Development and Validation of A New RP-HPLC Method for Simultaneous Estimation of N-acetylcysteine and L-Arginine in Combined Dosage Form

An isocratic, reversed phase-liquid chromatographic method was developed for quantitative determination of N-acetylcysteine and L-Arginine in combined dosage form. A Waters Xterra Rp8, (150*4.6*5μm) column with a mobile phase containing a Phosphate Buffer (pH 4.5) and Methanol (90: 10 v/v) was used. The flow rate was maintained at 0.8 ml/min, column temperature at 30°C and effluents were monitored at 248 nm. The retention times of N-acetylcysteine and L-Arginine were found to be 2.697 min and 3.156 min, respectively. The correlation co-efficient for N-acetylcysteine and L-Arginine was found to be 0.99 and 0.99, respectively. The proposed method was validated with respect to linearity, accuracy, precision, specificity, and robustness. Recovery of N-acetylcysteine and L-Arginine in formulations was found to be in the range of 97-103% and 97-103% respectively confirms the non-interferences of the excipients in formulation. Due to its simplicity, rapidness and high precision, this method can be successfully applied for estimation of N-acetylcysteine and L-Arginine in combined pharmaceutical dosage forms.

semiessential or conditionally essential amino acid, depending on the developmental stage and health status of the individual.Preterm infants are unable to synthesize or create arginine internally, making the amino acid nutritionally essential for them.There are some conditions that put an increased demand on the body for the synthesis of L-arginine, including surgical or other trauma, sepsis and burns.L-arginine may also play an important role in the treatment of cardiovascular disease due to its antiatherogenic, anti-ischemic, antiplatelet, and antithrombotic properties.It has been promoted as a growth stimulant and as a treatment for erectile dysfunction in men.
N-Acetylcysteine is a phar maceutical drug and nutritional supplement used primarily as a mucolytic agent and in the management of paracetamol (acetaminophen) overdose.Other uses include sulfate repletion in conditions, such as autism, where cysteine and related sulfur amino acids may be depleted.N-Acetylcysteine causes cleavage of disulfide bonds by converting them to two sulfhydryl groups.This action results in the breakup of mucoproteins in lung mucus, reducing their chain lengths and causes thinning of the mucus thus facilitates easy removal of the same and therefore improving conditions such as bronchitis and flu.N-acetyl cysteine is also used for preventing alcoholic liver damage; for protecting against environmental pollutants including carbon monoxide, chloroform, urethanes and certain herbicides; for reducing toxicity of ifosfamide and doxorubicin, drugs that are used for cancer treatment; as a hangover remedy; for preventing kidney damage due to certain X-ray dyes; and for human immunodeficiency virus (HIV) 1,2 .
A n u m b e r o f s p e c t r o p h o t o m e t r i c, c o l o r i m e t r i c , c h e m i l u m i n e s c e n c e , L i q u i d chromatographic methods with electrochemical, fluorimetric, mass and ultraviolet detectors and GC have been widely applied as the main methods of detection in biological samples and pharmaceuticals.Several liquid chromatographic (LC) methods involve spectrophotometric detection with pre or post-column derivatization, where time consuming extraction or long derivatization steps hamper the studies of this compound [3][4][5][6][7][8][9][10][11] .
The present work concerns the method development, method validation of L-Arginine and N-acetylcysteine in combined pharmaceutical dosage form.The developed Liquid Chromatographic method was validated with respect to specificity, limit of detection (LOD), limit of quantification (LOQ), linearity, precision, accuracy and robustness.These studies were performed in accordance with established ICH guidelines

Chemicals and Reagents
N -a c e t y l c y s t e i n e a n d L -A r g i n i n e were procured as gift samples from Dr.Reddy's Laboratories Ltd., Hyderabad.Methanol of HPLC grade was purchased from E. Merck (India) Ltd., Mumbai.MonoPotassium Phosphate of AR grade was obtained from S.D. Fine Chemicals Ltd., Mumbai and milli Q water.

HPLC Conditions
The mobile phase consisting of Phosphate Buffer (pH 4.5) : Methanol (HPLC grade) were filtered through 0.45µ membrane filter before use, degassed and were pumped from the solvent reservoir in a ratio of 90:10 v/v was pumped into the column at a flow rate of 0.8 ml/min.The column temperature was maintained at 30°C.The detection of effluents was performed at 248 nm and the run time was finalized as 6 min.The volume of injection was 10µl prior to injection of the drug solution the column was

N-Acetylcysteine
Accurately weighed quantity, 120 mg of N-Acetylcysteine and 160 mg of L-Arginine was transferred into 100 ml of volumetric flask.30 ml of mobile phase was added to this and sonicated for 15 min.Finally volume was made up to 100 ml with mobile phase.5ml above solution was transferred into 25ml volumetric flask and diluted to the mark with mobile phase.

Preparation of sample solution
Accurately weighed 500 mg of granules of dosage form was transfered into 100 ml of volumetric flask.25 ml of mobile phase was added to this and sonicated for 30 mins.Finally volume was made up to 100 ml with mobile phase.5ml above solution was transferred into 25ml volumetric flask and diluted to the mark with mobile phase.

Method Development
The analytical procedure for the estimation of L-Arginine and N-acetylcysteine in marketed formulation was optimized with a view to develop a precise and accurate assay method.Appropriate chromatographic separation was achieved on Waters Xterra Rp8, (150*4.6*5µm).Various mobile phase systems were prepared and used to provide an appropriate chromatographic separation, but the proposed mobile phase containing Phosphate Buffer (pH 4.5) and Methanol in the ratio of 900:100 (v/v) gave a better resolution.Using UV-visible PDA detector at 248 nm carried out the detection.Amongst the several flow rates tested, the flow rate of 0.8 ml/min was the best suited for both the drugs with respect to location and resolution of peaks.The retention time of L-Arginine and N-acetylcysteine was found to be 2.697 min and 3.156 min respectively.The chromatograms of standard and sample solution of L-Arginine and N-acetylcysteine were shown in Figure 3, 4 respectively.The asymmetry factor of L-Arginine and N-acetylcysteine was 1.353and 1.384 found to be respectively, which indicates symmetrical nature of the peak.The USP resolution of 7.957 was achieved between L-Arginine and N-acetylcysteine.The USP plate count of L-Arginine and N-acetylcysteine was 7190 and 8590 found to be respectively, which indicates column efficiency for separation.System suitability parameters such as Peak asymmetry, Resolution and Number of theoretical plates are meeting with ICH requirements.The results of analysis shows that the amounts of drugs estimated were in good agreement with the label claim of the formulations [12][13][14] .

System Suitability Studies:
System suitability was deter mined before sample analysis from duplicate injections of the standard solutions of L-Arginine and N-acetylcysteine.The column efficiency, resolution and peak asymmetry were calculated for the standard solutions.Resolution between L-Arginine and N-acetylcysteine peaks was found to be 7.957.USP tailing (Peak Asymmetry) for L-Arginine and N-acetylcysteine were found to be 1.353 and 1.384 respectively.Number of theoretical plates (USP plate count) for L-Arginine and N-acetylcysteine were found to be 5005 and 4588 respectively.
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 [15][16][17] .

Specificity
Specificity is the ability to assess unequivocally the analyte in presence of components which may be expected to be present.Typically these might include impurities, degradants, excipients etc. Placebo interference was evaluated by analyzing the placebo prepared by the test method.No peak due to placebo was detected at the retention time of Arginine and N-acetylcysteine.The specificity of the developed method was also conducted in presence of its degradation products.

Precision
The precision of method was verified by repeatability and intermediate precision.Repeatability was checked by injecting six individual sample preparations of Arginine and N-acetylcysteine.Percent relative standard deviation (RSD) of the area for each drug was calculated.The results of precision study are provided in Table 2.

Accuracy
The accuracy of the method was determined by recovery experiments.The recovery studies were evaluated in triplicate using three concentration levels 50%, 100% and 150%.The percentage recovery data was obtained, added recoveries of standard drugs were found to be accurate (Table III & IV).

Linearity and Range
The linearity of the method was determined at five concentration levels (50%, 75%, 100%, 125% and 150%).Linearity test solutions were prepared by diluting the stock solutions to the required concentrations.The calibration curves were plotted between the responses of peak area versus concentration of analyte.The slope and intercept value for calibration curve was y = 16616 x (r2=0.99)for N-Acetyl cysteine and y = 19288 x (r2=0.99)for L-Arginine.The result (Table V) shows that an excellent correlation exists between areas and concentration of drugs within the concentration range.Calibration curves are presented in Fig. 3.

Limit of detection & Limit of quantification (LOD & LOQ
Limit of quantification and detection were predicted by plotting linearity curve for different nominal concentrations of N-Acetylcysteine and L-Arginine (Table V).Relative standard deviation (ó) method was applied, the LOQ and LOD values were predicted using following formulas.Precision was established at these predicted levels.LOQ and LOD values for N-Acetylcysteine and L-Arginine were found to be 9.180, 2.754 and 9.677, 2.903 respectively of the method     System suitability results were given by Table-1 such as system suitability parameters are retention time, resolution, tailing and plate count were shown uniformity and %RSD was less than 1, so we can say system is suitable for analysis.There was no placebo and excipients peaks interfering with standard and analyte peak, so it proves method is selective.The result given in Table-2 says that the method precision is passed for both N-acetylcysteine and L-Arginine.The method accuracy was evaluated by recovery studies.N-acetylcysteine and L-Arginine recovery was found to be 100% as per ICH requirements such as 97%-103% and also percentage RSD was very low.So method is accurate as results shown in Table 3

Fig. 10 :
Fig. 10: Robustnes Chromatograms of Flow variation and Temperature was observed that there were no marked changes in the chromatograms, which demonstrates that the method developed is rugged and robust (Table VI & VII).
Method specificity was concluded by Figure-1 and Figure-2 those figures are N-acetylcysteine and L-Arginine Standard chromatogram and formulation.

& 4 .
Linearity calibration curves were given in Figure: 7 & 8. Linear correlation was found to be Y= 16616 for N-Acetylcysteine and y = 19288 for L-Arginine.Method robustness results were given inTable 5 & 6.Based on above results, the proposed