INTRODUCTION:

Acetaminophen (CH₃CONHC₆H₄OH) commonly known as paracetamol is chemically called N-(4-hydroxyphenyl) ethanamide^1,2. This substance is a regularly-used painkiller; it is prescribed as well as being available without prescription for treating headache, fever and other minor aches and pains². In developing countries, communities with low access to functioning modern healthcare service often used paracetamol drugs for all kind of diseases. Paracetamol formulations were obtained from licensed offices as well as from illicit market³.

The availability of substandard drugs in a number of developing countries is largely reported^3-8. Unfortunately these reports are generally devoid of quantitative data to support these claims⁸. In West Africa, populations often utilized substandard or fake medicaments because they are more affordable^5-8. The last ten years, these products have been mostly detected in the international medicines market but there is a lack of precise figures of the global situation.

It has been estimated that more than 10% of the globally traded medicines are counterfeits^5,9,10. In developing countries, because of the weakness of the medicines’ quality control mechanism, substandard and fake pharmaceuticals’ illegal trade has become more attractive. Substandard and fake medicines are usually sold on the irregular market circuit but, sometimes they interfere in the licensed pharmacies^5,9,10.

Many health problems are associated with the counterfeit medicines^4,6,8,11. Substandard and fake products can kill: for example 233 Bangladeshi children died in 1992 after taking paracetamol-based syrup polluted by antifreeze⁸. Moreover, more than 2500 children in Niger were died in 1995 after taking a fake meningitis vaccination⁸. It is also demonstrated that among the one million deaths that occur from malaria in Sub-Saharan Africa, more than 200,000 would be avoided, if the medicines available were of good quality and used correctly^5,8,11.

Paracetamol is available as tablets of 500 mg active ingredient for clinical practice, it is present in more than 60 pharmaceutical formulations^1,2. The quality control of those products is of paramount importance, especially the dosage of their active ingredient rate. Reviewed of literature showed that chromatographic (GC and HPLC) coupled with UV/MS detectors and NMR are the most used methods for drugs quality control^12-14. But those techniques are costly, relatively time-consuming and demand more qualification. To overcome these difficulties, simple, accurate and reproducible spectrophotometric methods were developed for paracetamol active ingredient analysis in different drug formulations^15-18. In this study, we have first developed an UV/Visible spectrophotometric method for paracetamol active ingredient rapid control. That method was then used to control the quality (active ingredient content) of four licensed market paracetamol products and four others products obtained from illicit vendors. Affordable instruments and cheap solvent (water ant ethanol) were used in this work. To our knowledge, this quantitative study is the first conducted in one of the West African countries.

EXPERIMENTAL:

Apparatus and conditions:

All absorbance measurements were performed at room temperature with a double beam ThermoSpectronic Helios Alpha spectrophotometer. A standard Hellma (Mulheim, Germany) quartz cuvette (1-cm pathlength) and a micropipette pipetman of 10–100 μL (Gilson, France) were used. All analytical measurements were carried out under the same conditions.

Figure 1: Normalized absorption spectrum of paracetamol

Reagents and standard solutions

All reagents and solvents used were of analytical grade. The standard paracetamol (acetaminophen analytical standard) is from Sigma Aldrich. Distilled water and ethanol (+99.7%) purchased from Hang Zhou Gao Jing (China) were used as solvents. The organic solvent was preliminarily distilled with a Rotavapor-R (Buchi, Switzerland) before utilization. Paracetamol stock solutions (1 mg/mL) were prepared in pure distilled ethanol. The diluted working standard solutions were freshly prepared in distilled water-ethanol (v:v, 20:1) before analysis. The obtained solution is filtered through a Labsman L-1 11CM filter paper (India) in order to match the real sample pre-treatment conditions. The paracetamol residue on the filter paper was washed two times with 10 mL of distilled ethanol. Absorption spectra were recorded over the range from 190 to 350 nm against a blank. Paracetamol absorption curve presented two peaks located at 193 and 245 nm. The last wavelength (245 nm) corresponds to the height peak of the intense and broad absorption band between 215 - 300 nm; it was selected for the absorption signal measurements (Figure 1).

Calibration curve

Working standard diluted solutions were used to prepare the calibration curve. Serial dilutions between 2 to 50 µg/mL were prepared in distilled water-ethanol (v:v, 20:1). The standard working solutions were scanned between 200 and 350 nm and the absorbance signal was measured at the height absorption wavelength (245 nm). All absorbance signal measurements were carried out in triplicate and the results were expressed as mean values. The curves displaying the variation of the absorption signal maxima versus paracetamol concentration is shown in Figure 2 and the curve displaying the variation of the absorbance signal maxima versus the standard solutions concentrations is plotted (Figure 2 inset). Microcal origin version 6.0 software was used for the statistical treatments of the data.

Figure 2: Curves displaying the variation of absorption signal versus paracetamol concentration

The treatment of the data by linear regression analysis between 2 to 40 µg/mL gives satisfactory correlation (r²=0.999). Statistical results and limits of detection (LOD) and quantification (LOQ) of the used UV/Visible spectrophotometric method are summarized in Table 1.

Table 1: Analytical figures of merit for the analysis of paracetamol by UV/Visible spectrophotometry in distilled water-ethanol.

Parameters	Results
Absorption maximum (nm)^a	245
Linearity range (µg/mL)^b	2 – 40
Correlation coefficient (r²)^c	0.999
Molar absorptivity (L mol^-1 cm^-1)^d	11012.08
Accuracy (% recovery ± RSD)^e	(99,2 ± 0,5)%
Precision^f	99.5% (intra-day) 98.9% (inter-days)
LOD (µg/mL)^g	0.65
LOQ (µg/mL)^h	2.18
ALOD (µg)ⁱ	1.63

^aAbsorption wavelength chosen for the analysis; ^bConcentration linear dynamic range (LDR); ^cCorrelation coefficient; ^dMolar absorptivity mean value calculated with triplicate absorbance measures in the LDR (e); ^eRecovery rate and relative standard deviation (n=6) for 15 µg a.i/mL spiked solution; ^fPrecision of intra-day analysis (n=6) and inter-days (one week); ^gLimit of detection (LOD) was defined as the amount of analyte giving a signal-to-noise ratio of 3; ^hLimit of quantification (LOQ) was defined as the amount of analytes giving a signal-to-noise ratio of 10; ⁱAbsolute limit of detection (ALOD), calculated using 2.5 mL sample^19,20.

The method accuracy is very significant (> 99%) and the results relative standard deviation (%RSD, n = 6) is very weak (±0.5%). The intra-day and inter-days analysis results comparison have shown that the method precision is excellent (≥ 99%).The observed limits of detection(LOD) and quantification (LOQ) are respectively 0.65 µg/mL and 2.18 µg/mL which indicate the adequate sensitivity of the method. This obtained method will be suitable for the qualitative and quantitative analysis of paracetamol active ingredient in drug formulations.

Formulation samples solutions:

Drugs were sampled in August and September, which are known to be the months of most intense drug sale in the West African area⁵. Eight (8) samples of most used paracetamol commercial products were collected from different places in Niamey (Niger). The drug samples were purchased from private pharmacies (licensed market) and street vendors (illicit market) as that can be done by a normal customer. At each sampling site, the most used paracetamol drugs were checked by asking the vendor about the most sold specimens. All the eight drug samples are obtained without any medical prescription. Each drug sample was then labelled (P_{i=1 – 8}) and put into a plastic bag. The drugs were stored in a dark, dry and air conditioned place in the laboratory (23°C). Their indicated active ingredients (a.i) contents, weights (mean value of three tablets) and origins (licensed or illicit market) are presented in Table 2.

Table 2: Paracetamol samples characteristics

Sample name	Indicated a.i content	Tablet weight	Obtained from
P₁	500 mg	600 mg	licensed market
P₂	500 mg	588 mg	licensed market
P₃	500 mg	576 mg	licensed market
P₄	500 mg	540 mg	licensed market
P₅	500 mg	540 mg	illicit market
P₆	500 mg	550 mg	illicit market
P₇	500 mg	605 mg	illicit Market
P₈	500 mg	560 mg	illicit Market

For each drug, eight (8) tablets were weighted (m₀) and grounded in an agate mortar; the supposed active ingredient weight in the as obtained powder is 4 g. In order to obtain a stock sample solution with supposed 1 mg a.i/mL, the corresponding amount of tablet powder was accurately weighted and transferred to a 50 mL calibrated flask containing about 30 mL of pure distilled ethanol. The flask content was shaken until complete dissolution or until effervescence ceased in case of effervescent tablets. After this dissolution process, the solution is filtered through a Labsman L-1 11CM filter paper (India). The residue was washed two times with 10 mL of distilled ethanol. Then, the calibrated flask volume is completed to 50 mL with distilled ethanol. The obtained stock samples were protected from light with aluminium foil to avoid their active ingredient photodegradation before analysis. For the estimation of paracetamol concentration in each sample, various dilutions were performed. The absorbance signal measures were done on the diluted water-ethanol working solutions. The sample corresponding concentration was calculated by using the calibration curve.

RESULTS AND DISCUSSION:

Eight paracetamol drugs were analyzed in this study. Those drugs were obtained from illicit market (50%) and licensed market (50%). Among the eight drugs, P₁ (licensed market drug) and P₇ (illicit market drug) have the same indicated name, manufacturer and distributor. Drugs P₃ (licensed market) and P₅ (illicit market) have also the same name but the indicated manufacturer and distributor are different. The drugs measured active ingredient (a.i) contents, their claimed a.i and their real a.i percentage when compared to the indicated a.i, are summarized in Table 3. The a.i percentage is calculated by using the following equation: Percentage of real a.i = (measured a.i / indicated a.i) x 100.

Table 3: Commercial paracetamol drug samples characteristics

Sample name	Indicated a.i content	Measured a.i	Observed % of real a.i
P₁	500 mg	501 mg	100.2%
P₂	500 mg	323 mg	64,6%
P₃	500 mg	428.64 mg	85.7%
P₄	500 mg	487.59 mg	97.5%
P₅	500 mg	137.75 mg	27.6%
P₆	500 mg	108.5 mg	21.7%
P₇	500 mg	415 mg	83.0%
P₈	500 mg	3.2 mg	0.6%

According to the above results, all the four (4) paracetamol drugs obtained from irregular market have substandard concentrations when compared to the claimed content (500 mg a.i). Amid those products, P₈ is a fake drug; it contains only 0.6% of the claimed a.i content. Among the four products obtained from licensed market, sample P₁ (100.2% of the indicated a.i) is the best; P₄ (97.5% of the indicated a.i) is a relatively good drug; P₂ and P₃ have substandard concentrations, they contain respectively 64.6 and 85.7% of the indicated 500 mg a.i. The presence of substandard paracetamol drugs in licensed pharmacies is probably due to the infiltration of the official market by substandard and fake medicaments.

Comparing the results obtained from P₁ and P₇ (same indicated name, manufacturer and distributor), the relatively high paracetamol contents in the illicit market formulation (83%) shows that, that product is probably not a counterfeit. Indeed, real licensed drugs also infiltrate the illicit market; sometimes drugs are subtracted from public health centers and sold to the illicit market. P₇ active ingredient loss of 17% is probably due to the bad conservation conditions of this drug in the illicit market. Thus, counterfeiting is not the only reason of drug poor quality. The active ingredient decomposition under bad conservation conditions was occasionally the cause of drugs poor quality in least developed countries. According to their corresponding active ingredient contents, P₃ (85.7%) and P₅ (27.6%) are probably two counterfeit paracetamol drugs. The first, with a relatively high a.i contents, infiltrates the official market and the second, with only 27.6% of the indicated a.i contents remains in illicit market. The infiltration of fake and substandard medicines in the official circuit is generally due to the absence of regular quality control mechanisms.

CONCLUSION:

Paracetamol is one of the most used medicines in the world. This study showed us the important presence of substandard and fake commercial paracetamol drugs in developing countries. In the eight investigated products, five have a substandard concentrations and one is a fake drug. According to the availability of these drugs and their related potential health risks, it’s urged to strengthen drugs quality control in the region. Infiltration of counterfeit medicines in the official market circuit will annihilate West African countries health systems efforts if strong regulatory actions are not taken.

ACKNOWLEDGEMENTS:

The authors are very thankful to Pr. Atanasse COLY, Laboratoire de Photochimie et d’Analyse (LPA), Faculté des Sciences et Techniques, Université Cheikh Anta Diop, Dakar-Sénégal for its invaluable support. Authors are also greatful to Moussa Souley and Garba Nissa, Technicians at Département de Chimie, Faculté des Sciences et Techniques, Université Abdou Moumouni de Niamey-Niger, for their help during this study.

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Received on 18.07.2011 Modified on 06.08.2011

Asian J. Research Chem. 4(12): Dec., 2011; Page 1877-1880