Chromatographic Application on Calixarene Bonded
Stationary Phases: A Stability Indicating Method
for Simultaneous Determination of Paracetamol,
Caffeine and Acetylsalicylic Acid in Excedrin Tablets
2010, 71, 31–35
Hisham Abdel-Aziz Hashem&
Analytical Chemistry Department, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt; E-Mail: hisham413@yahoo.com
Received: 12 May 2009 / Revised: 21 September 2009 / Accepted: 6 October 2009
Online publication: 5 November 2009
Abstract
A simple, rapid and accurate, routine-LC method is described for simultaneous determination of paracetamol, caffeine and acetylsalicylic acid in a tablet formulation. This study
represents a new application for the calixarene stationary phases. The chromatographic
separation of the three pharmaceuticals was achieved on a Caltrex BIIE column
(250 9 4 mm, 5 lm) using a binary mobile phase of 14% ACN and 86% 50 mM
NaH2PO4 pH 3.0 at 1 mL min-1 flow rate. Detection was at 214 nm. Separation was
achieved in less than 15 min. The method was validated for system efficiency, linearity,
accuracy, precision, limit of detection and quantification, specificity, stability and robustness. The limits of detection were 4.88, 9.77 and 78.13 ng per 10 lL of their injected
volumes, respectively. The recovery values of this method were between 94.63 and 101.85
and the reproducibility was within 3.88. The method could also be used for separation and
determination of salicylic acid which is considered the most important degradation product
of acetylsalicylic acid.
Keywords
Column liquid chromatography
Calixarene bonded LC-stationary phases
Cold medicaments
Introduction
The advances in packing materials,
which differ somewhat from the usual
RP stationary phases, may be considered
as one of the main reasons causing
the recent rapid development in LC.
Original
DOI: 10.1365/s10337-009-1390-7
0009-5893/10/01
Calixarene is an example of these packing materials. Calixarenes are macrocyclic molecules composed of phenolic
units linked by alkylidene groups. They
belong to the class of [1n] cyclophanes.
They are able to produce reversible
complexes with metals and organic
molecules [1, 2]. Nowadays, calixarene
stationary phases have several applications in LC [3–12].
The use of paracetamol alone or in
combination with other drugs, such as
caffeine and aspirin as analgesic and
antipyretic is well established in the
pharmaceutical formulations. Excedrin
tablet is a cold medication formulated
through a combination of 250 mg paracetamol, 250 mg aspirin and 65 mg caffeine. There are a few papers describing
the determination of cold medicaments,
most of them reporting the determination
of a combination of only two pharmaceuticals of these three [13–18]. A reversed
phase LC method has been published for
analysis of caffeine, aspirin and salicylic
acid in combination [18]. However, that
method utilized samples prepared in
MeOH which is known to accelerate the
degradation of aspirin in solution [19, 20].
Although the LC-separations of
pharmaceuticals were extensively studied
on the calixarene columns in the last
years, to the best of my knowledge there
is no work describing the use of calixarene columns for quantitative determination of cold medications.
This article describes the development and validation of a stability indicating method for the LC-determination
of paracetamol, caffeine and acetylsalicylic acid in tablets on calixarene
column.
Chromatographia 2010, 71, January (No. 1/2)
Ó 2009 Vieweg+Teubner | GWV Fachverlage GmbH
31
Table 1. System performance for the examined cold medicaments (n = 5)
Compound
tR ± SD (min)
N
k
Rs
a
Paracetamol
Caffeine
Salicylic acid
Aspirin
5.40
7.02
10.03
13.09
4,709
9,184
8,705
9,196
0.94
1.53
2.61
3.71
6.01
8.33
6.27
1.63
1.71
1.42
±
±
±
±
0.05
0.06
0.09
0.11
Table 2. Linearity and calibration (n = 4)
Compound
Detection
limit
(ng/10 lL)
Quantitation
limit
(ng/10 lL)
Paracetamol
4.88
9.76
Caffeine
9.76
19.53
Salicylic
acid
Aspirin
39.06
78.12
78.12
156.25
Range of
calibration
(ng/10 lL)
19.53
10 9 103
39.06
5 9 103
156.25
5 9 103
312.50
10 9 103
:
:
:
:
Slope
RSD
(%)
Intercept
RSD
(%)
Regression
coefficient
1.127
0.92
3.012
0.63
2.516
1.20
1.139
2.70
73.562
2.01
56.104
1.77
54.033
2.28
-36.860
1.55
0.9996
0.9997
Paracetamol
20.00
5.00
Caffeine
10.00
2.50
Salicylic acid
10.00
2.50
Aspirin
20.00
5.00
a
b
Intra-day (n = 4–5)
0.9995
Inter-day (n = 5)
Observed
amount
(lg) ± SD
CV
(%)a
Accuracy
(%)b
Observed
amount
(lg) ± SD
CV
(%)a
Accuracy
(%)b
19.25 ± 0.18
5.01 ± 0.08
0.94
1.60
96.25
100.20
19.35 ± 0.75
5.11 ± 0.13
3.88
2.54
96.75
102.20
9.65 ± 0.10
2.51 ± 0.04
1.04
1.59
96.50
100.40
9.72 ± 0.31
2.55 ± 0.07
3.19
2.75
97.20
102.00
9.56 ± 0.15
2.59 ± 0.02
1.57
0.77
95.60
103.64
9.59 ± 0.27
2.56 ± 0.05
2.82
1.95
95.90
102.40
19.43 ± 0.26
5.06 ± 0.13
1.34
2.57
97.15
101.20
19.56 ± 0.73
5.17 ± 0.19
3.73
3.68
97.80
103.40
Coefficient of variation (%) = SD 9 100/mean
Accuracy (%) = observed concentration 9 100/used concentration
Experimental
Chemicals
Acetonitrile (ACN) was LC grade and
purchased from LGC Promochem (Wesel,
Germany). Water was deionized and
doubly distilled. Mobile phase components were degassed before use. Sodium
hydroxide, phosphoric acid and sodium
dihydrogen phosphate were purchased
from Merck (Darmstadt, Germany).
Excedrin tablets (containing 250, 250
and 65 mg of paracetamol, aspirin and
32
Column
CALTREX BIIE column (250 9 4 mm,
5 lm) was friendly supplied by Synaptec
(Greifswald, Germany). The calixarene
stationary phase contains silica-bonded
calix[6]arene (basic silica: Kromasil Si
100 Å pore diameter, 5 lm particles;
manufacturer: EKA Chemicals (Bohus,
Sweden).
0.9994
Table 3. Reproducibility and precision (n = 4–5)
Injected
amount (lg)
Samples were injected with an SIL-10A
auto injector (Shimadzu).
pH-meter:
Knick
Elektronische
Meßgeräte (Berlin, Germany).
caffeine, respectively) were obtained
from the Egyptian market (produced by
Bristol-Myers Squibb, Cairo, Egypt).
Paracetamol, aspirin, caffeine and
salicylic acid raw materials were
obtained from Sigma.
Equipments
The liquid chromatograph consisted of
an LC-10AT pump (Shimadzu, Kyoto,
Japan) and a CBM- 10A system controller (Shimadzu).
Chromatography
The experiments were performed with
isocratic elution. The binary mobile
phase consisted of 1 mL min-1 14%
ACN and 86% 50 mM NaH2PO4, pH
3.0. The eluents were degassed before
running. In most cases, the column
temperature was set at 40 °C. The holdup times (t0) were determined via 100%
MeOH injection under each mobile
phase composition of each run and the
first disturbance in the baseline was taken as dead time peak. 20 lL (in most
cases) of each analyte in ACN solution
were injected each run. 214 nm was the
wavelength for estimations.
Preparation of Stock and
Standard Working Solutions
100, 100, 50 and 50 mg of paracetamol,
acetyl salicylic acid, salicylic acid and
caffeine, respectively, were dissolved in
25 mL ACN. The standard working
solutions were prepared by diluting aliquots of the stock solution to obtain
concentrations ranging from 0.244 to
2000 lg mL-1 for paracetamol and
acetyl salicylic acid and 0.488 to
1000 lg mL-1 for salicylic acid and
caffeine. The calibration graph was
constructed by plotting the peak areas
obtained at wavelength 214 nm versus
the corresponding injected concentrations for each analyte. For the range of
linearity and linear equations see
Table 2.
Chromatographia 2010, 71, January (No. 1/2)
Original
Sample Preparation
20 Excedrin tablets (each ca. 676.5 mg
containing 250 mg paracetamol, 250 mg
aspirin and 65 mg caffeine) were accurately weighed and ground to fine powder. To an accurately weighed portion of
the powder (270.5 mg) 25 mL ACN were
added then the solution was left in the
ultrasonic bath for 15 min. After that
the solution was filtered and the first
10 mL were rejected and 2 mL of the
following filtrate were quantitatively
taken and serial dilutions performed.
Results and Discussion
System Suitability
When a sample containing the four
analytes was injected into the LC apparatus under the mentioned isocratic
conditions, the tR values of the symmetrical peaks of paracetamol, caffeine,
salicylic acid and aspirin were
5.40 ± 0.05, 7.02 ± 0.06, 10.03 ± 0.09
and 13.09 ± 0.11 min and k values were
0.94, 1.53, 2.61 and 3.71 respectively.
The reproducibility tests for these analytes were less than 1% of the retention
times. Chromatographic parameters
such as number of theoretical plates (N),
resolution (Rs), selectivity (a), and
retention factor (k) were determined
(Table 1). These results indicate that the
described method showed adequate
column efficiency, good selectivity and
repeatability and could be applied for
simultaneous determination of the four
analytes.
Linearity and Calibration
Assay of each analyte was linear to a
concentration of 5–10 9 103 ng per
10 lL injection. The slope, intercept and
regression coefficient for each compound
were estimated (Table 2). The limit of
detection for each compound, defined as
the injected quantity giving an S/N of 3
(in terms of peak area), were found to be
4.88, 9.76, 39.06 and 78.12 ng per 10 lL
injection, respectively.
Original
Table 4. Assay results of paracetamol, caffeine and aspirin in Excedrin tablets
Paracetamol
Composition
(mg/tablet)
Found (mg/tablet)
Recovery (%) ± CV (%) n = 5
250
236.58
94.63 ± 3.87
66.20
101.85 ± 3.45
239.50
95.80 ± 3.22
Caffeine
65
Aspirin
250
Table 5. Accuracy of the described method applying standard addition technique (n = 5)
Paracetamol
Caffeine
Aspirin
Recovery (%) + SD
CV (%)
92.02 + 2.33
98.72 + 3.20
93.55 + 2.17
2.53
3.24
2.32
Precision and Accuracy
Specificity of the Method
Intra-day precisions were assessed
injecting standard solution four to five
times during a day (this solution was
extracted via the same procedure as the
tablets) of each analyte at two different
concentrations (a low and a high concentration). The resultant coefficients of
variation (CV) were lower than 3% for
all (Table 3). Under the higher concentration condition salicylic acid gave the
highest CV (=1.57) while under the
lower concentration condition, aspirin
gave the highest CV (=2.57). Inter-day
precision experiments were done after
treatment of the standard solution in the
same method of tablets extraction, and
then analyzed every day over 5 days
(Table 3). All CV were lower than 4%.
For the higher concentration, paracetamol had the highest CV (=3.88) while
under the lower concentration condition,
aspirin had the highest CV (=3.68).
The same techniques of extraction
and quantitative determination using LC
were applied for Excedrin tablets obtained from the Egyptian market. The
results indicated that each tablet contains about 94.63, 101.85 and 95.80% of
the labeled concentrations of paracetamol, caffeine and aspirin respectively
(Table 4).
Accuracy data is listed in Table 5.
Figure 1 reveals that there is no interference from the excipients. 15 compounds, which can be used or are present
in the analytes of this study, were tested
for interference. Only four have interfered with the separation. These are
theophylline, theobromine, aminophylline and antipyrine (Table 6).
Robustness of the Method
The robustness of the present method
was evaluated in the terms of temperature, wavelength of detection and content of ACN in the mobile phase.
Also the effects of slight changes, in
flow rate of the mobile phase, buffer
concentration and in pH of the mobile
phase on tR values and peak area were
studied (Table 7).
The slight variations in these six factors had no significant effect on resolution and peak shape. The temperature
was changed by increments of 2 °C from
38 to 42 °C and the effect of these
changes on peak area was studied. The
effect of changes in the flow rate on k
and tR values of the analytes was studied
by changing the flow rate in steps of
0.1 mL from 0.9 to 1.1 mL. As different
contents of ACN 13, 14 and 15% were
Chromatographia 2010, 71, January (No. 1/2)
33
700
650
600
Absorption (mAU)
550
CAF
500
PAR
450
400
ASA
350
300
250
200
150
100
50
0
0
2
4
6
8
10
12
14
16
Time (min)
three different wavelengths: 212, 214 and
216 nm, to examine the robustness of the
method. Three different pH values are
applied to see if the results will change
through the change in pH value (2.80,
3.00 and 3.20).
The results in Table 7 indicate that
the small changes in the examined factors led to slight changes in the peak
area, tR values and/or k values. The
changes in flow rate and detection
wavelength have the least effects, while
the changes in pH value have the highest
effect on the analysis. This means the
method is more sensitive to changes
in pH value than to changes in other
factors (Table 7).
Fig. 1. Separation of the three active constituents in Excedrin tablets
Stability Test
Table 6. Compounds tested for interference with the assay
Not interfered
Interfered
Sodium benzoate
Ascorbic acid
Ibuprofen
Indomethazin
Methyl paraben
Acetic acid
Ketoprofen
Diclofenac sodium
Ambroxol hydrochloride
Terbutaline sulfate
Methionin
Theophylline
Theobromine
Aminophylline
Antipyrine
CAF
The stability of both standard and
sample solutions was determined by
monitoring the peak areas of the components of Excedrin tablets over a period of 14 days at room temperature in
different solvents. The results (Fig. 2)
indicate that the presence of methanol in
the solvent enhances the degradation of
aspirin to salicylic acid and 100% ACN
is the best (this is why the samples and
standards were prepared in 100%
ACN).
PAR
Absorption (mAU)
SA
700
650
600
550
500
450
400
350
300
250
200
150
100
50
0
0
Conclusion
ASA
50% H 2 O: 50% MeOH
50% H O: 50% ACN
2
100% MeOH
100% ACN
2
4
6
8
10
12
14
16
Time (min)
Fig. 2. Effect of the extracting solvent composition on the stability of analytes (after 14 days)
used to study the effect of changes in the
ACN content on peak area. Buffer concentration was increased from 50 to
34
60 mM and decreased from 50 to
40 mM and the effect on peak area and
tR estimated. Also analyses were done by
With the proposed method a satisfactory
separation of the analytes, extended linear range and a rapid analysis time is
carried out. The three components of
Excedrin tablets adding to salicylic acid
were separated in less than 14 min. A
high recovery of each analyte was
achieved using CALTREX BIIE column
for determination. Recoveries, which
indicate good agreement with analytes
amounts as stated at the label, ranged
from 94 to 101%. The proposed LC
method ensures a precise, accurate, sensitive and selective determination of
aspirin, paracetamol and caffeine in
tablets. No interferences from the
excipients were noticed.
Chromatographia 2010, 71, January (No. 1/2)
Original
Table 7. Robustness of the method
Slight
changes in
pH (2.8,
3.0 and 3.2)
CV of
Peak area
Paracetamol
Caffeine
Salicylic acid
Aspirin
3.06
2.52
1.64
0.17
Detection by
(212, 214 and
216 nm)
Temp.
(38, 40 and
42 °C)
Flow rate
(0.9, 1.0 and
1.1 mL min-1)
ACN %
(13, 14
and 15%)
Buffer conc.
(40, 50
and 60 mM)
tR
tR
Peak area
tR
k
Peak area
Peak area
tR
2.93
3.06
9.25
3.03
1.41
1.37
1.01
0.88
2.43
2.56
1.45
4.10
2.56
2.63
2.27
2.66
1.16
0.78
1.19
0.81
2.49
1.86
2.52
2.38
1.74
1.87
1.72
1.83
0.90
0.86
2.79
1.50
Acknowledgment
The author thanks Synaptec GmbH
(Greifswald, Germany) for supplying the
calixarene column.
5.
6.
7.
References
1. Gutsche CD, Muthukrishnan R (1978)
J Org Chem 43:4905–4906. doi:10.1021/
jo00419a052
2. Böhmer V, McKervey MA (1991) Chem
Unserer Zeit 25:195–207. doi:10.1002/
ciuz19910250405
3. Park JH, Lee YK, Cheong NY, Jang MD
(1993) Chromatographia 37:221–223. doi:
10.1007/BF02275865
4. Glennon JD, Horne E, O0 Connor K,
Kearney G, Harris SJ, McKervey MA
Original
8.
9.
10.
11.
(1994) Anal Proc Incl Anal Commun
31:33–35
Friebe S, Gebauer S, Krauss GJ, Goermar G, Krueger J (1995) J Chromatogr
Sci 33:281–284
Glennon JD, Horne E, Hall K, Cocker D,
Kuhn A, Harris SJ, McKervey MA
(1996) J Chromatogr A 731:47–55. doi:
10.1016/0021-9673(95)01080-7
Lee YK, Ryu YK, Ryu JW, Kim BE,
Park JH (1997) Chromatographia
46:507–510. doi:10.1007/BF02496369
Gebauer S, Friebe S, Gübitz G, Krauss
GJ (1998) J Chromatogr Sci 36:383–387
Gebauer S, Friebe S, Scherer G, Gübitz
G, Krauss GJ (1998) J Chromatogr Sci
36:388–394
Healy LO, McEnery MM, McCarthy
DG, Hariss SJ, Glennon JD (1998) Anal
Lett 31:1543–1551
Kalchenko OI, Lipkowski J, Kalchenko
VI, Vysotzky MA, Markovsky LN (1998)
J Chromatogr Sci 36:269–273
12. Menyes U, Haak A, Sokoließ T, Jira Th,
Roth U, Tröltzsch Ch (1999) GIT Spez
Sep 1:17–19
13. Koshy KT (1964) J Pharm Sci 53:1280–
1282. doi:10.1002/jps.2600531044
14. Das Gupta V (1980) J Pharm Sci 69:110–
113. doi:10.1002/jps.2600690136
15. Ou CN, Frawley VL (1982) Clin Chem
28:2157–2160
16. Ramos-Martos N, Aguirre-Gomez F,
Molina-Diaz A, Capitan-Vallvey LF
(2001) J AOAC Int 84:676–683
17. Burge LJ, Raches DW (2003) J Liq
Chromatogr Relat Technol 26:1977–1990.
doi:10.1081/JLC-120021765
18. Jalal IM, Sasa SI (1984) Talanta 31:1015–
1017. doi:10.1016/0039-9140(84)80238-6
19. Montgomery ER, Taylor S, Segretario J,
Engler E, Sebastian D (1996) J Pharm
Biomed Anal 15:73–82. doi:10.1016/
0731-7085(96)01813-4
20. Xu X, Stewart JT (2000) J Liq Chromatogr Relat Technol 23:769–779. doi:
10.1081/JLC-100101488
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