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 C}18 ^{Column(250mm x 4.6mm)5µ}
Mobile Phase	Buffer: Methanol P^H2.5 (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 C}18 ^{Column (250mm x 4.6mm)5µg.}
Mobile Phase	Buffer: Methanol P^H2.5 (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 C}18 ^Column ^{(250mm x 4.6mm)5µg.}
Mobile Phase	Buffer: Methanol P^H2.5 (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; C}18 ^{Column (250mm x 4.6mm)5µg.}
Mobile Phase	Phosphate buffer: Methanol P^H2.5 (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 C}18 ^{Column (250mm x 4.6mm)5µg.}
Mobile Phase	Phosphate buffer: Methanol P^H2.5 (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 C}18 ^{Column (250mm x 4.6mm)5µg.}
Mobile Phase	Phosphate buffer: Methanol P^H2.5 (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 11. Chromatogram of Blank

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 19 Linearity Graph of Acetyl Cysteine

Figure 20. Linearity graph of Taurine.

Table 13. Preparation of working standard solutions for Linearity

Sample ID	Taurine		Acetyl cysteine
Sample ID	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

Figure 28 Chromatogram of Accuracy 50%

Figure 29 Chromatogram of Accuracy 50%

Figure 30 Chromatogram of Accuracy 50%

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

Table 19. Accuracy 50% values for acetyl cysteine

	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 36 Chromatogram of LOD

Figure 37 Chromatogram of LOQ

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

Asian J. Res. Pharm. Sci. 2018; 8(4):223-235.

DOI: 10.5958/2231-5659.2018.00038.3

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

	Name	Retention time (min)	*Area (μvsec)**	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

	Name	Retention time (min)	*Area (μvsec)**	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

	Name	Retention time (min)	*Area (μvsec)**	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

	Name	Retention time (min)	*Area (μvsec)**	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

Std. Replicate	Variation in flow rate		Variation in Mobile phase composition
Std. Replicate	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

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

Figure 11. Chromatogram of Blank

Figure 19 Linearity Graph of Acetyl Cysteine

Figure 28 Chromatogram of Accuracy 50%

Figure 29 Chromatogram of Accuracy 50%

Figure 30 Chromatogram of Accuracy 50%

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

Table 19. Accuracy 50% values for acetyl cysteine

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

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

Name

RT

Area

1

Acetyl

2.998

676367

2

Acetyl

3.002

673158