Quality Assurance and USP Certification of Baby
Aspirin Tablets using HPLC with UV detection
Badrinath Dhakal
10/21/2009
Abstract
High performance liquid chromatography (HPLC) with ultraviolet (UV) detection was used to
quantitatively measure the amount of acetylsalicylic acid per tablet of the baby aspirin. The separation
was performed using Shimadzu LC instrument with SPD-M 20 A Detector. The column was a C18, 5 µm,
150 mm length and 4.6 mm diameter and the mobile phase was a mixture of 60% methanol, 36%
deionized water and 4% glacial acetic acid at a flow rate of 1.5 mL / minute at 238 nm wavelength. The
average mass of ASA was found to be 57.76 mg/tablet which is only a 71.31 % of the claimed value.
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1. Introduction:
Aspirin is the acetate ester of salicylic acid. It is a white powder with a
acidic taste. It is poorly soluble in water and somewhat better soluble in ethanol. Acetylsalicylic
acid (aspirin) is one of the most commonly prescribed medicines to treat a variety of pain-related
conditions, including rheumatic diseases and arthritis. It is often used as analgesic, antipyretic,
anti-platelet and anti-inflammatory (1). It can be used to reduce the risk of death and lessen the
damaging effects of an acute heart attack. It is also used to reduce the risk of heart attack and
strokes in the persons who have already had a heart attack. It is a drug that permanently destroys
the enzyme cyclo-oxygenase on contact. Cyclo-oxygenase is important for the body’s production
of prostaglandins, which is a chemical that signals an injury or triggers pain. The use of aspirin has
been shown to reduce the risk of Alzheimer’s disease [2, 3]. Aspirin is also effective in the
prevention and treatment of colorectal cancer [4].It has anticoagulant properties and helps to
prevent strokes and heart attacks.
The use of aspirin has been shown to increase the risk of gastrointestinal bleeding,
cause ulcerations, abdominal burning, pain, cramping, gastritis and liver toxicity. Large doses of
salicylate have been proposed to cause tinnitus.
As a part of quality assurance in pharmaceutical industries, the amount of active
ingredient in a product is supposed to be determined on a regular basis. According to the protocol
of the US Pharmacopeia (USP), any product regulated by the Food and Drug Administration (FDA)
must contain the amount of active ingredient listed on the product label. The present study aimed
to simulate a uniformity of dosage study of the baby aspirin, using HPLC protocols. First, the HPLC
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was calibrated using the standard solution of acetylsalicylic acid and a linear range of calibration
curve was known. Then the concentration of acetylsalicylic acid in each of the aspirin tablets was
calculated using the calibration curve.
There are various techniques for the determination of ASA, salicylic acid and their
metabolites. Gas-Liquid Chromatography can be used to determine the ASA and its metabolites up
to 1 µg/mL. GC methods are time –consuming and have been replaced by HPLC methods with
ultra-violet detection, electrochemical detection, or mass spectrometry detection (5).
Spectrophotometry is the earliest and most widely used method for measuring serum salicylate
levels. In this technique, colorimetric determination of the salicylate is carried out using the
intense red color of salicylate-Fe+3 complex. Similarly, the salicylate can be easily determined by
Trinder method. This method involves the measurement of absorbance of the ferric ion-salicylate
complex. This technique has a disadvantage that spontaneous (pH dependent) and enzymatic
hydrolysis of aspirin though this problem can be solved by appropriate sample processing (1). The
content of ASA in the mixture of ascorbic acid and ASA has been determined by LC method [6, 7].
Similarly ASA in combination with other active compounds was determined by differential
spectroscopy (8), spectrofluorimetry (9), HPLC (10) Potentiometry (11) and Voltametry (12). The
high performance liquid chromatography (HPLC) separation and subsequent identification of the
spots by appropriate standards is the most frequently used technology. This method has several
advantages like simplicity, reproducibility, high sensitivity (detection limit of about 100 ng/mL) and
the possibility of simultaneous determinations of several aspirin metabolites together with aspirin
itself in one sample.
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2.
Experimental
2.1.
Reagents and materials
Acetylsalicylic acid (ASA) MW = 180.16 gmol-1 was used as a standard. The baby aspirin
sample was obtained from the St. Joseph (81 mg per tablet). Ten aspirin tablets were dissolved
separately in the 50 ml volumetric flasks using the methanol-water diluent (64:36). Both
standard and sample solutions were filtered using the luer-lock syringe filter before injecting
into the HPLC. The HPLC used was Shimadzu LC20AT with SPD-M20 uv-vis diode array detection
with the C18 stationary phase, 5 micrometer packing, column (4.6 mm internal diameter and
150 mm long). The mobile phase was made from 60% methanol, 36% deionized water and 4%
glacial acetic acid. The wavelength was adjusted at 238 nm and the flow-rate was set to 1.50 mL
per minute.
2.2. Preparation of standard solution
A 49.70 mg sample of acetyl salicylic acid was accurately weighed out and dissolved with
the diluent solution in a 50 mL volumetric flask. Using this solution five solutions of different
concentrations were prepared following the dilution scheme shown in table 1. Five solutions of
aproximately 1.0 mg/100 mL, 2.0 mg/100mL, 5mg/100mL, 8.0 mg/100mL and 10.0 mg/100mL
concentration were prepared from the standard solution by appropriate dilution.
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Table 1. Dilution of Aspirin Standards
Exact concentration
Volume of standard 1
Total volume
9.940 mg/100mL
5.0 mL
50 mL
7.952 mg/100mL
4.0 mL
50 mL
4.970 mg/100 mL
2.5 mL
50 mL
1.988 mg/100mL
1.0 mL
50 mL
0.994 mg/100mL
0.5 mL
50 mL
2.3. Preparation of samples
Ten sample solutions were prepared by dissolving each baby aspirin tablet in each of the
ten 50-mL volumetric flasks with the methanol-deionized water (64:36) diluent. Then diluted
solutions of the samples were prepared by taking 2.0 mL of each solution in 25 mL volumetric
flasks and diluted with the diluent solution.
2.4. Instrumentation and analysis
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The injection of standard solution was started with the solution of lowest concentration. A
100µL aliquot of standard solution was injected into sample injection port and read the
chromatogram of it on the computer. One chromatogram was obtained for each of the
standard solutions.
Similarly, a chromatogram for each of the ten unknown samples was obtained. Before
running the unknown samples and between the subsequent injections of samples the HPLC was
calibrated with the approximately 5.0 mg/100mL standard solution. Ultimately, the retention
time and peak area for each of the unknown samples were obtained.
Result and discussion
Table 2. The peak areas and retention times of standard solutions
Concentration of standards
Retention time
Peak area
0.994 mg/100mL
1.472 min.
128489
1.988 mg/100mL
2.404 min.
489338
4.970 mg/100mL
2.392 min.
1003061
7.950 mg/100mL
2.392 min.
1354373
9.940 mg/100mL
2.388 min.
1341161
blank
1.128 min.
819324
Table 2 shows that how the peak areas and retention time vary with the concentration
of the standard solution. These data were used to draw a calibration curve (Fig1). The slope and
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intercept of the line were recorded. Using the value of the slope and intercept, the exact
concentration of each of the standard solutions were reevaluated. Similarly, the exact
concentration of each of the unknown sample of baby aspirin was calculated by using the
Equation (1).
Mg of ASA/tablet = Peak area of unknown (4.97mg ASA Std.) 25×50
……… (1)
100×Peak area of std. ×2
Calibration curve for aspirin
y = 145043x + 236072
R² = 0.9806
1600000
1400000
peak area
1200000
1000000
Series1
800000
Linear (Series1)
600000
400000
200000
0
0
2
4
6
8
10
concentration, mg/100 mL
Fig 1. Calibration curve for the aspirin.
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Table 3. The peak areas and retention times of unknown samples (Baby aspirin)
Samples (Baby aspirin)
Retention times
Peak areas
D1
2.392 min.
1722197
D2
2.40 min.
1701991
D3
2.404 min.
1800832
D4
2.408 min.
1745001
D5
2.404 min.
1842195
D6
2.396 min.
1800516
D7
2.404 min.
1767297
D8
2.404 min.
1556586
D9
2.408 min.
1800357
D9
2.392 min.
1814338
D10
2.400 min.
1715173
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Fig 2. Chromatogram of the std. 5.0 mg/100mL.
Fig 3.Chromatogram of unknown sample of baby aspirin (D1)
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Fig 3 represents the chromatogram of the unknown aspirin sample D1.The chromatogram
seems to be almost similar to that of the 5.0mg/100mL standard sample (fig.2) which indicates
that the unknown sample has the concentration very close to that of the standard one.
Table 4. Mass of ASA in the different samples of baby aspirin.
Unknown samples
Peak area
Mg of ASA /tablet
% claim
D1
1722197
56.80
70.12%
D2
1701991
56.13
69.30%
D3
1800832
59.39
73.32%
D4
1745001
57.55
71.05%
D5
1842195
60.75
75.01%
D6
1800516
59.38
73.31%
D7
1767297
58.28
71.96%
D8
1556586
51.34
63.38%
D9
1800357
59.38
73.30%
10
1715173
56.57
69.83%
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Table 5. Statistical parameters determined for the unknown samples.
Average
Standard deviation
RSD
Mass per tablet
57.76 mg
2.6033
4.5068
% claim
71.31
0.0321
4.5068
The calibration curve indicates that the linearity of the line is satisfactory. There
are two peaks in all the chromatograms despite we normally expect only one peak.
Table 4 shows the masses of aspirin samples calculated from the peak area of
absorption, slope and intercept from the calibration curve. Table 5 summarizes the
result of the experiment in some statistical terms. The average mass per tablet of the
baby aspirin was found to be 57.76 mg which is only 71.31 % of the claimed value.
There might be a certain relation between the observation of more than one peak in
chromatograms and low value of the results. Another peak in the chromatogram is
similar to that of the salicylic acid. So, the sample might have been decomposed into
salicylic acid to some extent and this resulted in the lower value of the result. When we
take peak area of both the peaks and made the calculation, then the result came much
closer to the claimed value.
.
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Conclusion
High performance liquid chromatography (HPLC) with ultra-violet detection can be
successfully employed to pass the uniformity of dosage for the quality assurance and USP
certification of baby aspirin tablets. The result obtained in this experiment is lower than the
expected one. The discrepancy between the experimentally obtained result and the theoretical
value can be attributed to the fact that the aspirin sample
underwent decomposition to
salicylic acid to some extent. From the chromatograms, it is observed that there are clearly
distinguishable two peaks unlike our expectation.
Though HPLC with UV detection is most frequently used technique for routine analysis,
it suffers from the disadvantage that the sample could undergo decomposition because of the
length of time needed to prepare separately all the samples and consequently yields less
accurate results. So, I would like to do the same kind of work with more appropriate sampling
in the future. If the extent of decomposition of the aspirin is found out by any means, then the
calculation would be made closer to the true value and it would certainly allow us to make a
better decision.
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Acknowledgement
I would like to thank Dr. Ruth Ann Armitage for her valuable guidance and direction in
carrying out this work.
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