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Tizanidine hydrochloride is an α2-adrenergic agonist used for the symptomatic relief of spasticity associated with multiple sclerosis or with spinal cord injury or disease. The objective of this study was to develop an isocratic, robust and sensitive ultra-high performance liquid chromatography method using UV detection for use in a project to develop a transdermal therapeutic system to deliver tizanidine across the skin. Isocratic separation was achieved using a C18 column and a mobile phase comprising a 80:20 mixture of 0.004% trifluoroacetic acid in water and MeCN (pH* 3.2) at a flow rate of 0.2 mL min−1. Tizanidine eluted at 1.499 min and the total run time was 2 min. The method was specific, robust and the response was accurate, precise and linear from 17.4 to 290 ng mL−1. In contrast to existing methods, the method developed here was validated over a concentration range so as to include the low concentrations frequently observed in transdermal permeation studies and in samples extracted from the cutaneous matrix. Its suitability for use in transdermal permeation studies was subsequently tested and confirmed in preliminary experiments using porcine skin in vitro.
Tizanidine (5-chloro-N-(4,5-dihydro-1H-imidazol-2-yl)-2,1,3-benzothiadiazol-4-amine; Figure 1) is an α2-adrenergic agonist used for the symptomatic relief of spasticity associated with multiple sclerosis, spinal cord injury or disease (1). It is also used in the treatment of painful muscle spasm associated with musculoskeletal conditions and in the treatment of low back pain (2, 3). It has a short half-life (2.5 h) and requires multiple-dosing and there is a drive to exploit alternative routes of administration, such as transdermal delivery.
However, before any such study can be undertaken, robust, specific and reliable analytical methods must be developed to quantify the amounts of drug deposited in and permeated across the skin. Different analytical methods have been described to quantify tizanidine including spectrophotometry or HPLC with UV detection (4–9). However, they are not suited for transdermal delivery studies. For example, (i) all but one of the reported methods describe the determination of tizanidine in either a pharmaceutical dosage forms or the drug substance—only the method reported by Siddiqui et al. analyzes tizanidine in a biological matrix (human serum) and this requires sample pre-treatment before analysis (8) and (ii) they either lack the sensitivity to enable quantification at early time-points during transdermal delivery experiments or do not display a linear response over a sufficient concentration range (see below in the Discussion). Other analytical methods described in the literature include voltammetry (10), gas chromatography–mass spectrometry (11, 12) or thin layer chromatography (6, 13) but rely on complex instrumentation or complex sample preparation which may not be suitable for routine use with the large number of samples that can be generated in transdermal delivery studies.
The aim of this investigation was to develop and to validate a specific, robust and isocratic ultra-high performance liquid chromatography (UHPLC) method that could be used for the quantitative determination of tizanidine hydrochloride in transdermal delivery studies in vitro.
Tizanidine hydrochloride was purchased from Watson International Ltd (Kunshan, China). Trifluoroacetic acid (TFA) was acquired from Acros Organic (Geel, Belgium). Acetonitrile (MeCN) and sodium chloride (NaCl) were purchased from Sigma-Aldrich (Buchs, Switzerland). 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was obtained from Applichem GmbH (Darmstadt, Germany) and the polytetrafluoroethylene (PTFE) filters from VWR, Switzerland. All compounds were of at least of analytical grade. Deionized water was used in the preparation of all solutions (resistivity >18 MΩ cm; Milli-Q® system; Merck Millipore).
A 5.8 mg mL−1 stock solution of tizanidine hydrochloride was prepared in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (HEPES 20 mM, pH 5.2). Six standard solutions (17.4, 29, 72.5, 145, 218 and 290 ng mL−1) were prepared by further dilution of the tizanidine stock solution with buffer. Fresh standard solutions were used for the calibration curves.
The UHPLC analysis was carried out using a Waters Acquity UPLC® core system equipped with a Waters® Acquity UPLC® photodiode array eλ detector and an Acquity UPLC® BEH C18 (1.7 µm, 2.1 × 5.0 mm) column and pre-column. Chromatographic separation of the analyte was achieved at 27°C. The mobile phase consisted of an 80:20 mixture of 0.004% TFA in water and MeCN (pH* 3.2), and the flow rate was 0.2 mL min−1. The injection volume was 5 µL. The analyte was detected using its absorbance at 227 nm, and the tizanidine hydrochloride spectrum is shown in Figure 2. Prior to UHPLC analysis, all samples were centrifuged at 8,000 rpm for 20 min and 80% of the sample volume was recovered and placed in vials for analysis.
The method was validated according to ICH guidelines Q2 (R1) (14) with respect to linearity, limit of detection (LOD) and limit of quantification (LOQ), specificity, accuracy, precision and robustness.
The analytical method was validated using the six standard solutions described above. Calibration curves were obtained by least squares linear regression analysis of the peak area obtained as a function of drug concentration. Each concentration was assayed six times to determine intra-day reproducibility. In order to detect inter-day variation, the procedure described above was repeated on ten different days.
The LOD and LOQ were determined assuming a normal distribution of measured concentration values and were calculated as the signal equal to three and ten times the noise level (signal-to-noise ratio), respectively.
Absence of interference from endogenous compounds present in the skin was investigated by injecting 10 samples of porcine skin extract (15).
Accuracy expresses the closeness of agreement between the calculated value and the accepted reference value. It was defined as the relative error of the nominal solution concentrations. Measurements had to be within ±10% for all concentrations to be considered acceptable (16, 17). The precision of an analytical method expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous drug solution under fixed conditions. It provides information regarding random error. The variance of the repeatability and intermediate precision, and the corresponding relative standard deviation (RSD), were calculated from the estimated concentrations. To be considered acceptable, the RSD had to be lower than 10% at all of the concentrations analyzed (16, 17).
This was investigated by measuring any variation in the apparent concentration of a standard tizanidine solution (145 ng mL−1) following specific changes of (i) detector wavelength [±2 nm (225 nm–229 nm)], (ii) column temperature [±0.5°C (26.5–27.5°C)], (iii) mobile phase composition (75:25–85:15, v/v), (iv) mobile phase pH [±0.2 (pH* of 3.0–3.4)] and (v) flow rate (0.19–0.21 mL min−1).
Porcine ears were obtained from a local abattoir (CARRE; Rolle, Switzerland), the skin was excised (thickness 1.1 mm) with a surgical blade, wrapped in Parafilm™ and stored at −20°C for a maximum period of 2 months.
A tizanidine solution (290 ng mL−1; n = 3) was filtered using a PTFE filter (0.45 µm), and the concentration of the solution post-filtration was measured.
Skin samples were spiked with different amounts of tizanidine (2.5 µL of solutions at 145, 290 and 580 µg mL−1) (18). After solvent evaporation, the skin samples were cut into small pieces and immersed in 5 mL of phosphate buffered saline (PBS) for 12 h under constant stirring in order to extract the drug. Samples were filtered and analyzed using the validated UHPLC method.
Solution stability of tizanidine was tested by using a 100 ng mL−1 tizanidine solution in PBS stored at 4 ± 1°C and at ambient temperature (25 ± 2°C)—in both cases away from light. The samples were analyzed to determine the tizanidine concentration in solution 2, 5, 7, 31 and 65 days after preparation. The stability of tizanidine in contact with the skin was also evaluated. An aliquot of tizanidine (145 ng mL−1; 1 mL) was kept in contact with the dermal and epidermal skin surfaces, respectively, for 24 h (n = 6; mean ± SD) (19), and the drug concentration in solution determined.
Excised porcine ear skin was equilibrated for 30 min in 0.9% NaCl before being mounted in vertical Franz-type diffusion cells (area of 2 cm2). The receptor compartment (12 mL) was filled with PBS (pH 7.4). One milliliter of tizanidine hydrochloride (5.8 mg mL−1) in HEPES (20 mM, pH 5.4) was added to the donor compartment. Tizanidine permeation was determined by taking 0.9 mL samples from the receptor chamber hourly over 8 h. The volume removed was replaced immediately with fresh PBS buffer. The drug concentration in each sample was quantified using the optimized UHPLC method in order to calculate the cumulative permeation of tizanidine across the skin as a function of time.
At the end of the transdermal permeation experiments (8 h), the amount of tizanidine retained in the skin was determined following the previously validated protocol.
Statistical analysis was performed using either Student's t-test or by ANOVA.
Two tizanidine absorbance peaks were observed, at 227.4 and 319.5 nm (Fig. 2). The wavelength selected for detection was 227.4 nm because the signal was more intense (molar extinction coefficient was ~1.25-fold higher) and had the desired specificity to enable analysis of tizanidine in the samples collected in the transdermal permeation experiments.
A good correlation was observed between the response and the tizanidine concentration over the range of concentrations assayed (17.4–290 ng mL−1).
The LOD and LOQ were 1.26 and 3.83 ng mL−1, respectively.
Under the chromatographic conditions selected, the tizanidine retention time was 1.499 ± 0.008 min and the total run time was 2 min. The method was considered to be specific as there was no interference from endogenous compounds in the skin and the tizanidine peak was clearly separated from the solvent front (Fig. 3).
The results of the calculated intra-day and inter-day accuracy and precision are shown in Table I. Accuracy was within acceptable limits, as the variability of the values obtained for all concentrations was below 4% (16, 17). RSD values were calculated as a measurement of precision for each concentration and were less than 4% (16, 17).
The results of the test of robustness of the method are shown in Table II. Variation of device parameters, (detection wavelength and column temperature) or composition, pH and flow rate of the mobile phase had no significant effect on the retention time and chromatographic response of the method.
Adsorption of drug substances extracted from skin samples and their loss from solution can result in underestimation of drug deposition in the skin. The tizanidine concentration measured after filtration through a PTFE filter was 99.6 ± 0.9% (n ≥ 3; mean ± SD) of the initial value. The skin extraction procedure efficiency was 98.4 ± 5.2% (n = 6; mean ± SD).
The tizandine concentration in solution after 48 h at room temperature was 97.0 ± 0.4% of the initial value. Improved stability was observed after storage at 4°C where the solution concentration was 95.3 ± 0.8% of the initial value after storage for 65 days. The results are in agreement with published data (13). Tizanidine was considered to be stable in the presence of skin since the concentration measured after exposure for 24 h was 98.4 ± 4.7% of the initial value (n = 6; mean ± SD). The results are also in agreement with previously published data (19).
Cumulative permeation of tizanidine as a function of time is shown in Figure 4. The tizanidine transdermal flux was 0.26 ± 0.05 μg cm−2 h−1 (mean ± SD; n = 8), and the amount of drug retained in the skin after formulation application for 8 h was 78.2 ± 10.5 μg cm−2 (mean ± SD; n = 8).
The tizanidine concentrations in the different samples obtained from the permeation experiments in the present study are shown in Figure 5. These are plotted together with the concentration ranges of the published methods and the new analytical method described here. The method developed in this study is the only validated UHPLC-UV method that enables quantification of all the samples (89% are within the validated range). For comparison, with respect to the reported methods: (i) less than 10% of samples were above the LOQ and these were all below the lowest validated concentration (i.e. they were in the non-validated range) in the methods developed by Walash et al. (5) and Bhavsar et al. (9), (ii) all data points were below the range validated by Kaul et al. (6) and Qi et al. (7) and (iii) although the methods developed by Gandhimathi et al. (4) and Siddiqui et al. (8) were more sensitive, only 64 and 56%, respectively, of samples were inside the validated range.
The results demonstrate the feasibility of using this method for the routine determination of tizanidine hydrochloride in transdermal absorption studies.
The validated method was specific and robust and the response was accurate, precise and linear from 17.4 to 290 ng mL−1. The validated concentration range was suitable for quantification of tizanidine in samples from transdermal delivery studies. The preliminary results obtained during experiments with porcine skin demonstrated the feasibility of using the method for the determination of skin deposition and transdermal permeation of tizanidine in vitro. A complete investigation into the transdermal delivery of tizanidine is now underway.
The authors thank the University of Geneva, the “Generalitat Valenciana” (AP-114/09; AP-155/10; AP-175/11) and the “Universidad CEU Cardenal Herrera” for their financial support.
S.R. acknowledges Dr Brenda Rocamonde-Esteve for providing valuable comments.