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International Journal of Universal Pharmacy and Bio Sciences 3(5): September-October 2014
INTERNATIONAL JOURNAL OF UNIVERSAL
PHARMACY AND BIO SCIENCES IMPACT FACTOR 2.093***
ICV 5.13*** Pharmaceutical Sciences RESEARCH ARTICLE……!!!
DEVELOPMENT AND VALIDATION OF ACETYLCYSTEINE AND
TAURINE IN TABLET DOSAGE FORM BY RPHPLC
ASIYA BEGUM*
CMR college of pharmacy , Kandlakoya(v) , Medchal, Hyderabad.
KEYWORDS:
Taurine, Acetylcysteine,
HPLC, validation.
For Correspondence:
ASIYA BEGUM
Address:
CMR college of
pharmacy ,
Kandlakoya(v) ,
Medchal, Hyderabad..
E-mail:
asiya.begum889@gmail.
com
ABSTRACT
Objective: To develop and validate RP-high performance liquid
chromatographic (HPLC) method for Acetylcysteine and Taurine in
Tablet dosage form. Methods: An isocratic separation was carried
out using Inertsil ODS (250 x 4.6 mm, 5 μm) column and Potassium
dihydrogen OPA: Acetonitrile(60:40 v/v) as mobile phase With
quantification carried out at a wavelength of 248nm. Results: The
Retention time of Acetylcysteine and Taurine were observed to be
3.186and 4.142 minutes, respectively with theoretical plate count
and asymmetry as per the ICH limits. The % assay of Acetylcysteine
and Taurine were 99% and 100%. The flow rate was found to be
1ml/min .The linear regression analysis data for the calibration plots
showed a good linear relationship for Acetylcysteine and Taurine
over a concentration range of 1000-3000 μg/ml and 300-900μg/ml
with correlation co-efficient of 0.999 for Acetylcysteine and 0.999
for Taurine. The limit of detection and Quantitation were found to be
2.93, 2.76& 9.75,9.20 respectively. Conclusion: A new simple,
accurate, precise and selective high performance liquid
chromatographic (HPLC) method has been developed and validated
for the determination of Acetylcysteine and Taurine in tablet dosage
form.
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INTRODUCTION:
ACETYLCYSTEINE Acetylcysteine may decrease the viscosity of secretions by splitting of
disulphide bonds in mucoproteins. It also promotes the detoxification of an intermediate
paracetamol metabolite which is used in the management of paracetamol overdosage.
Acetylcysteine also possesses some anti-inflammatory effects possibly via inhibiting NF-κB and
modulating cytokine synthesis. N-Acetylcysteine have its effect on ameliorating oxidative
damage at the level of the glomerular apparatus and was useful in attenuating urinary albumin/
creatinine ratio (UACR).
Fig. 1: It Shows Structure of Acetylcysteine
TAURINE
An aminoacid, which is not part of human bodys structural proteins, but is most abundant free
aminoacid. The following indications are supposed to be based on limited studies. Manic
depression. Ischaemic heart disease, congestive heart failure, hypertension and
hypercholesterolemia. Type I diabetes mellitus. Hepatitis. Alcoholism.
.
Fig. 2: It Shows Structure of Taurine
Mechanism of Action: It is a major constituent of bile, It also acts as an antioxidant and protects
against toxicity of various substances (such as lead and cadmium).Additionally, supplementation
with taurine has been shown to prevent oxidative stress induced by exercise. In another study on
diabetic rats, taurine significantly decreased weight and decreased blood sugar in these animal
models. Likewise, a 2008 study demonstrated taurine administration to diabetic rabbits resulted
in 30% decrease in serum glucose levels. According to the single study on human subjects, daily
administration of 1.5g taurine had no significant effect on insulin secretion or insulin
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sensitivity.There is evidence that taurine may exert a beneficial effect in preventing diabetes-
associated microangiopathy and tubulointerstitial injury in diabetic nephropathy
MATERIALS AND METHODS
Instrumentation:
The separation was carried out on HPLC system with Waters 2695 alliance With binary HPLC
Pump, Waters 2998 PDA detector, Waters Empower2 software and Inertsil ODS column
(250mmx4.6mm, particle size5μm).
Reagents and Chemicals:
Acetylcysteine and Taurine pure drug samples were provided by Rainbow Pharma Training Lab
Hyderabad. OPA and Acetonitrile were of HPLC grade. Fixed dose combination Tablet (Brand
name: Nefrosave) containing 500mg of Taurine and 150 mg of Acetylcysteine were procured
from local pharmacy, Hyderabad, India.
Chromatographic Conditions:
The mobile phase consisting of orthophosphoric acid and acetonitrile (HPLC grade) were filtered
through 0.45μ membrane filter before use, degassed and were pumped from the solvent reservoir
in the ratio of 60:40v/v was pumped into the column at a flow rate of 1.0ml/min. The column
temperature was 30°C. The detection was monitored at 248nm and the run time was 8min. The
volume of injection loop was 3μl prior to injection of the drug solution the column was
equilibrated for at least 30 min. with the mobile phase flowing through the system.
Preparation of standard solution:
Accurately weigh 500mg of Taurine and 150mg of Acetylcysteine into a 50ml of volumetric
flask and dissolve the sample using water and sonicate it for 15min then finally make up the
volume to 50ml.Now pipette out 5ml of this solution into 25ml of volumetric flask and make up
the volume upto mark using mobile phase as shown in figure 3.
Preparation of sample solution:
Accurately weighed 2 tablets and calculated average weight of those tablets and crushed.
Transfer the tablet powder weigh about 818.6mg of sample into 50ml of volumetric flask added
with methanol and water and sonicate for 30mins and make up the volume with water and
filtered through the0.45μm Millipore filter paper Transfer above solution 5ml into 25ml
volumetric flask and make up the volume with mobile phase chromatogram is shown in figure 4.
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3.20
2
4.12
3
AU
0.00
0.10
0.20
0.30
0.40
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Fig. 3 Chromatogram of Trial-5
Table 1: Observations of Trial 5 Chromatogram
Name Retention Time USP Resolution USP Tailing USP Plate Count
1 Acetylcyst
eine
3.202 1.41 4977
2 Taurine 4.123 4.43 1.36 5578
Fig. 4: Standard Chromatogram – 1
Table 3: Observations of Standard Chromatogram-1
ID Name Retention
Time(min)
Area
(μV*sec)
USP Plate
Count
USP
Tailing
USP
Resolution
1 Acetylcysteine 3.186 3156252 4952 1.424 2 Taurine 4.142 3628057 5525 1.396 4.612
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Development and Validation of HPLC method [6, 8]
Present study was conducted to obtain a new, affordable, cost-effective and convenient method
for HPLC determination of Acetylcysteine and Taurine in tablet dosage forms. The experiment
was carried out according to the official specifications of ICH- 1996, Global Quality Guidelines-
2002. The method was validated for the parameters like system suitability, selectivity, linearity,
accuracy, precision, and robustness.
System suitability A standard solution was prepared using Saxagliptin and Metformin working
standard as per the test method and was injected six times into the HPLC system. The parameters
namely USP plate count, peak asymmetry factor and resolution for the standard solutions were
calculated.
Selectivity: Specificity was checked for the interference of impurities in the analysis of blank
solution and injecting sample solution under optimized chromatographic conditions to
demonstrate separation of both Saxagliptin and Metformin from impurities.
Linearity:
Linearity of the method was determined by constructing calibration curves. Standard solutions of
Acetylcysteine and Taurine at different concentrations level level (50%, 75%, 100%, 125%, and
150%) were used for this purpose. Before injection of the solutions, the column was equilibrated
for at least 30min with the mobile phase. Each measurement was carried out in six replicates to
verify the reproducibility of the detector response at each concentration level. The peak areas of
the chromatograms were plotted against the concentrations of Acetylcysteine and Taurine to
obtain the calibration curves. The five concentrations of the standard were subjected to
regression analysis to calculate calibration equation and correlation coefficients.
Accuracy
Accuracy is the closeness in agreement between the accepted true value or a reference value and
the actual result obtained. Accuracy studies are usually evaluated by determining the recovery of
a spiked sample of the analyte into the matrix of the sample to be analyzed.
Precision Method Precision was determined by injecting six replicates of drug sample solution.
The retention times and peak areas of six replicates are recorded. The precision is expressed as
the % RSD of Peak areas and it should not be more than 2%.
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Robustness The robustness of the method was assessed by altering the some experimental
conditions such as, by changing the flow rate from 0.9 to1.1 ml/min, amount of diluents (10% to
15%) the temperature of the column (28°C to 32°C) and pH of the mobile phase.
Limit of detection and limit of quantitation:
Limit of detection and limit of quantitation represent the concentration of analyte that would
yield signal to noise ratio of 3 for LOD and 10 for LOQ respectively. To determine LOQ and
LOD serial dilutions of mixed standard solution of Saxagliptin and Metformin was made from
standard solution. The samples were injected in LC system and measured signal from the
samples was compared with those of blank samples.
Fig. 3: It Shows the Chromatogram of Acetylcysteine and Taurine standard preparation
3.20
2
4.12
3
AU
0.00
0.10
0.20
0.30
0.40
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Fig. 4: It Shows the Chromatogram of Acetylcysteine and Taurine In sample preparation
RESULTS AND DISCUSSION
Method Development
System suitability: The system suitability tests were carried out to evaluate the resolution and
reproducibility of the system for the analysis. Table1 summarized the test results of system
suitability study.
Linearity: The linearity of the developed method was determined in triplicate at different
concentrations ranging from50%, 75%, 100%, 125%, and 150% were used for Acetylcysteine
and Taurine.
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Accuracy: was determined by the recovery studies at three different concentrations
(corresponding to 50,100,150 % of the test solution concentration )by addition of known amount
of standard to pre analysed sample preparation .For each condcentrations three sets were
prepared and injected. The recovery studies were carried out six timres and the regression
coefficient was 0.99.
Precision: Method Precision was determined by injecting six replicates of drug sample solution.
The retention times and peak areas of six replicates are recorded. The precision is expressed as
the % RSD of Peak areas and it should not be more than 2% shown in table 4.
Robustness As part of the robustness, deliberate changes in the flow rate and detection
wavelength were made to evaluate the impact on the method and retention times were
significantly changed.
Limit of detection and limit of quantification
Limit of detection and limit of quantification represent the concentration of analyte that would
yield signal to noise ratio of 3 for LOD and 10 for LOQ respectively. To determine LOQ and
LOD serial dilutions of mixed standard solution of Acetylcysteine and Taurine was made from
standard solution. The samples were injected in the system and measured signal from the
samples was compared with those of blank samples. LOD and LOQ was calculated from linear
curve using formulae LOD= 3.3 * σ / slope, LOQ= 10 * σ / slope (Where σ = the standard
deviation of the response and S = Slope of calibration curve) shown in table 7.
Table.1: It shows result of system suitability test of Acetylcysteine & Taurine
Parameter Acetylcysteine Taurine
Correlation Coefficient 0.999 0.999
LOD 2.93 2.76
LOQ 9.75 9.20
Theoretical plates 4952 5525
Tailing 1.42 1.39
Fig 5: It shows linearity curve of Acetylcysteine
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Fig 6: It shows Linearity curve of Taurine
Table 2: It shows recoveries of Acetylcysteine drugs
Accuracy of Acetylcysteine
Spiked
Level
Sample
Weight
Sample
Area
µg/ml
added
µg/ml
found
%
Recovery
% Mean
50% 409.30 1355496 990.000 990.07 100 100%
50% 409.30 1352609 990.000 987.96 100
50% 409.30 1354712 990.000 989.50 100
50% 409.30 1350788 990.000 986.63 100
50% 588.00 1359911 990.000 993.30 100
50% 588.00 1357607 990.000 991.61 100
100% 1175.35 2719861 1980.000 1986.62 100 100%
100% 1175.35 2715850 1980.000 1983.69 100
100% 1175.35 2719678 1980.000 1986.49 100
150% 1227.90 4079237 2970.000 2979.53 100 99.83%
150% 1227.90 4072198 2970.000 2974.39 100
150% 1227.90 4074412 2970.000 2976.00 100
150% 1227.90 4070091 2970.000 2972.85 100
150% 1227.90 4078872 2970.000 2979.26 100
150% 1227.90 4073289 2970.000 2975.18 100
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Table 3: It shows recoveries of Taurine drug
Fig. 7: Accuracy 50 % Chromatogram-1
ACCURACY OF TAURINE
Spiked
level
Sample
Weight
Sample
Area
µg/ml
added
µg/ml found %
Recovery
%
Mean
50% 409.30 1252598 300.000 297.36 99 99
50% 409.30 1253666 300.000 297.62 99
50% 409.30 1251543 300.000 297.11 99
50% 409.30 1256177 300.000 298.21 99
50% 409.30 1251190 300.000 297.03 99
50% 409.30 1252957 300.000 297.45 99
100% 818.60 2513293.00 600.000 596.65 99 99
100% 818.60 2514037.00 600.000 596.83 99
100% 818.60 2513733.00 600.000 596.75 99
150% 1227.90 3776217 900.000 896.46 100 100
150% 1227.90 3777124 900.000 896.68 100
150% 1227.90 3772325 900.000 895.54 100
150% 1227.90 3776773 900.000 896.60 100
150% 1227.90 3773862 900.000 895.90 100
150% 1227.90 3777790 900.000 896.84 100
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Fig. 8: Accuracy 50 % Chromatogram -2
Table 4 : Observations of Accuracy 50 % Chromatogram -1
Table 5 : Observations of Accuracy 50 % Chromatogram -2
ID Name RetentionTime (min) Area (μV*sec)
1 Acetylcysteine 3.202 1352609
2 Taurine 4.117 1253666
ID Name RetentionTime (min) Area (μV*sec)
1 Acetylcysteine 3.212 1355496
2 Taurine 4.117 1252598
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Table 6: It shows the method precision results of Acetylcysteine and Taurine
Table 7: Robustness test of Acetylcysteine
S.No Sample Weight Sample Area-1 Sample Area-2 % Assay % Assay
1 818.60 2714619 2514974 99 100
2 818.60 2712528 2514989 99 100
3 818.60 2719589 2510458 99 99
4 818.60 2717005 2518797 99 100
5 818.60 2714139 213023 99 99
6 818.60 2717791 2518939 99 100
Average
Assay:
99 100
STD 0.10 0.13
% RSD 0.10 0.13
Sample
Name
Peak Name RT AREA USP
TAILING
USP PLATE
COUNT
1 TEMP1 ACETYLCYSTEINE 3.206 3203781 1.407 5197
2 TEMP2 ACETYLCYSTEINE 3.199 3188463 1.353 5270
3 FLOW1 ACETYLCYSTEINE 3.993 3967081 1.469 5712
4 FLOW2 ACETYLCYSTEINE 3.199 2675037 1.353 4646
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Table 8: Robustness test of Taurine
Table 9:LOD & LOQ for Acetylcysteine and Taurine
DRUG LOD(μg/ml) LOQ(μg/ml)
Acetylcysteine 2.93 9.75
Taurine 2.76 9.20
CONCLUSION
The proposed RP-HPLC method was validated as per International Conference on
Harmonization (ICH) guidelines, and found to be applicable for routine quality control analysis
for the simultaneous estimation of Acetylcysteine and Taurine using isocratic mode of elution.
The results of linearity, precision, accuracy and specificity, proved to be within the limits. The
proposed method is highly sensitive, reproducible, reliable, rapid and specific. Hence, this
method can easily and conveniently adopt for routine quality control analysis of Acetylcysteine
and Taurine in its pharmaceutical dosage forms.
ACKNOWLEDGEMENT
The authors are thankful to Rainbow Pharma Training Lab, Kukatpally, Hyderabad.
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