The pharmaceutical activity of a drug formulation depends upon the chemical characteristics of drug molecules, thus any small variation in chemical properties and quantitative composition may lead to considerable variation in therapeutic effects. Aspirin, an antiinflammatory drug, produces analgesia both peripheral and CNS effects and inhibits platelet aggregation by irreversible inhibition of platelet cyclooxygenase, inhibits the generation of thrombooxygenase A2, relieves headaches, neuralgia, rheumatism, a powerful inducer of platelet aggregation and vascoconstriction.

Aspirin overdose has potentially serious consequences leading to significant morbidity and death. Patients with mild intoxication frequently have nausea and vomiting, abdominal pain, lethargy, ringing in the ears, and dizziness. More significant signs and symptoms occur in more severe poisonings and includes high body temperature, fast breathing rate, respiratory alkalosis, metabolic acidosis, low blood potassium, low blood glucose, confusion, seizure cerebral edema, hallucinations, and coma. The most common cause of death following an aspirin overdose is cardiopulmonary arrest usually due to pulmonary edema [1]. A proper assay and quantification technique to ascertain aspirin content in a multi-component pharmaceutical dosage and its stability is indeed necessary, to avoid overdose, vital and appreciated in order to avoid overdose.

Several methods have been put forward for determination of aspirin content as an individual or combined dosage in commercial tablets using various techniques as described [2-7]. 6pecific analytical methods for determining aspirin in multi-component drug based on liquid chromatographic techniques has also been reported [8]. More rapid and specific methods have also been developed based on hyphenated chromatographic techniques [9,10]. and UV spectrophotometric flow through multi-parameter sensor [11]. Stability studies using HPTLC method for determination of aspirin and clopidogrel bisulphate in combine dosage form has been presented [12,13]. but have not taken into consideration effects of acidic excipients on the final assay (Figure 1).

Figure 1: Structure of Aspirin: (Physical State-White crystalline powder; Nature-Weakly acidic; Molecular formula-C9H804; Molecular mass-180.157 g/mol; Melting point-135°C; Boiling point-140°C).

Titrimetry or wet analytical classical techniques and UV spectrophotometric analyzes are oіen preferred for quality control testing of aspirin in most pharmaceutical and chemical laboratories due to their broad availability, easy to operate and cost effectiveness [4]. Most of these pharmaceutical analytical techniques targets mainly the active ingredient but does not give much attention to effects of the drug constituents that plays other roles which facilitates drug metabolism activities and stability of active substance(s) within the drug formulation. Нis research compares techniques used in analyzing and quantifying aspirin in various pharmaceutical analgesic formulations and concludes on the best method that can be relied upon to give good resolution and best results within the accepted pharmacopeia limits.

Instruments and reagents used for the study

Glass ware for titrimetry analysis, UV-VIS Double beam Shimadzu 1800 (tra-0% to 400%), FT-IR 8101 M Shimadzu Spectrophotometer (4600–400 cm^-1) HPLC Knauer [auto sampler 3900 comp., column LI C18 (ODS), sonycate/karlkotl vibrator, UV-VIS detector]. Reagents used in HPLC are HPLC grade, while for titrimetry are analytical grade (AR) and spectroscopic grade in case of UV –VIS spectroscopic Double beam machine.

Drug samples

The six pharmaceutically formulated drug samples containing aspirin for this study were collected from various pharmaceutical shops within the market are reported in the Table 1.

Table 1: Drug samples containing aspirin for this study.

Preparation of pure Aspirin

Pure aspirin used for quantitative assay was prepared, purified, dried and characterized [14].

Preparation of pure Aspirin stock solution for UV-VIS spectroscopy

50 mg of well dried (2 h, at 105°C, cooled in vacuum desiccators) sample of pure aspirin weighed, transferred into 100 ml volumetric flask, added 70 ml of 0.1 N of NaOH, heated on water bath for 10 min, cooled to room temperature, filtered (Whatmann filter paper No. 41) and the filtrate further diluted to 100 ml using the 0.1 N to give a concentration of 50 mg/100 ml.

Preparation of Aspirin drugs samples working stock solutions for titrimetric analysis

Ten tablets of each drug were powdered and dried in an oven for a half an hour, an equivalent amount of 50 mg aspirin from the dried sample accurately weighed into a 100-ml volumetric flask diluted with standardized NaOH (0.093 N), heated on a water bath for 10 min, cooled to room temperature, filtered, the filtrate further diluted to 100 ml using 0.093 N NaOH.

Preparation of HPLC pure aspirin standard stock solution

25 mg of pure aspirin weighed, added into a 25-ml volumetric flask containing diluent (HPLC solvent mixture of acetonitrile and formic acid [99:1]), volume made to the mark using the diluents to give a stock solution of concentration 1 mg/ml.

Preparation of HPLC sample solutions

20 tablets of each drug powdered separately, dried for 2 h at 105°C, cooled in a dessicator, quantity equivalent to 50 mg of aspirin transferred into 100 ml volumetric flask added the diluents, sonicated and volume made to the mark using the diluents.

Qualitative Analysis of the Pure Aspirin Synthesized

The pure Aspirin prepared was characterized qualitatively by physical constants, ferric chloride test, esterification and IR spectroscopic studies.

IR spectral studies

The spectra of the pure drug as KBR pellets in the range 4600–400 cm^-1 shown below (Figure 2).

Figure 2: The spectra of the pure drug as KBR pellets in the range 4600–400 cm^-1.

The 3500–2500 cm^-1 represents carboxylic acid –OH str, the peak at 1687 shows the characteristic carbonyl of carboxylic acids. The peaks at 1577 and 1581 are aromatic carbon double bond, 1300 cm^-1 represent C-O str, 1380 cm^-1 represents CH₃ sym bending, peak at 755 cm^-1 represents. ortho substituted aromatic ring C-H.

The peak at 1754 cm^-1 represents carbonyl str. of the ester. Assignment of the peaks made on consultations with the reference from IR data base confirms Aspirin.

Ferric chloride test

0.5 gm of pure Aspirin taken into 10 ml test tube, added 10 ml of 5N NaOH, boiled for 3 min, cooled, added 10 ml of sulphuric acid, a white precipitate obtained, filtered and dissolved in cold distilled water and added 1 ml ferric chloride solution gave a deep violet coloration confirming the presence of aspirin.

Esterification of Aspirin

0.5 gm of pure Aspirin taken into 10 ml test tube, added 10 ml of 5N NaOH, boiled for 3 min, cooled, added 10 ml of sulphuric acid, a white precipitate obtained, filtered., to the filtrate added 3 ml of alcohol and 3 ml of sulphuric acid, then warmed. The odor of ethyl acetate perceptible from the reaction confirms the presence of aspirin.

Physical parameter

Conformity test: A white crystalline powder, weakly acidic, m.p. 140°C, molecular mass C₉H₈O₄, exp λ_max= 297.4 nm at E^1%1cm 24.35 and λ_max= 220.6 nm at E^1%1cm 47.6. For this study, λ_max= 297.4 nm at E^1%1cm 24.35 with absorbance at 0.487 taken since it is close to the reported value of λ_max= 296 nm at E^1%1cm 25.

Identification of Aspirin from the drug samples

Ferric chloride tests and Esterification test [2] were conducted on the drug samples and all gave positive results confirming the presence of Aspirin in each drug.

Procedures for Quantitative Determination of Aspirin

Titrimetric assay

The excess alkali in each drug stock solution was titrated with 0.055N HCl using phenolphthalein as the indicator.

UV spectroscopic assay

0.1 ml, 0.2 ml, 0.9 ml was pipette out from the pure aspirin stock solution into 10 ml volumetric flasks and diluted to the mark using 0.1 N NaOH. Absorbance of each solution noted at λ_max=297.4 nm. This procedure was repeated three times and average results taken to plot a calibration curve.

0.4 ml of each drug stock solution was pipette into 10 ml volumetric flask and diluted to the mark using 0.1 N NaOH and absorbance was recorded at λ_max=297.4 nm to estimate the concentration for each. Microsoft excel was used to tabulate the measurement data and perform the linear regression analysis.

HPLC assay

From the HPLC pure aspirin solution, different volumes (0 ml, 1 ml, 2 ml, 6 ml) were taken into different 10 ml volumetric flasks and diluted to 10 ml with the diluents and filtered through a 0.45 µm membrane filter then each dilution injected into HPLC.

The flow rate time of 1.9 ml/min, running time of 7 min, injection volume 20 µl and detector wavelength 297.4 nm (UV-Visible detector. The mobile phase used was prepared by dissolving HPLC grade sodium heptanesulfonate 2 g in a mixture of acetonitrile 150 ml and DI water 850 ml, the pH adjusted to 3.4 using glacial acetic acid. The area under the peak noted.

Each of the drug samples is quantified using the same HPLC under the same conditions as above for pure aspirin and the area under the peaks recorded.

Titrimetric assay

The Table 2 below shows results of quantitative determination of aspirin from drug samples. The influence of citric acid also stated since it also consumes NaOH solution in the ratio of 3:1 stoichiometrically.

Table 2: Quantitative determination of aspirin from drug samples.

UV-VIS spectroscopic assay

The Table 3 and Figure 3 below shows the absorbance data collected at λ_max = 297.4 nm for the pure aspirin and its standard calibration curve. *Y_cal – mean absorbance, Y_corr – corrected mean absorbance*

Table 3: Absorbance data collected at λ 297.4 nm for the pure aspirin and its standard calibration curve. * Ycal – mean absorbance, Ycorr – corrected mean absorbance*.

Figure 3: The estimation of Aspirin from samples using calibration curve method.

Different quantities ranging from 15 mg to 35 mg of dried powdered sample of each drug weighed accurately on a watch glass, transferred into 100 ml volumetric flask added 0.1N NaOH to the mark, boiled on a water bath for 10 min, filtered, filtrate diluted to 100 ml using NaOH. 0.4 ml of each solution taken into 10 ml volumetric flask and diluted to the mark with 0.1 N NaOH, then absorbance recorded at λ_max= 297.4 nm. The results obtained are shown in the table 4 below.

Table 4: Estimation of Aspirin from samples(Weight of the samples taken: x gm; Concentration of Aspirin in the solution: Y mg/ml; Concentration of Asprin from absorbance: Z mg/ml; Percentage error:[(Y – Z)/Y] × 100).

Estimation based on HPLC instrumentation

Figure 4 below shows the chromatogram of 0.5 mg/ml of pure aspirin solution shown below, flow rate 1.9 ml/min.

Figure 4: Chromatogram of 0.5 mg/ml of pure aspirin solution.

The Table 5 below shows the results obtained using area under the peaks of dilutions from pure Aspirin stock solution at different concentrations.

Table 5: Area under the peaks of dilutions from pure Aspirin stock solution at different concentrations.

The calibration curve drawn below Figure 5 by plotting the concentration of aspirin versus the area under the peak (Aup).

Figure 5: Concentration of aspirin versus the area under the peak (Aup).

Each sample solution injected into HPLC machine and their area under the peaks which reflects aspirin content in the drug is elucidated and with the help of the calibration curve concentration of the aspirin content in the pharmaceutical formulations was determined. The general method used for calculating estimated weight, percentage error and recovery shown below Table 6:

Table 6: Estimated weight, percentage error and recovery.(Calculated weight of the sample (Wt): Wt : [(conc of the sample × 100)/50] × 100; Error % : [(Wt-Std. ntWt)/Std. ntWt] × 100; Recovery %: (Ws/StdntWt) × 100; *Stdnt. Refers to standard weight*).

From the data obtained, as shown in the table below, high errors in estimation was realized in titrimetric analysis than in either UV-VIS spectroscopic or HPLC techniques (Table 7).

Table 7: High errors in estimation was realized in titrimetric analysis than in either UV-VIS spectroscopic or HPLC techniques.

In drug formulations matrix, in addition to the active ingredients, there are also chemical additives such as binders, diluents, disintegrants, stabilizers, lubricants, coloring and flavoring agents, vehicles and bases which mainly helps in dissolution of the drug activity in our body mechanism and stability. These chemical additives, and other excipients, some of which have acidic properties interfere with estimation particularly while using a strong alkali like NaOH in quantification exercise. Their structural formulations and a wellbalanced equation with the estimating reagent (alkali e.g. NaOH, etc) is required for the stoichiometric calculations in order to counter check their interference or effects in estimating aspirin in-case of titrimetric assay. This is indeed a grueling exercise encompassed with mathematical manipulations and errors are unavoidable. Hence such methods involving neutralization processes in wet classical analytical techniques for estimations should be avoided and discouraged particularly in cases where there are excipients which are acidic in nature.

In case of UV-VIS spectroscopic technique, interference arises mainly when there are absorbing electrons from the drug matrix near the same region with the analyte resulting into significant errors like in the case of Disprin tablet which has carboxylic groups within the drug matrix. Such can be avoided, if realized, by isolating the active analyte and quantify, best done with a hyphenated technique or estimated with respect to a calibrated standard curve. With all hyphenated spectroscopic techniques, implication concept of multivariate calibration curve is still unavoidable.

HPLC techniques has proven to be the best according to the data obtain with very good recovery. The peak purity test of aspirin at the stress conditions has revealed that the method was stability indicating and specific. No other peaks at the retention time of aspirin were realized indicating that excipients used in formulations or even its degrading product, be it existing, do not interfere with its estimation. However, with all these techniques, excellent recovery can be achieved if assay is performed on placebo in triplicates before engaging in quantification exercise. MS/GC proves to be comparable with HPLC but rather laborious and time consuming due to extraction and isolation processes.

The study finds it necessary for manufacturers to indicate the nature of exicipients whether acidic or not, and should have quantitative analytical data of the drug matrix on the literature inserts.