Spectrophotometric Estimation of Carbocisteine in Bulk and Pharmaceutical Dosage Form by First Order Derivative and Area Under Curve Methods
Rele Rajan V.*, Patil Sachin S.
Central Research Laboratory, D.G. Ruparel College, Matunga, Mumbai 400016.
*Corresponding Author E-mail: drvinraj@gmail.com
ABSTRACT:
A simple and precise first order derivative and area under curve [AUC] UV- spectrophotometric methods have been developed and validated for the estimation of carbocisteine in bulk and its tablet formulation. The standard and sample solutions of carbocisteine were prepared in 0.1 N HCl. Carbocisteine was estimated at 198.4 nm for the first order derivative UV-spectrophotometric method (A), while in area under curve (AUC) method (B) the zero order spectrum of carbocisteine was measured in between 197.4 nm to 210.2 nm. Beer’s law was obeyed in the concentration range of 10 to 140 μg / ml with coefficient of correlation value 0.9999 for first order derivative method. Similarly in AUC method, Beer’s law was obeyed in the concentration range of 10 to 140 μg / ml with coefficient of correlation value 0.9999. These methods were tested and validated for various parameters according to ICH guidelines. The precision expressed as relative standard deviation, which was within the range of 0.02560 % to 0.13375 % for the above two methods. The proposed methods were successfully applied for the determination of carbocisteine in pharmaceutical formulation. Results of the analysis were validated statistically and were found to be satisfactory. The proposed methods are simple, easy to apply, low-cost and require relatively inexpensive instruments.
KEYWORDS: Carbocisteine, Derivative spectroscopy, Area under curve method.
In this communication the present work proposes UV spectrophotometric methods for assay of carbocisteine from bulk drug and pharmaceutical formulation. Its chemical name is (2R)-2-amino-3-[(carboxy-methyl) sulphanyl] propanoic acid. Carbocisteine is a mucolytic drug, which breaks down mucus in the body so that it can be more easily cleared from the body. In chronic obstructive pulmonary disease (COPD) symptoms involve the over secretion of mucus, mucolytic have great potential for treatment of this disease. Additional characteristics of COPD include airflow limitation oxidative, stress and airway inflammation. The structure of carbocisteine is shown in Fig.1.
Fig. 1: Chemical structure of carbocisteine
Carbocisteine is official in British Pharmacopoeia1 and European Pharmacopoeia2. In literature survey HPLC3-4, UPLC5 and Ion-Chromatography6 methods were reported. This method can be used for the routine analysis and research organization. In the proposed work optimization and validation of these methods are reported.
MATERIALS AND METHODS:
Instrument and reagents
Spectral scan was made on a Shimadzu UV-spectrophotometer, model 1800 (Shimadzu, Japan) with spectral band width of 0.5 nm with automatic wavelength corrections by using a pair of 10 mm quartz cells. All spectral measurements were done by using UV-Probe 2.42 software. Reference standard of carbocisteine was obtained from reputed firm with certificate analysis.
Preparation of standard drug solution
100 mg standard carbocisteine was weighed accurately and transferred to a 100 ml volumetric flask and sonicated with 30 ml of 0.1 N HCl for 15 minutes. The volume was made up to the mark with 0.1 N HCl to give a stock solution of concentration 1000 μg /ml. From this solution, 10 ml of solution was pipetted out and transferred into 100 ml volumetric flask. The volume was made up to mark with 0.1N HCl to give a working standard solution of concentration 100 μg/ml.
Estimation from tablets
Twenty tablets were weighed accurately and average weight of each tablet was determined. Powder equivalent to 10 mg of carbocisteine was weighed and transferred in 100 ml of volumetric flask. A 30 ml of 0.1N HCl was added and sonicated for 15 minutes and filtered. The filtrate and washing were diluted up to the mark with 0.1N HCl to give concentration as 100 μg /ml. Such solution was used for analysis.
EXPERIMENTAL:
Method A: First order derivative method
For the selection of analytical wavelength, 100 μg/ml solution of carbocisteine was scanned in the spectrum mode from 400 nm to 190 nm by using 0.1 N HCl as blank. The first order derivative spectrum was obtained by using derivative mode by UV probe 2.42 software. From the spectrum, the amplitude of the derivative spectrum was measured between 200 nm to 198 nm (Fig. 2).
Fig. 2. First order derivative spectrum of carbocisteine (100 μg/ml) showing absorbance at 198.4 nm
Fig. 3. Calibration curve for carbocisteine at 198.4 nm by first order derivative spectroscopy
Into series of 10 ml graduated flask, varying amount of sample solutions of carbocisteine were pipette out and volume was adjusted with 0.1N HCl. Solutions were scanned between 400 nm to 190 nm in spectrum mode. The first order derivative spectra were obtained by using derivative mode. Amplitudes of the resulting solutions were measured at between 200 nm to 198 nm by using 0.1N HCl as blank. The calibration curve was prepared in the concentration range of 10 to 140 μg/ml (Fig. 3).
Method B: Area under curve (AUC) method
Area under curve method involves the calculation of integrated value of absorbance with respect to the wavelength between two selected wavelengths such as λ1 and λ2. The area under curve between λ1 and λ2 were calculated by UV probe 2.42 software. In this method, 100 μg/ml solution of carbocisteine was scanned in the spectrum mode from 400 nm to 190 nm. From zero order spectrum the AUC calculation was done. The AUC spectrum was measured between 197.4 nm to 210.2 nm (Fig. 4).
Fig. 4. Area under curve spectrum of carbocisteine ( 100 μg/ml) showing area from 197.4nm to 210.2 nm.
Into series of 10 ml graduated flask, varying amount of sample solutions of carbocisteine were pipette out and volume was adjusted with 0.1N HCl. Solutions were scanned between 400 nm to 190 nm in spectrum mode. The AUC calculations were done and the calibration curve for carbocisteine was plotted in the concentration range of 10 to 140 μg/ml (Fig. 5). Results of analysis are given in table 1.
Table 1: Values of results of optical and regression of drug
|
Parameter |
First order derivative method |
Area under curve (AUC) method |
|
Detection Wavelength (nm) |
198.4 |
197.4-210.2 |
|
Beer Law Limits (µg/ml) |
10-140 |
10-140. |
|
Correlation coefficient(r2) |
0.9999 |
0.9999 |
|
Regression equation (y=b+ac) |
||
|
Slope (a) |
0.002 |
0.03 |
|
Intercept (b) |
0.0003 |
0.001 |
Fig. 5. Calibration curve for carbocisteine by area under curve spectroscopy
VALIDATION:
Accuracy
Accuracy of the proposed methods was carried as on the basis of recovery studies. It is performed by the standard addition method. Recovery studies were performed by adding standard drug at different levels to the pre-analyzed tablets powder solution and the proposed method was followed. From the amount of the drug estimated, the percentage recovery was calculated. The results of the analysis are shown in table (2, 3).
Precision
The method precision was established by carrying out the analysis of homogenous powder blend of tablets. The assay was carried out of drug by using proposed analytical method in seven replicates. The values of relative standard deviation lie well within the limits indicated the sample repeatability of the method. The results obtained are tabulated in table 4.
Inter-day and intra-day precision
An accurately weighed quantity of tablets powder equivalent to 10 mg of carbocisteine was transferred to 100 ml of volumetric flask. A 30 ml of 0.1N HCl was added and sonicated for 15 minutes and filtered. The filtrate and washing were diluted up to the mark with 0.1N HCl to give concentration as 100 μg /ml. Such solution was used for analysis.
For first order derivative method
Solution was scanned between 400 nm to 190 nm in spectrum mode. The first order derivative spectrum was obtained by using derivative mode.
Table 2: Results of recovery of carbocisteine for first order derivative method
|
Amount of sample added (µg/ml) |
Amount of standard added (µg/ml) |
Total amount recovered (µg/ml) |
Percentage recovery(%) |
Standard deviation |
Percentage of relative standard deviation (C.O.V.) |
|
20 |
0 |
19.9782 |
99.891 |
00.0496 |
00.2484 |
|
20 |
20 |
39.9927 |
99.981 |
00.0543 |
00.1361 |
|
20 |
40 |
60.0217 |
100.036 |
00.0496 |
00.0826 |
|
20 |
60 |
80.0072 |
100.009 |
00.0543 |
00.0678 |
Table 3: Results of recovery of carbocisteine for area under curve (AUC) method
|
Amount of sample added (µg/ml) |
Amount of added standard (µg/ml) |
Total amount recovered (µg/ml) |
Percentage recovery (%) |
Standard Deviation |
Percentage of relative Standard deviation |
|
10 |
0 |
10.0004 |
100.004 |
0.00522 |
0.05215 |
|
10 |
10 |
20.0019 |
100.009 |
0.00689 |
0.03446 |
|
10 |
20 |
30.0004 |
100.013 |
0.0023 |
0.00768 |
|
10 |
30 |
39.9985 |
99.996 |
0.00324 |
0.00812 |
Table 4: Precision- method precision
|
Experiment no |
Weight of carbocisteine taken in mg. |
Content in mg. of carbocisteine |
|
|
First order derivative method |
Area under curve method |
||
|
1 |
20 |
20.00 |
20.00 |
|
2 |
20 |
20.00 |
19.9966 |
|
3 |
20 |
19.8982 |
20.0033 |
|
4 |
20 |
19.9491 |
19.9966 |
|
5 |
20 |
20.0508 |
20.00 |
|
6 |
20 |
20.00 |
20.00 |
|
7 |
20 |
19.9491 |
20.0166 |
|
|
Standard deviation = |
0.04965 |
0.00689 |
|
%RSD = |
0.24847 |
0.03446 |
|
Table 5: Summary of validation parameter for intra-day and inter-day
|
Sr. no. |
Parameters |
First order derivative method |
Area under curve (AUC) method |
|
(A) |
Intra-day precision ( n=3) |
99.60 % |
99.45% |
|
|
Amount found ± |
|
|
|
|
%RSD |
0.24847 |
0.03446 |
|
(B) |
Inter-day precision ( n=3) |
98.484 % |
98.762 % |
|
|
Amount found ± |
|
|
|
|
%RSD |
0.13607 |
0.00768 |
|
(C) |
Ruggedness |
100.12 % |
99.87 % |
|
|
Analyst to analyst (n=3) |
|
|
|
|
%RSD |
0.06786 |
0.00812 |
Amplitude of the resulting solution was measured at between 200 nm to 198 nm by using 0.1N HCl as blank. The amplitude of final solution was read after 0 hr., 3 hrs. and 6 hrs. in 10 mm cell at 198.4 nm for first order derivative (method A). Similarly the amplitude of the same solution was read on 1st, 2nd and 5th day. The amount of carbocisteine was estimated by comparison with standard at 198.4 nm for first order derivative, table 5.
For area under curve method
Solution was scanned between 400 nm to 190 nm in spectrum mode. The area under curve of resulting solutions was measured at between 210.2 nm to 197.4 nm by using 0.1N HCl as blank. The area under curve of final solutions was read after 0 hr., 3 hrs. and 6 hrs. in 10 mm cell at 210.2 nm to 197.4 nm (method B). Similarly area under curve of the same solution was read on 1st, 2nd and 5th day. The amount of carbocisteine was estimated by comparison with standard at 210.2 nm to 197.4 nm, table 5.
Limit of Detection (LOD) and Limit of Quantification (LOQ)
The limit of detection (LOD) is defined as the lowest concentration of an analyte that an analytical process can reliably differentiate from back-ground levels. In this study, LOD and LOQ were based on the standard deviation of the response and the slope of the corresponding curve using the following equations-
LOD = 3.3 σ/S and LOQ = 10 σ/S
Where σ is the standard deviation of the signal to noise ratio of the sample and S is the slope of the related calibrations graphs.
The limit of quantification (LOQ) is defined as the lowest concentration of the standard curve that can be measured with an acceptable accuracy, precision and variability .The values of LOD and LOQ are given in table 6.
Table 6: Values of results of LOD and LOQ
|
Parameter |
First order derivative method |
Area under curve (AUC) method |
|
Limit of Detection (μg/ml) |
0.16102 |
0.01731 |
|
Limit of Quantification (μg/ml) |
0.49879 |
0.05246 |
Ruggedness
The ruggedness of the method is defined as degree of reproducibility of results obtained by analysis of carbocisteine sample under variety of normal test conditions such as different laboratories, different analysts and different lots of reagents. Quantitative determination of carbocisteine was conducted spectrophotometrically on one laboratory. It was again tested in another laboratory using different instrument by different analyst. The assays obtained in two different laboratories were well in agreement. It proved ruggedness of the proposed methods.
RESULTS AND DISCUSSION:
The first order derivative and area under curve UV-spectroscopic methods are useful for routine analysis of carbocisteine in bulk drug and formulation. The derivative spectroscopy method applied has the advantage that it locates hidden peak in the normal spectrum. It eliminates the interference caused by the excipients and the degradation products present, if any, in the formulation. The method was validated according to International Conference on Harmonization guidelines for validation of analytical procedures 7-9. Carbocisteine has the absorbance maxima at 198.4 nm (method A) and in the AUC spectrum method areas were measured between 197.4 nm to 210.2 nm (method B). The polynomial regression data for the calibration plots showed good linear relationship in the concentration range of 10 to 140 μg/ml and given in table1. Recovery studies were carried out by adding the pure drug to the previously analyzed tablet powder sample and shown in table 2, 3. The percentage recovery value indicates non interference from excipients used in formulation. The reproducibility and accuracy of the method were found to be good, which was evidenced by low standard deviation.
CONCLUSION:
The most striking features of two methods are its simplicity and rapidity, not requiring tedious sample solutions preparations which are needed for other instrumental methods. From the results obtained it can be concluded that the proposed methods are fully validated and found to be simple, sensitive, accurate, precise, reproducible, rugged and robust and relatively inexpensive. So, the developed methods can be easily applied for the routine quality control analysis of carbocisteine in pharmaceutical formulation.
ACKNOWLEDGEMENT:
Authors express sincere thanks to the Principal, Dr. Tushar M. Desai of D. G. Ruparel College, Mumbai.
REFERENCES:
1. British Pharmacopoeia, Her Majesty’s Stationary Office, London. Volume I, II, and III, 1983, 2009.
2. European Pharmacopoeia 5.0. Volume-2; 2005.
3. Rele RV, Patil SP. Reversed phase high pressure liquid chromatography technique for determination of carbocisteine from pharmaceutical formulation. J.Chem.Parm.Res. 2(4); 2010: 24-30.
4. Archana Nadimintri, Ashwini Gunda, Karnaker Reddy Tupally, Abbaraju Prasanna Lakshmi, Kedarnath Jakka, Aravind Sai Nagubandi. Simultaneous estimation of amoxillcine trihydrate and carbocisteine drug present in formulation by RP-HPLC method and its validation. Journal of pharmacy research. 5(4); 2012: 1889-1895.
5. Ravindhar Burugu, G.Venkateshwarlu. A stability indicating ultra performance liquid chromatography (UPLC) method for the determination of assay of carbocisteine in various formulation products. International journal of pharmacy and pharmaceutical sciences. 4(4); 2012: 653-656.
6. Megoulas NC, Koupparis MA. Ion-chromatographic determination of carbocisteine in pharmaceuticals based on non-suppressed conductometric detection. J.chromatography A. 1026 (1-2); 2004: 167-74.
7. ICH, Q2A Text on Validation of Analytical Procedures; 1994.
8. ICH, Q2B Validation of Analytical Methodology; 1996.
9. ICH, Q2 (R1) Validation of Analytical Procedures: text and methodology; 2005.
Received on 15.02.2013 Modified on 01.03.2013
Accepted on 06.03.2013 © AJRC All right reserved
Asian J. Research Chem. 6(3): March 2013; Page 236-240