A Validated High Performance Liquid Chromatography Method for the Simultaneous Analysis of Guaifenesin , Ambroxol and Loratidine in Bulk and Liquid Dosage form

Article history: Received on: 15/09/2015 Revised on: 20/10/2015 Accepted on: 01/11/2015 Available online: 27/12/2015 A simple and sensitive HPLC method for simultaneous quantification of guaifenesin, ambroxol and loratidine in bulk and liquid dosage form was developed and fully validated. The separation and quantification was performed using a Kromasil C8 (250 × 4.6 mm, particles 5 μm) HPLC column. Isocratic elution mode with a flow rate of 1.2 mL/min was used, and the injection volume was 10 μL. The detector was set to a wavelength of 290 nm and the column oven was maintained at 30 °C. Orthophosphoric acid (0.1%) and acetonitrile in the ratio of 60:40 v/v was used as the mobile phase. Guaifenesin, ambroxol and loratidine were eluted with retention time of 3.045 min, 5.489 min and 13.981 min, respectively. The method was validated in accordance with ICH guidelines and the results of all the validation parameters were found to be within the acceptable limits. The calibration plots were linear over the concentration ranges from 50-150 μg/mL, 30-90 μg/mL and 5-15 μg/mL for guaifenesin, ambroxol and loratidine, respectively. Developed method was successfully applied for the quantification of the above three drugs in liquid dosage form. The excipient did not interfere with drug peaks.


Apparatus
The Waters HPLC system, consisted of a binary HPLC pump model 2695, photodiode-array (PDA) detector model 2998 and a vacuum degasser, all controlled by a Waters Empower2 software was used in the present investigation.

Chromatographic conditions
Kromasil C8 (250 × 4.6 mm; 5 µm particle size) analytical column was used for separation and analysis of guaifenesin, ambroxol and loratidine.The column temperature was maintained at 30 ± 1 o C. Mobile phase consisted of 0.1% orthophosphoric acid and acetonitrile in the ratio of (60:40 v/v, respectively).The separation was done under isocratic elution with flow rate maintained at 1.2 mL/min.The injection volume was 10 μL.The guaifenesin, ambroxol and loratidine were analyzed using a PDA detector set at 290 nm.

Standard solutions
Reference standard of guaifenesin, ambroxol and loratidine were obtained from Lara Drugs Private Limited, Telangana.A combined standard stock solution of accurately weighted guaifenesin (50 mg), ambroxol (30 mg) and loratidine (5 mg) was prepared in 100 mL volumetric flask and dissolved in mobile phase.5 mL aliquot of guaifenesin, ambroxol and loratidine stock solution were added to a 25 mL volumetric flask, and diluted in mobile phase, yielding a final concentration of 100, 60 and 10 µg/mL, respectively.

Sample solution
Commercial syrup (Lorfast AM Syrup, Cadila Pharmaceuticals Ltd., Ahmadabad: labeled to contain 50 mg guaifenesin, 30 mg ambroxol and 5 mg loratidine per 5 mL of syrup) was purchased from local pharmacy store.The syrup was shaken thoroughly to make homogenous mixture.A volume of the syrup equivalent to 50 mg guaifenesin, 30 mg ambroxol and 5 mg loratidine was transferred accurately into a 100 mL volumetric flask containing 30 mL of mobile phase.The contents of the flask was shaken for about 10 min and diluted to volume with the mobile phase.The solution was then filtered through a 0.45 µm millipore filter.The above solution was appropriately diluted with the mobile phase to get a final concentration of 100, 60 and 10 µg/mL of guaifenesin, ambroxol and loratidine, respectively.

Method development
In the present work, we proposed a simple, sensitive, precise and accurate high performance liquid chromatographic method for simultaneous estimation of guaifenesin, ambroxol and loratidine.For optimization of the chromatographic conditions and to obtain symmetrical peaks with better resolution and with acceptable system suitability results, various chromatographic conditions such as composition of mobile phase, flow rate and two different analytical columns were applied to guaifenesin, ambroxol and loratidine combination.Among the tested analytical columns [ACE C8 (150 mm x 4.6mm, 5 μm particle size) and Kromasil C8 (250 × 4.6 mm; 5 µm particle size)] during preliminary investigations, Kromasil C8 (250 × 4.6 mm; 5 µm particle size) was the most appropriate column for simultaneous analysis of guaifenesin, ambroxol and loratidine.In the preliminary trials different compositions of mobile phases consisting of 0.1 M dipotassium hydrogen phosphate/acetonitrile and 0.1% orthophosphoric acid/acetonitrile, different ratios and different flow rates of these solutions were employed to achieve the best system suitability results.Finally, the mobile phase composition of 0.1% orthophosphoric acid: acetonitrile in the ratio of 60:40 v/v with a flow rate of 1.2 mL/min was shown to have good resolution with minimal tailing factor in acceptable range.The column temperature of 30 o C and detector wavelength set at 290 nm was chosen as suitable condition.Under the mentioned chromatographic conditions highly symmetrical and sharp peaks of guaifenesin, ambroxol and loratidine were obtained at retention times of 3.045 min, 5.489 min and 13.981 min, respectively (Fig. 2).

Method validation
The developed method was validated for system suitability, linearity, sensitivity, precision, accuracy and robustness following the ICH guidelines (International Conference on Harmonization, 2005).

System suitability
Earlier to each analysis, the chromatographic system must satisfy suitability test requirements.System suitability test was performed from five replicate injections of a standard solution containing 100, 60 and 10 µg/mL of guaifenesin, ambroxol and loratidine, respectively.All peaks were well resolved.The precision of injections for all peaks were acceptable.The percent relative standard deviations of the peaks area responses were measured.The USP tailing factor, USP resolution and USP plate count were also calculated.The results of system suitability in association with the required limits are presented in Table 1.The developed method met requirements within the accepted limits.

Linearity
The linearity test was performed using five different amounts of guaifenesin, ambroxol and loratidine in the range 50-150 µg/mL, 30-90 µg/mL and 5-15 µg/mL, respectively.Solutions corresponding to each concentration level were injected in duplicate and linear regression analysis of the guaifenesin, ambroxol and loratidine peak area vs guaifenesin, ambroxol and loratidine concentration were calculated.The results are summarized in Fig. 3, 4 & 5.The results show a good correlation between the peak area of drugs and their concentrations with R 2 value > 0.9998.

Sensitivity
The sensitivity of the developed method was assessed by determining Limit of quantification (LOQ) and detection (LOD).The LOQ and LOD were predicted by the following formulae (a) LOQ = 10 σ / S (b) LOD = 3.3 σ / S Where σ = standard deviation of response; S = slope of the calibration curve.
The LOD was found to be 0.754 μg/mL, 0.231 μg/mL and 0.145 μg/mL, whereas LOQ was found to be 2.513 μg/mL, 0.769 μg/mL and 0.483 μg/mL for guaifenesin, ambroxol and loratidine, respectively.The results reveal satisfactory sensitivity of the developed method.

Precision
The precision of the developed method was demonstrated by intra-day variation studies.For this purpose, six repeated injections of standard solutions (guaifenesin-100 μg/mL; ambroxol-60 μg/mL; loratidine-10 μg/mL) were made.The response of guaifenesin, ambroxol, loratidine and their percentage relative standard deviation (%RSD) were calculated.From the results, the developed method was considered to be precise (Table 2).

Accuracy
The accuracy of the method was determined via recovery experiments.The accuracy of the proposed method was demonstrated by preparing samples spiked with 50%, 100%, and 150% of the test concentration of guaifenesin, ambroxol and loratidine.Each concentration level was analyzed thrice.
Mean percent recovery and percent RSD were calculated for each concentration.Recovery of individual components was well within the acceptable limit (Table 3).From the data obtained, added recoveries of drugs were found to be accurate.

Robustness
Robustness of the method was determined by making slight changes in the chromatographic conditions.In all the deliberate varied chromatographic conditions, the parameters like tailing factor, peak area and theoretical plates were not much affected, which shows that the method is robust.The results are shown in Table 4.

CONCLUSION
The optimal chromatographic conditions for separation and simultaneous quantification of guaifenesin, ambroxol and loratidine were achieved on an Kromasil C8 (250 × 4.6 mm; 5 µm Cencentration of loratidine (ug/mL) Peak area particle size) analytical column with a isocratic elution at a flow rate of 1.2 mL/min, using 0.1% orthophosphoric acid:acetonitrile (60:40 v/v) as mobile phase and detection set to a wavelength of 290 nm.The method was simple and does not require preparation of buffer.The proposed method has the advantages of being sensitive, high resolution factor and less tailing factor than the reported HPLC methods (Sameena et al., 2014;Vani et al., 2014).
All measured parameters of the validation reveal the suitability of developed HPLC method for the simultaneous analysis of guaifenesin, ambroxol and loratidine in bulk and liquid pharmaceutical preparation.

Table 2 :
Results of precision of the method.

Table 3 :
Results of accuracy of the method.

Table 4 :
Results of robustness.