Reverse Phase High Performance Liquid Chromatography Method for Simultaneous Estimation of Aspirin and Caffeine in Pure and Tablet

 

B. Sudhakar1*, Palaparthi Srivalli1, Ramya Sri. S2

1Department of Pharmaceutical Analysis, Samskruti College of Pharmacy,

Affiliated to JNTUH University, Hyderabad 501301, Telangana, India.

2Department of Pharmacy, University College of Technology, Osmania University,

Hyderabad, Telangana, 500007, India.

*Corresponding Author E-mail: sudhakarspkg@gmail.com

 

ABSTRACT:

A new, simple, rapid, accurate and precise Reverse Phase High Performance Liquid Chromatographic method has been developed for the validated of Aspirin and Caffeine, in Active Pharmaceutical Ingredient form as well as in combined tablet dosage form. Chromatography was carried out on Symmetry ODS C18 (4.6mm × 250mm, 5µm) column using a mixture of Methanol: Acetonitrile (35:65v/v) as the mobile phase at a flow rate of 1.0ml/min, the detection was carried out at 273nm. The retention time of the Aspirin and Caffeine, was 2.085, 5.262±0.02min respectively. The method produces linear responses in the concentration range of 30-70mg/ml of Aspirin and 6-14mg/ml of Caffeine. The mean % assay of marketed formulation was found to be 100.04%, and % recovery was observed in the range of 98-102%. Relative standard deviation for the precision study was found <2%. The developed method is simple, precise and rapid, making it suitable for estimation of Aspirin and Caffeinein API and combined tablet dosage form. The method is useful in the quality control of bulk and pharmaceutical formulations.

 

KEYWORDS: Aspirin and Caffeine, RP-HPLC, Validation, ICH Guidelines.

 

 


INTRODUCTION:

High performance liquid chromatography (HPLC) is a technique used for analysis of drug substance, drug product and determination and quantification of known as well as unknown impurities at lower level, food and drug administration (FDA) also trust on the purity method of analysis by using HPLC, because of high accuracy and reproducibility of results1.

 

The importance of chromatography is increasing rapidly in pharmaceutical analysis for the exact differentiation, selective identification, quantitative determination of structurally closely related compounds.

 

 

Another important field of application of chromatographic methods is the purity testing of final products and the intermediates2.

 

Aspirin, 2-(acetyloxy) benzoic acid, acts as an inhibitor of cyclooxygenase which results in the inhibition of the biosynthesis of prostaglandins. It also inhibits platelet aggregation and is used in the prevention of arterial and venous thrombosis3. Aspirin is also used for long-term, at low doses, to help prevent heart attacks, strokes, and blood clot formation in people at high risk for developing blood clots4.

 

 

Fig 1: Chemical Structure of Aspirin5

Caffeine (C8H10N4O2) is the common name for trimethylxanthine (systematic name is 1, 3, 7-trimethylxanthine or 3, 7-dihydro-1, 3, 7-trimethyl-1H-purine-2, 6-dione)6. Caffeine is a xanthine alkaloid found in non-alcoholic beverages (e.g. tea, coffee, and cocoa), which may boost mood, metabolism, mental and physical performance7. Hence, it is added to some commercially available paracetamol tablets to enhance the pain-killing effects of paracetamol8. Massive caffeine ingestion resulting in death9.

 

Fig 2: Chemical Structure of Caffeine10

 

MATERIALS AND METHODS:

Caffeine from Sura labs, Aspirin from Sura labs, Water and Methanol for HPLC from Lichrosolv (Merck)

Acetonitrile for HPLC from Merck.

 

HPLC Method Development:

Preparation of standard solution:

Accurately weigh and transfer 10mg of Caffeine and Aspirin working standard into a 10ml of clean dry volumetric flasks add about 7ml of Methanol and sonicate to dissolve and removal of air completely and make volume up to the mark with the same Methanol.

 

Further pipette 0.5ml of the above Aspirin and 0.1ml of Caffeine stock solutions into a 10ml volumetric flask and dilute up to the mark with Methanol.

 

Procedure:

Inject the samples by changing the chromatographic conditions and record the chromatograms, note the conditions of proper peak elution for performing validation parameters as per ICH guidelines.

 

Mobile Phase Optimization:

Initially the mobile phase tried was Methanol: Water and Water: Acetonitrile and Methanol: TEA Buffer: ACN with varying proportions. Finally, the mobile phase was optimized to Methanol: Acetonitrile in proportion 35:65 v/v respectively. 

 

Optimization of Column:

The method was performed with various columns like C18 column, Symmetry and Zodiac column. Symmetry ODS C18 (4.6mm × 250mm, 5µm) was found to be ideal as it gave good peak shape and resolution at 1ml/min flow.

 

Optimized Chromatogram (Standard):

Mobile phase: Methanol: Acetonitrile (35:65v/v)

Column: Symmetry ODS C18 (4.6mm × 250mm, 5µm)

Flow rate: 1ml/min

Wavelength: 273nm

Column temp: Ambient

Injection Volume: 20µl

Run time: 10minutes

 

Fig-1: Optimized Chromatogram

 

 

Table 1: - Peak Results for Optimized Chromatogram

Peak Name

Rt

Area

Height

USP Resolution

USP Tailing

USP plate count

Aspirin

2.085

289658

3526

 

1.65

6745

Caffeine

5.262

4658749

28547

8.59

1.82

8638

 

Observation:

From the above chromatogram it was observed that the Aspirin and Caffeine peaks are well separated and they shows proper retention time, resolution, peak tail and plate count. So it’s optimized trial.

 

Optimized Chromatogram (Sample)

 

Figure 2: Optimized Chromatogram (Sample)


 

 

 

 

Table 2: Optimized Chromatogram (Sample)

S. No.

Peak Name

Rt

Area

Height

USP Resolution

USP Tailing

USP plate count

1

Aspirin

2.089

298698

3658

 

1.68

6859

2

Caffeine

5.327

4758695

29586

8.64

1.85

8789

 

Table 3-: Results of system suitability for Aspirin

S No

Name

Rt

Area

Height

USP plate count

USP Tailing

1

Aspirin

2.090

289854

3526

8659

1.82

2

Aspirin

2.090

285745

3541

8642

1.83

3

Aspirin

2.089

289587

3612

8674

1.82

4

Aspirin

2.089

285466

3584

8692

1.83

5

Aspirin

2.085

285987

3572

8654

1.82

Mean

 

 

287327.8

 

 

 

Std. Dev

 

 

2194.024

 

 

 

% RSD

 

 

0.763596

 

 

 

 

Table 4-: Results of system suitability for Caffeine

S No

Name

Rt

Area

Height

USP plate count

USP Tailing

USP Resolution

1

Caffeine

5.289

4658745

28564

8659

1.82

 

2

Caffeine

5.289

4652587

28457

8647

1.83

 

3

Caffeine

5.338

4674833

28952

8632

1.82

 

4

Caffeine

5.327

4685825

28754

8645

1.83

 

5

Caffeine

5.262

4652145

28964

8694

1.82

 

Mean

 

 

4664827

 

 

 

 

Std. Dev

 

 

14905.35

 

 

 

 

 

Validation methods procedures followed as per ICH guidelines11-14.

 

Assay (Standard):

Table 5-: Peak Results for Assay Standard

S No.

Name

Rt

Area

Height

USP Resolution

USP Tailing

USP plate count

Injection

1

Aspirin

2.090

289654

3566

 

1.65

6785

1

2

Caffeine

5.289

4685784

28654

8.60

1.83

8659

1

3

Aspirin

2.089

289745

3598

 

1.66

6798

2

4

Caffeine

5.338

4658975

286598

8.59

1.82

8624

2

5

Aspirin

2.089

285687

3589

 

1.65

6782

3

6

Caffeine

5.327

4658798

254875

8.60

1.83

8695

3

 

Assay (Sample):

Table 6-: Peak Results for Assay sample

S. No.

Name

Rt

Area

Height

USP Resolution

USP Tailing

USP plate count

Injection

1

Aspirin

2.088

296852

3659

 

1.66

6859

1

2

Caffeine

5.276

4785658

29865

9.75

1.83

8754

1

3

Aspirin

2.087

298545

3698

 

1.67

6874

2

4

Caffeine

5.268

4788982

29863

9.82

1.82

8785

2

5

Aspirin

2.085

296854

3674

 

1.67

6857

3

6

Caffeine

5.262

4789856

29865

9.78

1.83

8795

3

 

%ASSAY =

  Sample area        Weight of standard     Dilution of sample     Purity      Weight of tablet

 ___________ ×   ________________ × _______________×_______×______________×100

  Standard area      Dilution of standard    Weight of sample       100          Label claim

 

The % purity of Aspirin and Caffeine in pharmaceutical dosage form was found to be100.04%.

 


Linearity:

Chromatographic Data for Linearity Study:

Aspirin:

Concentration mg/ml

Average Peak Area

0

0

30

185658

40

245475

50

309658

60

365847

70

428698



Figure 3: Linearity for Aspirin

 

Caffeine

Concentration mg/ml

Average Peak Area

0

0

6

2658795

8

3556974

10

4458749

12

5265874

14

6169886

 

Figure 4: Calibration Curve for Caffeine

 


 

Repeatability:


Table 7: Results of Repeatability for Aspirin:

S No

Name

Rt

Area

Height

USP plate count

USP Tailing

1

Aspirin

2.086

289658

3569

6789

1.65

2

Aspirin

2.083

289547

3526

6758

1.66

3

Aspirin

2.083

285698

3598

6792

1.65

4

Aspirin

2.081

284579

3547

6749

1.66

5

Aspirin

2.081

285698

3598

6742

1.65

Mean

 

 

287036

 

 

 

Std. Dev

 

 

2387.328

 

 

 

% RSD

 

 

0.831717

 

 

 

 

Table 8-: Results of Repeatability for Caffeine:

S. no

Name

Rt

Area

Height

USP plate count

USP Tailing

USP Resolution

1

Caffeine

5.178

4685982

28569

8659

1.83

8.60

2

Caffeine

5.199

4698547

28574

8695

1.82

8.60

3

Caffeine

5.235

4658754

28598

8654

1.82

8.60

4

Caffeine

5.202

4635981

26985

8678

1.82

8.60

5

Caffeine

5.206

4658798

26857

8692

1.83

8.60

Mean

 

 

4667612

 

 

 

 

Std. Dev

 

 

24754.3

 

 

 

 

% RSD

 

 

0.530342

 

 

 

 

 

Accuracy:

Table 9-: The Accuracy Results for Aspirin

% Concentration

(at specification Level)

Area

Amount Added

(ppm)

Amount Found

(ppm)

% Recovery

Mean Recovery

50%

153851

25

24.985

99.94%

100.00%

100%

306722.7

100

49.981

99.962%

150%

460175.7

150

75.071

100.094%

 

Table 10-: The Accuracy Results for Caffeine

%Concentration

(at specification Level)

Area

Amount Added

(ppm)

Amount Found

(ppm)

% Recovery

Mean Recovery

50%

233866.3

5

4.963

99.26%

99.94%

100%

455388.3

10

9.994

99.94%

150%

680034

15

15.095

100.633%

 


Limit of Detection:

The detection  limit  of  an  individual  analytical  procedure  is  the  lowest  amount  of analyte in a sample which can be detected but not necessarily quantitated as an exact value.

 

LOD= 3.3 × σ / s

Where 

σ = Standard deviation of the response   

S = Slope of the calibration curve

 

Result:

Aspirin:

=0.7µg/ml

 

Caffeine:

=2.8µg/ml

 

Limit of Quantitation:

The  quantitation  limit  of  an  individual  analytical  procedure  is  the  lowest  amount  of analyte  in  a  sample  which  can  be  quantitatively  determined. 

 

LOQ=10×σ/S

Where 

σ = Standard deviation of the response   

S = Slope of the calibration curve

 

RESULT:

Aspirin:

= 2.1µg/ml

 

Caffeine:

= 6.3µg/ml

 

Robustness

Table 11-: Results for Robustness

Aspirin:

Parameter used for sample analysis

Peak Area

Retention Time

Theoretical plates

Tailing factor

Actual Flow rate of 1.0 mL/min

289658

2.090

6745

1.65

Less Flow rate of 0.9 mL/min

298659

2.736

6854

1.69

More Flow rate of 1.1 mL/min

275478

1.673

6685

1.62

Less organic phase

265397

2.736

6635

1.64

More organic phase

245876

1.673

6425

1.67

 

Acceptance criteria:

The tailing factor should be less than 2.0 and the number of theoretical plates (N) should be more than 2000.

 

Table 12-: Results for Robustness

Caffeine:

Parameter used for sample analysis

Peak Area

Retention Time

Theoretical plates

Tailing factor

Actual Flow rate of 1.0 mL/min

4658749

5.289

8638

1.82

Less Flow rate of 0.9 mL/min

4875985

6.746

8759

1.81

More Flow rate of 1.1 mL/min

4525321

4.032

8452

1.80

Less organic phase

4425643

6.746

8695

1.83

More organic phase

4258675

4.032

8239

1.84

 

CONCLUSION:

In the present investigation, a simple, sensitive, precise and accurate RP-HPLC method was developed for the Quantitative estimation of Caffeine and Aspirin in bulk drug and pharmaceutical dosage forms.

 

This method was simple, since diluted samples are directly used without any preliminary chemical derivatisation or purification steps.

 

Caffeine sodium is freely soluble in ethanol, methanol, and water and practically insoluble in Acetonitrile.

 

Aspirin is freely soluble in water, soluble in methanol, insoluble in acetone.

 

Methanol: Acetonitrile (35:65v/v) was chosen as the mobile phase. The solvent system used in this method was Economical.

 

The %RSD values were within 2 and the method was found to be precise.

 

The results expressed in Tables for RP-HPLC method was promising. The RP-HPLC method is more sensitive, accurate and precise compared to the Spectro photometric methods.

 

This method can be used for the routine determination of Caffeine and Aspirin in bulk drug and in Pharmaceutical dosage forms.

 

ACKNOWLEDGEMENT:

Thе Authors arе thankful to the Management and Principal, Department of Pharmacy, Samskruti College of Pharmacy, Hyderabad, for extending support to carry out the research work. Finally, the authors express their gratitude to the Sura Pharma Labs, Dilsukhnagar, Hyderabad, for providing research equipment and facilities.

 

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Received on 17.10.2022         Modified on 26.11.2022

Accepted on 23.12.2022   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2023; 13(2):81-86.

DOI: 10.52711/2231-5691.2023.00016