cross-polarised images were frequently taken of the drug microarray, and the imaging process lasted approximately 30 minutes before the slides were returned to the humidity chamber. Raman Analysis Raman spectra for the drug microarray were recorded using a LabRAM HR system (HORIBA Jobin Yvon, France) and Raman data were acquired using LabSpec 5 software. The instrument was supplemented with an automated X-Y-Z slide holder and was calibrated with the Rayleigh line in the zero position and to position 520.7 cm-1 using a standard silicon wafer. The drug samples were illuminated using a HeNe (785 nm) laser, and the confocal hole was set to 300 µm, and a grating of 600 lines/mm was selected. The spectral range measured was 20-2000 cm-1. Drug microarray micro-dots were located using the automated stage and a 10× microscope. A Raman spectrum was acquired from a single point and multiple points within a micro-dots or mapping of a micro-dot using the 100× objective lens. The spectrum for each micro-dot is the mean of four acquisitions, where each acquisition time/grating position was 5 sec.



Raman mapping data were processed using multivariate curve resolution (MCR) analysis, where the large volume of spectroscopic data was decomposed to individual MCR components. To perform the MCR analysis, the MCR-ALS (alternating least square) module was installed in the open source R program, and median centring and data scaling were performed before applying the MCR analysis to mitigate the effects of variable sample volumes probed by Raman laser spot as a function of position and to focus the analysis on chemical and structural differences. The MCR results provide a "loadings" trace (corresponding to spectral profile) and a "score" (corresponding to concentration or amount of that component) at each XY position for each of the components specified by the operator. The MCR analysis was run repeatedly using randomly generated starting models for the measured data and always converged to give near-identical model parameters for a given number of MCR components, which provides confidence that the results are robust and reproducible.

Results Paracetamol micro-dots on polystyrene (PS) substrate This preliminary experiment was intended to investigate the physical form of the printed drug micro-dots as a function of micro-dot size and to assess their physical stability, employing a hydrophobic substrate as this is expected to give compact final micro-dots of the drug due to the high contact angle of the printed droplets on the surface18. A paracetamol microarray (three repeats of micro-dots of drug mass 14, 73, 147 and 294 nano-grams) was printed on a PS slide using the ink-jet printing method described, and the physical appearance was viewed under an optical microscope after the solvent had totally evaporated. The visual appearance of all the paracetamol micro-dots as-printed on day zero was featureless, glassy and transparent, which indicated that the micro-dots were in the solid amorphous form, as seen in the example of the 147 ng micro-dot shown in Figure 2 A. On the third day of storage at 33% RH, the appearance of one of the three 147 ng micro-dots had changed to a less transparent, more roughened appearance, as shown in Figure 2 B, although at this time none of the 14, 73, or 294 ng microdots had changed in appearance. This observation, coupled with the clear birefringence observed when the micro-dot was viewed through cross polars, indicates that this micro-dot had


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crystallised. On the fifth day, one of the 294 ng micro-dot similarly changed its appearance, again suggesting recrystallisation had occurred (Figure 2 B).

Figure 2 Investigation of the recrystallization of an amorphous paracetamol micro-dot. (A) Bright-field image and MCR analysis (low-wavenumber region) for 147 ng paracetamol amorphous micro-dot. (B) Bright-field image and MCR analysis (low-wavenumber region) for recrystallized 147 ng paracetamol micro-dot. Raman surface mapping was used to explore any had differences in the whole area of the paracetamol micro-dot. Two 147 ng paracetamol micro-dots, one that crystallised at day 3 and one still having a glassy appearance were mapped. 100×100 µm area of the 147 ng amorphous micro-dot and surrounding background (Figure 2 A) was mapped twice (total 1250 spectra and the data acquisition for each spectrum was 0.25 sec) (Figure 2 B). Similar Raman surface mapping was performed on the 147 ng crystallised micro-dot. The Raman mapping was performed first



using the low wave-number region (30-400 cm-1) and then again using the mid-wavenumber region (400-1600 cm-1). For each of the two micro-dot, MCR (multi-variate curve resolution) analysis was employed to simplify the Raman mapping spectroscopic data into two spectral components, assuming that there was a drug micro-dot and the substrate in the area of Raman mapping. Three components were also trialled but provided physically meaningless results (e.g. negative peaks in the loadings trace), which suggests that the two-component model is preferred (this is the analysis presented below). The results from the MCR analysis are presented as loadings which present the spectral information about a specific phase, and the MCR scores are plotted to provide the spatial distribution of the specific phase in the mapped area. In the MCR scores plot, the red colour indicates that the specific phase is present at its maximum and the blue colour indicates the minimum. For the MCR analysis for the glassy 147 ng micro-dot using the low-wavenumber region, both MCR1 and MCR2 showed a broad peak feature (Figure 2 A), which is a universal indicator for amorphous materials [28]. Due to the similarity in the MCR loadings for the two components, the scores plot (Figure 2 A) exhibit an unclear spatial distribution for the paracetamol micro-dot on the PS slide background (PS is also amorphous), and this suggests a strong interference of the PS substrate with the signal from the paracetamol sample. This was supported by a set of singlepoint measurements undertaken on all spots and the substrate, which are available in the supporting information ( Figure S 1 A and B). The MCR analysis of the recrystallised 147 ng micro-dot at the 5-day time-point using the lowwavenumber region (Figure 2 B) showed that the MCR1 loading exhibited a broad peak feature, indicative of amorphous materials, whilst the MCR2 loading exhibited several peaks at low wavenumber, which are indicative of crystalline materials as a result of the periodic arrangement of the molecules within the crystals19. The MCR2 loading showed a similar pattern to the singlepoint measurements described earlier (Figure S 1 C ) and similar pattern to the paracetamol form II reference Raman spectrum presented in the support information (Figure S 1 D). Also The MCR scores plots are shown in Figure 2 B, where MCR1 is the PS slide background and MCR2 is


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associated with the crystalline paracetamol micro-dot. The colour distribution for MCR2 was concentrated in the middle of the micro-dot and gradually decreased towards the edge of the micro-dot, as it has a spherical cap shape and the ratio paracetamol: substrate changes radially across it. The spatial anti-correlation between MCR1 and MCR2 ties in with this interpretation. Data for the Raman mapping using the mid-wavenumber spectral region are shown in Figure 3 for the same two micro-dots (147 ng). Both MCR1 and MCR2 share a dominant peak at 1001 cm-1 that is associated with the PS slide background (see also supporting information Figure S 1 A)20,21. For the glassy micro-dots, the MCR scores plots (Figure 3 A) showed a clear spatial distribution, where the MCR1 scores are concentrated on the background area and the MCR2 scores on the area of the paracetamol amorphous micro-dots. The two MCR loadings both appear to both contain a strong contribution from the PS substrate, with the MCR2 exhibiting a greater number of peaks than MCR1 and therefore also including a contribution from the (amorphous) paracetamol. The MCR analysis for the Raman mapping using the mid-wavenumber region for the glassy microdot (Figure 3 B) showed features associated with PS background in the MCR120, while MCR2 presented features that are similar to the paracetamol form II spectrum (Figure S 1 C and D), such as the dominant peak at 1323 cm-1 which is related to C N stretching vibrations22. The MCR score plots for the crystal micro-dot (Figure 3 B) exhibited a clear spatial distribution of the PS background (MCR1) and the paracetamol (crystalline, MCR2). Similar to the amorphous paracetamol micro-dot, the MCR1 and MCR2 for the crystalline one shared the 1001 cm-1 peak that is associated with the PS background, but this is less intense for MCR2. The use of the strongly Raman-active PS as a substrate clearly causes some significant issues with the analysis, especially for the case where the printed micro-dot is itself amorphous (147 ng glassy), and therefore its spectrum exhibits strong overlap with many of the PS substrate peaks. The presence of a small number of negative peaks in the loading trace for MCR2 (147 ng, glassy) also indicates some problems with the analytical approach and correlating the loadings output directly with real-world reference spectra.



Figure 3 Investigation of the recrystallization of an amorphous paracetamol micro-dot on a polystyrene substrate. (A) Bright-field image and MCR analysis (mid-wave number region) for the 147 ng paracetamol amorphous micro-dot. (B) Bright-field image and MCR analysis (mid-wave number region) for the recrystallized 147 ng paracetamol micro-dot. A similar analysis was carried out for the larger, 294 ng micro-dots, and similar results were obtained; data for the low-wavenumber spectral region are presented in Supporting information (Figure S 2). Upon further storage of the sample (2-3 weeks), all printed micro-dots exhibited a changed appearance and birefringence using cross-polarised optical microscopy, suggesting that they had all crystallised in this time-frame. During the storage period, Raman single point measurements were conducted on randomly selected recrystallized paracetamol micro-dots. Three examples of


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Raman spectra are presented in the SI (Figure S 3). All Raman spectra obtained from the paracetamol micro-dots in the PS substrate were assigned to form II. The precipitation of amorphous paracetamol from the printing step appears reasonable upon consideration of the printing method. The experiment began by dissolving paracetamol form I in water and spotting it at room temperature onto a PS slide. Due to the small volume (nano-litre), the evaporation of the deposited droplets led to fast precipitation of the paracetamol in less than 1 minute. The optical and Raman results presented above indicate that this rapid evaporation produced solid micro-dots which were initially amorphous. Recrystallisation of the two larger ones after several days led not to the most stable polymorph, form I, but to the meta-stable form II. This has been reported to be the form which is produced from uncovered recrystallisation of the amorphous form13,23 and its appearance in our experiments seems to indicate that the crystallisation occurred from the amorphous solid, rather than from solution in the initial dispensed droplets. Any solution-state crystallisation, or indeed the presence of any form I seed crystals in the stock solution, would have been expected to lead to from I, rather than the form II which was observed. A simple stepwise process: solution → (rapid evaporation) → amorphous micro-dot → (humidity and storage) → crystalline form II, therefore, appears to be the simplest explanation of the data observed. Paracetamol micro-dots on gold-coated glass substrate In the previous experiment, the use of a PS substrate resulted in significant contamination of the Raman signal, particularly for amorphous paracetamol. Therefore, in the experiment reported below the substrate was change to a gold coated glass slide, as initial tests indicated that this would provide a very low background substrate with a suitable water contact angle to encourage the formation of compact micro-dots upon solvent evaporation. Gold is also known to potentially enhance the Raman signal via the SERS effect24,25. The same micro-array was printed as earlier, i.e. three repeats of micro-dots having masses 14, 73, 147 and 294 nano-grams. The gold coating reduced the light illumination through the slide but the paracetamol microarray was still visible under a bright-field microscope. The bright-field images (Figure 4) showed glassylooking, featureless paracetamol micro-dots the first day after the printed droplets had



completely dried. Cross-polarised microscopy (also Fig 4) indicated that no birefringence was evident, suggesting that these solid micro-dots are amorphous. On the fifth day of the experiment, the first amorphous paracetamol micro-dot to change appearance was one of the 73 ng repeats, the second to change was one of the 294 ng repeats at day 12, followed by another 294 ng micro-dot at day 13. The cross-polarised images of the recrystallized paracetamol microdots showed clear birefringence for all areas, except for the last crystallised micro-dot at day 13, which at the time of imaging was only partially birefringent (again Figure 4). This is taken as evidence of recrystallisation of these micro-dots. The bright-field images showed that accidentally-included particle contaminants do not obviously influence the nucleation process and start crystal growth in an amorphous micro-dot, as highlighted by the yellow circle shown in Figure 4 which highlights an accidental impurity (black) surrounded by glassy-looking paracetamol, with no detectable crystal growth adjacent.


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Figure 4 Systematic monitoring of an amorphous paracetamol micro-dot recrystallized during the time of the experiment, on a gold-coated glass substrate. Recrystallization of paracetamol was started at day 5. Bright-field images are shown at the left-hand side and the cross-polarised images are shown at the right-hand side.

After the last optical microscope images were taken of the paracetamol microarray at day 13, the slide was taken for further analysis via Raman spectroscopy. Initially, a Raman spectrum was obtained from the gold coated slide (i.e. no paracetamol) which showed two peaks: a very intense one at 44.9 cm-1 and a much weaker one at 56 cm-1 in the low-wavenumber region (very close to the low-wavenumber cut-off in the instrument used), with no noticeable peaks at the molecular region (Figure 5 A) or higher than 56 cm-1. In this respect, the Raman signal from this gold-coated glass substrate is far cleaner than that for the PS substrate described earlier. A



Raman spectrum was then obtained from a 14 ng paracetamol micro-dot which exhibited a glassy visual appearance. The low-wavenumber region in the Raman spectrum (Figure 5 B) displayed the peak associated with the gold slide, then a broad peak (boson peak) indicative of amorphous material. The intra-molecular region displayed the characteristic Raman peaks for amorphous paracetamol without interference from the substrate23. Therefore for the gold-coated glass substrate, only minimal interference with the sample Raman signal is noted, and this is restricted to only two easily-identified peaks at very low wavenumber. This is a significant improvement in this respect upon the polystyrene substrate used in the initial experiments. An additional Raman spectrum was obtained from one of the recrystallized 74 ng paracetamol micro-dots (Figure 5 C). The phonon region exhibited the intense peak associated with the gold slide, then multiple peaks indicative of crystalline material which were more intense than the second, relatively low intensity peak from the gold substrate noted earlier. Interestingly, the peak pattern is obviously different to that observed for crystalline micro-dots in the polystyrene substrate experiments (Figure 2) which were assigned to form II paracetamol, whereas the trace observed for this 74 ng micro-dot when using the gold-coated glass as a substrate showed similar characteristic peaks of the paracetamol form III reference presented in Figure 5 D. Comparison of the data at higher wavenumber support this conclusion, for example the peaks around 1600 cm-1 are quite different for the crystalline samples from the PS and gold-coated substrates (both data sets being also notably different to the spectrum from the amorphous samples), and match well with the reported spectra for forms II and III respectively (e.g. see data in Nanubolu & Burley, 201223). No evidence of any SERS effect was observed in any of the experiments, which is presumably due to a lack of appropriate surface asperities on the gold coating.


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Figure 5 Raman spectra obtained from gold coated glass slide. (A) Gold coated glass slide, (B) 14 ng amorphous paracetamol micro-dots, (C) 73 ng recrystallized paracetamol micro-dots, (D) Paracetamol form III Raman spectrum reference. Left-hand side presents the phonon region and the right-hand side presents the molecular region.



The bright-field and the cross-polarised image for the 294 ng micro-dot on day 13 (Figure 6 A) show that, at the time of imaging, only the upper right corner had crystallised. After removing the sample from the optical microscope and transferring to the Raman system (ca. 5 minutes across campus), the micro-dot appeared to have fully recrystallized (Figure 6 B). Another brightfield and cross-polarized image were taken for the same micro-dot after the complete crystallization which shows an overlapping between two or more crystallisation processes (Figure 6 C). Raman surface mapping was employed in order to further understand how this two-step recrystallization influenced the resultant paracetamol crystal form (Figure 7). Three MCR components were found to be the minimum number required to explain the data. The MCR loadings, as shown in Figure 7, exhibited three distinct areas. MCR1 picked up the gold coated background. Comparison with literature indicated two crystal forms within the micro-dot, form II on MCR2 and form III on MCR3 (Figure 7). The three MCR score plots showed a clear spatial distribution of the substrate and the two paracetamol crystal forms. The distribution of form II (MCR2) was concentrated in the middle of the micro-dot, while that for form III (MCR3) was concentrated around the edges (Figure 7). Intriguingly, this distribution of polymorphs does not appear to link directly with the visual morphology of the micro-dot, in which context we note that external crystal morphology is not always a good predictor of internal crystal structure26,27. This micro-dot was only one in which evidence of form II paracetamol was found, all others recrystallised on the gold-coated slide were found to comprise form III only. Upon further storage of the sample (2-3 weeks) all printed micro-dots exhibited a changed appearance and birefringence using cross-polarised optical microscopy, suggesting that they had all crystallised in this time-frame. During the storage period, Raman single point measurements were conducted on randomly selected recrystallized paracetamol micro-dots. Three examples of Raman spectra are presented in the Supporting information (Figure S 4). All Raman spectra obtained from the paracetamol micro-dots in the gold coated substrate were assigned to form III.


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Figure 6 Bright-field and cross-polarized images showing the recrystallization behaviour of a 294 ng paracetamol micro-dot. (A) Paracetamol micro-dot partially recrystallized before Raman mapping (Figure 7). (B) Optical image taken by the Raman spectrometer at the time of Raman mapping. (C) Bright-field and cross-polarized microscope image after the Raman analysis.



Figure 7 MCR mapping of a paracetamol micro-dot showing the different metastable paracetamol forms in the same 294 ng micro-dot. MCR1 shows the spectrum features of the gold coated glass slide, MCR2 correlates to paracetamol form II, and MCR3 is correlated to paracetamol form III.


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The reason for the appearance of the typically elusive form III paracetamol in the majority of crystallisations for the experiment on the gold-coated slide is not clear. While the experimental conditions employed are certainly novel, given the fact that a similar experiment on the polystyrene substrate produced only form II, the appearance of form III on the gold-coated slide is unexpected. The form III appears to have crystallized directly from the amorphous form. Previous reports of form III have required either crystallisation under a cover slip (or similar)13,23,28, or the presence of both lactose monohydrate and HPMC29. It is not immediately clear that either of these situations might apply in the current case. It is possible that the posited templating effect of form II on gold occurs due to a close match between the form II "b" lattice parameter (reported as 17.17 A at room temperature)30 and a 6x multiple of the atomic spacing in gold (17.24 A, i.e. 3 x sqrt(2) of gold lattice parameter31. However, significant further work would be required to address this.

Conclusions For the first time, nano-gram scale crystallisation of paracetamol has been carried out using a pico-litre inkjet-printing method. An addressable micro-array format was used for the experiments. Using paracetamol as a model drug, the amorphous solid form and the meta-stable crystalline forms II and III were isolated and characterised by low-wavenumber Raman spectroscopy, the stable and most easily accessible form I was not found in any of our experiments. A solid-state crystallisation pathway, with an initial amorphous solid recrystallising, seems to be compatible with our observations. Form II was isolated from experiments using a polystyrene substrate, form III was prevalent when a gold-coated glass slide was used as a substrate, although a single example of a mixed II/III micro-dot was found on the gold-coated substrate. The limit of detection for analysis of these nano-gram scale micro-dots using optical and Raman microscopy was shown to be well below 14 ng but was not explicitly determined.



The main novelty of this work lies in the very small amounts of sample screened for solid-form behaviour (as low as 14 ng in an individual micro-dot), which if developed further may allow for very early-stage solid-form screening of compounds in an industrial drug-discovery pipeline. Significant further development of the outlined micro-array approach is required prior to any translation of this work into pharmaceutical development, with key areas including substrate effects, limits of detection and analysis, and investigation of a wider range of drug molecules than reported here. However, this initial simple investigation appears promising and illustrates the viability of miniaturising solid form screening by several orders of magnitude beyond anything reported previously.

Supporting information The single point Raman measurements of the 147 ng paracetamol micro-dot on PS and the MCR analysis of the recrystallized 249 ng paracetamol micro-dot are included in the supporting information. In addition, the single point measurements of the randomly selected recrystallized paracetamol micro-dots on PS and gold-coated slide.

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For Table of Contents Use Only Establishing a new method to evaluate the recrystallization of nano-gram quantities of paracetamol printed as a microarray using ink-jet printing Mohammed S N Algahtani, Martyn C Davies, Morgan R Alexander, Jonathan C Burley

Synopsis We propose a new method to track and evaluate the recrystallization of a nano-gram quantity drug printed as a microarray (paracetamol as a drug model). The paracetamol microarray was produced by the ink-jet printing technology resulted in amorphous spots. The recrystallization was tracked and evaluated using bright-field and cross-polarised microscopy and evaluated using Raman spectroscopy.


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Establishing a new method to evaluate the recrystallization of nano

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