Method Development and Validation of Taurine and Acetyl Cysteine by Using RP -HPLC Method
R Anusha Naik1*, Ajay Kumar D.1, M. Venkatesh2
1Gyana Jyothi College of Pharmacy, Uppal Bus Depot, Hyderabad-500098.
2Princeton College of Pharmacy, Ghatkesar, Medchal-500088.
*Corresponding Author E-mail: anushanaik2107@gmail.com
ABSTRACT:
An accurate, precise and sensitive HPLC method was developed and validated for the simultaneous estimation of Acetyl Cysteine and Taurine in tablet dosage form. An isocratic separation was carried out using Inertsil (250×4.6mm, 5µ) ODS C-18 RP-column and Phosphate Buffer: Methanol pH 2.5 (25:75 v/v) mobile phase carried out at a wavelength of 254nm.The Retention time of Taurine and Acetyl cysteine were found to 2.589 ± 0.004 min and 3.711 ± 0.005 min, respectively with theoretical plate count and asymmetry as per the ICH limits. The % assay of Acetyl cysteine and Taurine were 99.3 % for both the drugs. The flow rate was found to be 1ml/min .The linear regression analysis data for the calibration plots showed better linear relationship for Taurine and Acetyl cysteine over a concentration range of 20 to 60 μg/ ml and 10 to 30 μg/ml, with regression values of 0.9979 and 0.9999, respectively. The limit of detection and Quantitation of Taurine was found to be 0.001µg/ml and 0.004µg/ml and Acetyl cysteine was found to be 0.005 µg/ml and 0.015µg/ml respectively.
KEYWORDS: Taurine, Acetyl cysteine, phosphate buffer, methanol, Inertsil ODS C-18 RP-column
INTRODUCTION:
Analytical chemistry plays a vital role in maintaining the quality of drugs. It consists of Qualitative and Quantitative estimations. Taurine Dioxygenase is an enzyme which is used for manic depression, ischemic heart diseases, congestive heart failure, hypertension and hypercholesterolemia. Type I diabetes mellitus, hepatitis and alcoholism. Acetyl cysteine may decrease the viscosity of secretions by splitting of disulphide bonds in mucoproteins.
It’s used to help thin and loosen mucus in the airways due to the certain lung diseases such as emphysema, bronchitis, cystic fibrosis and pneumonia. Various methods are available to estimate Taurine and acetyl cysteine such as UV spectroscopic method, HPLC, LC-MS etc. By using various solvents like water, Acetonitrile, buffers etc. an attempt was made to develop simple, precise HPLC method development to quantify Taurine and Acetyl Cysteine in table dosage form and to validate the method as per ICH guidelines.
MATERIALS AND METHODS:
Chemicals and regents: Taurine and Acetyl Cysteine are procured from KP Laboratories, Hyderabad. Commercial pharmaceutical preparation was purchased from local market. Phosphate buffer, methanol and water used were of analytical grade (Merck). All other chemical used were analytical grade until otherwise indicator.
Instrumentation:
The proposed research was carried out on a Waters 2690 separation module (photo diode array detector). A fast clean Analytical Technologies Limited- Ultrasonic cleaner was used for degassing the mobile phase.
Selection of chromatographic condition:
Proper selection of the method depends upon the nature of the sample, its molecular weight and solubility. The drugs selected in the present study are polar in nature and hence reversed phase or ion-pair or ion exchange chromatography method may be used. The reversed phase HPLC was selected for the separation because of its simplicity and suitability.
Selection of detection wavelength:
The sensitivity of method that uses UV- Vis detector depends upon the proper selection of wavelength. An ideal wavelength is that gives maximum absorbance and good response for both the drugs to be detected. Standard solutions of Taurine and Acetyl cysteine were scanned in the UV range (200- 400nm) and the spectrums obtained were overlaid and the overlain spectrum was recorded. From the overlain spectrum, 254 nm was selected as the detection wavelength for the present study.
Preparation of Buffer:
About 7.0g of potassium dihydrogen orthophosphate was dissolved in 1000ml of HPLC grade water and pH 2.5 was adjusted with orthophosphoric acid. It was filtered through 0.45µm nylon membrane filter and degassed with sonicator. It was used as a diluent for the preparation of sample and standard solution.
Preparation of mobile phase:
Mobile phase consist of buffer: Methanol of pH 2.5 (25:75) was taken sonicate and degassed for 10min and filtered through 0.45 µm nylon membrane filter
Standard Preparation:
Weigh accurately 10 mg Taurine Working Reference Standard and 10mg of Acetyl cysteine Working Reference Standard is taken in to 100ml volumetric flask and then it was dissolved and diluted to volume with mobile phase up to the mark. After that 50ml of the above solution was taken into 100ml standard flask and made up with mobile phase. (Stock solution) Further pipette 0.5ml of the above stock solution in to a 10ml volumetric flask and dilute up to the mark with diluent. Preparation of samples for Assay Standard preparation: Weigh accurately 10mg Taurine Working Reference Standard and 10mg of Acetyl cysteine Working Reference Standard is taken in to 100ml volumetric flask and then it was dissolved and diluted to volume with mobile phase up to the mark. After that 50ml of the above solution was taken into 100ml standard flask and made up with mobile phase. (Stock solution) Further pipette 0.5ml of the above stock solution in to a 10ml volumetric flask and dilute up to the mark with diluent. Sample preparation: 10 tablets were weighed and calculate the average weight of each tablet then the weight equivalent to 10 tablets was transferred into a 100ml standard flask. A volume of mobile phase was added and sonicate for 30min.Then the solution was cooled and diluted to volume with mobile phase and filtered through 0.45µm membrane filter. (Stock solution) Further pipette 0.25ml of Taurine and Acetyl cysteine of the above stock solution in to a 10ml volumetric flask and dilute up to the mark with diluent.
Assay procedure:
20µl of the standard and sample solutions of Taurine and Acetyl cysteine were injected into the HPLC system and the chromatograms were recorded. Amount of drug present in the capsules were calculated using the peak areas.
VALIDATION:
Validation of an analytical method is the process to establish by laboratory studies that the performance characteristic of the method meets the requirements for the intended analytical application. Performance characteristics were expressed in terms of analytical parameters. After development of RP-HPLC method for estimation of Taurine and Acetyl cysteine, validation of the method was carried out according to ICH guidelines
SYSTEM SUITABILITY:
A Standard solution of Taurine and Acetyl cysteine working standard was prepared as per procedure and was injected five times into the HPLC system. The system suitability parameters were evaluated from standard Chromatograms obtained by calculating the % RSD of retention times, tailing factor, theoretical plates and peak areas from five replicate injections.
LINEARITY:
The linearity of an analytical method is its ability to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to the concentration of analyte in samples within a given range. Serial dilutions of Taurine and Acetyl cysteine (20-60µg/ml and 10-30 µg/ml) were injected into the column and detected at a wavelength set at 254 nm. The calibration curve was obtained by plotting the concentration vs. peak area.
SPECIFICITY:
ICH defines specificity as “the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically this might include impurities, degrades, matrix, etc.
PRECISION:
The precision of the method was demonstrated by intra-day and inter-day precision studies. Intra-day studies were performed by injecting three (3) repeated injections within a day. Peak area and %RSD were calculated and reported. The chromatograms of intra-day precision studies were shown. Inter-day precision studies, was done by injecting three (3) repeated injections for three consecutive days. Peak area and %RSD were calculated and reported.
INTERMEDIATE PRECISION:
Intermediate precision of the analytical method was determined by performing method precision on another day by different analysts under same experimental condition. Assay of all six replicate sample preparations was determined and mean %assay value, standard deviation and %RSD was calculated.
ACCURACY:
Accuracy of the method was determined by recovery experiments. There are mainly 2types of recovery studies are there.
Standard addition method:
To the formulation, the reference standard of the respective drug of known concentration was added, analyzed by HPLC and compared with the standard drug concentration.
Percentage method:
For these assay method samples are prepared in three concentrations of 50%, 100%, and 150% respectively.
Acceptance criteria:
The mean % recovery of the Taurine and Acetyl cysteine at each level should be not less than 95.0% and not more than 105.0%.
LIMIT OF DETECTION AND LIMIT OF QUANTIFICATION:
The Sensitivity of measurement of Taurine and Acetyl cysteine by use of the proposed method was estimated in terms of the Limit of Detection (LOD) and the Limit of Quantitation (LOQ).
ROBUSTNESS:
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. For the determination of a method’s robustness, deliberate change in the Flow rate was made to evaluate the impact on the method.
RESULTS:
Table 1. Chromatographic condition Trail -1
Parameters |
Description |
Flow rate |
1ml min-1 |
Column |
Agilent C18 Column(250mm x 4.6mm)5µ |
Mobile Phase |
Buffer: Methanol PH 2.5 (30:70 v/v) |
Buffer |
Potassium dihydrogen orthophosphate PH 2.5 adjust with Orthophosphoric acid |
Detector |
PDA |
Column temperature |
Ambient |
Wavelength |
254 nm |
Type of elution |
Isocratic |
Injection volume |
20µl |
Run time |
10min |
Fig.1 Chromatogram of Trial-1
Table 2. Chromatographic condition Trail-2
Parameters |
Description |
Flow rate |
1ml min-1 |
Column |
Agilent C18 Column (250mm x 4.6mm)5µg. |
Mobile Phase |
Buffer: Methanol PH 2.5 (30:70 v/v) |
Buffer |
Potassium dihydrogen orthophosphate ph2.5 adjusted with Orthophosphoric acid |
Detector |
PDA |
Column temperature |
Ambient |
Type of elution |
Isocratic |
Wavelength |
254nm |
Injection volume |
20µl |
Run time |
10min |
Fig.2 Chromatogram of Trial-2
Table 3. Chromatographic condition Trail-3
Parameters |
Description |
Flow rate |
1ml min-1 |
Column |
X bridge C18 Column (250mm x 4.6mm)5µg. |
Mobile Phase |
Buffer: Methanol PH 2.5 (60:40 v/v) |
Buffer |
Potassium dihydrogen orthophosphate PH 2.5 adjusted with OPA |
Detector |
PDA |
Column temperature |
Ambient |
Type of elution |
Isocratic |
Wavelength |
254 nm |
Injection volume |
20µl |
Run time |
10min |
Fig3. Chromatogram of Trial-3
Table 4.Chromatographic condition Trail – 4
Parameters |
Description |
Flow rate |
1ml min-1 |
Column |
Thermosil; C18 Column (250mm x 4.6mm)5µg. |
Mobile Phase |
Phosphate buffer: Methanol PH 2.5 (20:80 v/v) |
Buffer |
Potassium dihydrogen orthophosphate PH 2.5 adjust with orthophosphoric acid |
Detector |
PDA |
Column temperature |
Ambient |
Type of elution |
Isocratic |
Wavelength |
254 nm |
Injection volume |
20µl |
Run time |
10min |
Fig 4. Chromatogram of Trial-4
Table 5. Chromatographic condition Trail-5
Parameters |
Description |
Flow rate |
1ml min-1 |
Column |
Inertsil C18 Column (250mm x 4.6mm)5µg. |
Mobile Phase |
Phosphate buffer: Methanol PH 2.5 (55:45 v/v) |
Buffer |
Potassium dihydrogen orthophosphate PH 2.5 adjust with Orthophosphoric acid |
Detector |
PDA |
Column temperature |
Ambient |
Type of elution |
Isocratic |
Wavelength |
254 nm |
Injection volume |
20µl |
Run time |
10min |
Fig 5.Chromatogram of Trial-5
Table 6: Chromatographic condition Trail-6
Parameters |
Description |
Flow rate |
1ml min-1 |
Column |
Agilent C18 Column (250mm x 4.6mm)5µg. |
Mobile Phase |
Phosphate buffer: Methanol PH 2.5 (25:75 v/v) |
Buffer |
Potassium dihydrogen orthophosphate PH 2.5 adjusted with Orthophosphoric acid |
Detector |
PDA |
Column temperature |
Ambient |
Type of elution |
Isocratic |
Wavelength |
254 nm |
Injection volume |
20µl |
Run time |
10min |
Fig 6.Chromatogram of Trail-6
Fig 7. Chromatogram of standard
Fig 8 . Chromatogram of standard
Table 7: Peak results of Standard chromatogram– Taurine
|
Name |
RT |
Area |
USP Rate count |
USP Tailing |
USP Resolution |
1 |
Taurine |
3.525 |
810802 |
3527.8 |
1.0 |
2.4 |
2 |
Taurine |
3.528 |
808790 |
3566.0 |
1.0 |
2.3 |
Mean |
|
|
809796 |
3547.0 |
1.0 |
|
Std. Dev. |
|
|
1422.2 |
|
|
|
% RSD |
|
|
0.18 |
|
|
|
Table 8: Peak results of Standard Chromatogram – Acetyl Cysteine
|
Name |
RT |
Area |
USP Plate Count |
USP Tailing |
1 |
Acetyl |
2.984 |
681469 |
3115.4 |
1.1 |
2 |
Acetyl |
2.989 |
683696 |
3209.7 |
1.1 |
Mean |
|
|
682582 |
3162.5 |
1.1 |
Std. Dev. |
|
|
1575.2 |
|
|
% RSD |
|
|
0.23 |
|
|
Fig 9. Chromatogram of Test
Fig 10. Chromatogram of Test
Table 9. Peak results of Test Chromatograms - Taurine
|
Name |
RT |
Area |
1 |
Taurine |
3.527 |
828933 |
2. |
Taurine |
3.528 |
810493 |
Mean |
|
|
819713 |
Std .Dev. |
|
|
13039.2 |
%RSD |
|
|
1.59 |
Table.10. Peak results of Test Chromatograms - Acetyl Cysteine
|
Name |
RT |
Area |
1 |
Acetyl |
3.003 |
687178 |
2 |
Acetyl |
3.003 |
682217 |
Mean |
|
|
684698 |
Std. Dev. |
|
|
3507.7 |
% RSD |
|
|
0.51 |
Table 11: Results of Assay
Parameters |
Taurine |
Acetyl cysteine |
Standard peak area |
810802 |
681469 |
Test peak area (mean) |
828933 |
687178 |
Average Weight |
694.2mg |
694.2mg |
% Purity of Standard |
99.50 |
99.58 |
Amt obtained |
399.88 mg |
150.10 mg |
% Assay |
99.77% |
100.12% |
Figure 12. Chromatogram of sample system suitability
Figure 13. Chromatogram of standard system suitability
Figure 14. Chromatogram of linearity – Taurine and Acetyl cysteine
Figure 15. Chromatogram of linearity – Taurine and Acetyl cysteine
Figure 16. Chromatogram of linearity – Taurine and Acetyl cysteine
Figure 17. Chromatogram of linearity – Taurine and Acetyl cysteine
Figure 18. Chromatogram of linearity – Taurine and Acetyl cysteine
Table 12. Linearity results of Taurine and Acetyl
|
Name |
RT |
Area |
Height(μv) |
1 |
Taurine |
2.996 |
226418 |
26134 |
2 |
A cysteine |
3.519 |
277182 |
28872 |
3 |
Taurine |
3.003 |
432920 |
50127 |
4 |
A cysteine |
3.528 |
521695 |
54273 |
5 |
Taurine |
3.005 |
677256 |
78323 |
6 |
A cysteine |
3.529 |
808274 |
83849 |
7 |
Taurine |
2.998 |
869825 |
100093 |
8 |
A cysteine |
3.522 |
1033875 |
106297 |
9 |
Taurine |
2.987 |
1095759 |
125962 |
10 |
A cysteine |
3.510 |
1285804 |
132354 |
Figure 20. Linearity graph of Taurine.
Table 13. Preparation of working standard solutions for Linearity
Sample ID |
Taurine |
Acetyl cysteine |
||
Concentration (mcg/ml) |
Area |
Concentration (mcg/ml) |
Area |
|
20% of operating concentration |
20 |
226418 |
10 |
277182 |
40% of operating concentration |
30 |
432920 |
15 |
521695 |
60% of operating concentration |
40* |
677256 |
20* |
808274 |
80% of operating concentration |
50 |
869825 |
25 |
1033875 |
100% of operating concentration |
60 |
1095759 |
30 |
1285804 |
Correlation Coefficient |
0.999 |
Figure 21. Chromatogram of precision – Taurine and Acetyl cysteine
Figure 22. Chromatogram of precision – Taurine and Acetyl cysteine
Table 14. Results for chromatogram of precision – Taurine
|
Name |
RT |
Area |
1 |
Taurine |
3.557 |
819305 |
2 |
Taurine |
3.547 |
807157 |
3 |
Taurine |
3.544 |
804070 |
4 |
Taurine |
3.537 |
808474 |
5 |
Taurine |
3.534 |
804505 |
Mean |
|
|
808702 |
Std. Dev. |
|
|
6203.7 |
% RSD |
|
|
0.77 |
Table 15. Results for chromatogram of precision – Acetyl Cysteine.
|
Name |
RT |
Area |
1 |
Acetyl |
3.019 |
691143 |
2 |
Acetyl |
3.011 |
685431 |
3 |
Acetyl |
3.004 |
683543 |
4 |
Acetyl |
20997 |
683564 |
5 |
Acetyl |
20994 |
683532 |
Mean |
|
|
685443 |
Std. Dev. |
|
|
3289.7 |
% RSD |
|
|
0.48 |
Figure 23. Chromatogram of Intermediate precision – Taurine and Acetyl cysteine
Figure 24. Chromatogram of Intermediate precision – Taurine and Acetyl cysteine
Figure 25. Chromatogram of Intermediate precision – Taurine and Acetyl cysteine
Figure 26. Chromatogram of Intermediate precision – Taurine and Acetyl cysteine
Figure 27. Chromatogram of Intermediate precision – Taurine and Acetyl cysteine
Table 16. Intermediate Precision Results for Taurine.
|
Name |
RT |
Area |
1 |
Taurine |
3.524 |
813507 |
2 |
Taurine |
3.533 |
817673 |
3 |
Taurine |
3.533 |
815189 |
4 |
Taurine |
3.517 |
815816 |
5 |
Taurine |
3.530 |
815356 |
Mean |
|
|
815508 |
Std. Dev. |
|
|
1492.7 |
% RSD |
|
|
0.18 |
Table 17. Intermediate Precision Results for Acetyl cysteine
|
Name |
RT |
Area |
1 |
Acetyl |
3.001 |
673725 |
2 |
Acetyl |
3.009 |
672535 |
3 |
Acetyl |
3.010 |
676216 |
4 |
Acetyl |
2.997 |
679037 |
5 |
Acetyl |
3.007 |
677101 |
Mean |
|
|
675723 |
Std. Dev. |
|
|
2611.5 |
% RSD` |
|
|
0.39 |
Table 18. Accuracy 50% values for Taurine
|
Name |
RT |
Area |
1 |
Taurine |
3.530 |
641412 |
2 |
Taurine |
3.519 |
644644 |
3 |
Taurine |
3.517 |
648238 |
Mean |
|
|
644765 |
Std. Dev. |
|
|
3414.8 |
% RSD |
|
|
0.52 |
|
Name |
RT |
Area |
1 |
Acetyl |
2.994 |
544164 |
2 |
Acetyl |
2.986 |
542589 |
3 |
Acetyl |
2.985 |
547381 |
Mean |
|
|
544711 |
Std. Dev. |
|
|
2442.4 |
% RSD |
|
|
0.44 |
Figure 31 Chromatogram of Accuracy 100%
Figure 32 Chromatogram of Accuracy 100%
Table 20. Accuracy 100% values for Taurine
|
Name |
RT |
Area |
1 |
Taurine |
3.528 |
798842 |
2 |
Taurine |
3.533 |
803075 |
3 |
Taurine |
3.530 |
809247 |
Mean |
|
|
803722 |
Std. Dev. |
|
|
5232.4 |
% RSD |
|
|
0.65 |
Table 21. Accuracy 100% values for acetyl cysteine
|
Name |
RT |
Area |
1 |
Acetyl |
2.998 |
676367 |
2 |
Acetyl |
3.002 |
673158 |
3 |
Acetyl |
3.002 |
678282 |
Mean |
|
|
675935 |
Std. Dev. |
|
|
2588.9 |
% RSD |
|
|
0.38 |
Figure 33 Chromatogram of Accuracy 150%
Figure 34 Chromatogram of Accuracy 150%
Figure 35 Chromatogram of Accuracy 150%
Table 22. Accuracy 150% values for Taurine
|
Name |
RT |
Area |
1 |
Taurine |
3.517 |
960574 |
2 |
Taurine |
3.521 |
964089 |
3 |
Taurine |
3.521 |
964089 |
Mean |
|
|
962917 |
% RSD |
|
|
0.2 |
Table 23. Accuracy 150% values for acetyl cysteine
|
Name |
RT |
Area |
1 |
Acetyl |
2.991 |
813332 |
2 |
Acetyl |
2.993 |
812480 |
3 |
Acetyl |
2.993 |
812480 |
Mean |
|
|
812764 |
% RSD |
|
|
0.64 |
Table24. Accuracy Study of Taurine
Sample Id |
Conc found (µg/ml) |
Concn Obtained (µg/ml) |
% Recovery |
Mean recovery |
Statistical Analysis |
50% |
5 |
5.01 |
100.2 |
|
%RSD= 0.505 |
50% |
5 |
4.96 |
99.2 |
99.73 |
|
50% |
5 |
4.99 |
99.8 |
|
|
100% |
10 |
9.95 |
99.5 |
|
%RSD=0.66 |
100% |
10 |
9.87 |
98.7 |
98.8 |
|
100% |
10 |
9.82 |
98.2 |
|
|
150% |
15 |
14.64 |
97.6 |
|
%RSD=1.45 |
150% |
15 |
14.76 |
98.4 |
98.8 |
|
150% |
15 |
15.06 |
100.4 |
|
Table 25. Accuracy Study of Acetyl cysteine
Conc (µg/ml) |
Concn Obtained(µg/ml) |
%Recovery of drug |
Mean accuracy |
%RSD |
5 |
4.92 |
98.0 |
99.2 |
1.2 |
5 |
4.96 |
99.2 |
||
5 |
5.02 |
100.4 |
||
10 |
9.95 |
99.5 |
99.5 |
0.2 |
10 |
9.94 |
99.4 |
||
10 |
9.98 |
99.8 |
||
15 |
14.78 |
98.6 |
99.0
|
0.053 |
15 |
14.94 |
99.6 |
||
15 |
14.83 |
98.8 |
Figure 37 Chromatogram of LOQ
Table 26. LOD and LOQ Data of Taurine and Acetyl cysteine
Taurine |
Acetyl Cysteine |
|||||
Conc.(x) (µg/ml) |
Peak Areas (y) |
Statistical Analysis |
Conc.(x) (µg/ml) |
Peak Areas (y) |
Statistical Analysis |
|
40 |
2004682 |
S = 39092 |
20 |
1184227 |
S = 39092 |
|
c =369381 LOD:0.005 |
||||||
40 |
2004587 |
20 |
1186425 |
|||
c = 618048 |
||||||
LOD: 0.001µg/ml |
µg/ml |
|||||
|
LOQ: 0.004µg/ml |
|
LOQ: |
|||
0.015µg/ml |
Figure.38 Representative Chromatogram at Flow rate of 0.8 ml/min
Table 27 representative Chromatogram at Flow rate of 0.8 ml/min.
|
Name |
Retention time (min) |
Area (μv*sec) |
Height (μv) |
USP plate count |
USP Tailing |
USp Resolution |
1 |
Taurine |
3.276 |
740721 |
73704 |
2690.4 |
0.9 |
|
2 |
Acetyl |
3.847 |
903225 |
78896 |
2716.2 |
0.9 |
1.9 |
Figure.39. Representative Chromatogram at Flow rate of 1.2 ml/min
Table28. Representative Chromatogram at Flow rate of 1.2 ml/min
|
Name |
Retention time (min) |
Area (μv*sec) |
Height (μv) |
USP plate count |
USP Tailing |
USp Resolution |
1 |
Taurine |
2.747 |
623847 |
75117 |
2503.3 |
0.9 |
|
2 |
Acetyl |
3.220 |
756748 |
80446 |
2658.4 |
0.9 |
1.9 |
Figure.40.Representative Chromatogram for Mobile phase composition (Buffer: Methanol: 40:60)]
Table 29.Representative Chromatogram for Mobile phase composition (Buffer: Methanol: 40:60)]
|
Name |
Retention time (min) |
Area (μv*sec) |
Height (μv) |
USP plate count |
USP Tailing |
USp Resolution |
1 |
Taurine |
2.743 |
623812 |
75134 |
2707.1 |
1.1 |
|
2 |
Acetyl |
3.221 |
756795 |
80412 |
3001.8 |
1.0 |
1.9 |
Figure.40.Representative Chromatogram for Mobile phase composition (Buffer: Methanol: 30:70)
Table 30 Representative Chromatogram for Mobile phase composition (Buffer: Methanol: 30:70)
|
Name |
Retention time (min) |
Area (μv*sec) |
Height (μv) |
USP plate count |
USP Tailing |
USp Resolution |
1 |
Taurine |
3.275 |
740841 |
73795 |
2818.9 |
1.1 |
|
2 |
Acetyl |
3.846 |
903365 |
78845 |
3107.7 |
1.0 |
1.9 |
Table.31. Robustness data for Taurine
Std. Replicate |
Variation in flow rate |
Variation in Mobile phase composition |
||
Flow Rate 0.8ml/min |
Flow Rate 1.2ml/min |
Buffer: Methanol (40:60) |
Buffer: Methanol (30:70) |
|
Tailing factor |
0.9 |
0.9 |
1.1 |
1.1 |
Theoretical plates |
2690 |
2503 |
2707 |
2818 |
Table.31. Robustness data for Acetyl cysteine
Parameter |
Variation in flow rate |
Variation in Mobile phase composition |
||
Standard |
Flow Rate 0.8ml/min |
Flow Rate 1.2ml/min |
Buffer: Methanol (40:60) |
Buffer: Methanol (30:70) |
Tailing factor |
0.9 |
0.9 |
1.0 |
1.0 |
Theoretical plates |
2716 |
2685 |
30018 |
3107 |
DISCUSSION:
In RP-HPLC method, the conditions were optimized to obtain an adequate separation of eluted compounds. Initially, various mobile phase compositions were tried, to separate title ingredients. Mobile phase and flow rate selection was based on peak parameters (height, tailing, theoretical plates, capacity or symmetry factor), run time and resolution. The mobile phase containing mixture of orthophosphoric acid buffer solution: Methanol (25:75v/v, pH 2.45) with a flow rate of 1.0 ml/min is quite robust. The optimum wavelength for detection was 254 nm at which better detector response for both the drugs was obtained. The retention times for Taurine and Acetyl cysteine was found to be 2.589 ± 0.004 min and 3.711 ± 0.005 min, respectively. To ascertain its effectiveness, system suitability tests were carried out on freshly prepared stock solutions. The calibration was linear in concentration range of 20 to 60 μg/ ml and 10 to 30 μg/ml, with regression 0.9979 and 0.9999, Taurine and Acetyl cysteine respectively. The low values of %R.S.D indicate the method is precise and accurate. The mean recoveries were found above 99.3 % for both the drugs. Robustness of the proposed method was determined by varying various parameters, the %RSD reported was found to be less than 2 %. The proposed method was validated in accordance with ICH parameters and the applied for analysis of the same in marketed formulations.
CONCLUSION:
The proposed HPLC method was found to be simple, specific, precise, accurate, rapid and economical for simultaneous estimation of Taurine and Acetyl cysteine in tablet dosage form. The developed method was validated in terms of accuracy, precision, linearity, robustness and ruggedness, and results will be validated statistically according to ICH guidelines. The Sample recoveries in all formulations were in good agreement with their respective label claims. From literature review and solubility analysis initial chromatographic conditions Mobile phase ortho phosphoric acid buffer: Methanol 25:75 were set (Buffer pH2.45 adjusted with Triethylamine), Inertsil C 18 (250×4.6mm, 5µ) Column, Flow rate 1.0 ml/min and temperature was ambient, eluent was scanned with PDA detector in system and it showed maximum absorbance at 254 nm. As the methanol content was increased Taurine and Acetyl cysteine got eluted with good peak symmetric properties. The retention times for Taurine and Acetyl cysteine was found to be 2.589 min and 3.711 min respectively. System suitability parameters were studied by injecting the standard five times and results were well under the acceptance criteria.
ACKNOWLEDGEMENT:
The authors are grateful to the management of Gnan Jyothi College of Pharmacy Hyderabad and Princeton College of Pharmacy Hyderabad. Authors sincerely thank KP Labs Hyderabad for helping in carrying out the research work.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 31.08.2018 Modified on 20.09.2018
Accepted on 10.10.2018 © A&V Publications All right reserved
Asian J. Res. Pharm. Sci. 2018; 8(4):223-235.
DOI: 10.5958/2231-5659.2018.00038.3