A RP-HPLC Method for Simultaneous Estimation of Ambroxol Hydrochloride and Loratidine in Pharmaceutical Formulation

 

Krishna Veni Nagappan*, Meyyanathan SN, Rajinikanth B Raja, Suresh Reddy, Jeyaprakash MR, Arunadevi S Birajdar and Suresh Bhojraj

 

Department of Pharmaceutical Analysis, J S S College of Pharmacy, Rocklands, Ootacamund-643 001 Tamilnadu, India.

*Corresponding Author E-mail:  krisath@yahoo.com

 

ABSTRACT

A simple, selective, rapid, precise and economical reverse phase HPLC method has been developed for the simultaneous estimation of Ambroxol Hydrochloride and Loratidine from pharmaceutical dosage forms. The method was carried out on a Phenomenex Gemini C18 (25 cm x 4.6 mm i.d., 5 µ) column with a mobile phase consisting of acetonitrile: 50mM Ammonium Acetate (50:50 v/v) at a flow rate of 1.0 mL/min. Detection was carried out at 255 nm. Hydrochlorthiazide was used as an internal standard. The retention time of Ambroxol Hydrochloride, Loratidine and Hydrochlorthiazide was 5.419, 15.549 and 3.202 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.

 

KEY WORDS                                 Loratidine, Ambroxol Hydrochloride, RP HPLC and Pharmaceutical dosage form.

 


INTRODUCTION:

Loratidine is chemically 4-[8-chloro-5, 6-dihydro-11H-benzo [5, 6] cyclohepta [1, 2-b] pyridin-11-ylidene]-1-piperidinecarboxylic acid ethyl ester1 with a potent antihistaminic activity. Ambroxol hydrochloride is chemically, 1 ({[2 – Amino – 3, 5 dibromo phenyl] – methyl} amino) cyclohexanol monohydrochloride1 which is a semi synthetic derivative of vasicine from the Indian shrub “Adhatoda vasica”. It is a mucolytic agent. Ambroxol hydrochloride is an N – desmethyl metabolite of bromohexine. Many methods have been described in the literature for the determination of Loratidine2-9 and Ambroxol Hydrochloride10-18 individually and in combination with other drugs. However, there is no HPLC method reported for the simultaneous estimation of these drugs in combined dosage forms. Fixed dose combination containing Ambroxol Hydrochloride (60 mg) and Loratidine (5 mg) is available in the tablet form in the market.

 

The aim of this work was to develop an RP-HPLC method with ultraviolet detection for the simultaneous determination of Ambroxol Hydrochloride and Loratidine in pharmaceutical dosage forms. The present RP-HPLC method was validated following the ICH guidelines19-20.

 

MATERIALS AND METHODS:

Acetonitrile HPLC grade was procured from E. Merck (India) Ltd, Mumbai. Ammonium acetate AR grade was procured from Qualigens fine chemicals, Mumbai. Water HPLC grade was obtained from a Milli-QRO water purification system. Reference standards of Loratidine and Ambroxol Hydrochloride are procured from Franco-Indian Pharma, Mumbai, India and Hydrochlorothiazide were procured from Apex Pharmaceuticals, Andhra Pradesh, India.

 

Chromatographic separation was performed on a Waters® liquid chromatographic system equipped with a Waters 1515 isocratic HPLC system (pump), Waters 2487 dual wavelength detector, Rheodyne 7725i injector with 20mL loop volume. Waters Breeze version 3.01 data station was applied for data collecting and processing. A Phenomenex Gemini C18 column (25 cm x 4.6 mm i.d., 5µ) was used for the separation.

 

Preparation of mobile phase and standard solutions

The mobile phase used was a mixture of acetonitrile and 50 mM Ammonium acetate (50:50 v/v). It was filtered through a 0.2 µ membrane filter and degassed. Standard stock solutions of 1mg/mL of Ambroxol Hydrochloride, Loratidine and Hydrochlorothiazide were prepared separately using a mixture of water and acetonitrile in the ratio 1:1 v/v. From the standard stock solution, mixed standard solution was prepared to contain 100 µg/mL of Ambroxol Hydrochloride, 10 µg/mL of Loratidine and 100 µg/mL of Hydrochlorothiazide as internal standard.  The mobile phase was delivered at a flow rate of 1 mL/min with detection at 255 nm. The injection volume was 20 ml; Analysis was performed at ambient temperature.

 

TABLE I. RESULTS OF ANALYSIS OF PHARMACEUTICAL DOSAGE FORM AND RECOVERY STUDIES

 

 

Drug

Amount mg/ tab

% Label claim*

% Recovery*

Labeled

Found *

Ambroxol HCl

60

59.5181± 0.0317

99.19 ±0.0316

98.78 ±0.0068

Loratadine

5

5.0431± 0.0184

100.86± 0.0184

99.20 ± 0.011

*Average of 6 determinations ± standard deviation; Cozy AM- each tablet containing 60 mg of Ambroxol Hydrochloride and 5 mg of Loratidine

 

Preparation of sample solutions

Twenty tablets, each containing 60 mg of Ambroxol Hydrochloride and 5 mg of Loratidine were weighed and finely powdered; a quantity of powder equivalent to 60 mg of Ambroxol Hydrochloride and 5 mg of Loratidine was weighed and transferred to a sintered glass crucible. To this 12.5 mL of 1 mg/mL solution of Hydrochlorothiazide was added and the drugs were extracted with three quantities, each of 20 mL of methanol. The combined extracts were made up to 100 mL with methanol. The solution was filtered and were made to get a concentration of 12 µg/mL of Ambroxol Hydrochloride, 1 µg/mL of Loratidine  and  25 µg/mL of Hydrochlorthiazide (internal standard) in acetonitrile: water mixture (1:1 v/v). This solution was used for the estimation.

 

Assay method

With the optimized chromatographic conditions, a steady baseline was recorded, the mixed standard solution was injected and the chromatogram was recorded. The retention time of Ambroxol Hydrochloride, Loratidine and Hydrochlorothiazide was found to be 5.4, 15.5 and 3.2 min, respectively. This procedure was repeated for the sample solution obtained from the formulation. The response factor (peak area ratio of standard peak area and internal standard peak area) of the standard solution and sample solution were calculated. The analyte concentration of the drugs were calculated (Table 1) using following formula,

Analyte Concentration of drugs = (Response factor of the sample) x (Concentration of standard                                           Response factor of the standard)

 

Fig.1 Typical Chromatogram of Sample Solution

 

RESULTS AND DISCUSSION:

Estimation of Ambroxol Hydrochloride and Loratidine in dosage forms by RP-HPLC method was carried out using optimized chromatographic conditions. The standard and sample solutions were prepared. The chromatograms were recorded. The typical chromatogram of sample solution with internal standard is given in Fig 1. The peak area ratio of standard and sample solutions was calculated. The assay procedure was repeated for six times and mean peak area ratio and mean weight of standard drugs were calculated. Table I shows the result of analysis of pharmaceutical dosage form (tablet) and the recovery studies. The results of analysis shows that the amount of drugs was in good agreement with the label claim of the formulation.

The method was validated as per ICH guidelines. The accuracy of the method was determined by recovery experiments. The recovery studies were carried out six times by standard addition method. The percentage recovery and standard deviation of the percentage recovery were calculated and presented in Table 1. From the data obtained, added recoveries of standard drugs were found to be accurate.

 

The precision of the method was demonstrated by inter day and intra day variation studies. In the intra day studies, six repeated injections of standard and sample solutions were made and the response factor of drug peaks and percentage RSD were calculated and presented in Table 2. In the inter day variation studies, six repeated injections of standard and sample solutions were made for three consecutive days and response factor of drug peaks and percentage RSD were calculated and presented in Table 2. From the data obtained, the developed HPLC method was found to be precise.

 

The linearity of the method was determined at seven concentration levels ranging from 3.0 to 21.0 µg/mL for Ambroxol Hydrochloride and 0.25 to 1.75µg/mL for


TABLE 2. INTRADAY AND INTERDAY PRECISION STUDIES

Intraday studies

Interday studies

RF* of AmbroxolHCl

Mean

± (% RSD*)

RF of Loratidine

Mean

±(% RSD)

Day

RF of Ambroxol Hydrochloride

Mean

± (% RSD)

RF of Loratidine

Mean

± (% RSD)

0.4312

0.4554

0.4479

0.4510

0.4447

0.4314

 

0.4436

±

0.0229

 

0.0938

0.0958

0.0935

0.0940

0.0948

0.0956

 

0.0946

±

0.0102

 

Day 1

0.4313

0.4552

0.4479

0.4511

0.4463

0.4298

0.4436

±

0.0238

 

0.0938

0.0956

0.0950

0.0940

0.0948

0.0953

0.0948

±

0.0074

 

Day 2

 

0.4329

0.4554

0.4479

0.4513

0.4464

0.4315

0.4442

±

0.0221

 

0.0940

0.0949

0.0953

0.0941

0.094

0.0957

0.0949

±

0.0069

 

Day 3

0.4330

0.4539

0.4494

0.4514

0.4466

0.4298

0.4440

±

0.0228

 

0.0941

0.0951

0.0960

0.0941

0.0949

0.0959

 

* RF-Response Factor, % RSD- Relative standard deviation

 

TABLE 3. LINEARITY AND RANGE

Internal standard peak area (100µg/mL Hydrochlorothiazide)

Ambroxol Hydrochloride

Loratidine

Concentration

(µg/mL)

Peak area

Response factor

Concentration

(µg/mL)

Peak area

Response factor

 

 

642960

 

 

 

3.0

6.0

9.0

12.0

15.0

18.0

21.0

73252

135821

215703

281962

348170

403374

502455

0.1139

0.2112

0.3355

0.4479

0.5415

0.6274

0.7815

0.250

0.500

0.750

1.000

1.250

1.500

1.750

24606

34601

48936

60953

71439

85017

99637

0.0383

0.0538

0.0761

0.0948

0.1111

0.1322

0.1550


 

Loratidine (Table 3). The calibration curve was constructed by plotting response factor against concentration of drugs. The slope and intercept value for calibration curve was y = 0.0364x + 0.0011 (R2 = 0.9974) for Ambroxol Hydrochloride and y = 0.0774x + 0.0171 (R2 = 0.9979) for Loratidine. The results show that an excellent correlation exists between response factor and concentration of drugs within the concentration range indicated above.

 

The Limit of Detection (LOD) and Limit of Quantification (LOQ) of the developed method were determined by injecting progressively low concentrations of the standard solutions using the developed RP-HPLC method. The LOD is the smallest concentration of the analyte that gives a measurable response (signal to noise ratio of 3). The LOD for Ambroxol Hydrochloride and Loratidine was found to be 5 ng/mL and 25 ng/mL, respectively. The LOQ is the smallest concentration of the analyte, which gives response that can be accurately quantified (signal to noise ratio of 10). The LOQ was 15 ng/mL and 75 ng/mL for Ambroxol Hydrochloride and Loratidine, respectively (Table 4).

 

 

 

The ruggedness of the method was determined by carrying out the experiment on different instruments like Shimadzu HPLC (LC-10AT), Agilent HPLC and Water’s Breeze HPLC by different operators using different columns of similar type like Hypersil C18, Phenomenex LUNA C18 and Hichrom C18. Robustness of the method was determined by making slight changes in the chromatographic conditions. No marked changes in the chromatograms demonstrated that the HPLC method developed are rugged and robust.

 

TABLE 4. VALIDATION AND SYSTEM SUITABILITY STUDIES

S.No.

Parameters

Ambroxol HCl

Loratidine

1

Linearity range

3.0 to 21.0 µg/mL

0.250 to 1.750µg/mL

2

Regression equation

Y = mx + c

y = 0.0364x - 0.0011

y = 0.0774x + 0.0171

3

Correlation coefficient

0.9974

0.9979

4

Theoretical plate/meter

25478

29784

5

Resolution factor

1.32

1.32

6

Asymmetric factor

0.91

1.02

7

LOD (ng/mL)

5

25

8

LOQ (ng/mL)

15

75

In order to demonstrate the stability of both standard and sample solutions during analysis, both solutions were analyzed over a period of 5 h at room temperature. The results show that for both solutions, the retention time and peak area of Ambroxol Hydrochloride and Loratidine remained almost unchanged (% R.S.D. less than 2.0) and no significant degradation within the indicated period, thus indicated that both solutions were stable for at least 5 hr, which was sufficient to complete the whole analytical process.              

 

The column efficiency, resolution and peak asymmetry were calculated for the standard solutions (Table 4). The values obtained demonstrated the suitability of the system for the analysis of this drug combinations, system suitability parameters may fall within ± 3 % standard deviation range during routine performance of the method.

 

Thus the proposed RP-HPLC method for the simultaneous estimation of Ambroxol Hydrochloride and Loratidine in combined dosage forms is accurate, precise, linear, rugged, robust, simple and rapid. Hence the present RP-HPLC method is suitable for the quality control of the raw materials, formulations and dissolution studies.

ACKNOWLEDGEMENT:

The Author’s thank to M/s. Franco Indian Pharma, Mumbai for providing gift samples of Loratidine and Ambroxol Hydrochloride and M/s. Apex Pharmaceuticals Andhra Pradesh for providing a gift sample of Hydrochlorothiazide. The Author’s are grateful to His Holiness Jagadguru Sri Sri Shivarathree Deshikendra Mahaswamigalavaru” of Sri Suttur Mutt, Mysore for providing facilities to carry out this work.

 

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Received on 16.07.2008       Modified on 05.08.2008

Accepted on 10.08.2008      © RJPT All right reserved

Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008;Page 366-369