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 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

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

 


 

 

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