INTRODUCTION:

Acebrophylline, 7-(1,3-dioxalan-2-ylmethyl)theophylline is an anti-inflammatory and air-way mucus regulator drug. It contains ambroxol and theophylline-7-acetic acid (acefylline), former facilitates the biosynthesis of pulmonary surfactant while later rises blood levels of ambroxol there by stimulating surfactant production. Chemically acebrophylline is- 1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H purine-7-acetic acid with trans-4-[(2-amino-3,5-dibromophenyl)methyl]amino]cyclohexanol.

Literature survey releaved that various analytical methods like spectrophotometric, HPLC and HPTLC methods has been reported for determination of ambroxol HCl and theophylline-7-acetic acid, individually and combination with some other drugs. No HPLC methods for determination of Acebrophylline in single dosage form have so far reported. The review of literature prompted to develop a rapid, accurate and precise HPLC method for the estimation of Acebrophylline in pharmaceutical dosage.

Fig.1: Chemical structure of Acebrophylline.

In view of the above fact, a simple analytical method is needed for its quantitative estimation.

So, an attempt was made to develop a new spectrophotometric method for its estimation in bulk and in capsule dosage form with good accuracy, specificity, precision and economy.

MATERIALS AND METHODS:

A reverse phase high performance liquid chromatographic model of Waters 2695 separation model with Waters 2487 detectors with dual λ-absorbance and Shimadzu LC-2010 CHT chromatograph and another detector is Shimadzu UV. Data acquisition was performed on Empower-2 software. Pure grade of Acebrophylline, Acefylline and Ambroxol were procured from Pretiwell, Mumbai. Brophyle capsule used for the formulation analysis contains Acebrophylline Manufactured by Tirupati Medicare Ltd., Paonta Sahib, Himachal Pradesh. Acetonitrile and methanol used were HPLC grade. Ammonium orto-phosphate, Ammonium tetra borate tetra hydrate,Sodium acetate,Glacial acetic acid,Orthophosphoric acid, Hydrogen Peroxide, Hydrochloric acid used were analytical grade.

Method development:

Optimization of mobile phase:

The mobile phase must be optimized showing chromatograms using different organic solvents as modifiers of mobile phase. HPLC chromatogram by using mobile phase buffer: acetonitrile. Buffer uses 0.1% ammonium tetra borate tetra hydrate gives chromatogram in which acefylline peak is eluted in void volume of column. Further change in mobile phase composition to 50:50 gives ambroxol peak broadening. Next increase in buffer concentration to 92% gives acefylline peak out of void volume but ambroxol peak moves to 5 min. Change to gradient flow system from 0 to 40 min. with variable buffer: Acetonitrile conc. leads to give peak purity of ambroxol failing due to co-elution of interfering compound. Then change in buffer:Acetonitrile conc. as 45:55 gives good resolution but peaks fails in specificity. Then change into buffer to Sodium acetate and Glacial acetic acid with methanol and Acetonitrile as solvent gives peaks of ambroxol not satisfactory. Next buffer used in solvent instead of Acetonitrile buffer: solvent 50:50 leads to tailing of peaks. Then change in buffer (0.01 M) ammonium phosphate buffer pH 7 with Orthophoshoric acid and used in gradient program gives ambroxol peaks did not elutes out. Then next introduce methanol in gradient flow and water used as diluent gives satisfactory results and peaks of chromatogram.

Preparation of Mobile Phase:

The mobile phase was composed of buffer solution (0.01 M Ammonium ortho-phosphate (pH 7): acetonitrile: methanol in gradient program given in following table.

Table.1. Table showing gradient program flow of mobile phase for acebrophylline.

Time(min.)	Buffer	Acetonitrile	Methanol
0 min.	85	5	10
10min	85	5	10
15min	65	25	10
40min	65	25	10
45min	60	30	10
50min	60	30	10

Preparation of Standard solution:

Standard solution of acebrophylline was prepared by dissolving 25mg of Acebrophylline working standard to 25ml of water in 25ml volumetric flask. Further diluted 10ml of above solution to 25ml of volumetric flask and make up the volume.

Preparation of Sample solution:

Sample solution of acebrophylline was prepared by weighing 20 capsules. Calculate its average net content. Mix powder and weigh powder (equivalent to 100mg of Acebrophylline) and transfer to 100ml volumetric flask. Make up the volume with water. Further dilute 10ml of solution with 25ml of water.

Preparation of Placebo solution:

Placebo solution of Acebrophylline. Placebo solution was prepared by calculation of placebo content in each capsule.

Placebo content = Average net content of capsule – weight of API per capsule:

Make same dilutions as of Standard and Stock solutions. Prepare solution.

Chromatographic conditions:

An Inertsil ODS-3V (250mm x 4.6mm, 5µ) column was used at ambient temperature, with UV detection 240 nm, injection volume of 20 µl and a flow rate of 1.0 ml/min. As mentioned in previous section, the mobile phase consisted of buffer solution (0.01M Ammonium ortho-phosphate (pH 7): acetonitrile: methanol. The peak areas of responses were used for quantitation.

RESULTS:

Linearity:

The linearity of the HPLC methods for determination of Acebrophylline was evaluated by analyzing a series of different concentrations. In this study nine concentrations were chosen, ranging between 200ppm to 600ppm. This approach will provide information on the variation of peak areas. The high value of the correlation coefficient and the intercept value that was not statistically (P<0.05) different from zero validated the linearity of calibration graphs (Fig 1).

Fig.1 Figure showing calibration curve of Acebrophylline working standard from 200ppm to 600ppm concentration.

Fig.2. Figure showing HPLC chromatogram of acebrophylline standard.

Table.2. Table shows linearity relationship between 200 to 600 ppm for acebrophylline.

Concentration in %	Conc. of Std in ppm	Area		Average Area	Slope	23964
Concentration in %	Conc. of Std in ppm	Acephylline	Ambroxol	Average Area	Intersept	337376
Linearity-50.0 %	198.899	1546913	3395535	4942448	Correl	0.99904
Linearity-60.0 %	238.679	1944099	4207259	6151358
Linearity-70.0 %	278.459	2203567	4802937	7006504
Linearity-80.0 %	318.239	2513455	5479355	7992810
Linearity-90.0 %	358.019	2876789	6252378	9129167
Linearity-100.0 %	397.799	3044861	6645659	9690520
Linearity-110.0 %	437.579	3431466	7522112	10953578
Linearity-125.0 %	497.249	3787091	8347758	12134849
Linearity-150.0 %	596.698	4582309	10053571	14635880

Table.3. Table shows the method repeatability/intermediate precision for acebrophylline.

Identity	Wt. taken in mg	Area			Amount mg/Capsule	% LC
Identity	Wt. taken in mg	Injection 1	Injection 2	Average	Amount mg/Capsule	% LC
Precision-1	265.36	8988704	8976327	8982516	99.5	99.5
Precision-2	270.36	8939519	8936095	8937807	97.1	97.1
Precision-3	267.50	8973990	8951304	8962647	98.5	98.5
Precision-4	266.36	8935763	8919487	8927625	98.5	98.5
Precision-5	266.00	8936887	8932510	8934699	98.7	98.7
Precision-6	260.36	8623695	8636198	8629947	97.4	97.4
					Average	98.3
					Std dev.	0.886
					% RSD	0.902

Table.4. Table shows Specificity for acebrophylline.

Identity	Wt. taken in mg	Area			Amount mg/Capsule	% LC	Average Precision Results	% Degradation
Identity	Wt. taken in mg	Area of Acephylline	Area of Ambroxol	Sum	Amount mg/Capsule	% LC	Average Precision Results	% Degradation
Acid (0.1N HCl)	278.30	1296136	3386800	4682936	100.2	100.2	98.3	----
Base ( 0.1N NaOH)	269.56	1156854	2771041	3927895	86.8	86.8		11.5
Peroxide(3% H2O2)	268.36	1218823	2989976	4208799	93.4	93.4		4.9

Fig.3. Figure showing HPLC chromatogram of acebrophylline sample.

Precision:

Precision was validated using repeatability, intermediate precision and reproducibility. The repeatability measures by preparing two acebrophylline standards and were injected using different injections. Six samples were prepared and injected in duplicate for precision. Intermediate precision were determined by using inter day and intraday precision. The percentage relative standard deviation (%RSD) was calculated. The %RSD should be less than 2% of six sets of sample preparation.

Specificity:

Specificity was validated using stress conditions such as acid (0.1N HCl), base (0.1N NaOH) and oxidant (3%H₂O₂). The forced degradation of Acebrophylline API, Acebrophylline standard, Acebrophylline capsule sample was tested. The presence of peaks in Placebo and diluents at the Rt of the principal peaks of Acefylline and Ambroxol was also checked in untreated sample and standard. The specificity was validated with no interference of other peaks at the Rt of main peak.

Accuracy.

This method was validated by using adding Acebrophylline working standard to fixed concentration of Placebo to calculate accuracy and range of the method. The placebo and concentration of standard prepared by using 50% to 150% of concentration standard added. The % recovery and % RSD were calculated for three sets of samples and found within limits.

Ruggedness.

Ruggedness was validated by using- different analyst, different column and different system were used and all the parameters for chromatography remain same. The five replicate standards were injected and then samples were run. The ruggedness will be established on the basis of %RSD which should not be more than 2% for all six ruggedness sets and six precision sets of sample preparation. Six samples were prepared and injected for establishment.

Table.5. Table shows Accuracy/recovery for acebrophylline.

Sample Identity	Amount Added in mg	Area			Actual Amount Added in mg	Amount Recovered in mg	% Recovered
Sample Identity	Amount Added in mg	Area of Acephylline	Area of Ambroxol	Sum	Actual Amount Added in mg	Amount Recovered in mg	% Recovered
Accuracy-50 % Set-1	51.63	1296450	3533313	4829763	51.32	51.540	100.4
Accuracy-50 % Set-2	51.41	1317474	3581788	4899262	51.10	52.281	102.3
Accuracy-50 % Set-3	51.78	1308130	3534781	4842911	51.47	51.680	100.4
Accuracy-100 % Set-1	99.62	2507923	6783717	9291640	99.02	99.154	100.1
Accuracy-100 % Set-2	100.36	2522690	6789976	9312666	99.76	99.378	99.6
Accuracy-100 % Set-3	100.56	2523523	6771759	9295282	99.96	99.193	99.2
Accuracy-150 % Set-1	149.25	3729920	9986357	13716277	148.35	146.370	98.7
Accuracy-150 % Set-2	150.15	3717212	10039699	13756911	149.25	146.804	98.4
Accuracy-150 % Set-3	149.76	3700686	10013688	13714374	148.86	146.350	98.3
						Average	99.7
						Stdev	1.265
						% RSD	1.269

Table.6. Table shows ruggedness for acebrophylline.

Sample Identity	Wt. taken in mg	Area			Amount mg/Tablet	% LC
Sample Identity	Wt. taken in mg	Acephylline	Ambroxol	Sum	Amount mg/Tablet	% LC
Precision-1						99.5
Precision-2						97.1
Precision-3						98.5
Precision-4						98.5	Average	98.3
Precision-5						98.7	Std dev.	0.886
Precision-6						97.4	% RSD	0.902
Ruggedness-1	278.52	3503837	6684552	10188389	98.59	98.6
Ruggedness-2	279.36	3504765	6685721	10190486	98.31	98.3
Ruggedness-3	272.45	3507475	6302080	9809555	97.04	97.0
Ruggedness-4	273.60	3476253	6596715	10072968	99.22	99.2
Ruggedness-5	270.36	3270713	6215705	9486418	94.57	94.6	Average	97.4
Ruggedness-6	274.57	3401416	6451445	9852861	96.71	96.7	Std dev.	1.672
					Average	97.8	% RSD	1.717
					Std dev.	1.4
					% RSD	1.387

Fig.3 Figure showing HPLC chromatogram of Acebrophylline standard for accuracy parameter.

Fig.4. HPLC chromatogram of Acebrophylline standard for ruggedness parameter by using Shimadzu u.v. detector.

Fig.5. HPLC chromatogram of Acebrophylline standard for ruggedness parameter by using Shimadzu u.v. detector.

CONCLUSIONS:

The linearity of acebrophylline peak area was demonstrated, from approximately 50 to 150% of the analytical concentration 0.40 mg/ml, by a correlation coefficient 0.999. The precision of acebrophylline peak area of responses, calculated for two replicates injections of capsule sample solution showed RSD of 0.902 %. The accuracy/recovery of sample was tested using three concentration run in triplicate manner. The mean % label claim was found to be 99.72% and RSD of 1.269. Recoveries of triplicate preparation at levels corresponding to 50, 100 and 150% of the nominal analytical concentration of 0.40 mg/ml acebrophylline in capsule samples were 99.72% (RSD 1.269) as mean of all three levels. Forced degradation studies on sample preparation shows degradation in basic and oxidative environment.

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Received on 29.06.2011 Modified on 17.07.2011

Research J. Pharm. and Tech. 4(10): Oct. 2011; Page 1542-1546