Feasibility of 1H-High Resolution-Magic Angle Spinning NMR

Mar 28, 2013 - Feasibility of 1H-High Resolution-Magic Angle Spinning NMR Spectroscopy in the Analysis of Viscous Cosmetic and Pharmaceutical ...
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Feasibility of 1H‑High Resolution-Magic Angle Spinning NMR Spectroscopy in the Analysis of Viscous Cosmetic and Pharmaceutical Formulations Mattia Marzorati, Peter Bigler, Michel Plattner, and Martina Vermathen* Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland S Supporting Information *

ABSTRACT: The feasibility of 1H-High Resolution-Magic Angle Spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy for the direct analysis of viscous cosmetic and pharmaceutical formulations such as creams, gels, and pastes is presented. Three examples are described: (i) the detection of chitosan in toothpaste, (ii) the analysis of dexamethasone acetate (DMA) in a cream, and (iii) the analysis of the local anesthetics, lidocaine and prilocaine, in a gel and a cream. All active components could be directly detected in their original commercial formulations without the need for laborious sample preparation steps. In addition, the possibility for HR-MAS-based quantifications and the analysis of dynamic properties of active components in different formulations applying HR-MAS diffusion-ordered NMR spectroscopy are shown.

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tool in the detection and quantification of specific active ingredients. Three examples are presented from the cosmetic and pharmaceutical field, which are aimed at demonstrating the potential of HR-MAS NMR as a direct, fast, and minimally invasive method: (1) The detection of chitosan, a natural polysaccharide, in toothpaste, (2) the detection of dexamethasone acetate, a synthetic glucocortico-steroid, in a cream, and (3) the analysis of local anesthetics in a gel and a cream with respect to their quantity and formulation.

iscous, semisolid samples are characterized by restricted molecular mobility, resulting in broad NMR lines and not well-resolved NMR spectra. High resolution-magic angle spinning (HR-MAS) NMR spectroscopy is a relatively new technique in the field of NMR, which allows for one to obtain high-resolution proton spectra of such samples.1,2 To date, 1HHR-MAS NMR has been applied in several research areas, where semisolid, viscous, and heterogeneous materials are involved, such as biopsy samples,2,3 food material,4−6 solid supports for chemical reactions,7,8 or lipid membranes.9,10 In cosmetics and pharmaceutics, a lot of formulations such as creams, ointments, and toothpaste fall into the regime of viscous, heterogeneous materials well-suited for HR-MAS NMR analysis. However, to date, there are hardly any reports in the literature concerning HR-MAS applications in this field.11 The main advantage of the HR-MAS technique is that it provides direct access to the chemical composition of a sample in its original form without the need of any laborious sample preparation steps. Thus, qualitative and quantitative spectral information on different components in a mixture can be obtained at the same time. This is, in particular, useful in the determination of active ingredients where usually a series of time-consuming and selective steps like extraction or chromatographic separation are required prior to the classical, mostly spectroscopic, experiment. Moreover, extraction or separation procedures can potentially induce unintended modifications in qualitative or quantitative sample composition. The goal of the present work was to probe the feasibility of HR-MAS NMR applied to viscous multicomponent samples like emulsions, tooth paste, and gels as a potential analytical © 2013 American Chemical Society

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EXPERIMENTAL SECTION Materials. Deuterated water (D2O, D 99.9%) and DCl (20% in D2O) were obtained from Cambridge Isotopes Laboratories, Inc. Trimethyl-silyl-3-propionic acid-d4 sodium salt (TSP-d4, D 98%), obtained from Euriso-Top, was used as an internal 1H NMR reference. Lidocaine HCl monohydrate and dexamethasone acetate (DMA) were purchased from Sigma/Aldrich. Kamistad Gel (STADA GmbH, Bad Vilbel, Germany), Emla cream (AstraZeneca GmbH, Wedel, Germany), and Excipial cream (Spirig Pharma GmbH, Egerkingen, Switzerland) were bought in a pharmacy (for detailed composition see the Supporting Information). Excipial cream was spiked with concentrations of 0.01%, 0.05%, and 0.1% w/w DMA. Toothpaste samples with and without chitosan and a chitosan reference were kindly made available to us by GABA International AG, Switzerland. Received: February 21, 2013 Accepted: March 28, 2013 Published: March 28, 2013 3822

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Sample Preparation. For all 1H-HR-MAS measurements, ∼300 mg of the viscous sample (toothpaste, cream, and gel) were mixed with 30 μL D2O (1 mM TSP-d4). Fifty microliters of the mixture was then packed into a 4 mm MAS rotor using a syringe. Toothpaste, Chitosan: a chitosan reference solution was prepared by dissolving 2.5 mg chitosan in 1 mL slightly acidic D2O (0.2% DCl). For toothpaste extracts, ∼500 mg of toothpaste was mixed in a vial with 3 mL of acidic D2O (0.2% DCl) containing 1 mM TSP-d4. The suspension was stirred overnight at room temperature. Successively, the liquid part was separated from the insoluble toothpaste components by centrifugation (5 min at 13000 rpm). Five-hundred and fifty microliters of the clear supernatant was used for the liquid state 1 H NMR analysis. For standard addition experiments, 2 mg of chitosan were directly added to the NMR tube containing a toothpaste extract. Excipial cream, dexamethasone acetate: Chloroform extracts of cream samples were obtained by adding CDCl3 to an aliquot of cream. After vigorous mixing, the samples were filtered and the clear solutions were submitted to liquid state NMR spectroscopy. Kamistad Gel, lidocaine: For quantitative 1H-HR-MAS NMR measurements, an exact amount (∼300 mg) of Kamistad Gel was mixed with an exact amount (∼30 mg) of D2O (1 mM TSP-d4) and the homogeneous mixture was then packed into a 4 mm MAS rotor. For the calibration curve, 5 lidocaine reference solutions of known concentrations in the range between 0.5 and 5% w/w were prepared in D2O. For 1H-HR-MAS measurements, the exact amount of sample transferred to the rotor was determined by controlling the weight difference of the filled and the empty MAS rotor. The integral values and the calibration curve are shown in Tables S-1 and S-2 and Figure S-1 of the Supporting Information. HR-MAS NMR Spectroscopy. All HR-MAS NMR experiments were performed on a Bruker Avance II spectrometer, operating at a resonance frequency of 500.13 MHz for 1H nuclei and 125.77 MHz for 13C nuclei. The instrument was equipped with a 4 mm HR-MAS dual inverse 1H/13C probe with a magic angle gradient. The samples were spun at 5000 Hz, and the temperature was set to 303 K, if not mentioned differently. Liquid State NMR Spectroscopy. The liquid state NMR experiments were performed on Bruker Avance II spectrometers operating at resonance frequencies of 400.13 and 500.13 MHz for 1H nuclei, respectively, each equipped with a 5 mm dual probe (BBI) for inverse detection with a z-gradient coil. All pulse programs were obtained from the Bruker pulseprogram library. Detailed acquisition parameters for HR-MAS and liquid state NMR spectra are given in the Supporting Information. Postprocessing of all spectra included Fourier transformation of the coadded free induction decays (FIDs) after exponential multiplication (line broadening factor typically 1 Hz), phase, and baseline correction.

a qualitative detection method of chitosan as novel active ingredient in toothpaste, the aim of this study was to set up a procedure using 1H NMR spectroscopy. Both techniques, liquid state 1H NMR of a toothpaste extract and 1H-HR-MAS NMR of toothpaste were applied and compared. As stabilized suspension of inorganic solids in a liquid phase, toothpaste is a mixture of components with different solubility properties. Analysis of chitosan by liquid-state NMR required an extraction with slightly acidic water (see Experimental Section for details). Two otherwise identical toothpaste samples without and with 0.5% (w/w) chitosan (samples A and B, respectively) were analyzed. Compared with a chitosan reference solution, the resonances overlapped with those of the toothpaste, except for one isolated singlet at ∼2 ppm (Figure S2 of the Supporting Information), which was assigned to the methyl protons of the N-acetyl group.16 This resonance was also visible in the spectra of the water-soluble fraction of chitosan containing toothpaste (sample B) and a blank toothpaste spiked with chitosan, while it was absent in the chitosan-free sample A (Figure 1A). Applying a 2D NOESY-

Figure 1. (A) Details of the liquid state 1H NMR spectra of the watersoluble fraction of (a) toothpaste A (without chitosan), (b) toothpaste B (with chitosan), (c) toothpaste A spiked with chitosan, and (d) chitosan reference solution in acidic D2O (from bottom to top). (B) Details of the 1H-HR-MAS NMR spectra of (e) toothpaste A (without chitosan), (f) toothpaste B (with chitosan), and (g) chitosan reference solution in acidic D2O (from bottom to top). The chitosan N-acetylmethyl resonance region is highlighted.

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RESULTS AND DISCUSSION Chitosan. Chitosan, a natural linear polysaccharide composed of D-glucosamine and N-acetyl-D-glucosamine units, is an interesting ingredient for toothpaste formulation because of its rheological properties, its tendency to adhere to negatively charged surfaces like dental enamel, and for its known beneficial tissue regenerating and wound healing properties.12−15 Due to the lack of chromophores, saccharides including chitosan are not directly accessible by the commonly applied UV spectroscopic methods. Inspired by the demand for

based NMR-fingerprinting method previously developed for structurally similar heparin compounds,17 additional sugar protons of chitosan, correlating with the N-acetyl protons, were detected to further prove the presence of chitosan (Figure S-3 of the Supporting Information). In a second approach, HR-MAS NMR was assessed for the direct analysis of toothpaste, omitting the need of any sample preparation steps except for the addition of a small amount of lock solvent commonly used for magnetic field stability. In 3823

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Figure 2. Structure of dexamethasone acetate (DMA) with ring-A protons visible in the 1H NMR spectra indicated. (A) Liquid state 1H NMR spectrum of a CDCl3 extract of cream B (with 0.1% DMA) recorded at RT, (B) 1H-HR-MAS NMR spectra recorded at RT of cream B (with 0.1% DMA) and at (C) 343 K of cream A (without DMA), (D) cream B (with 0.1% DMA), (E) cream C (with 0.05% DMA), and (F) cream D (with 0.01% DMA).

Figure 1B, the detailed 1H-HR-MAS NMR spectra of toothpaste samples A and B and a chitosan reference are shown (see Figure S-2 of the Supporting Information for a larger spectral range). The toothpaste spectra are identical apart from the resonance at 1.9 ppm present in sample B (with chitosan) but not in sample A (without chitosan). On the basis of the liquid-state NMR experiments and a corresponding HRMAS 2D NOESY (Figure S-3 of the Supporting Information), this resonance, assigned to the N-acetyl-methyl protons of chitosan, can be used as a directly accessible marker for the presence of chitosan. The one-step HR-MAS analysis was tested on additional chitosan-containing toothpaste samples. In all samples, the chitosan marker peak could readily be detected (Figure S-4 of the Supporting Information). Thus, HR-MAS may provide a fast screening method for chitosan in toothpaste, which may be useful in monitoring production processes or in quality control of the products. At the same time, other toothpaste components giving rise to proton signals can be measured in their original environment without the need for extraction. Even an NMR-based quantification involving a deconvolution step as previously reported for heparin compounds18 might be possible. Dexamethasone Acetate. Dexamethasone acetate (DMA) is a synthetic glucocorticoid with anti-inflammatory effects, which can be used for topical applications as an active component in creams or ointments. Such pharmaceutical cream formulations containing topical steroids are widespread and play an important role in dermatology. The most frequently used analytical method for measuring steroids in pharmaceut-

ical drugs is RP-HPLC, sometimes with the need of additives, combined with UV-spectrometric detection. For ointment and cream formulations, the analysis is usually preceded by extraction with different solvents.19 In search of rapid screening methods in this field, liquid state NMR has recently been shown as an effective analytical method to monitor counterfeit corticosteroids in creams following extraction.20 To probe the potential of HR-MAS NMR, test samples have been prepared using a commercial cream (Excipial) based on an oil-in-water emulsion. From this control cream (cream A), 3 samples were taken and spiked with 0.1% (cream B), 0.05% (cream C) and 0.01% w/w (cream D) of dexamethasone acetate. For comparison, chloroform extracts were prepared from aliquots of creams A and B. In Figure 2A, a part of the liquid state 1H NMR spectrum obtained from the CDCl3 extract of cream B is shown. From comparison with the extract of the nonspiked control sample (Figure S-5 of the Supporting Information), the resonances highlighted in the spectrum were assigned to the ring-A protons of DMA (Figure 2). The 1H-HR-MAS NMR spectra directly measured on the neat cream samples are shown in Figure 2B− F. At room temperature, the resonances of DMA could not be detected in the spiked cream B. In addition, the resonances appeared relatively broad (Figure 2B). However, increasing the temperature to 343 K yielded a much better resolution and peak narrowing (Figure 2C−F), and the DMA peaks appeared in the spiked cream samples B, C, and D (Figures 2D−F). Assignment of the steroid ring-A protons in the cream were confirmed by 2D HR-MAS TOCSY and HMQC experiments 3824

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Figure 3. 1H-and DOSY HR-MAS NMR spectra at RT of (A) Kamistad Gel and (B) Emla cream. The resonances of lidocaine and prilocaine are marked with a star and rhombus, respectively. The aromatic signals of lidocaine (highlighted) were used for lidocaine quantification in Kamistad Gel. Red arrows indicate lidocaine or prilocaine resonances with diffusion coefficients along the red line in the DOSY spectra. P: polymer, L: lipid signals.

Lidocaine and Prilocaine. Lidocaine and prilocaine are amino-amide-type local anesthetics (LA) which are used in creams and gels for topical application. They bear ionizable groups, thus, their solubility can be controlled toward either aqueous or more hydrophobic phases. As opposed to the predominantly hydrophobic steroids like DMA, the local anesthetics still give rise to well-resolved NMR spectra at room temperature.10,23,24 In this study, two different LA formulations were analyzed. One is a water-based gel (Kamistad Gel) containing lidocaine in its protonated water-soluble form. The other is an oil-in-water emulsion-based cream (Emla cream) containing a eutectic mixture of lidocaine and prilocaine associated with an acrylic acid polymer (carbomer 974P) for transdermal drug delivery. Both formulations were directly submitted to HR-MAS NMR spectroscopy. The corresponding 1 H-spectra are shown in Figure 3. In the gel (Figure 3A), the lidocaine resonances could readily be identified and assigned based on the literature data25 and reference spectra (Figures S-7 and S-8 of the Supporting Information). Likewise, in the cream (Figure 3B), the resonances of both lidocaine and prilocaine could be unambiguously detected and assigned based on literature23 and 2D COSY data (Figures S-9 and S-10 of the Supporting Information). Direct Quantification of Lidocaine in the Gel. Since NMR peak areas are linearly dependent on the concentration of the respective compounds, NMR spectroscopy may also serve as a direct quantitative tool.26,27 This is particularly of interest in cases where nonoverlapping peak regions exist. Thus, the lidocaine content in the gel can be determined by peak integration of its aromatic proton resonances highlighted in Figure 3A. While in liquid state NMR, exact volumes can be transferred to the NMR tube, MAS rotors are usually filled and then closed with an insert that allows releasing an overflow to

at increased temperature (Figure S-6 of the Supporting Information) and were in agreement with the literature data.21 The remaining DMA resonances were not visible in the cream samples, most likely due to overlapping with resonances of other cream components. The NMR invisibility of DMA resonances at room temperature must be attributed to the DMA encapsulation in the lipid phase. Due to its hydrophobic properties DMA is associated with lipid droplets or liposomes in the o/w emulsion, which is a common formulation for topical corticosteroid creams.22 Temperature increase to 343 K destabilizes the emulsion, most likely resulting in drug release. Both, drug-carrier dissociation and increased thermal mobility contribute to the NMR signal narrowing and resolution enhancement. Thus, simple one-dimensional 1D 1H-HR-MAS NMR at elevated temperature allows the direct detection of DMA in a lipid-encapsulated cream formulation along with the other cream components in a short amount of time (i.e., ca. 10 min). For 0.05% w/w DMA, which is a common concentration in commercial steroid creams (0.05−0.1%),20 a very good signal-to-noise ratio could be obtained within a 25 min measurement time, corresponding to the acquisition of 256 scans (Figure 2E). Doubling the experiment time allows detecting DMA amounts down to 0.01% w/w unambiguously (Figure 2F). The NMR analysis may also be well-suited for quantification of DMA via the peak integration method, since there is no signal overlap in the spectral region between 6 and 6.5 ppm. In their liquid state NMR study, McEwen et al. were able to differentiate 16 different corticosteroids based on their signals appearing in the spectral region between 6 and 7 ppm.20 As a follow up to this work, HR-MAS NMR allows analyzing the same resonances without the need for prior extraction or separation, thereby avoiding potential modifications or quantitative losses. 3825

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required for evaluation in each individual case, the examples presented in this paper demonstrate the feasibility of HR-MAS NMR for the direct analysis of active components in creams, pastes, and gels with respect to their identification, quantification, and formulation. Despite its relatively low sensitivity, NMR is well-suited to detect sample amounts within reasonable measuring times, at least down to 0.01% w/ w, which is sufficient for the common concentration range of active components27 (0.01−0.2% w/w) in most viscous cosmetic and pharmaceutical formulations. Evolved from successful applications of liquid state NMR, HR-MAS NMR may thus become a promising new tool in this field.

avoid air enclosures. Therefore, known amounts of the gel and lock solvent (∼10% D2O) were mixed homogenously before being transferred to the MAS rotor. The sample amount inside the rotor was then controlled via its weight. Finally, 1H-HRMAS NMR spectra were recorded, and the absolute integral values of the signals between 7.05 and 7.25 ppm were determined. Three gel samples were analyzed, yielding 23.21, 20.00, and 20.97 mg/g lidocaine, based on linear regression of a lidocaine calibration curve (Figure S-1, Tables S-1 and S-2 of the Supporting Information). The resulting average lidocaine content of 21.40 ± 1.65 mg/g (average ± SD) was in good agreement with the value declared by the producer (20 mg/g). While additional measurements would be required for the validation and evaluation in each specific case, our basic approach demonstrates the feasibility of HR-MAS NMR for quantitative analyses in analogy to existing methods based on liquid state NMR.26,27 Dynamic Properties of Lidocaine and Prilocaine in Different Formulations. As exemplified by the two different samples investigated in this study, a water-based gel and cream, LAs are prepared in different formulations, depending on their field of application. While the application to the gingiva (as in Kamistad Gel) requires a water-soluble formulation for immediate disposure of the LA, sustained release of the LA over a certain period of time is often desired in creams applied to the skin (as in Emla cream). The latter can be achieved by drug delivery vehicles like liposomes or polymeric hydrogels, such as carbomer 974P, an acrylic acid-based polymer component in Emla cream. The application of HR-MAS diffusion-ordered NMR spectroscopy (DOSY) allows one to directly determine the dynamic properties (i.e., the diffusion coefficients of the drug compounds in their original environment). In Figure 3, the HR-MAS DOSY spectra of (A) Kamistad Gel and (B) Emla cream are shown with the diffusion coefficients (D) given on the ordinate. In the gel, the lidocaine signals have D values of 0.6 × 10−9 m2/s, while the polymer signals (marked with “P”) clearly diffuse slower (D values of 0.15 × 10−9 m2/s). In the cream, diffusion coefficients of polymers, lipids, and the drug molecules lidocaine and prilocaine are all on the same order (D values of 0.15 × 10−9 m2/s). This indicates that the LAs are encapsulated in the polymer phase adopting similar slow diffusion properties. Thus, HR-MAS DOSY is an efficient tool to obtain direct information on the formulation of a drug as opposed to time-consuming separation techniques such as size exclusion chromatography.

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +41 31 631 3948. Notes

The authors declare no competing financial interest.

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REFERENCES

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CONCLUSION Compared to conventional methods usually requiring sample preparation steps like extraction or separation procedures, HRMAS NMR allows the direct analysis of materials like creams and ointments, as shown with three exemplary studies. While NMR spectrometers equipped with HR-MAS probes are not yet a common part of the equipment in most analytical laboratories, it is a relatively small step going from the conventional liquid state to HR-MAS NMR, since the same magnet systems, similar operation procedures, and pulse programs can be used. Thus, HR-MAS NMR combines its feature as a minimally invasive, direct method with the advantages of conventional NMR, like providing access to structural and dynamic properties, the possibility for quantitative analyses, or the detection of single components in a mixture and the simultaneous nontargeted detection of potential adulterations. While additional studies would be 3826

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(18) Beyer, T.; Diehl, B.; Randel, G.; Humpfer, E.; Schaefer, H.; Spraul, M.; Schollmayer, C.; Holzgrabe, U. J. Pharm. Biomed. Anal. 2008, 48 (1), 13−19. (19) Görög, S. J. Pharm. Biomed. Anal. 2011, 55 (4), 728−743. (20) McEwen, I.; Elmsjo, A.; Lehnstrom, A.; Hakkarainen, B.; Johansson, M. J. Pharm. Biomed. Anal. 2012, 70, 245−250. (21) Ohta, T.; Zhang, H. Y.; Torihara, Y.; Furukawa, I. Org. Process Res. Dev. 1997, 1 (6), 420−424. (22) Pierre, M. B. R.; Costa, I. D. M. Arch. Dermatol. Res. 2011, 303 (9), 607−621. (23) Cabeça, L. F.; Pickholz, M.; de Paula, E.; Marsaioli, A. J. J. Phys. Chem. B 2009, 113 (8), 2365−2370. (24) Cabeça, L. F.; Figueiredo, I. M.; de Paula, E.; Marsaioli, A. J. Magn. Reson. Chem. 2011, 49 (6), 295−300. (25) Fernandes Fraceto, L.; Spisni, A.; Schreier, S.; de Paula, E. Biophys. Chem. 2005, 115 (1), 11−18. (26) Holzgrabe, U.; Deubner, R.; Schollmayer, C.; Waibel, B. J. Pharm. Biomed. Anal. 2005, 38 (5), 806−812. (27) Malet-Martino, M.; Holzgrabe, U. J. Pharm. Biomed. Anal. 2011, 55 (1), 1−15.

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