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

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.

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

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• 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

605

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

606

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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