Validation of Liquid Chromatographic Analytical Method for Determination of Aspirin, Caffeine and Paracetamol in some Pharmaceutical Tablets Form in Iraqi market

 

Ahmed Mahdi Saeed, Omer Jasim Mohammed, Nada Ghaaeb Hussein

College of Education for Pure Science, Chemistry Department, Diyala University.

 

ABSTRACT:

In this research, an accurate, efficient and reproducible isocratic reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and validated for the determination of aspirin (ASP), caffeine (CAF), and Paracetamol (PCM). A mixture of drugs as standard material and in formulation tablets were separated using an phenomenex C18 column, (L, 15cm, I.D, 0.46cm, and Size of particle, 5μm), with the shimadzu RP – HPLC, model LC–20 - A, Japan. The eluent phase was optimized through the design, experiment. Elution was done by an eluent phase composed of water (H2O), methanol (MOH) and acetonitrile (ACN) mixture have a ratio of (60: 20: 20, V/V), with adjusted pH of 4.0 with acetic acid, have a pumped flow rate of 0.5mL/min. The drugs, separation were performed at 254nm using a UV-VIS - detector for 8 min. The time of elution for the drugs was recorded at (3.980, 4.366, and 6.894 min) for PCM, CAF and ASP respectively. The validation of the method was conducted in a range of concentration (1 - 100μg/mL) for PCM, CAF and ASP. The method was found to be robust, specified and resisting against the small experimental changes in the value of the flow rate, pH, and wavelength. The LOD were found to be 0.01μg/mL, 0.03μg/mL and 0.05μg/mL for PCM, CAF and ASP respectively. The values of LOQ were 0.033μg/mL, 0.099μg/mL and 0.165μg/mL for PCM, CAF and ASP respectively.

 

 

 

INTRODUCTION: 

Analytical procedure refers to a performing way of the analysis. Which must describe in detail the all steps that necessary for the analytical test. The validation objective of the analytical procedures for the demonstrate if they are suitable for its destined purpose. It is very rare, or there is no single remedy to remediation all pain forms, as there is no typical analgesic, as each factor has the disadvantages and advantages recognize it from other analgesics to treat pain. Every mechanical analgesic have its own assignment to the pain prevent. For anti-inflammatory nonsteroidal drugs, the action mechanism is in their ability to the (COX) enzyme inhibit, which in turn is primarily responsible for the prostaglandin synthesis, which is playing a key role in the group which is acting as pain converters¹.

 

Aspirin (ASP) is known as acetylsalicylic acid, is a non-steroidal anti-inflammatory drug (NSAIDs) and shows inhibition of the enzyme cyclooxygenase, and it is often used as an analgesic, antipyretic, anti-inflammatory. ASP also has an antiplatelet effect by inhibiting the production of thromboxane, which under normal circumstances binds platelet molecule together to create a patch over damage on the walls within blood vessels². PCM is classified as part of this group of materials³. PCM was used for pain joint, pain in the middle ear, headache from analgesic effect, neuralgia, toothache, and aches generate from cold, tumor and flue. Caffeine, which is deemed as alkaloid of Purina group⁴. CAF (1,3,7-trimethyxanthine), is soluble partially in water because of its moderate polarity. Caffeine is an excitant of the central system of the nerve. Since it is widely humans consumed, it is considered a most used in the world as a psychoactive substance. It was used both as a medicine and as a recreational. It was caused the vigilance increased and focusing and improved overall the coordination of body⁵. The combination of drugs has ultimate effective when the agents act out of different analgesic mechanization and synergistically active⁶. The analgesics like paracetamol is generally combined to increase the effectiveness of the analgesic⁷. Various methods of analysis are announcing to determine these drugs in formulating drugs like HPLC^8,9,10, SP-FT - Raman¹¹, electromagnetic¹², spectroscopy^13,14,15, GC-Mass¹⁶, Electrode ion¹⁷ and GC - ion trap¹⁸. The work objective is to evaluate a new accurate and easy chromatography analytical method for the estimation of the drug content in tablet formulated samples manufactured by different pharmaceutical corporation which available in the pharmaceutical market in Iraq, to tool up information about the different products, which may enforce or not enforce with the requirements of the formal method or other standard methods.

 

MATERIALS AND METHODS:

Chemicals and reagents:

ASP, CAF and PCM standard powder were from SDI- Iraq. Methanol and acetonitrile (HPLC-grade) were from BDH. Sodium hydrogen phosphate, sodium borate, sodium acetate, boric acid, acetic acid, and phosphoric acid were from BDH. Deionized water, freshly prepared was used.              

 

Instrumentation and Conditions of chromatographic:

HPLC (shimadzu - LC – 20 - A, Japan), Germany Sartorius - balance, Karl – Kolb - Ultrasonic bath - Germany), Shaking bath water (Taiwan) and Memmert - oven – Germany, were used in this study. ASP, PCM and CAF were separated on column type C-18 phenomenex -  (L, 15cm, I.D, 0.46cm -  and size of particle, 5-µm). Separation was utter at room - temperature (~25^oC) and the run time was 8 min under Reversed -  Phase conditions. The elution phase was methanol (MOH) acetonitrile (ACN) and water (H₂O) in the ratio of (20: 20: 60 V/V) adjusted pH with acetic acid at 4.0. The rate of flow was 0.5mL/min, and an 10 μL injector loop was used for injecting samples and detection was done at 254nm. The eluent phase was degassing using the sonicator type - ultrasonic cleaner, power -  sonic- 420, and then filtered over a 0.45μm filter of nylon. The identity established of the compound was done through the comparing of the standard compound solution retention time with those of a sample compound solution. Chromatography was complete in temperature column that maintained at 25±2°C. The UV- spectrums of ASP, CAF and PCM  selecting the detection working wavelength were taken by the Jasco - V-650 – Japan, double - beam UV-VIS - spectrophotometer has 10mm length path quartz cells, which was used for the analytical object.

 

 

Preparation of solutions:

Standard stock solution:

The standard stock solutions have a 1000μg/mL of the ASP, CAF and PCM  were prepared in the mixture of MOH, ACN and H₂O using standard material of drugs. Transfer 10mL of the stock solution 1000μg/mL into 100mL volumetric calibrated flask and make up to the mark with elution phase for giving a standard working solution having a100μg/mL concentration.

 

Diluent:

From the stock solution of 100μg/mL, additional dilutions were conducted through withdrawing a different volume (0.1 – 10) from the standard solution of ASP, PCM and CAF  into the series of 10mL volumetric calibrated flasks and all were complete to the mark with eluent phase to prepare standard working solutions have concentrations of (0.01 – 100μg/mL).

 

Procedure for drugs assay in pharmaceuticals tablets:

Ten tablets of the ASP, CAF and PCM  drug’s, formula were accurately weighed and finely powdered. An accurately quantity weighed of tablets, powder which equivalent to (100mg) of ASP, CAF and PCM  drugs were conveyed to a (100mL) volumetric flask and then diluted with (H₂O: MOH: ACN 60: 20: 20 V/V), the content were ultra - sonicated for 25 min. The drugs solutions volume were completed to the mark and mixed well with solvent. The solutions were filtered again using no. 1 whatman filter paper for the removing of unwanted materials particulate. A filtered solution was appropriately further diluted with the elution phase to produce a sample solution for analysis. The amount of the ASP, CAF and PCM  present in the solution sample was estimated using the standard calibration graphs.

 

RESULTS AND DISCUSSION:

Estimation of detection wavelength:

A drugs solution of the 50μg/mL concentration was scanned at the range of 200 to 400nm wavelength. It was observed that ASP, CAF and PCM  solutions have shown enormous, sharp, and maximum absorbance at 238, 272.6 and 244.8nm wavelength respectively. Therefore, it was selected as the detection wavelength in the analysis. The spectrum study revealed that ASP, CAF and PCM  solutions were indicating a well - defined λ_max at 238 ,  272.6 and 244.8nm as clear in (Fig. 1).

 

 

Fig: 1: UV spectra of  CAF.

Method Development  and System Suitability Test:

Various tests were conducted to get reasonable  resolution – separation of ASP, CAF and PCM  using different eluent phases with different ratios of water, organic solvent and buffer. An ideal eluent phase was found to be the mixture of water, methanol and acetonitrile. This eluent phase used in ratio (60: 20: 20 V/V) gave a good and satisfactory resolution of ASP, CAF and PCM. The pH value (4.0) of the eluent phase, increasing or decreasing by ±0.2, did not indicate a worthy change in the analyte retention time. The time of retention using analytical column was estimated at a rate of flow with 0.5mL/min. The volume of injection was 10μL. The retention time of sample and standard for ASP, CAF and PCM  was well pleased with high resolution in formulating sample. This labour was converging on optimization of the conditions for the rapid, simple, low cost, and effective analysis, involving a selection of the eluent phase to take out satisfactory results. Solvent strength, solvent type (organic solvent volume fraction in the eluent phase and pH of the mobile phase solution), the wavelength of  detection and rate of flow were varied to estimation the Chromatographic conditions which were given the good separation. The optimized of eluent phase conditions was conducted so there no solvent interference and excipients. The entire predicate chromatographic optimum conditions and the notice values of column efficiency, resolution and factor tailing were mentioned in Table 1. The  chromatograms of standard ASP, CAF and PCM  and mixture of standard drugs applied optimum condition are revealed in (Fig. 2 A and B).

 

 

 

 Fig: 2 A: HPLC chromatograms for standard drugs.

 

Fig: 2 B: HPLC chromatograms for standard drugs mixture.

 

Table: 1: The predicate optimum parameters  and system suitability of HPLC method.

Predicate  optimum parameters results
Compassion of eluent phase, H2O: MOH: ACN 60: 20: 20 V/V	Column Type, ODS, (15 – 0.46) cm, 5µm
	Sample Temperature, ambient
Rate of flow, 0.5mL/ min.	Column Temperature, 25 ±2°C
Injection volume, 10µL	Run Time min, 8.00
Detection wavelength,  nm 254	Retention Time min,  3.890 PCM, 4.364 CAF, and 6.894 ASP
System  Suitability results
Parameters  of system Suitability	Results	Acceptance  criteria
Retention  time	3.890 PCM, 4.364 CAF, and 6.894 ASP
RSD% for area of nine injections of standard drug solution	0.318 PCM, 0.335 CAF, and 0.328 ASP	NMT  2.0
Peak  talling factor	1.213 PCM, 1.165 CAF, 1.154 ASP	NMT  2.0
Theoretical  plates	4165 PCM, 3998 CAF, 3876 ASP	NLT 2000

 

Preparation of Calibration graph:

From the standard stock solution, posterior dilutions were done with eluent phase to gain a series of standard solutions have a range of concentration with (0.01 – 100 μg/mL) of drugs. The solutions were injected using injector loop of 10μL and chromatograms were recorded. A graphs were plotted by taking a concentration on X-axis and the area under the peak on Y-axis which gave a straight line.

 

Analytical method validation:

Validation of progress method was conducted as per ICH Q₂ R₁ guideline¹⁹. Parameters such as accuracy, linearity, precision, specificity, LOD and LOQ, robustness and ruggedness were taken in considering testing for the analytical validation method.

 

Linearity and Range:

The proposed RP-HPLC method was shown a good linearity in range concentration of (1 up to 100μg/mL) for ASP, CAF and PCM  respectively were represented in (Fig. 3). The straight line equations are y = 13317 x + 8183.2 (R² = 0.9993) for ASP, y = 59143 x + 5172.4 (R² = 0.9997) for PCM and y = 56701 x + 6450.9 (R² = 0.9993) for CAF. The results are satisfactory, because there is a significant correlation between concentration of drugs and response factor within the concentration range.

 

 

Fig: 3: Linearity of the standard drugs

 

Precision:

The developed method intraday precision of the was evaluated by analysing ASP, CAF and PCM  samples of different concentrations three times in the same day and RSD% was estimated. The precision inter day was estimated through the samples analysing have variable concentrations of ASP, CAF and PCM  in different three days and RSD% was estimated. Evaluated of Repeatability was conducted by  injecting the standard drugs solutions of (15μg/mL) five time in the one day and the RSD% value were calculated. The obtained results are tabulated in Table 2.

 

LOD and LOQ:

LOD and LOQ were estimated by the gradual dilution for lowest concentration, and 3.3 LOD respectively. The obtained results are tabulated  in Table 2.

 

Table: 2: parameters validation summery.

Sr. No.	Validation  parameters	Results	Standard values
1	Linearity Range	1 – 100µg/mL for ASP, CAF and PCM	-
2	Straight line equation	y = 13317 x + 8183.2 (R² = 0.9993) for ASP   y = 59143 x + 5172.4 (R² = 0.9997) for PCM y = 56701 x + 6450.9 (R² = 0.9993) for CAF	-
3	Correlation Coefficient	0.9993 ASP, 0.9993 CAF, 0.9997 PCM	≥ 0.9990
4	Precision (% R.S.D.)		≤ 2.0 % R.S.D.
	Repeatability Intraday Interday	0.314 PCM, 0.265 CAF 0.388 PCM, 0.402 CAF 0.764 PCM, 0.716 CAF
5	Mean % Recovery		95 – 105%
6	Specificity	Specific
7	LOD  (µg/mL)	0.05, 0.03, 0.01 µg/ml for ASP, CAF and PCM	-
8	LOQ  (µg/mL)	0.165, 0.099, 0.013 µg/ml for ASP, CAF and PCM	-
9	Ruggedness	Complies	≤ 2.0 % R.S.D.
10	Robustness		≤ 2.0 % R.S.D.
	Flow rate change Wavelength change Solution pH change	Complies

 

Accuracy:

This study was carried out to assure the closeness of the test results obtained by the analytical method to the true value²⁰. For this method,  ASP, CAF and PCM  were measured at three selected different concentrations within the limits of Beer’s law 5, 25, 45μg/mL.  The results are tabulated in Table 3, which revealed that the suggested method for detection of   interesting and quite convenient with respect to the methods and parameters calculated. The recoveries of standard drugs are between 99.00 – 101.12%.

 

Table: 3: proposed method accuracy of drugs determination.

ASP µg/mL		% Recovery		% Error	R.S.D n =3
Taken	Found
5	5.06	101.12	Mean = 100.38 S.D. = 0.710 R.S.D. 0.707	1.12	0.11
25	24.98	99.92		-0.08	0.13
45	45.01	100.02		0.02	0.09
CAF µg/Ml		% Recovery
Taken	Taken			%Error	R.S.D n =3
5	4.95	99.00	Mean = 99.65 SD = 0.570 R.S.D. 0.572	- 1.00	0.20
25	24.98	99.92		-0.08	0.14
45	45.02	100.04		0.04	0.21
PCM µg/Ml		% Recovery
Taken	Taken			%Error	R.S.D n =3
5	4.97	99.40	Mean= 99.86 S.D. = 0.41 R.S.D. 0.411	- 0.60	0.12
25	25.01	100.04		0.04	0.08
45	45.07	100.15		0.15	0.11

 

Specificity:

Specifically, is the analyte ability to unequivocally assays in the presence of  other components. Which, may be expected to be present. These components might include degrades, impurities, etc. The placebo solution of eluent phase was injected. The obtained chromatogram revealed there is no inferring peaks at the drug retention time. The obtained placebo chromatogram was compared with those obtained from the ASP standard solution. The correlation (in terms of t_R and area) was good, which indicate the specificity of the method. The specificity Chromatograms and for the standard ASP were shown in (Fig. 4, a and b).

 

Fig: 4: (a) specifity chromatogram for blank placebo in H₂O: MOH: CAN (60: 20: 20 V/V) and (b) standard ASP (50 µg/ Ml).

 

Ruggedness and Robustness:

The proposed method ruggedness was carried out by analysis of aliquots of sample  solution (15μg/Ml PCM) by two analysts using same operational and environmental conditions. The method robustness were evaluated by changing the rate of flow by ± 0.1Ml/min. (0.6 Ml/min and 0.4 Ml/min), changing the Ph by ±0.2 (4.2 and 3.8) for eluent phase and the wavelength detection changing by ± 2nm (256nm and 252nm). The results obtained are shown in Table 4.

 

Table: 4: The ruggedness and robustness results of the proposed method.

Ruggedness results
	Analyst 1			Analyst 2
Mean % Assay* ± SD	98.97 ± 0.19			99.61 ± 0.11
% R.S.D.	0.192			0.110
Robustness results
Method Robustness Parameters		Mean*	S.D.		%R.S.D.
Flow rate change  0.5 ± 0.1 Ml/min.		99.83	0.37		0.371
Mobile  phase Ph change  4.0 ± 0.2		100.15	0.35		0.349
Detection wavelength change  254 ± 2 nm		99.45	0.41		0.412

* n = 3

 

 

Analytical assays:

Four formulated samples were analysed for ASP, CAF, and PCM uses a validated high-performance liquid chromatography (HPLC) method with UV detection at 254nm. A 10μL of sample were injected to HPLC analysis under the optimum separation conditions. Eluent phase H₂O: MOH: ACN (60: 20: 20 V/V) was delivered at a flow rate of 0.5mL/min with UV detection at 270nm. The column was Phenomenex C-18 (15cm × 0.46cm I.D) and 5µm particle size. Analysis was performed at room temperature (~25^oC) and the total run time was 8 min. The results obtained are tabulated in Table 5. Figures 5, A and B were shown the separation chromatograms of the drugs in different formulating samples. The recoveries of drugs in samples were between 99.16 – 100.20%.

 

Fig: 5 A: Separation chromatogram of drugs in different formulating samples. 

 

 

Fig: 5 B: Separation chromatogram of drugs mixture in (Excedrin) formulating sample.

Table: 5: Estimated quantity of drugs in different formulating samples

Name and Company	Drugs type Contain	Claim Label Amount mg/ tab.	Found Mean Amount mg/tab.	% Found Mean Amount	R.S.D n = 3
Metanol MVC	PCM	500	500.88	100.18	0.32
Aspirin Avenzop Syria	ASP	81	80.32	99.16	0.33
Panadol Irland	PCM CAF	500 25	499.77 24.94	99.95 99.76	0.15 0.29
Excedrin Syria	PCM CAF ASP	250 65 250	250.50 64.68 249.31	100.20 99.51 99.72	0.22 0.14 0.26

 

CONCLUSION:

The RP – HPLC validated methods appoint here steady to be accurate, fast, simple, robust, and precise, so it can used in the routine analysis of ASP, CAF and PCM as standard and in formulating form.

 

ACKNOWLEDGEMENT:

The Authors wish to swift his grace to the Diyala University, College of education for pure science, Chemistry department for providing the facilities of research and lab for the completion of this work.

 

CONTRIBUTIONS OF AUTHORS:

The authors are all have equally contributed

 

INTERESTS CONFLICT:

Pronounced none

 

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Accepted on 13.02.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(1):215-220.