Nuclear Quadrupole Resonance: A Technique to Control Hydration

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Nuclear Quadrupole Resonance: A Technique to Control Hydration Processes in the Pharmaceutical Industry Silvina Limandri,† Claudia Vis~novezky,† Silvina C. Perez,† Clemar A. Schurrer,† Alberto E. Wolfenson,*,† Maribel Ferro,‡ Silvia L. Cuffini,‡ Joel Gonc-alves de Souza,§ F. Armani Aguiar,§ and C. Masetto de Gaitani§ †

Facultad de Matematica, Astronomía y Física, Universidad Nacional de Cordoba, IFEG-CONICET, Medina Allende s/n, Ciudad Universitaria, 5000 C ordoba, Argentina ‡ Subsecretaría CEPROCOR, Ministerio de Ciencia y Tecnología-Cordoba, Alvarez de Arenales 230, Cordoba, Argentina § Departamento de Ci^encias Farmac^euticas, Faculdade de Ci^encias Farmac^euticas de Ribeir~ao Preto, Universidade de S~ao Paulo, Av. do Cafe s/n Ribeir~ao Preto-SP CEP 14040-900 ABSTRACT: Pharmaceuticals can exist in many solid forms, which can have different physical and chemical properties. These solid forms include polymorphs, solvates, amorphous, and hydrates. Particularly, hydration process can be quite common since pharmaceutical solids can be in contact with water during manufacturing process and can also be exposed to water during storage. In the present work, it is proved that NQR technique is capable of detecting different hydrated forms not only in the pure raw material but also in the final product (tablets), being in this way a useful technique for quality control. This technique was also used to study the dehydration process from pentahydrate to trihydrate.

T

he vast majority of pharmaceuticals are administered as solid dosage forms. The active pharmaceutical ingredient (API) can exist in many different solid forms (polymorphs, solvates, amorphous, and hydrates),1,2 having different physical and chemical properties. Differences in crystal packing energies produce different dissolution rates and solubilities, which may have implications on the absorption of the active drug. Particularly, it is well-known that pharmaceutical solids can be in contact with water during the manufacturing process (crystallization, wet granulation, aqueous film coating, etc.) and can also be exposed to water during storage (atmosphere containing water vapor).1,3 This water contact could give rise, in some cases, to a solid-solid transition to a hydrate form even under standard conditions (25 °C and relative humidity (RH) even below 60%). Not long ago, it was showed that nuclear quadrupole resonance (NQR) can be used to identify and quantify different polymorphs in pharmaceutical agents.4-7 It can be used to detect signals from solids containing nuclei with spin quantum number I > 1/2 (14N, 35Cl, 79Br, 127I, 23Na). NQR is nondestructive, highly specific, and noninvasive and it requires no static magnetic field.8 Moreover, excipients do not modify the NQR spectrum in most cases as it happens in powder XRD and solid-state NMR as well. In the present work, this technique is used to detect hydrated forms, not only in pure raw materials, but also in the final product (tablets), showing that NQR is a technique that can be used in quality control during the manufacturing process or storage. To demonstrate the potentiality of this technique, diclofenac sodium was used as a test drug because it can exist in different solid forms9,10 (anhydrous, trihydrate, 4.75 pentahydrate, and r 2011 American Chemical Society

acid), but the active pharmaceutical ingredient is the anhydrous form (DS). Besides, the trihydrate form was accidentally found on the Italian market as the active pharmaceutical ingredient, suggesting that this lot probably originated from an improper control during manufacturing.10 Finally, it is demonstrated that NQR can also be used to study dehydration processes in order to determine the temperature at which the process is more effective.

’ MATERIALS AND METHODS Diclofenac sodium tablets of 50 mg, under study in this work, were purchased from pharmacies or were provided by pharmaceutical companies in blister packs. They belong to nine Argentinean and three Brazilian companies. In some cases, the raw material used in the manufacturing process was provided by the company to be analyzed. Diclofenac sodium 4.75 hydrate (DSH) and diclofenac sodium acid (DSA) were obtained from dichlofenac sodium provided by Calao Resfar Division, following well-known procedures.11,12 Diclofenac sodium trihydrate (DSH3) was obtained by heating 2 g of DSH (in a Petri cell) in an oven at 45 °C for 45 min and then by keeping the sample at 27 °C for 1 day. The samples did not need a specific preparation for NQR measurements. Raw material containers are glass cylinders of 2 cm in length and 1 cm in diameter, while the commercial tablets were wrapped with Teflon tape in packs of 5 tablets each. Received: November 25, 2010 Accepted: January 7, 2011 Published: February 11, 2011 1773

dx.doi.org/10.1021/ac103106y | Anal. Chem. 2011, 83, 1773–1776

Analytical Chemistry

ARTICLE

Figure 1. (a) NQR spectra and (b) X-ray diffraction patterns corresponding to pure DS, DSH, DSA, and DSH3.

Figure 2. (a) NQR spectra and (b) X-ray diffraction patterns of AP1, AP2, AP3, and BP1 samples.

Table 1. NQR Frequencies of Pure Solid Forms of Diclofenac Sodium compound 35

Cl NQR frequency [MHz]

DS

DSH

DSA

34.972

35.459

35.625

35.253

35.496

DSH3 35.472 35.547

35.429 35

Cl NQR measurements were done using a Fourier transform pulse spectrometer with a Tecmag NMRkit II multinuclei observe unit and a Tecmag Macintosh-base real time NMR station. The line shape was obtained using spin-echo Fourier transform mapping spectroscopy.13 The measurements were made upon the echo using the standard two pulse sequence (π/2 - 100μs - π). The average number was 10 000 and π/2 = 15 μs. The repetition rate was 50 ms, and all measurements were done at 300 K. X-ray diffraction (XRD) patterns were recorded on a Bruker D8 Advance diffractometer using a CuKR tube (λ = 1.5418 Å). Dissolution profiles of enteric diclofenac sodium tablets (none of them with controlled release declared) were done using a Hanson Research Dissolutor with the Dissolution Apparatus I employing 900 mL of dissolution medium at 37 ( 0.5 °C and at 50 rpm. Two dissolution media were chosen in such a way to cover pH physiological conditions: simulated intestinal fluid (SIF) without pancreatin (pH 6.8) and phosphate buffer solution, 0.2 mol/L (pH 4.5), according to the The United States Pharmacopoeia (USP).14 Each dissolution experiment was performed in duplicate. Quantitative analysis of dissolution in diclofenac sodium samples was performed using a Shimadzu chromatograph (Japan), composed of a pump model LC10AS, a Rheodyne model 7125 injector with 20 μL sampler, a detector for absorption in the UV-vis, model SPD-10A, operating in 282 nm, and a Shimadzu integrator model C-R6A (Japan), using the software Class-VP for obtaining and analyzing data. For these analyses was used a RP-18 column (125 mm  46 mm, 5 mm particles) (LiChroCART, 125-4, Merck) protected by a column RP-18 (4.0 mm  4.0 mm) capped (Merck).

’ RESULTS AND DISCUSSION The characteristic NQR spectra of DS, DSH, DSH3, and DSA are shown in Figure 1a and their frequencies are depicted in Table 1. Figure 1b shows the X-ray powder diffraction patterns of diclofenac sodium in these different solid forms. These diffraction

Figure 3. NQR spectra corresponding to samples (a) AP4, AP5, AP6, AP7 and (b) AP8, AP9, BP2, BP3.

patterns were consistent with those previously reported,10,15,16 and they are sufficiently distinct to characterize each crystalline form. According to the characteristics of their NQR spectra, the samples from Argentina and Brazil, called as AP and BP respectively, can be classified in three groups. In Figure 2a are shown the NQR spectra of those samples (AP1, AP2, AP3, and BP1), where the frequencies detected correspond only to DS. In AP1 and BP1 samples, a very small amount of API is in DSH form. The corresponding X-ray diffraction patterns are also shown in Figure 2b. From the NQR spectra it is clear that the active agent is found in the anhydrous form, while this identification is not possible from X-ray patterns because they are affected by the presence of excipients. In Figure 3a, the NQR spectra of the samples AP4, AP5, AP6, and AP7 show other peaks besides the peaks corresponding to the frequencies of DS. These spectra indicate the presence of a mixture of solid forms in the final product. It can be concluded then that the active agent in the samples AP4 and AP7 is a mixture of DS and DSH3, while the AP5 and AP6 samples are a mixture of DS and DSH. Figure 3b shows the NQR spectra of the samples AP8, AP9, BP2, and BP3. In AP8, the active agent is a mixture of DSH and DSH3, while in samples AP9 and BP2, the API is completely in the form DSH3. The BP3 spectrum shows a single resonance peak whose frequency corresponds to a DSA. In all these samples the anhydrous form is not detected The analysis of NQR spectra in Figure 4 shows some interesting facts: (a) Although AP4 and BP2 are manufactured by the 1774

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Analytical Chemistry

Figure 4. (a) NQR spectra of samples AP4 and BP2 produced by the same company in different countries and two different batches of the AP9 sample. (b) NQR spectra of tablets with different doses of API produced by the same company. AP8_75A: spectrum after a drying process of 60 h at 60°. AP8_75B: spectrum after a drying process of 48 h at 70°.

Figure 5. NQR line intensity of DSH as a function of time: (b) T = 50 °C, (1) T = 49 °C, and (9) T = 48 °C.

same company in different countries, the API in them is in different solid forms. In AP4, a mixture of DS and DSH3 is observed, while in BP2 only the DSH3 is observed(see Figure 4a). (b) A comparative study of the NQR spectrum of pills corresponding to two different batches (called AP9_A and AP9_B), together with the raw material used in their manufacturing, shows that the final product is quite different even when the manufacturing process is the same. Although the raw material is in the anhydrous form, the API in sample AP9_A is in the DSH3 form and the API in sample AP9_B is a mixture of DS and DSH3. Thus, it is possible to assume that the hydration process takes place at some stage of the manufacturing process of the tablets (Figure 4a). (c) Figure 4b shows the NQR spectra of tablets, with different doses of diclofenac sodium, which were produced by the same company (AP8_50 mg and AP8_75 mg). It can be observed that the API is found in different solid forms, even when they were produced using the same excipients and following the same manufacturing process. The API in AP8_50 mg tablets is a mixture of the DSH and DSH3 forms and the API in AP8_75 mg is completely in the DSH3 form.

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Figure 6. Dissolution profiles of tablets at (a) pH 6.8 (0) AP1, (Δ) AP4, (() AP5, (9) AP8, (b) BP1, (1) BP2, (O) BP3 and (b) pH 4.5 (9) AP8, (b) BP1, (1) BP2.

The above results demonstrate the ability of NQR spectroscopy to detect hydrated forms in final products, showing that it could be used for quality control in the pharmaceutical industry. Other useful application of NQR is the possibility to study hydration or dehydration processes in order to know the conditions at which they take place. As an example of this, the dehydration process from DSH to DSH3 has been studied in the present work. Curves of isothermal transformation for three different temperatures were analyzed. It is well-known that the area under the NQR line shape is proportional to the number of the observed nuclei and its central frequency is typical of the crystalline phase; in this case it characterizes the hydration degree.17 In an isothermal experiment, the NQR line width does not change over time, then the line amplitude is proportional to the volume fraction transformed. In the present work, the amplitude of the pure DSH signal was measured as a function of time, at three different temperatures. In Figure 5 it is possible to observe that the transformation from DSH to DSH3 is an exponential process with a dehydrating time constant of 30 min at 50 °C, 101.5 min at 49 °C, and 1128 min at 48 °C. The amount of sample used in each case was 600 mg. In a similar way it is possible to study the dehydration process from the DSH3 to the DS. Finally we would like to point out that the ability of NQR technique to detect solid forms in the final products could be of great use in combination with studies of dissolution profiles. For example, in the case of diclofenac sodium, intrinsic dissolution studies reported by Bartolomei9 have demonstrated that, at pH 4.5 and pH 6.8, the anhydrous form has a higher dissolution rate than the hydrated forms, with the DSH3 more soluble than DSH. On the other hand, studies on the dissolution profiles of diclofenac sodium prolonged release tablets have shown that the release characteristics change considerably among different manufacturers and that even identical formulations show dissimilar release profiles.18 In these cases NQR could be useful to see if these changes in dissolution rates are related with the API hydration. For example, Figure 6a shows the average release profiles for tablets AP1, AP4, AP5, AP8, BP1, BP2, and BP3 in SIF pH 6.8 between 0 and 60 min. It is observed that API release in AP8 and BP2 is higher than in all the other samples up to 40 min. This result is interesting, since these two samples only have hydrate forms in its composition, while the other samples have mainly the anhydrous form and, as it was stated before, the hydrated forms are expected to be less soluble than the anhydrous form. This inversion in the dissolution behavior between anhydrous and hydrated forms observed in tablets was also seen in the dissolution profiles at pH 4.5 (see Figure 6b). 1775

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’ CONCLUSIONS From the analysis of 35Cl NQR spectra, different hydrated forms and mixtures of them were identified in diclofenac sodium tablets. This enables to show that the use of the NQR technique not only makes it possible to distinguish different polymorphs (as it was stated in previous works4,5) but also identifies different degrees of hydration and mixtures in the final product. It is also shown that this technique can be useful to determine the best condition at which hydration or dehydration takes place and that it could be of great help to explain the differences in dissolution rates that sometimes are observed in final products. These properties make NQR a useful technique for quality controls of API during the manufacturing process, from raw material up to the final product. This is relevant in preformulation’s studies and final products in order to ensure identical batch-to-batch quality, bioavailability, and bioequivalence of finished products.

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(17) Meriles, C.; Perez, S.; Schurrer, C.; Brunetti, A. Phys. Rev B 1997, 56, 14374–14379. (18) Bertocchi, P.; Antoniella, L.; Valvo, L.; Alimonti, S.; Memoli, A. J. Pharm. Biomed. Anal. 2005, 37, 679–685.

’ AUTHOR INFORMATION Corresponding Author

*Phone: 54 351 4334051. Fax: 54 351 4334054. E-mail: wolf@ famaf.unc.edu.ar.

’ ACKNOWLEDGMENT A.E.W. and S.L.C. are fellows of CONICET, Argentina. The authors want to thank SeCyT-UNC, CONICET, Fapesp, CNPq, Fundacion Sauberan, and Ministerio de Cienciay Tecnología de Cordoba for financial support. ’ REFERENCES (1) Guillory, J. K.; Britain, H. G. Polymorphism in Pharmaceutical Solids; Marcel-Dekker: New York, 1999; pp 202-208. (2) Hilfiker, R.; Blatter, F.; von Rauner, M. Polymorphism in the Pharmaceutical Industry; Wiley-VCH: Weinheim, Germany, 2006; pp 1-19. (3) Haleblian, J. K. J. Pharm. Sci. 1975, 64, 1269–1288. (4) Perez, S.; Cerioni, L.; Wolfenson, A.; Faudone, S.; Cuffini, S. Int. J. Pharm. 2005, 298, 143–152. (5) Balchin, E.; Malcolme-Lawes, J.; Poplett, I. J. F.; Rowe, M. D.; Smith, J. A. S.; Pearce, G. E. S.; Wren, S. A. C. Anal. Chem. 2005, 77, 3925–3930. (6) Latosinska, J. N. Expert Opin. Drug Discovery 2007, 2, 225– 248. (7) Tate, E.; Althoefer, K.; Barras, J.; Rowe, M. D.; Smith, J. A. S. Anal. Chem. 2009, 81, 5574–5576. (8) Das, T. P.; Hahn, E. L. Nuclear Quadrupole Resonance Spectroscopy, Solid State Physics, Suppl. 1; Academic Press: New York, 1958. (9) Bartolomei, M.; Bertocchi, P.; Antoniella, E.; Rodomonte, A. J. Pharm. Biomed. Anal. 2006, 40, 1105–1113. (10) Bartolomei, M.; Rodomonte, A.; Antoniella, E.; Minelli, G.; Bertocchi, P. J. Pharm Biomed. Anal. 2007, 45, 443–449. (11) Gabboun, N. H.; Najib, N. M.; Ibrahim, H. G.; Assaf, S. Int. J. Pharm. 2001, 212, 73–80. (12) O’Brien, F. E. M. J. Sci. Instrum. 1948, 25, 73–76. (13) Bussandri, A.; Zuriaga, M. J. Magn. Reson. 1998, 131, 224– 231. (14) The United States Pharmacopeia, Vol. 30; The United States Pharmacopeia Convention, Inc.: Rockville, MD, 2008. (15) Llinas, A.; Burley, J. C.; Box, K. J.; Glen, R. C.; Goodman, J. M. J. Med. Chem. 2007, 50, 979–983. (16) Perlovich, G. L.; Surov, A. O.; Hansen, L. K.; Bauer-Brandl, A. J. Pharm. Sci. 2007, 96, 1031–1042. 1776

dx.doi.org/10.1021/ac103106y |Anal. Chem. 2011, 83, 1773–1776