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Indian Journal of Chemical Technology Vol. 10, November 2003, pp. 603-606
Articles
Determination of acetylsalicylic acid and related substances in pharmaceutical preparations and bulk drugs by capillary electrophoresis
R Sekar*, P Ravi Prasad & M Vairamani
Analytical Chemistry Di vision, Indian Institute of Chemical Technology, Hyderabad 500 007 , India
Received 28 August 2002; revised received I 5 April 2003; accepted I I June 2003
Micellar electrokinetic chromatography (MEKC) using sodium dodecyl sulphate (SDS) in separation buffer has been employed for the separation and determination of acetylsalicylic acid and related compounds in tablets and bulk drugs. Base line separation was achieved by using 12.5 mM sodiumtetraborate decahydrate, 15 mM boric acid (pH 9.0) and 50 mM SDS. Analytical characteristics such as migration time, relative migration time and relative response factor were determined. Acetaminophen was used as internal standard to correct and compensate errors due to injection and evaporation losses. Drug content in formulations and bulk drugs was determined and the recovery was between 98.6 and 101.5%. All the preparations and bulk drugs examined were found to contain the hydrolyzed and starting material of salicylic acid, in varying amounts.
Acetylsalicylic acid (Aspirin, AS), is an analgesic and antipyretic drug. It is also used for initial treatment of cardiac vascular disorders such as angina pectoris and myocardial infraction 1• AS is prepared from salicylic acid (SA) by heating with acetic anhydrate. Salicylic acid is synthesized either by Kolbe-Schemitt process of phenolate carboxylation and rearrangement2 or from benzoic acid. A small quantity of salicylic acid (SA), benzoic acid (BA), phenol (PL) and phenyl salicylate (PS) present as an impurity in the raw material and pharmaceutical formulations may cause hypersensitive, corrosive, nausea vomiting, sweating and diarrhea. Acetylsalicylic acid itself has a relatively high risk of inducing poisoning following improper use, especially in young children3
. Many methods have been described in literature for the determination of ASA and SA in biological fluids and pharmaceutical preparations using liquid chromatography and gas chromatography4
-7
.
Capillary Electrophoresis (CE) is extensively used as a separation technique in the pharmaceutical industrl·9. CE methods have been developed not on ly to determine the drug content in pharmaceuticals but also for the separation and determination of drugrelated impurities of various nature 10
. It is generall y considered as a complimentary or an alternative technique to liquid chromatography for analys is of pharmaceuticals. Further, it provides many advantages,
*For correspondence (E-mail: sekar@iict.ap.nic.in)
such as high efficiency, speed of analysis, ease of automation, reduced overall costs, minimal environmental impact and small sample volume 11
-13
• Therefore, CE has been chosen as the method of choice for the present study.
A few methods have been reported for the determination of aspirin and salicylic acid in pharmaceutical and biological fluids using CE technique. Boonerd et al. 14 reported a CE method for the simultaneous determination of AS, SA in a multicomponent analgesic tablet formulations. However, none of those methods have been attempted for the separation of the above mentioned impurities in bulk drugs and formulations. Micellar electro-kinetic chromatography (MEKC) is a special mode of CE separation, based on the partitioning of the analytes between the buffer solution and a pseudo-phase and has been widely employed for separating analytes which are difficult to separate by capillary zone electrophoresis (CZE).
The aim of present work is to develop an analytical method that allows simultaneous detection and quantification of aspirin and related compounds.
Experimental Procedure
Materials and methods All reagents were of analytical grade. Benzoic acid,
phenol , aceton itrile and sodium hydroxide were purchased from S. D. Fine Chern. Ltd (Mumbai , India). Acetoaminophen, acety lsalicylic acid were obtained
Articles
from Sigma (St. Louis , MO, USA) . Boric acid and sodium tetraborate were supplied from Aldrich (St. Louis, MO, USA). Bulk drugs and tablets were obtained from local firms.
A Prince Technologies CE system (Model 460, The Netherlands) equipped with Lambda 1010 UV-Yis detector and Dax software was used. An uncoated fused silica capillary with 75 11m inner diameter (ID) and 375 11m outer diameter (OD) with an effective length of 95 em (l 06 em total length) was employed. Capillary temperature was kept constant at 25 °C and UV detection was performed at 208 nm. Samples were introduced hydrodynamically by applying a pressure of 50 mbar for 6 s. A voltage of +15 kV was applied during analysis.
A new capillary was conditioned by rinsing with 1.0 M NaOH for 20 min followed by water for I 0 min. Before each injection, the capillary was conditioned with 0.1 M NaOH for 2 min followed by run buffer for 3 min. The run buffer was replaced for every I 0 injections.
Preparation of standard solutions Stock solution of acetylsalicylic acid, internal stan
dard and related compounds ( 1.0 mg/mL) were prepared in acetonitrile. The working standards were prepared by diluting the stock solution with water at the time of analysis.
Tablets Five aspirin tablets were powdered and homoge
nized. A portion of the powder equivalent to 20 mg of aspirin was taken in a 20 mL calibrated glass tube. 10 mL of acetonitrile was added and shaken for 3 min and made up to the required volume with the same solvent. An appropriate proportion of sample and IS were mixed and diluted with water at the time of analysis. The optimum concentration should be around 0.5 mg/mL of each. Standard solutions were stored in a refrigerator at 4°C when it is not used.
Separation buffer Background electrolyte was prepared by mixing
12.5 mM sodiumtetraborate decahydrate, 15 mM boric acid (pH 9.0) and 50 mM sodium dodecyl sulphate (SDS) in de-ionized water. All the solutions and buffers were filtered through 0.45 11m membrane filter.
Results and Discussion All the compounds studied in this work were pre
pared in acetonitrile and further I part of solution was diluted with 20 parts of water at the time of analysis. This hydro-organic mixture increases the solubility
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Indian J. Chem. Techno!. , November 2003
and avoids evaporation. The limits of detection are generally poorer in CE, compared to those obtained in HPLC. This is due to small volume of sample loading (approximately 5-8 nL) and narrow path length (75 11m). However, it is possible to use lower wavelength in CE detection as 190 nm, where many compounds have strong UV absorption.
UY spectrum of acetylsalicylic acid and related compounds were recorded in running buffer solution. All the compounds have sufficient UV absorption at wavelength 208 nm. Thus, a wavelength of 208 nm is chosen for analysis of all diluted compounds.
Factors that affect separation in CE are pH of running buffer, ionic strength, surfactant and applied voltage. The running buffer consists of sodiumtetraborate and boric acid of different strengths ( 10 to 20 mM) and pH range of 8 to 10. Even at varying buffer composition and pH, all the compounds could not be separated. The PS is not eluted within 25 min. Therefore, the effect of surfactant is investigated for further studies on separation of all the compounds. An anion or cation surfactant above its critical micellar concentration (cmc) is normally used for the separation of the components, which are not resolved by CZE (earlier mentioned buffer without surfactant). In the present study SDS micelles have been used. These micelles have a hydrophobic interior and hydrophilic exterior, which allows partitioning of analyte on the basis of hydrophobicit/ 5
. Different concentrations of SDS (25 to 50 mM) are mixed to a buffer of 12.5 mM sodiumtetraborate and 15 mM boric acid of pH 9.0. The pH of a micellar system affects the ionization of a solute as well as its retention in the capillary . Usually a pH below 7 is not applicable for MEKC because of the unfavourable partition of solute in the micellar phase due to this poor resolution. A micellar system consisting of 50 mM SDS is sufficient to separate all the analytes. The pKa values of ASA and SA are 3.5 and 3.0 respectively which ionizes 14 completely at the running buffer pH of 9.0.
Synthetic mixture containing known amount of authentic AS and related compounds along with IS are analysed under the conditions mentioned in Fig.1. The electropherogram (Fig. I) shows the separation of all the compounds in the optimized MEKC system. The analytes get separated due to the combination of both electrophoretic mobility and differential interaction of analyte with negatively charged SDS .
In CE, internal standard is preferable to correct and compensate errors due to injection voiume and evapo-
Sekar eta/.: Determination of acetylsalicylic acid in bulk drugs by capillary electrophoresis Articles
Table !-Analyt ical characteristics of aspirin and related compounds
Compound+ Migration time Relative Relative Range Linear (min) migration time response factor (~-tg/mL) coefficient (R2
)
AP (IS) 11.86 1.00 1.00
PL 12.90 1.09 0.92 1.2-12 0.9986
AS 14.13 1.19 0.87 30-150 0.9995
BA 16.47 1.39 1.30 1.5-15 0.9998
SA 17.28 1.45 2.15 0.8-8.0 0.9996
PS 21.60 1.78 1.12 2.0-20.0 0.9975
+ = AP (IS), acetaminophen (lS); PL, phenol ; AS, acetylsalicylic acid; BA, benzoic acid; SA, salicylic acid; PS , phenylsalicylate.
Table 2-Assay of aspirin in tablets and bulk drugs
Sample Label claims Taken Found Recovery RSD (%)
Tablet
Bulk drugs
0.010-
-
~ c: 0.005-co € 0 VJ .0 < -
o.ooo-
-
I
0
(mg)
A 50
B 50
c 350
A
B
c
SA
AS
IS
Pl BA
I I
Migration time (min)
I 20
Fi g. I-CE separation of aspirin and related compounds. Synthetic mixture of five compounds. Cond iti ons: 12.5 mM sodiumtetraborate decahydrate, 15 mM boric ac id (pH 9.0) and 50 mM sodium dodecy l sulphate (S DS ); capillary. LOS em x 50 ~-tm ID (9 1 em to detector) ; detecti on wavelength 208 nm
(I! g)
60.5
30.8
75.2
40.2
55 .8
69.4
(I! g) (%) (n=5)
61.40 101.5 2.05
30.43 98.8 1.87
74.14 98.6 2.08
39.99 99.5 1.98
55.91 100.2 1.85
69.26 99.8 1.66
ration losses of solvents. Several compounds are screened and finally acetoaminophen is selected, because of its migration time being close to those of analytes and its easy availability in pure form. In addition, its response factor (1.0) being close to that of acetylsalicylic acid (1.19), at the detection wavelength of 208 nm.
Upon optimization of the above-referenced separation conditions, analytical performance of the method is also investigated. The peaks were identified by injecting authentic standard sample. The migration behaviour of the solute depends on several properties, including hydrophobicity and degree of dissociation in the soluti on. PS gave longer migration time, presumably owing to its higher hy~rophobicity. The parameters such as migration time (MT ), relative migration time (RMT), and relative response factor (RRF) and peak area in relation to concentration are examined. The linear regression coefficient (R2
) of aspiri n and related compounds are found to be~ 0.99. Results
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Articles Indian J. Chem. Technol., November 2003
Table 3-Analysis of impurities in different sources of aspirin table ts and bulk drugs
Sample* Tablets(%) Bulk drugs(%)
A B c A B c BA NO NO NO NO NO NO
PL NO NO NO NO NO NO
SA 0.7 1.0 1.2 0.8 1.5 0.6
UK 0.2 0.8 0.4
ND= Not detectable; UK= Unknown impurity (RMT 1.49) represented in relative percentage. *For expansion refer Table I.
0.04- AS
-
-
Q)
'-' -c "' ~ 0.02- IS
"' .0 ~ -
-
- SA BA I
I PL \ J 0.00 L_r--~ ~
I 0 Migration time (min) 20
Fig. 2-CE assay of aspirin bulk drug
are given in Tablel. The detector response for SA is two-fold higher as compared to AS.
Using this method, quantification of the active ingredient in pharmaceutical formulations and bulk drugs has been carried out. Results are summarized in Table 2. From the table it is observed that the recovery of aspirin in tablets (98.6-10 1.5%) and bulk drugs (99.5-100.2%) are found to be in good agreement with a set of standard limits of 95-105%. During the electrophoresis, excipients used for the preparations of tablets are not interfered with the substances examined.
The present work is primarily developed for the analysis of aspirin, but can also be used for the detection and determination of some starting materials and hydrolysis products. The limit of detection of all the impurities are determined by MEKC on series of diluted standard solutions and is found to be in the range of 0.6- 1.8 ).!g/mL. Three tablets and bulk drugs are analysed for the detection and determination of impurities. All the preparations and bulk drugs examined are found to contain, in varying amounts, the hydrolyzed and starting material of SA. An unknown
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impurity is present in most of samples with RMT 1.49. The percentage of impurities detected in tablets and bulk drugs are tabulated in Table 3. None of the tablet formulations and bulk drugs examined is found to contain BA and PL. Fig . 2 shows the electropherogram of aspirin bulk drug spiked with 0.8 and 0.5 % BA and PL, respectively.
Thus, it can be inferred that the proposed CE method is simple, fast and economical and there is no need for extensive sample preparations.
Acknowledgement Authors are grateful to Dr K Y Raghavan, Director,
Indian Institute of Chemical Technology, Hyderabad, for his encouragement.
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