Development and validation of a reverse phase high performance liquid chromatography ( HPLC ) method for determination of tizanidine in human plasma

A simple and cost effective high performance liquid chromatography (HPLC) method was developed for determination of tizanidine in human plasma using liquid extraction technique. The assay of tizanidine was performed after extraction of drug from plasma using diethyl ether as extraction solvent. The isocratic elution was performed in Agilent, Zorbax SB-C18, 4.6 × 150 mm column maintained at 30°C with mobile phase containing acetonitrile and ammonium acetate in a ratio of 15:85 v/v, respectively. The linear relationship was found within the concentration range of 0.25 to 8 ng/ml, with a flow rate of 1 ml/min and detector wavelength of 230 nm. The evaluated validation parameters were found within the acceptable range. Use of simple HPLC technique with short retention time makes this method a convenient choice for assay of tizanidine in human plasma.


INTRODUCTION
Tizanidine hydrochloride is an imadazoline derivative that acts on centrally located alpha 2 receptors for producing monolytic response on skeletal muscles (Wagstaff and Bryson, 1997).It is used for the treatment of multiple sclerosis or spinal cord injury or spasticity associated with diseased condition (Sweetman, 2009).Tizanidine is also used for the relieving of pain with disorders like myofacial pain (Meythaler et al., 2001), refractory pain, neuropathic pain, chronic tension type headache and chronic daily headache (Saper et al., 2002).Tizanidine is widely absorbed in gastro intestinal (GI) tract.Peak plasma concentration is achieved in 1 to 2 h after oral administration.Tizanidine protein binding is 30% and it undergoes extensive first pass metabolism (Shanker et al., 2009).Tizanidine is chemically [5-chloro-4-(2imidazolin-2-ylamino)-2,1,3-benzothiadiazole] and demonstrate basic and lipophilic properties.The drug is ionised in acidic environment and soluble in water (Qi et al., 2003).
Many researchers reported method for determination of tizanidine in human plasma.Lee et al. (2002) evaluated tizanidine by using gas chromatography-mass spectrometry.*Corresponding authors.E-mail: tariqali155@yahoo.com.Tel: +923002266740; harrisshoaib2000@yahoo.com; Tel: +923332264798.Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License (Lee et al., 2002).Momo et al. (2010) determined tizanidine in human plasma and urine with use of liquid chroma-tography-tandem mass spectrometry in presence of maxiletine (Momo et al., 2010).Ulu et al. (2012) utilized a spectroflourimetric method based on dervitization for estimation of tizanidine in plasma, urine and dosage forms (Ulu et al., 2012).
In the current study, tizanidine hydrochloride was analyzed using simple high performance liquid chromatography (HPLC) method.The low amount of drug in plasma was extracted by liquid-liquid extraction technique.The method overcomes the issue of determination of low concentration of tizanidine in human plasma.The described method is specific and sensitive with short retention time (4.4 min) that makes it cost effective (Figure 1 and Table 1).

Mobile phase
The mobile phase consisted of acetonitrile and 0.1 M ammonium acetate in a ratio of 15:85 v/v.The mobile phase was filtered and sonicated before use.

Preparation of stock solutions and working standard
The standard stock solution was prepared in mobile phase by dissolving accurately weighed quantity of tizanidine to make 1 mg/ml.The standard samples for calibration curve in plasma were prepared by spiking standard stock solution in 1 ml of blank plasma for preparation of secondary stock solution of 100 ng/ml by serial dilution.Secondary stock solution of 100 ng/ml was diluted in plasma for 0.25, 0.5, 1, 2, 4 and 8 ng/ml concentrations.

Sample preparation for drug determination in plasma using HPLC
Sodium fluoride (20 mg) and 0.4 ml of sodium hydroxide (5 mol/L) were added in 1 ml of spiked plasma and vortex for 1 min.Diethyl ether (5 ml) was added and vortex for 10 min for extraction, later centrifuged for 5 min at 4500 rpm.Supernatant was transferred into another test tube and evaporated to dryness using gentle stream of nitrogen at 40°C.The sample was reconstituted in 70 µl of mobile phase, then vortex for 2 min and again centrifuged for 5 min at 4500 rpm.A quantity of 50 µl of the final sample was injected into the HPLC for analysis.

Chromatographic conditions
In the current study, the evaluation of tizanidine was performed at a flow rate of 1 ml/min with detection wavelength of 230 nm and column maintained at a temperature of 30°C.

Method validation
The reported method for determination of nicergoline metabolite in human plasma in which Zheng et al. (2012) used tizanidine hydrochloride as internal standard, was modified for estimation of tizanidine hydrochloride in plasma.The proposed method was validated as per International Conference on Harmonisation (ICH) guidelines (ICH, 2005) for selectivity, linearity, accuracy, precision, sensitivity and stability.

Selectivity
Selectivity is helpful for differentiation between drug and other components present in the sample.For selectivity of method, six different blank samples of plasma were run, and it was established at lower limit of quantification (LLOQ) of drug.

Linearity and calibration curves
The linearity was assessed between different concentrations of drug molecule and response of detector by calibration curve (Lister, 2005).The samples with concentrations of 0.25, 0.5, 1, 2, 4, and 8 ng/ml were analyzed in triplicate and calibration curve was constructed, and coefficient of correlation (r 2 ) was determined.

Intraday and interday accuracy and precision
Five samples of four different concentrations in plasma were analyzed at different time in the same day for intraday accuracy and precision determination and analyzed for three consecutive days for interday accuracy and precision.The concentrations were calculated using standard calibration curves.

Lower limit of quantification (LLOQ) and limit of detection (LOD)
Lower limit of quantification (LLOQ) and limit of detection (LOD) were established by analysis of different low concentrations (0.05, 0.1, 0.15 and 0.25 ng/ml).For lower limit of quantification, the signal to noise ratios was observed for the lowest concentration and considered acceptable when response was 7 times of the noise.
Lowest detectable and quantifiable concentration against standard was considered as LOQ.The concentration when signal to noise ratio was observed as 3 times has been considered as LOD.

Analytical recovery of method
The absolute recovery was established by comparative study of drug spiked in plasma and in mobile phase.Three different concentrations (2, 4 and 6 ng/ml) each with 5 replicates were examined for determination of recovery.

Plasma stability of the drug
Freeze-thaw and long term stabilities were carried out for the drug in plasma.The freeze and thaw stability was evaluated with selected low (1 ng/ml) and high (7 ng/ml) concentrations with 20 samples of each concentration.Samples were frozen at -20°C for 24 h.A set of all concentrations was thawed and assessed while remaining samples were frozen for the next 24 h.Other two sets of five samples of each concentration were analyzed with the same procedure while the last set was refrozen for next day analysis to complete the three freeze-thaw cycles.These samples were evaluated with reference to freshly prepared samples.The long term stability was performed by preparing fifteen samples of low and high concentrations in plasma.The samples were stored at -20°C.Five samples of each concentration were analyzed at the end of second week and the next five at the end of third week of storage with respect to its initial concentration.

RESULTS AND DISCUSSION
In the current method, tizanidine was extracted from plasma using liquid extraction which is extensively for determination of low drug concentrations in biological fluids (Ciccolini et al., 2001;Esrafili et al., 2007;Xiong et al., 2009).Gan et al. (2002) developed and validated the estimation of tramadol in human plasma using HPLC followed by liquid-liquid extraction.Nirogi et al. (2006) quantified tizanidine in human plasma after liquid-liquid extraction with liquid chromatography tandem mass spectrometry in the range of 50 to 5000 pg/ml.Siddiqui et al. (2011) validated simple HPLC method for simultaneous determination of paracetamol, tizanidine and diclofenac in biological fluids and found a linearity for tizanidine in the concentration range of 120 to 10,000 ng/ml.

Selectivity
Six blank plasma samples were run and no peak at the retention time of drug was detected in plasma.The drug sample was run in same condition and no interference was found.No interfering plasma peak was observed at the drug retention time proved a good selectivity of the method.Selectivity of the method was shown in Figure 2.

Linearity and calibration curves
Evaluations of samples with concentration 0.25, 0.5, 1, 2, 4 and 8 ng/ml were performed in triplicate.The standard calibration curve was linear with a mean r 2 of 0.9989 with percent accuracy between 90 to 104.6%.The linearity chromatogram and linearity curve of tizanidine are shown in Figure 3 and 4.

Accuracy and precision
The HPLC method was also validated for intraday and interday accuracy and precision.The intraday accuracy was found in a range of 90 to 96% while interday accuracy with value of 84 to 92% was observed as shown in Table 2.All the values complied with standard acceptable range of ±15 % for bioanalytical method accuracy and precision.

Lower limit of quantification and limit of detection
The five samples of each concentration (0.05, 0.1, 0.15 and 0.25) were analyzed for LLOQ and LOD determination.The concentration 0.05 ng/ml was not detectable while the lower limit of detection was found as 0.1 ng/ml, and lower limit of quantification (LLOQ) value was 0.25 validated with accuracy of 94.4% that is within the specified limit of 20% and presented in Table 3.

Analytical recovery
Recovery of the method was performed for low, medium and high concentration within the calibration curve range.The method was found with good recovery with the mean analytical recovery of 97.135% for three selected concentrations of 2, 4 and 6 ng/ml as shown in Table 4.

Plasma stability of drug
Freeze and Thaw stability of the drug in plasma were evaluated for three freeze-thaw cycles and estimated accuracy were found to be 99.4,97.8 and 98.2% for low concentration (1 ng/ml) and 98.66, 98.37 and 98.31% for high selected concentration (7 ng/ml) for freeze thaw cycle 1, 2 and 3, respectively.The average degradation of drug in three FT cycles was found to be 2.268 and 1.040% for concentration of 1 and 7 ng/ml, respectively.Long term stability of the drug was performed for three weeks for low and high concentrations.More than 95% drug was found in the samples after three weeks that represent good long term stability of the drug in plasma.
The mean degradation of drug for both the concentrations of drug in plasma after three weeks was 3.695%.The results of freeze thaw stability and long term stability studies are presented in Tables 5 and 6.

Conclusion
The method has been validated successfully for the determination of tizanidine in human plasma sample.
Validation parameters such as selectivity, linearity, accuracy, precision and stability showed good results and complied with standard acceptable range.Hence, this liquid extraction based HPLC method can be used effectively for the estimation of tizanidine in human plasma.

Figure 2 .
Figure 2. Chromatogram showing selectivity of the method.

Figure 4 .
Figure 4. Linearity of different concentration of tizanidine.

Table 2 .
Accuracy and precision of tizanidine in plasma.

Table 3 .
Limit of detection of tizanidine in plasma.

Table 4 .
Results of recovery studies.

Table 5 .
Freeze and thaw stability of tizanidine.

Table 6 .
Long term stability of tizanidine in plasma.