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Asian J. Pharm. Res. Vol 5, Issue 3, 143-150, 2015.

e-ISSN 2231 363X

Print ISSN 2231 3621
Asian Journal
of
PHARMACEUTICAL RESEARCH
Journal homepage: - www.ajprjournal.com

SIMULTANEOUS ESTIMATION OF ACEBROPHYLLINE AND

ACETYLCYSTEINE IN TABLET DOSAGE FORM BY RP HPLC
METHOD
A. Geetha Susmita1*, G. Aruna2, S. Angalaparameswari2, M. Padmavathamma3
1
Department of Pharmaceutical Analysis, SV University, Tirupati, A.P, India.
2
Department of Pharmaceutical Analysis, Krishna Teja Pharmacy College, Tirupati, A.P, India.
3
Department of Pharmaceutical chemistry, Sri Padmavathi Womens Polytechnic, Tirupati, A.P, India.

ABSTRACT
A simple, economic, precise and accurate Reverse Phase High Performance Liquid Chromatographical method has
been developed for the simultaneous estimation of Acebrophylline and N-Acetylcysteine in tablet dosage form. An enable
Hypersil BDS, C18, 100 x 4.6 mm, 5 particle size column was used as stationary phase. The mobile phase consisting of a
mixture of buffer solution and acetonitrile (90:10) was pumped isocratically at a flow rate of 1 ml/min with detection at 260nm.
The retention time of Acebrophylline and N-Acetylcysteine were found to be 5.5 min and 2.3 min respectively. The calibration
curves were linear over a concentration range of 25-150 g/ml with coefficient regression (r2) = 0.9995 for Acebrophylline and
(r2) = 0.9996 for N-Acetylcysteine. The limits of detections were 0.18 g/ml and 1.50 g/ml for Acebrophylline & N-
Acetylcysteine respectively. The selectivity, specificity, system suitability, ruggedness and robustness were performed as per
ICH guidelines. In quantitative and recovery studies was 100.37% and 100.8% for Acebrophylline and N-Acetylcysteine
respectively. The percentage RSD was found to be less than 2. Due to the simplicity, rapidity and accuracy of the method with
believe that the method will be useful for routine quality control analysis of Acebrophylline and N-Acetylcysteine in
pharmaceutical formulation.

Key words: Acebrophylline-ACB, N-Acetylcysteine-NAC, Accuracy, RP-HPLC and Validation.

INTRODUCTION
Acebrophylline is an anti-inflammatory and phase. It increases the mucociliary clearance by stimulating
airway mucus regulator. It contains ambroxol and cilia motility. Acebrophylline inhibits phospholipase A,
theophylline-7-acetic acid are facilitates the biosynthesis of and phosphatidylcholine leading to lesser production of the
pulmonary surfactant while later raises blood levels of powerful pro-inflammatory substances like leukotrienes
ambroxol, by stimulating surfactant production [1]. and tumour necrosis factor. By inhibiting the synthesis and
Chemically acebrophylline (Fig 1) is (1, 3- dimethyl-2, 6- release of these inflammatory mediators, acebrophylline
dioxo-1, 2, 3, 6- tetrahydro-7H-purine-7yl) acetic acid- reduces inflammation, a key factor in airway obstruction,
4[((2-amino-3, 5-dibromophenyl) methyl) amino] especially in chronic forms [2].
cyclohexanol. It is a salt obtained by reaction of equimolar Acetylcysteine (NAC), ((2R)-2-(acetylamino)-3-
amounts of theophylline-7-acetic acid and ambroxol [2]. sulfanylpropanoic acid) is a mucolytic agent used in
Theophylline-7-acetate has a bronchodilator effect due to bronchitis, pulmonary diseases and respiratory disorders
inhibition of the intracellular phosphodiesterases, followed associated with active cough. It depolymerises
by an increase of adenosine monophosphate cyclic levels, mucopolysaccharides, reduces the viscosity of pulmonary
which promote the relaxation of bronchial muscles. secretions. Besides mucolytic effect it also has anti-oxidant
Ambroxol modifies the mucous gel phase of secretions by and antiinflammatory effects and it is used as an antidote in
decreasing the viscosity and increasing the serous gel Paracetamol poisoning [3]. More recently, animal and

Corresponding Author :- A. Geetha Susmita Email:- [email protected]

1
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human studies of NAC have shown it to be a powerful Preparation of Sample solution

antioxidant and a potential therapeutic agent in the 20 tablets were weighed and powdered.
treatment of cancer, heart disease, HIV infection, heavy Calculated the average weight of each tablet and weighed
metal toxicity, and other diseases characterized by free equivalent to 50 mg was transferred into a 500 ml
radical and oxidant damage [3]. volumetric flask. 50ml of diluent was added and sonicated
The literature survey reveals that there was no for 30 min; further the volume was made up to with diluent
analytical method for the simultaneous estimation of and filtered through 0.45 nylon filters. From the above
Acebrophylline and Acetylcysteine in tablet dosage form. filtrate 1ml was pipette out into a 10 ml volumetric flask
Sravani Takkarusu et al [1] had developed a precise and and made upto 10ml with diluent. The desired
accurate RP-HPLC method for the determination of concentrations for the drug were obtained by accurate
Acebrophylline in bulk and capsule dosage form. G. dilution, and the analysis was followed up as in the general
Lalitha et al [2] had developed a UV-visible analytical procedure.
spectroscopical estimation for Acebrophylline and N-
Acetylcysteine in bulk and capsule dosage form. So their is RP-HPLC Method Development and optimization of
a need to develop a simple, less time consuming and chromatographic conditions
economical method for the estimation of Acebrophylline The method development was conducted on a
and N-Acetylcysteine in tablet dosage form. Hence an Hypersil BDS, C18, 100 x 4.6 mm, 5 particle size column
attempt was made to develop and validate a simple, precise used for separation. The chromatographic conditions were
and accurate RP-HPLC method as per ICH guidelines [9, optimised with respect to specificity, resolution and time of
10]. analysis. The optimal experimental conditions fixed with a
mixture of mobile phase consist of buffer and acetonitrile
MATERIALS AND METHODS in ratio of 90:10 and a flow rate of 0.9ml/min with
Reagents detection at 260nm. The ACB and NAC depicted a well
All the reagents and chemicals used were of defined chromatographic separation within a run time of 8
HPLC grade. Acebrophylline and N-Acetylcysteine min. The retention times of ACB and NAC were 5.5min
working standards were obtained from Sun Pharma, 0.32 and 2.3 min 0.35, respectively. Fig 2 shows
Mumbai and Torrent Pharmaceuticals, Ahmadabad chromatograms of standard and sample of Acebrophylline
respectively. The Pulmoclear Tablets were obtained from and Acetylcysteine.
Fourrts (India) Laboratories Pvt. Ltd. HPLC grade
Acetonitrile was procured from SDFCL Mumbai, India. RP-HPLC Method Validation
All other chemical reagents were of analytical grade. The developed method was validated according to
ICH guidelines [4] with respect to specificity, accuracy,
Instrumentation precision, linearity, limit of detection (LOD), limit of
A Waters Alliance 2695 with Empower 2 quantification (LOQ) ruggedness, robustness and system
software HPLC system provided with PDA detector of suitability
model 2998 and an automatic injector were used. The
chromatographic analysis was performed on Hypersil Specificity of the method
BDS, C18, 100 x 4.6 mm, 5 particle size column. It is an ability to measure the accurate
concentration of analyte in the presence of all other
Preparation and selection of mobile phase excipient. The 10.0mg of placebo was transfer into a 100
Buffer: (0.02M KH2PO4) ml volumetric flask and added sufficient amount of
Accurately weighed and transferred 2.72gm of diluents. The sample solution was sonicated to dissolve
Potassium dihydrogen orthophosphate in a 1000ml of and make up the volume with diluents. Further dilute 1 ml
volumetric flask, add about 900ml of milli-Q water and of the above solution to 10 ml with diluents. The final
1ml of triethylamine then sonicated to degasses the solution of placebo was injected in to a chromatographic
solution, finally the volume make up with water. The pH system and chromatogram was observed.
adjusted to 3.2 with dil. ortho phosphoric acid solution.
Accuracy 50%, 100% and 150%
Mobile phase Accurately weighed 10mg of acebrophylline and
Buffer and Acetonitrile taken in the ratio 90:10. 12.5mg of acetylcysteine were transferred into a10 ml
volumetric flask. The compounds were first dissolved in 7
Preparation of Standard solution ml of diluent and it was sonicated for 5 minutes then the
Accurately Weighed and transferred 10mg of volume was made up to the mark with diluents and mixed
Acebrofylline and 12.5mg of N-Acetylsteine working well. Further 0.5 ml, 1ml and 1.5ml were diluted to 10 ml
standards into a 10 ml clean dry volumetric flask, add 7ml in different volumetric flasks with diluents to get 50%,
of diluents, sonicated for 30 minutes and make up to the 100% and 150% solutions respectively and mixed well,
volume with diluents (methanol). filtered through 0.45 nylon filter. The accuracy of the
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method was computed by the determination of recovery for concentrations that are within the specified range that 25%,
three concentrations [5]. The amount of ACB and NAC 50%, 75%, 100%, 125%, 150% of Acebrophylline and
was recovered and then percentage of drug content was Acetylcysteine. The calibration curves were plotted
calculated. between certain concentrations [5] and average peak areas.
The results were shown in fig 5.
Precision
Precision was studied to find out system Robustness
precision, method precision and interday variation. In Robustness study with effect of flow rate
method precision, a homogenous sample of a single batch Robustness was carried by small deliberate
was analyzed six times. The % RSD of the assay value for changes in chromatographic conditions at three different
six determinations was calculated. The system precision levels and retention time was noted. The factors selected
was checked by measuring six successive injections of were flow rate and pH. To carry out the test method with
Acebrophylline and Acetylcysteine standard to ensure that variation of flow rate ( 0.2 ml/minute of set value, i.e. 0.8
the analytical system was precise [7]. The percentage RSD and 1.2 ml/minute) and PH (+ 0.2 of set value). Prepare
was calculated for ACB and NAC shown in table 4. standard preparation and perform analysis as per test
method and evaluate the system suitability parameters [8].
Ruggedness
The standard stock solution and sample stock System suitability test
solution were prepared by different analysts on different To establish the chromatographic conditions, a
days and the resulting solutions were injected and system suitability test during the development and
chromatograms are recorded. optimization of the method was performed. The test was
Precision was studied to find out system performed by injecting standard mixture to the
precision, method precision and interday variation. The chromatographic system and the various parameters,
percentage RSD was calculated for ACB and NAC, Table retention time, tailing factor and theoretical plates were
(4) shows the results of the test obtained by running the computed as reported by the International conference
samples for two days by various analysts. harmonized guidelines [4, 6].
Linearity
Linearity was determined by the regression Limit of Quantification (LOQ) and Limit of Detection
analysis and was studied by preparing standard solution at (LOD)
different concentration levels (25-150%). Acebrophylline The limit of quantification (LOQ) and limit of
and Acetylcysteine standard solutions are prepared across detection (LOD) were calculated using the signal-to-noise-
the range of the analytical method with a minimum of five ratio of 10 and 3 the results were tabulated III.

Table 1. Data showing assay of tablet formulation

Component Label claim % Amount Mean S.D %RSD
Acebrophylline 100mg 100.09 0.56 0.566
Acetylcysteine 600mg 99.75 0.51 0.512

Table 2. Results of Accuracy

Mg added(ppm) Mg Recovered(ppm) %Recovery
S.No Spike level
ACB NAC ACB NAC ACB NAC
1 50% 50 300 49.93 303.0 99.8 101.02
2 50% 50 300 50.09 302.7 100.1 100.9
3 50% 50 300 50.17 299.1 100.3 99.7
4 100% 100 600 100.6 595.2 100.6 99.2
5 100% 100 600 102.1 600.9 102.1 100.1
6 100% 100 600 101.7 597.3 101.7 99.5
7 150% 150 900 152.02 909.5 101.3 100.06
8 150% 150 900 150.4 901.2 100.2 100.1
9 150% 150 900 151.9 909.6 101.2 100.07

Table 3. Results of intermediate precision

Assay%
Sample Acebrohylline Acetylcysteine
1 100.6116 99.82471
2 99.57081 99.69537
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3 100.667 100.2917
4 100.1357 99.32941
5 101.262 99.71991
6 100.4335 98.77111
AVG 100.4468 99.60537
SD 0.566493 0.512353
%RSD 0.56 0.51

Table 4. Linearity Results, Limit of Detection (LOD) and Limit of Quantification (LOQ)
Parameters Acebrophylline Acetylcysteine
Number of samples 5 5
Correlation range ( g /mL) 25-150 (g/mL) 25-150 (g/mL)
Regression coefficient 0.9995 0.9996
Limit of Quantification (g/mL) 0.568100676 4.558335662
Limit of Detection (g/mL) 0.187473223 1.504250768

Table 5. Summaries of Validation Parameters

Validation Results Acceptance
S.No. Requirement
Parameters Acebrophylline Acetylcysteine Criteria
1. Specificity No Interference Pass Pass No Interference
Correlation
2. Linearity 0.9999 0.9998 NLT 0.999
Coefficient
3. Accuracy %Recovery 100.8% 100.37% 100 2%
Method Precision 0.51 0.51 NMT 2%
4. System Precision %RSD 0.6 0.2 NMT 2%
Ruggedness 0.78 0.72 NMT 1%
5. Robustness %RSD 0.97 0.57 NMT 1%
RT 5.564 2.401 -
Tailing Factor 0.99 1.09 NMT 2
6. System Suitability
Plate Count 5945 4448 NLT 4000
Assay Value 100.44% 99.6% 100 2%
Concentration of
7. LOQ 0.56 4.55 -
analyte
Lowest
8. LOD 0.18 1.5 -
concentration

Fig 1-2. chromatogram of Acebrophylline and N-Acetylcysteine sample and standard

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Fig 3. Chromatogram for system suitability

Fig 4. Linearity plot of Acebrophylline and Acetylcysteine

Fig 5. Chromatogram for linearity concentration at 25%, 50%, 75%, 100%, 125%, 150%
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Fig 6. Accuracy 50% level

Fig 7. Accuracy 100% level

Fig 8. Accuracy 150% level

Fig 9. Method Precision chromatogram

Fig 10. Ruggedness chromatogram for day1and day2

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Fig 11. Ruggedness chromatogram by analyst 1 and analyst 2

RESULT AND DISCUSSION not interfere in the drug peak (Fig 2&3). The ruggedness
The chromatogram of sample and standard are study of standard solution (Fig 10-11) indicated that the
shown in the figure 1 and 2 indicating retention time for retention times for both the drugs were not so different and
ACB was 5.504 and 5.509 respectively. Similarly the relative standard deviations of drugs are less than 2. This
retention time for NAC was 2.370 and 2.370 respectively might be due to proper system suitability (Table 4). Table
which is not so different it can be conclude that the 5 Indicated that system suitability parameters for drugs
developed RP-HPLC method is accurate and precise Acebrophylline and Acetylcysteine. The resolution for
without any interference and overlapping (Fig 3). The both the drugs was found to be more than 2. Hence it can
assay of acebrophylline and acetylcysteine was calculated be concluded that the RP-HPLC method shown the
and mentioned in Table I. This data of both the drugs reproducibility for the method systems.
complies with the standard specifications of 50%, 100%
and 150% recovery data for acebrophylline and CONCLUSION
acetylcysteine are shown in Table II .The % recovery of From the above results, method was found to be
sample was observed between 99 to 101% and relative accurate, precise, linear, specific, system suitable, robust
standard deviation was 0.5 and 0.4 for NAC, ACB proved to be sensitive, convenient and cost effective for the
respectively. Hence the data shown under accuracy estimation of Acebrophylline and Acetycysteine in tablet
parameters were found within the prescribed limits, dosage form. The proposed method has a run time of
indicating that the developed method was accurate and 8mintues, which makes the method simple, cost effective
prcised (Fig 6-9). The linearity parameter and and suitable for the routine analysis of Acebrophylline and
corresponding regression data indicated excellent linear Acetylcysteine in tablet dosage form.
relationship (R2 =0.9999) for ACB and (R2 =0.9998) for
NAC (table 3). Fig.4 indicates ACB and NAC were ACKNOWLEDGEMENT
obeying Beer Lamberts law limit. The LOD and LOQ for The author thank Fourrts (India) Laboratories Pvt.
the ACB were found 0.18 (g/mL) and 0.56 (g/mL), Ltd, India for making Acebrophylline and Acetylcysteine
while for NAC was 1.50 (g/mL) and 4.55 (g/mL) and marketed pulmoclear tablet preparation available for
respectively. This specificity test of the proposed method experiments and also thanks to Krishna Teja Charities
demonstrated that the excipients of tablet formulation do Trust for providing facility to take over this project work.

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