Development and Validation of a New Rp-Hplc Method For Simultaneous Estimation of N-Acetylcysteine and L – Arginine in Combined Dosage form

Arginine is an α-amino acid. The L-form is one of the 20 most common natural amino acids. At the level of molecular genetics, in the structure of the messenger ribonucleic acid mRNA, CGU, CGC, CGA, CGG, AGA, and AGG, are the triplets of nucleotide bases or codons that code for arginine during protein synthesis. In mammals, arginine is classified as a 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 anti atherogenic, anti-ischemic, anti platelet, and anti thrombotic properties. It has been promoted as a growth stimulant and as a treatment for erectile dysfunction in men.

N-Acetylcysteine is a pharmaceutical 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 number of spectrophotometric, colorimetric, chemiluminescence, Liquid 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 [3-11].

 

Fig1:  Structure of L–ArginineClick here to View Figure

 

Fig2:  Structure of N-acetylcysteine  Click here to View Figure

 

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

EXPERIMENTAL

Instrumentation

The separation was carried out on HPLC system with Waters e2695 alliance with binary HPLC pump, Waters 2998 PDA detector, Waters Empower2 software, Waters Xterra Rp8, (150*4.6*5µ) column.

Chemicals and Reagents

N-acetylcysteine  and L-Arginine 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 equilibrated for at least 15 min. with the mobile phase flowing through the system.

Preparation of standard solution

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.

RESULTS AND DISCUSSION

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 [12-14].

Parameters	N-Acetyl cysteine	L-Arginine
Resolution
Theoretical plates	4588	5005
Tailing	1.353	1.384
Correlation Coefficient	0.99	0.99
Regression Equation	y = 16616x	y = 19288x
LOD	2.87	2.98
LOQ	9.56	9.94

Table 1. System suitability parameters

 

Figure3 : Standard chromatogram for N-acetylcysteine  and L – Arginine  Click here to View Figure

 

Figure4: Formulation chromatogram for N-acetylcysteine  and L – ArginineClick here to View Figure

 

3.2.Method Validation

Method validation of above set method is established by performing the below listed validation parameters as per ICH guidances.

System Suitability Studies

System suitability was determined 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 [15-17].

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.

Table 2: Precision studies of L-Arginine and N-acetylcysteine

S.No	Sample Wt(mg)	Area(L-Arginine)	Area(N-Acetyl cysteine)	%Assya(L-Arginine)	%Assya(N-Acetyl cysteine)
1	500	3453165	5883357	99	100
2	500	3450236	5889593	99	100
3	500	3452627	5886255	99	100
4	500	3451253	5885959	99	100
5	500	3452239	5883805	99	100
6	500	3450246	5880174	99	100

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 Figure III.

 

Figure5: Overlay chromatograms of Linearity for N-acetylcysteine and L-Arginine   Click here to View Figure

 

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 (σ) method was applied, the LOQ and LOD values were predicted using following formulas. Precision was established at these predicted levels.

(a) LOQ = 10 σ / S

(b) LOD = 3.3 σ / S

Where σ = residual standard deviation of response

S = slope of the calibration curve.

LOQ and LOD values for N-Acetylcysteine and L-Arginine were found to be 9.180, 2.754 and 9.677, 2.903 respectively.

 

Figure6: Accuracy Chromatograms-50%, 100% and 150% of N-acetylcysteine and L-Arginine  Click here to View Figure

 

Figure7: Linearity Curve for N-Acetylcysteine  Click here to View Figure

 

Figure8: Linearity Curve for L-Arginine  Click here to View Figure

 

Table 3: Accuracy for N-Acetylcysteine 

Spiked Level	Sample Weight	Sample Area	µg/ml added	µg/ml found	% Recovery	Mean
50%	250.00	1724734	118.800	118.64	100	100
50%	250.00	1722164	118.800	118.46	100
50%	250.00	1727440	118.800	118.83	100
50%	250.00	1721876	118.800	118.44	100
50%	250.00	1720592	118.800	118.36	100
50%	250.00	1729189	118.800	118.95	100
100%	500.00	3453345	237.600	237.55	100	100
100%	500.00	3456638	237.600	237.78	100
100%	500.00	3450512	237.600	237.35	100
150%	750.00	5188152	356.400	356.88	100	100
150%	750.00	5184469	356.400	356.63	100
150%	750.00	5183991	356.400	356.60	100
150%	750.00	5184381	356.400	356.62	100
150%	750.00	5176017	356.400	356.05	100
150%	750.00	5179664	356.400	356.30	100

 

Table 4: Accuracy for L-Arginine

Spiked level	Sample weight	Sample area	µg/ml added	µg/ml found	% recovery	Mean
50%	250.00	2942271	160.000	159.52	100	100
50%	250.00	2948345	160.000	159.85	100
50%	250.00	2949836	160.000	159.93	100
50%	250.00	2940368	160.000	159.42	100
50%	250.00	2940916	160.000	159.45	100
50%	250.00	2949928	160.000	159.94	100
100%	500.00	5880059.00	320.000	318.81	100	100
100%	500.00	5882977.00	320.000	318.96	100
100%	500.00	5886713.00	320.000	319.17	100
150%	750.00	8820491	480.000	478.23	100	100
150%	750.00	8820071	480.000	478.21	100
150%	750.00	8828925	480.000	478.69	100
150%	750.00	8821166	480.000	478.27	100
150%	750.00	8821638	480.000	478.29	100
150%	750.00	8828376	480.000	478.66	100

 

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