Research Article ISSN: 0974-6943
Trivedi Aditya et al. / Journal of Pharmacy Research 2010, 3(6),1398-1401
Available online through www.jpronline.info
Development of modified Spectrophotometric and HPLC method for simultaneous estimation of Ambroxol hydrochloride and Cetirizine hydrochloride in tablet dosage forms Trivedi Aditya, Banerjee Lopamudra *. Pharmaceutical Chemistry Research Laboratory,Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar (M.P.) ,470003
Received on: 11-03-2010; Revised on: 19-04-2010; Accepted on:25-05-2010 ABSTRACT A high-performance liquid chromatographic and an UV Spectrophotometric method were developed and validated for the quantitative determination of two drugs, Ambroxol hydrochloride and Cetirizine hydrochloride in tablets. Ultraviolet-visible (UV-Vis) spectroscopy, have been used to estimate the drugs by using water as a solvent at 20oC. Simultaneous equation method developed by UV spectrum measurements indicates the concentration of Ambroxol hydrochloride and Cetirizine hydrochloride in tablets. A simple, selective, rapid, precise and economical reverse phase HPLC method has been developed for the simultaneous estimation of Ambroxol hydrochloride and Cetirizine hydrochloride in dosage forms (tablets) by using acetonitrile, methanol and water in the ratio 10:20:70 as a solvent system. The method was carried out on a Phenomenex Gemini C18 (25 cm x 4.6 mm i.d., 5 µ) column, at a flow rate of 1.0 mL/ min. Detection was carried out at 244,230 nm.The retention time of Ambroxol hydrochloride and Cetirizine hydrochloride was -4.2 and 9.3 min, respectively. The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantitation and solution stability. The proposed method can be used for the estimation of these drugs in combined Pharmaceutical dosage forms.
Keywords: Ambroxol hydrochloride, Cetirizine hydrochloride, simultaneous equation,HPLC,UV INTRODUCTION Cetirizine is the carboxylated metabolite of hydroxyzine, and it has high specific affinity for histamine H1 receptor. Cetirizine is chemically known as 2-[4(4-chlorobenzhydryl) piperazine-1-yl] ethoxy acetic acid. Ambroxol hydrochloride is semi synthetic derivative of vasicine obtained from Indian shrub Adhatoda vasica. It is the metabolic product of bromhexine.It is official in Martin Dale-The Extra Pharmacopoeia.[1] Chemically it is trans- 4-(2-amino3,5-dibromobenzylamino) cyclohexanol hydrochloride. It acts as a bronchosecretolytic and expectorant drug. It stimulates the transportation of the viscous secretions in the respiratory organs and reduces the accumulation of the secretions. Several Spectrophotometric methods [2, 3] have been used for the qualitative and quantitative determination of Ambroxol hydrochloride in pharmaceutical formulations. Different high-performance liquid chromatographic (HPLC) methods have been reported for determination of Ambroxol hydrochloride in pharmaceutical formulations and biological fluids. Several Spectrophotometric methods have been reported for determination of Cetirizine in pharmaceutical formulations and in human plasma. Different HPLC methods have been reported for determination of Cetirizine in pharmaceutical formulations and biological fluids. Literature survey reveals that high-performance thin-layer chromatography has been reported for the simultaneous determination of Ambroxol hydrochloride and Cetirizine hydrochloride in pharmaceutical formulations Fig. 1.
2. MATERIALS AND METHODS 2.1. REAGENTS Ambroxol hydrochloride and Cetirizine hydrochloride were kindly donated by Fourrts Limited (India.) HPLC grade methanol and acetonitrile, and water were all purchased from Fisher scientific (Mumbai, India) Water was purified in a Purite system (Oxon, UK). 2.2. UV-Vis spectroscopy A UVPROB type 1700 ultraviolet-visible spectrometer (Shimadzu) with matched 1 cm quartz cells was used in all the UV experiments. Analyses were performed using both direct modes over a wavelength range of 200–400 nm. Standard solutions of 100µg of Ambroxol hydrochloride and Cetirizine hydrochloride in water proportions were prepared individually. Different standard solution of Ambroxol hydrochloride and Cetirizine hydrochloride was scanned against the same concentration of individual drug solutions placed in the reference cell. The spectra were then compared with the spectra of individual drug samples at the same concentration scanned against water below 200c. 2.3. HPLC The HPLC system consisted of a model
LC-20A (Prominence) Simadzu, Japan. Separation was achieved using a Phenomenex Gemini C18 (25 cm x 4.6 mm i.d., 5 µ) column The column Fig. 1. Chemical structures of (a) Ambroxol hydrochloride, (b) Cetirizine temperature was maintained at 40°C using a model 7716 HPLC column block heater CTO-20AC.The mobile phase was a mixture of acetonitrile-methanolhydrochloride demonized water (10:20:70 v:v) . Before use the solvent was filtered through *Corresponding author. a 0.45 mm filter (Millipore, Watford, UK). The analysis was carried out at a Banerjee Lopamudra flow rate of 1 ml/min. The injection volume was 20 µ ml and detection was by Pharmaceutical Chemistry Research Laboratory,Department of Pharmaceuti- UV (SPD-20AV) at 230,244 nm . The sensitivity of the detector was kept cal Sciences, Dr. H. S. Gour University, Sagar (M.P.),470003 fixed throughout the experiment. Data handling was carried out using a CTel.: + 91-9827118551 R3A Chromatopac (Shimadzu, Kyoto, Japan) system. Telefax: +91-07582264236 CE
AMB
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Trivedi Aditya et al. / Journal of Pharmacy Research 2010, 3(6),1398-1401 2.4. PREPARATION OF STANDARD SOLUTIONS 2.4.1 UV method Standard stock solution containing Ambroxol hydrochloride (AMB) and Cetirizine hydrochloride (CE) was prepared by dissolving 100 mg of AMB and CE separately in 100 mL of water(below 250C); and then final volume of both the solutions was made up to containing 100 µg mL-1 of AMB and 100 µg mL-1 of CE in two different 100-mL volumetric flasks. Solutions containing 20 µg mL-1 of AMB and CE were prepared and scanned in the UV region separately. The wavelengths selected were 230 and 244 nm for simultaneous determination of CE and AMB.By appropriate dilution of standard drug solutions with distilled water, ten working standard solutions containing 5, 10, 15, 20, 25, 30, 35, 40, 45, and50 µg mL-1 of AMB and CE were prepared separately and scanned in the range of 200 to 400 nm. The values of absorbance were recorded at the selected wavelengths, and the absorptivity and molar absorptivity values were determined for AMB and CE. 2.4.2. HPLC method Mixed standard solutions consisting of 100 µg/mL Ambroxol hydrochloride and Cetirizine hydrochloride were prepared. Typically, 1-100 µg/mL of Ambroxol hydrochloride and Cetirizine hydrochloride analytical working standard were taken into a 10 ml volumetric flask. Ten milliliters of HPLC grade acetonitrile, 20mLof HPLC grade methanol and 70 ml of deionised water were added separately to the mixed solution. Each sample was sonicated for 10 min to dissolve the solutes. The solutions were made up to the final volume with premixed HPLC grade acetonitrile-methanol- deionised water (10:20:70 v:v).
was determined by assaying six sample solutions of the highest test concentration (20µg/ml for HPLC method and, 50µg/ml respectively for AMB and CE for the UV method). 3.3. Specificity The method specificity was assessed by comparing the chromatograms (HPLC) and scans (UV) obtained from the drug and the most commonly used excipients mixture with those obtained from blank (excipients solution in water without drug). The excipients chosen are the ones used commonly in tablet formulation, which included lactose, starch, microcrystalline cellulose, PVP, and magnesium stearate. The drug to excipients ratio used was similar to that in the commercial formulations. 3.4. LOD AND LOQ The limit of detection (LOD) is defined as the lowest concentration of an analyte that an analytical process can reliably differentiate from background levels. The limit of quantification (LOQ) is defined as the lowest concentration of the standard curve that can be measured with acceptable accuracy, precision and variability [7]. The LOD and LOQ were calculated as LOD = 3.3s /S and LOQ = 10s /S Where s is the standard deviation of the lowest standard concentration and S is the slope of the standard curve. Table 1.The chromatographic conditions optimized for analysis of ambroxol hydrochloride and cetirizine hydrochloride by RP-HPLC Drug
Mobile phase
AMB+ CE
Acetonitrile:Methanol :Water (10:20:70)
3. Method validation 3.1. LINEARITY
Flow rate (ml/min)
Detection wavelength (nm)
Injection volume (µl)
1
230
20
The methods were validated according to International Conference on Harmonization Q2B guidelines [4] for validation of analytical procedures in order to determine the linearity, sensitivity, precision and accuracy for each analyte [4,5]. Six point calibration curves were generated with appropriate volumes of working standard solutions for both UV and HPLC methods. In case of UV the range was optimized at 5-50µg/ml for AMB and CE respectively.
Although the ?max AMB and CE are 244 and 230 nm, respectively,230 nm was used for the quantification by HPLC, since this wavelength was successfully used for the simultaneous drug estimation in combinations.
The calibration range was 1–10 _g/ml in the HPLC methods of analysis for both the drugs. The linearity was evaluated by the least square regression method using un weighted data.
A RP-HPLC method was developed for AMB and CE, which can be conveniently employed for routine quality control in pharmaceutical dosage forms. The chromatographic conditions were optimized in order to provide a good performance of the assay. The mobile phase for each drug was selected based on its polarity. Different ratios of acetonitrile: methanol: water combinations were tried and the final working mobile phases are listed in Table 1. The retention times of AMB and CE were 4.2 and 9.3 min, respectively. The total run time was short for all the three drugs. The chromatograms have been shown in Fig. 2. The methods were specific as none of the excipients interfered with the analytes of interest. Hence, the methods were suitably employed for assaying the commercial antiretroviral individual formulations. The slopes were calculated using the plot of drug concentration versus area of the chromatogram. The developed HPLC methods were accurate, precise, reproducible and very sensitive. All the validation parameters of the three drugs were shown to be within the specified limits (Table 2). Accuracy and precision were determined by elaboration of three standard calibration curves, two from the same day (intra-day) and third one from a different day (interday).The intra- and inter-day precision (%R.S.D.) at different concentration levels were found to be less than 5%. AMB showed 100.126 ±0.00358
3.2. PRECISION AND ACCURACY Both precision and accuracy were determined with standard quality control samples (in addition to calibration standards) prepared in triplicates at different concentration levels covering the entire linearity range. Precision is the degree of repeatability of an analytical method under normal operational conditions. The precision of the assay was determined by repeatability (intra-day) and intermediate precision (inter-day) and reported as %R.S.D. for a statistically significant number of replicate measurements [5]. The intermediate precision was studied by comparing the assays on 3 different days and the results documented as standard deviation and %R.S.D. Accuracy is the percent of analyte recovered by assay from a known added amount. Data from nine determinations over three concentration levels covering the specified range was determined [6]. The repeatability of the method
4. RESULTS AND DISCUSSION 4.1. HPLC method
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Trivedi Aditya et al. / Journal of Pharmacy Research 2010, 3(6),1398-1401 and CE 100.115 ±0.0096% recoveries from the commercial formulations when assayed with the developed HPLC methods (Table 3). Moreover the %R.S.D. (less variation) shows good precision of the developed HPLC methods. The calculated LOQ and LOD concentrations confirmed that the methods were sufficiently sensitive. The methods were specific as none of the excipients interfered with the analytes of interest. Hence, the methods were suitably employed for assaying the commercial AMB and CE formulations.
4.2. UV method The development of simple, rapid, sensitive and accurate analytical method for routine quantitative determination of samples will reduce unnecessary tedious sample preparations and cost of materials and labor. The correlation coefficient of the standard curves for AMB and CE was greater than 0.97. The stock solutions and working standards were made in aqueous media (ultra pure water below 250C). The ?max of the drugs for analysis was determined by taking scans of the drug sample solutions in the entire UV region (200–400 nm) Fig. 3. All the method validation parameters are well within the limits as specified in the ICH Q2B guidelines [9] as shown in Table 4. Table 3 lists the percent recovery (content uniformity) of drugs in the commercial formulations by the developed methods. The commercial dosage forms showed 100.115 %for CE and 100.126 for AMB % recovery by this method which were within the specified limits of content uniformity. Moreover, the UV method offers a cost effective and time saving alternative to HPLC method of analysis.
Fig. 2. Typical chromatograms showing the elution of AMB and CE
Table 2.Validation parameters of the HPLC method of Ambroxol HCl and Cetirizine HCl Method characteristic
Ambroxol HCl
Cetirizine HCl
Linear range (µg mL-1) Correlation coefficient
5-50 0.993
5-50 0.995
Standard deviation Theoretical plates Resolution Asymmetry Accuracy (%) LOD (µg mL-1) LOQ (µg mL-1) Tailing factor Precision RSD (%) Repeatability day 1 Intermediate precision day 2
0.0022363 5386 0.210 0.99 98.5 0.015 0.043 1.01
0.012126 10085 1.344 1.00 99.6 0.014 0.047 0.99
4.6 5.5
6.7 5.2
Figure 3: Overlay spectra of AMB and CE Table 4.Validation parameters for UV method of analysis of Ambroxol HCl and Cetirizine HCl
Table 3.Results from analysis of formulations and from recovery studies HPLC Formulation Ambet tablets[Fourrts] Ambroxol HCl Cetirizine HCl
Labelled amount (mg per dose)
Amount found (mg per dose)
%RSD
60 5
60.05 5.03
8.33% 17.33%
Table.4.Results from analysis of formulations and from recovery studies UV Formulation
Labelled amount (mg per dose)
Amount found (mg per dose)
Recovery (%, mean ± SD, n = 6)
60 5
59.97 5.00575
100.126 ±0.00358 and
Ambet tablets[Fourrts] Ambroxol HCl Cetirizine HCl
Validation parameters
Cetirizine HCl
Ambroxol HCl
Range (µg/ml)
5-60
5-70
Regression equation slope Correlation coefficient (r2) Limit of quantification (_g/ml) Limit of detection (_g/ml)
y = 0.009X+0.012 0.009 0.991 10.73 3.54
y = 0.004X+0.008 0.004 0.974 20.85 6.88
5. CONCLUSIONS The proposed RP-HPLC and UV methods are simple, reliable and selective providing satisfactory accuracy and precision with lower limits of detection and quantification. Moreover the shorter duration of analysis for Cetirizine and Ambroxol make these reported methods suitable for routine quantitative analysis in pharmaceutical dosage forms. The recoveries achieved are good by both the methods. REFERENCES 1. 2. 3. 4.
Reynolds JE, et al. Martin dale-the extra pharmacopoeia, 31st ed. London: Royal Pharmaceutical Society; 1996. p. 1062. Reddy MN, Rao KV, Swapna M, Sankar DG, Spectrophotometric determination of Ambroxol. East Pharm 1998; 48:125-6. Indrayanto G, Handayani R, Quantitative determination of Ambroxol hydrochloride in tablets. J Pharma Biomed Anal 1993; 11:781-4. Anonymous, ICH Guidelines: Validation of Analytical Procedures: Methodology Q2 (B) (2003).
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M.E. Swartz, I.S. Krull, Pharm. Technol. 22 (1998) 104. M.V.S. Varma, N. Kapoor, M. Sarkar, R. Panchagnula, J. Chromatogr. B 813 (2004) 347. A.P. Argekar, J.G. Sawant, J. Pharm. Biomed. Anal. 21 (1999) 221.
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Source of support: Nil, Conflict of interest: None Declared
Journal of Pharmacy Research Vol.3.Issue 6.June 2010
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