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	R_t	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	R_t	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 guidelines^11-14.

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%.

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

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

DOI: 10.52711/2231-5691.2023.00016

Concentration mg/ml	Average Peak Area
0	0
30	185658
40	245475
50	309658
60	365847
70	428698

Concentration mg/ml	Average Peak Area
0	0
6	2658795
8	3556974
10	4458749
12	5265874
14	6169886