Chemometric-assisted RP-HPLC method for the simultaneous determination of ambroxol hydrochloride, terbutaline sulfate, and guaiphenesin in combined dosage form

Chemicals and reagents

The active pharmaceutical ingredients (API) of AMB (98.9%), TER (98.7%), and GUA (98.7%) were supplied by Raffles Pharmaceuticals Pvt Ltd., Tirupati. HPLC-grade methanol, acetonitrile, and analytical reagent grade potassium dihydrogen phosphate (KH₂PO₄) and orthophosphoric acid were acquired from S.D fine chemical Ltd., Mumbai, India, and HPLC grade water from Millipore were used in the analysis. NORVENT expectorant containing AMB 15 mg, TER 1.25 mg, and GUA 50 mg was purchased from the local medical store.

Figure 1. Chemical structures of analyte. [Click here to view]

Instrumentation and software

HPLC system used was Shimadzu Prominence LC system with LC-20AT pump with UV-VIS detector and with 20 μl loop volume Rheodyne injector. The software used in data processing is LC Solution. An Elico pH meter (Model LI-120) was used to measure and adjust the pH of the buffer. Ultra-sonicator (PCI Analytics) was used for degassing the solutions. Design-Expert, version 11.0.0. (Stat-Ease Inc., Minneapolis, MN) software was used to select the design for optimization of the data and for further processing.

Chromatographic conditions

RP-HPLC method was developed using a Phenomenex C18 analytical column (4.5 × 250 mm, 5 μ) at room temperature with a mobile phase containing methanol (MN), acetonitrile (CAN), 50 mM KH₂PO₄ buffer_, pH adjusted to 5 in the ratio 55:10:35 (v/v/v) . The flow rate of the mobile phase was 0.8 ml/min. and the detection was carried using a UV detector and wavelength selected was 220 nm. The injection volume was 20 μL.

Preparation of stock and standard solutions

Stock solutions of AMB, TER, and GUA containing 1 mg/ml were prepared individually by dissolving 50 mg in 50 ml of methanol. A mixture containing the three standard solutions were prepared from the stock solution to get a concentration of AMB (20 μg/ml), TER (20 μg/ml), and GUA (20 μg/ml), respectively, using mobile phase.

Preparation of phosphate buffer

6.8 gm of potassium di-hydrogen orthophosphate (50 mM) was dissolved in sufficient water (HPLC grade) and volume was made up to 1,000 ml. The solution was kept for sonication and pH was adjusted to 5.0 using orthophosphoric acid.

Sample preparation

Sample enrichment technique was used in sample preparation. Syrup sample equivalent to 20 mg of GUA (2 ml of syrup), 0.5 mg of TER, and 6 mg of AMB was transferred to a 50 ml volumetric flask containing 20 ml of the mobile phase. To this 19.5 mg of TER API, 14 mg of AMB API were added and this sample solution was kept for sonication for 15 minutes and then the volume was adjusted up to the mark with mobile phase to get a concentration of 400 μg/ml of the three drugs. Filtration was carried out by using vacuum filter using 0.45 μ filter papers. 0.5 ml of the filtrate was taken and transferred into a 10 ml volumetric flask and the volume was made up to the mark with the mobile phase to get a concentration of AMB (20 μg/ml), TER (20 μg/ml), and GUA (20 μg/ml), respectively.

Design of experiments

The widely used chemometric model in the optimization of RP-HPLC method is the central composite design which is a subclass of response surface methodology (Giriraj et al., 2014; Sivakumar et al., 2007; Sree Janardhanan et al., 2016). In central composite design (CCD), three factors at two levels were used to optimize HPLC separation. Based on the trials performed, the factors were selected and in the variation ranges of 55–65(% v/v) of MN concentration (A), 4–5 of mobile phase pH (B), and 0.6–1.0 ml/minute of flow rate (C), respectively. The retention time of first peak (tR₁), resolution of 2nd and 3rd peak (Rs_2,3), and theoretical plates of the first peak (TP₁) were selected as responses and measured in 20 runs in randomized order according to the CCD.

Method validation

Accuracy

Accuracy in terms of % recovery was performed by using standard addition method for 70%, 100%, and 130% levels of test concentration.

Table 1. Central composite design of experiments and responses. [Click here to view]

Development and optimization

The factors A, B, and C and their ranges were given as input to the CCD employed. The software generated 20 runs in total and all the experiments were performed. The experiments conducted and their obtained responses are shown in Table 1. Three responses were selected based on their effect on the separation of drugs. The design model chosen was Quadratic mathematical model as its p-value is less than 0.05 and this model was suggested by the design. Different statistical parameters, such as adjusted r², adequate precision, and coefficient of variation (C.V.) obtained from analysis of variance (ANOVA) are shown in Table 2 and all the values were found to be within the acceptance criteria (Beg et al., 2003; Lundstedt et al., 1998; Parajo et al., 1992).

Interaction between the factors and responses and the impact of factors on responses were given by the design in the form of Perturbation plots and response surface plots which are shown in Figures 2 and 3, respectively (Myers et al., 1995). Perturbation plots revealed that factor C has the most important effect on tR₁ (Fig. 2A), whereas factors A, B, and C had the effects on Rs_{2, 3} and TP₁ (Figs. 2B and C). The interaction of the three factors on responses was predicted by response surface plots as shown in Figure 3 (% MN concentration in Perturbation plots and response surface plots revealed that factors B and C had a large significant effect on separation, whereas factor A, i.e., is MN concentration, is of little significance.

Derringer’s desirability function (D) is used as a critical parameter for optimization. D value near to 1 state that response values are near to the criteria values (Derringer et al., 1980).

Table 2. Statistical parameters obtained from ANOVA for CCD. [Click here to view]

The criteria for each response which was used in optimization are presented in Table 3. The diagrammatic representation of the response surface obtained for the global desirability function according to the criteria is shown in Figure 4. The maximum desirability value (D = 0.944) produced by the conditions (MN concentration of 55%( v/v), mobile phase pH of 5, and flow rate at 0.8 ml/minute) was chosen for optimization of the method. The experiment was performed using optimized conditions and the obtained and predicted responses are shown in Table 4 and the chromatogram is shown in Figure 5.

Method validation

The optimized method was validated according to ICH guidelines. Specificity was performed by comparing the chromatograms of the sample and blank and it was found that there were no interferences. Hence, the observed peaks in the sample solution revealed that the method was specific for the three drugs. Linearity was constructed at five levels in the range of 10–30 μg/ml for all the three drugs with R² of more than 0.999. Standard addition method was used to determine the accuracy and the percent recovery values were found to be 101.62%, 99.42%, and 99.52% for TER, GUA, and AMB, respectively, which were within acceptable ranges of 100 ± 2%. System precision and method precision were performed and the % RSD values were found to be less than 2.0. The experiments conducted as per the CCD and their responses revealed the information about robustness. The results obtained by the validation parameters revealed that the developed and optimized method was specific, suitable, linear, precise, accurate, and robust for the simultaneous determination of ambroxol hydrochloride, terbutaline sulfate, and guaiphenesin in their combined dosage form.

Figure 2. Perturbation plots. [Click here to view]

Figure 3. Response surface plots. [Click here to view]

Table 3. Criteria for optimization of individual responses. [Click here to view]

Figure 4. Response surface obtained for the global desirability function. [Click here to view]

Table 4. Comparison of experimental and predictive values under optimum condition. [Click here to view]

Figure 5. Test sample chromatogram. [Click here to view]

Table 5. Assay of marketed formulation by proposed methods. [Click here to view]

Application of the developed method to combined dosage form

The developed and validated chemometric-assisted RP-HPLC method was applied for simultaneous determination of AMB, TER, and GUA in the syrup dosage form. The sample solutions (n = 3) were injected into the HPLC system and their assay results were calculated. The assay results obtained were found to be within the acceptable limits for all the three drugs and are presented in Table 5.