Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 108 (2013) 20–25

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Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

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A rapid method for the simultaneous determination of L-ascorbic acidand acetylsalicylic acid in aspirin C effervescent tablet by ultraperformance liquid chromatography–tandem mass spectrometry

Saikh Mohammad Wabaidur, Zeid Abdullah Alothman, Mohammad Rizwan Khan ⇑Advanced Materials Research Chair, Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia

h i g h l i g h t s

" UPLC–MS/MS method wasdeveloped for analysis of L-ascorbicand acetylsalicylic acid.

" The method was applied to theaspirin C effervescent tablet.

" Method was reproducible andsuitable for quantitative analysis ofAA and ASA.

" Comparison of UPLC–MS/MSperformance with HPLC–PDA wasstudied.

1386-1425/$ - see front matter � 2013 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.saa.2013.01.070

⇑ Corresponding author. Tel.: +966 14674198; fax:E-mail addresses: swabaidur@ksu.edu.sa (S.M. Wa

g r a p h i c a l a b s t r a c t

UPLC–MS/MS chromatogram of studied compounds (a) L-ascorbic acid and (b) acetylsalicylic acid in aspi-rin C effervescent tablet.

a r t i c l e i n f o

Article history:Received 1 December 2012Received in revised form 21 January 2013Accepted 27 January 2013Available online 6 February 2013

Keywords:Aspirin C effervescent tabletL-ascorbic acidAcetylsalicylic acidUltra performance liquidchromatography–tandemmass spectrometry

a b s t r a c t

In present study, a rapid and sensitive method using ultra performance liquid chromatography–tandemmass spectrometry (UPLC–MS/MS) was developed for the simultaneous determination of L-ascorbic acidand acetylsalicylic acid in aspirin C effervescent tablet. The optimum chromatographic separation wascarried out on a reversed phase Waters� Acquity UPLC BEH C18 column (1.7 lm particle size,100 mm � 2.1 mm ID) with an isocratic elution profile and mobile phase consisting of 0.1% formic acidin water and acetonitrile (75:25, v/v, pH 3.5) at flow rate of 0.5 mL min�1. The influences of mobile phasecomposition, flow rate and pH on chromatographic resolution were investigated. The total chromato-graphic analysis time was as short as 2 min with excellent resolution. Detection and quantification ofthe target compounds were carried out with a triple quadrupole mass spectrometer using negative elec-trospray ionization (ESI) and multiple reaction monitoring (MRM) modes. The performance of themethod was evaluated and very low limits of detection less than 0.09 lg g�1, excellent coefficient corre-lation (r2 > 0.999) with liner range over a concentration range of 0.1–1.0 lg g�1 for both L-ascorbic acidand acetylsalicylic acid, and good intraday and interday precisions (relative standard deviations(R.S.D.) <3%), were obtained. Comparison of system performance with traditional liquid chromatogra-phy–photo diode array detector (HPLC–PDA) was made with respect to analysis time, sensitivity, linearityand precisions. The proposed UPLC–MS/MS method was found to be reproducible and appropriate forquantitative analysis of L-ascorbic acid and acetylsalicylic acid in aspirin C effervescent tablet.

� 2013 Elsevier B.V. All rights reserved.

ll rights reserved.

+966 14675992.baidur), mrkhan@ksu.edu.sa (M.R.

Khan).

Time (min)

1 20

1001.66

0.49

Rel

ativ

e ab

unda

nce

(%)

0

(a)

(b)

O

HOOH

H

H

H

H

OH

O OH

O C H

O O H

O

3

Fig. 1. UPLC–MS/MS chromatogram of 5 lL injected (a) L-ascorbic acid(0.5 lg mL�1) and (b) acetylsalicylic acid (0.5 lg mL�1) standards in MRM mode.

S.M. Wabaidur et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 108 (2013) 20–25 21

Introduction

L-ascorbic acid, [(5R)-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxy-furan-2(5H)-one] (AA), is a water-soluble vitamin, known as ananti-scorbutic food factor [1]. It is essential for the formation ofintercellular material, collagen, and for the healing of wounds[2]. It also facilitates to maintain the softness of the skin, helpthe absorption of iron and recovers resistance to infection. Dueto its anti-oxidative effect, it is also used in the treatment of scurvyand to prevents the occurrence and development of cancer [3,4].The deficiency of this vitamin may increase the level of oxidativestress [5], hemorrhage, anemia [6], and ecchymosis [7]. Fatigue,easy bruising, and bleeding gums are early signs of vitamin C defi-ciency that occur long before frank scurvy develops. Nevertheless,the overdoses of vitamin C may cause diarrhea and other gastroin-testinal disturbances [8]. Also the intake of large amounts of AAcan deplete copper in the body an essential element [9,10].

Acetylsalicylic acid, [2-acetoxybenzoic acid] (ASA) is a salicylatedrug which usually known as aspirin is often used as an analgesic,antipyretic and anti-inflammatory medication [11]. Once swal-lowed, ASA is rapidly hydrolyzed in the human body, which is pri-marily responsible for the pharmacological activity of thiscompound [12]. ASA has been shown to be effective in reducingcardiovascular morbidity and mortality in high-risk patients withmyocardial infarction or stroke [13]. The over dosage of aspirin in-clude dizziness, sweating, nausea, vomiting, altered glucosemetabolism, hyperventilation, respiratory alkalosis, metabolic aci-dosis, etc. [14]. Therefore, in order to achieve a better curative ef-fect and a lower toxicity, it is very important to rapidly controlthe content of ASA and its impurities in pharmaceuticalformulations.

On the other hand, a combination of AA and ASA have beenwidely employed in pharmaceutical formulations for pain and fe-ver relief including fever associated with colds and flu and mostlikely they are the major consumed drugs in the world [15,16].As a result, with the increase of the manufacture and consumptionof drugs that employ AA and ASA together, it becomes highly inter-esting to develop new analytical methods for their simultaneousdetermination. Several works have appeared in the literature forthe quantification of these drugs based on differential spectropho-tometry [17,18], spectrofluorimetry [19], voltametry [20], potenti-ometry [21], derivative spectrophotometry (DS) [22], ratio-spectraderivative and partial least squares methods (PLSs) [23] and liquidchromatography coupled with different detectors such as photodiode array detector (PDA) and mass spectrometry (MS) [18,24–27]. However, the familiar spectrophotometric methods have somewell-known limitations for the simultaneous determination ofthese drugs, such as, the spectral overlap of the main maxima[23]. The DS technique contains a variety of drawbacks includingfair resolution of mixture spectra due to the diminished peakamplitude with higher order derivation procedure. PLSs and otherchemometric methods need to use an abstract mathematical con-tent and it is not easy to apply and understand the calibration the-ory of these chemometric techniques. Voltammetry methods aresensitive but need long time for electrode cleaning and the appli-cation of high potential can be responsible for damage of the elec-trode surface. On the other hand HPLC–PDA/MS methods aresensitive but took longer analysis time.

Therefore, the aim of this work was to develop an analyticalmethod for simultaneous determination of these drugs with theadvantages of short time analysis, highly sensitive, accurate quan-tification and low sample injection volume. In this work a newlydeveloped method based on UPLC–MS/MS has been proposed toquantitate the amount of active components such as AA andASA in commercial pharmaceutical effervescent tablet formula-tions. The method is simple, rapid, precise, reproducible and

highly sensitive in addition with other advantage that the quan-titation may be accurately carried out without the use of an inter-nal standard.

Materials and methods

Reagents

L-ascorbic and acetylsalicylic acid were provided by Fluka (Buc-hs, Switzerland). Gradient-grade acetonitrile was purchased fromMerck (Darmstadt, Germany) and formic acid was supplied by Pan-reac (Bercelona, Spain). Water was purified through a Milli-Qwater purification system from Millipore (Bedford, MA, USA). Aspi-rin C effervescent tablets were obtained from Bayer pharmaceuti-cal company (AG, Germany), containing 240 mg AA and 400 mgASA per tablet. The chemical structures of the AA and ASA areshown in Fig. 1.

The individual stock solutions of AA and ASA standards(100 lg g�1) were prepared by dissolving in ultra-purified Milli-Qwater, and used for further dilution. The working standard solu-tions for linear calibration were prepared by diluting the stocksolutions to the concentration sequences of 0.1, 0.3, 0.5, 0.7,1.0 lg g�1. Both the stock and working solutions were freshly pre-pared and filtered before being injected into the UPLC–MS system.

Sample preparation

The procedure of sample preparation was according to our pre-viously reported work with some modifications [24]. Briefly, 10aspirin C effervescent tablets were weighed using analytical bal-ance and powdered in a mortar. A tablet amount was transferredto a 100 mL calibrated flask and dissolved with Milli-Q water andthen stirred and sonicated simultaneously until the effervescencetablet particles dissolved completely. The final solution was

22 S.M. Wabaidur et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 108 (2013) 20–25

diluted to the working range for application of the proposed meth-ods. After dilution, the samples were filtered through a 0.22 lmdisposable nylon filter (Bellefonte, PA, USA) into amber glass autosampler vials for UPLC–MS analysis.

Instrumentation

Ultra performance liquid chromatography (UPLC)The chromatographic separation of drugs was carried out on a

Acquity UPLC BEH C18 column (2.1 mm � 100 mm, 1.7 lm particlesize) (Waters�, Milford, MA, USA), maintained at temperature25 �C. The column was equipped with Waters� Acquity UPLC sys-tem (Waters�, Manchester, UK) consisting of an Acquity UPLC bin-ary solvent manager, sample manager and column heater. Theoptimum chromatographic separation was achieved in isocraticmode using a binary mobile phase mixture of acetonitrile andwater with 0.1% formic acid, (75:25, v/v) at a flow rate of0.5 mL min�1. The sample volume injected was 5 lL.

Tandem mass spectrometry (MS/MS)A Quattro Premier triple-quadrupole mass spectrometer from

Micromass Company Inc. (Manchester, UK) equipped with an ESIsource was used for drug detection. An Oerlikon rotary pump,model SOGEVAC SV40 BI (Paris, France) provided the primary vac-uum to the mass spectrometer. MS/MS detection was performedwith electrospray negative ionization mode. Monitoring conditionswere optimized for each drug. The optimized specific cone voltagewas chosen for the formation of each precursor ion and the multi-ple reaction monitored (MRM) conditions were optimized to ob-tain the transition precursor ion-product ion and collision energyfor the analysis of drugs (Table 1). The optimum source conditionswere as follows: capillary voltage 3.5 kV, source temperature120 �C and desolvation temperature 400 �C. The desolvation andcone gas flows were 800 and 80 L h�1, respectively. High-puritynitrogen was used as nebulising, desolvation and cone gas pro-duced by a Peak Scientific NM30LA nitrogen generator (Inchinann,UK). High-purity Argon (99.99%) supplied by Speciality Gas Center(Jeddah, Saudi Arabia) was used as collision gas. The collision gasflow was set at 0.10 mL min�1. Data acquisition was carried outby MassLynx V4.1 software (Micromass, Manchester, Lancashire,UK).

Validation study

For the validation study, following parameters were evaluatedin order to ensure the method quality: linearity, recovery, preci-sion, limits of detection (LOD) and limits of quantification (LOQ).Linearity of the method was estimated by analysis of five standardcalibration solutions, in duplicate, in the range 0.1–1.0 lg g�1 forboth L-ascorbic acid and acetylsalicylic acid. Recoveries were calcu-lated from the slope of the linear regression obtained between theadded analyte concentration and the measured analyte concentra-tion. To evaluate intraday precision, five replicate of spiked sam-ples were carried at same day. To assess interday precision,

Table 1Retention time (RT) and data acquisition parameters of MRM transitions for each analyte

Analyte RT(min)

Precursor ion [M–H]+ (m/z)

Quantification transition

Product ion (m/z)

Cone voltage(V)

AA 0.49 175 115 25ASA 1.66 179 137 10

Ionization mode: ESI�.a Dwell time was 0.1 s in all cases.

fifteen replicate of the same samples were performed over threeconsecutive days (five replicates each day). LOD and LOQ weredetermined by spiked samples based on signal to noise ratio of3:1 for detection limit and 10:1 for quantification limit.

Results and discussion

Optimization of chromatographic separation conditions

The development of a high throughput analytical technique forthe analysis of AA and ASA in aspirin C effervescent tablet using re-versed phase liquid chromatography is of high awareness. One ofthe major benefits of the UPLC columns, where particle size is<2.0 lm, is that the efficiency does not drop when increasing theflow rates. A flow rate of 0.5 mL min�1 was found to be optimalfor the analysis of AA and ASA, conditions that made possible toachieve an analysis time <2 min. Additionally, owing to isocraticconditions equilibration of the column was not necessary. Fig. 1shows the chromatograms of AA and ASA (0.5 lg mL�1) standardsin MRM mode obtained with the optimal working conditions. Iso-cratic elution by means of single eluent such as water, methanoland acetonitrile, and a mixture of two eluents (water/methanol,water/methanol containing 0.1% formic acid, acetonitrile/water,and acetonitrile/water containing 0.1% formic acid) of differentcomposition at different pH range (3–7) were compared. The sig-nal-to-noise ratio could be improved using acetonitrile/water con-taining 0.1% formic acid (75:25, v/v, pH 3.5) as mobile phase, thus,selected as mobile phase. The chromatographic separation of AAand ASA using different columns (C8, C18 and amide) was also eval-uated. The AA and ASA separation with good symmetry has beenachieved with C18 column. To minimize the carryover, after eachsample injection, methanol was injected as a quality control. How-ever, the auto sampler of the UPLC system repeatedly washes theinjector with strong wash and weak wash solvents after each sam-ple injection. Therefore, there is minute possibility to found resi-dues in the injector which is responsible for carryover peakduring the experiments.

Optimization of ESI–MS/MS conditions

The ESI–MS/MS conditions were optimized by infusing stan-dards of 5 lg g�1 of AA and ASA in both positive and negative ion-ization modes to desolvate efficiently the aqueous/organic mobilephase and provide the maximum analyte response. In positivemode, no analytes signal was appeared under different ion sourceparameters. Then the optimization was carried out in negative ion-ization mode, and highly abundant analytes signals were appeared,thus the negative ionization mode was chosen for mass spectro-metric detection. The narrow chromatographic peaks (5 s width)required a fast scanning analyzer to define the peaks with enoughpoints such as the triple quadrupole used in this work. The effect ofESI–MS conditions such as capillary voltage (2.5–4.5 kV), cone volt-age (5–90 V), source temperature (100–150 �C), desolvation tem-perature (200–450 �C) and desolvation gas (400–800 L h�1) were

used in UPLC–MS/MS.a

Confirmation transition

Collision energy(eV)

Product ion (m/z)

Cone voltage(V)

Collision energy(eV)

10 87 25 2412 93 10 26

m/z160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190

%

0

100 MRM of 4 Channels ES-175

179

O C H

O O H

O

3

O

H OO H

H

H

H

H

O H

O O H

m/z80 82 84 86 88 90 92 94 96 98 100 102 104 106 108 110 112 114 116 118 120 122 124 126 128 130 132 134 136 138 140

%

0

100 MRM of 4 Channels ES-137

115

8793

O C H

O O

O

H +

C O C H 2

3

(-4 2 )

O

HOOH

H

H

HH

OH

O

H

CHOCH OHO

+

(-60)

23 3

O CH

O O

O

H+

3

C O H (-86)

O

HOOH

H

H

HH

OH

O

H

O

COCHOCH OH2

+

(-88)

(a)

(b)

Fig. 2. (a) UPLC–MS/MS mass spectra of precursor ions of L-ascorbic acid (m/z 175) and acetylsalicylic acid (m/z 179) in MRM mode and (b) UPLC–MS/MS spectra of productions of L-ascorbic acid (m/z 175) and Acetylsalicylic acid (m/z 179) in MRM mode.

S.M. Wabaidur et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 108 (2013) 20–25 23

studied. The maximum analyte response was obtained with 3.5 kVof capillary voltage, 30 V of cone voltage, 120 �C of source temper-ature, 400 �C of desolvation temperature and 800 L h�1 of desolva-tion gas flow rate. The experiments point out the effects of the ESI–MS parameters exceeded higher values, were insignificant. Fullscan mass spectra were recorded in order to select the most abun-dant m/z value. The relative intensity for the most abundant m/zvalue was used to evaluate the performance of each ionizationand the cone voltage value. The most abundant precursors are m/z 175 (AA) and179 (ASA) which is displayed in Fig. 2a. Multiplereaction monitoring for two transitions per compound was ac-quired while the most sensitive transition was used for quantifica-tion and the other one for confirmation analysis (Fig. 2b). TheOptimized MS parameters (precursor and product ions, cone volt-ages and collision energies) are shown in Table 1.

Method validation

Linearity, limits of detection and limits of quantificationLinearity of the proposed method was estimated by investigat-

ing the detection signals as a function of analyte concentration,with the aid of a regression line by the method of least squares.

Table 2Validation parameters of UPLC–MS/MS method.

Analyte UPLC–MS/MS

LODa

(lg g�1)LOQb

(lg g�1)Linearity(r2)

Intraday(RSD%)c

Interday(RSD%)c

AA 0.09 0.29 0.999 1.7 2.6ASA 0.01 0.04 1.000 1.3 2.1

a Limit of detection was estimated at a signal-to-noise ratio of 3.b Limit of quantification was estimated at a signal-to-noise ratio of 10.c Relative standard deviation (n = 5).

The calibration curve for the determination of AA and ASA intablets by UPLC–MS/MS analysis was linear over the range of0.1–1.0 lg g�1. The correlation coefficient (r2) obtained was>0.999. The LOD of the quantitative analysis indicates the lowestlevel of the analyte that can be measured with definable statisticalcertainty in a sample, and equivalents to three times the standarddeviation of noise on analysis, while the LOQ is calculated from theconcentration of the analytes that provided signals equal to 10times the signal to noise on analysis. These LOD and LOQ valueswere calculated analyzing three replicates of a blank sample(Milli-Q water) spiked with AA and ASA at low concentration lev-els. The LOD and LOQ evaluated by the statistical MassLynx V4.1software. The LOD was 0.09 and 0.01 lg g�1 and the LOQ was0.29 and 0.04 lg g�1 for AA and ASA respectively, which ade-quately satisfied the quantification of AA and ASA in pharmaceuti-cal tablets by the UPLC–MS/MS method. The obtained results arepresented in Table 2.

Precision

The precision of proposed method was evaluated by the intra-day and interday determination of L-ascorbic and acetylsalicylic

HPLC–PDA [24]

LODa

(lg g�1)LOQb

(lg g�1)Linearity(r2)

Intraday(RSD%)c

Interday(RSD%)c

0.51 1.7 0.997 3.3 4.10.43 1.4 0.997 3.0 3.8

Table 3Assays of AA and ASA in pharmaceutical formulations.

Formulations Formulations claimed (mg) UPLC–MS/MS HPLC–PDA [24]

Conc. found (mg) ± SDa Recovery (%) ± SDa Conc. found (mg) ± SDa Recovery (%) ± SDa

Aspirin C (Bayer) L-ascorbic acid, 240 239.42 ± 0.01 99.75 ± 0.01 238.23 ± 0.03 99.26 ± 0.01

Acetyl-salicylic acid, 400 399.12 ± 0.03 99.78 ± 0.01 393.49 ± 0.05 98.37 ± 0.03

Conc. = Concentration.a Standard deviation (n = 5).

Table 4Recovery study of AA and ASA added to tablet samples.

Analytes Added (lg g�1) Found (lg g�1) ± SDa Recovery (%)

AA 0.1 0.099 ± 0.03 99.020.5 0.498 ± 0.01 99.601.0 0.999 ± 0.01 99.90

ASA 0.1 0.099 ± 0.04 99.000.5 0.496 ± 0.03 99.201.0 0.998 ± 0.01 99.81

SD = standard deviation.a Mean of three measurements.

24 S.M. Wabaidur et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 108 (2013) 20–25

acid. The intraday was estimated from five replicate injections of aspiked sample (0.5 lg g�1) on the same day and interday was mea-sured by five replicate injections of the aforementioned spikedsample along three consecutive days. These determinations wereevaluated on the basis of the calculation of the relative standarddeviation of the obtained concentration values. The intraday andinterday RSD values were obtained between 1.3% and 2.6% for AAand ASA (Table 2). The outcomes of the intraday and interday pre-cision data showed good reproducibility for the UPLC–MS/MS anal-ysis of AA and ASA in aspirin C effervescent tablet.

L-ascorbic acid

Rel

ativ

e ab

unda

nce

(%)

0.51

Intensity: 8.93e3 cps

175 > 114

0

100

)nim(emiT

0 1 2

Fig. 3. UPLC–MS/MS chromatogram of studied c

Comparison between UPLC–MS/MS and HPLC–PDA methods

A HPLC–PDA analytical technique for the quantification of AAand ASA in commercial pharmaceutical effervescent tablet formu-lations was reported in our previous work [24]. In aforementionedwork, AA and ASA have been simultaneously analyzed in commer-cial pharmaceutical effervescent tablet formulations using re-versed phase Betasil C18 column with particle size of 3 lm,150 mm � 4.6 mm I.D. with analysis time of 4.89 min, LOD be-tween 0.43 and 0.5 lg g�1 and intraday and interday precision val-ues from 3.0% to 4.1%. The data has been shown in Table 2. In thepresent work, the performance of UPLC and MS has been combinedto attain a fast simultaneous analysis of AA and ASA in isocraticconditions, with an analysis time of less than 2 min. The high sen-sitivity and selectivity provided by the MS lead to achieve a LODbetween of 0.01 and 0.09 lg g�1 and intraday and interday preci-sion values from 1.3% to 2.6% (Table 2) with negligible matrix ef-fect. As a whole, the UPLC–MS based method has offeredsubstantial improvements in sensitivity, analytical speed and reso-lution. The results of the quality parameters, analysis time and res-olution achieved with this system, confirm that they are sufficientto propose it as a new methodology for the fast and reliable simul-taneous analysis of AA and ASA in commercial pharmaceuticaleffervescent tablet formulations.

)nim(emiT

1.68

0 1 2

0

100

Intensity: 2.14e4 cps

179 > 137

Rel

ativ

e ab

unda

nce

(%)

Acetylsalicylic acid

ompounds in aspirin C effervescent tablet.

S.M. Wabaidur et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 108 (2013) 20–25 25

Application: analysis of real sample

In order to assess the applicability of the UPLC–MS/MS methodfor the simultaneous determination of AA and ASA in aspirin Ceffervescent tablets, 10 single tablets of the same batch were ana-lyzed. The amount of AA and ASA was found as an average of239.42 mg and 399.12 mg tablet�1, respectively. The obtained val-ues are given in table 3. The achieved experimental values andamount claimed by the manufacturer (AA = 240 mg andASA = 400 mg) of the commercial effervescent preparation are ingood agreement. The recovery was obtained between 99.75% and99.78%, depending on the sample (Table 3). To validate the ob-tained results in the pharmaceutical formulation, the recoveriesof AA and ASA were estimated by spiking both standards of threeconcentration levels to tablet samples. The recoveries were ob-tained 99.00–99.90% (Table 4). Fig. 3 shows the UPLC–MS/MS chro-matograms of AA and ASA in aspirin C effervescent tablets. Thechromatograms show good resolution and did not show any inter-ference, as no detectable matrix peak was eluted in the retentiontime of the analyzed compounds. Nevertheless, there are slightchanges in the retention time of the analyzed compounds; thismight be due to the matrix effect of the column. The mass spectraof the corresponding compound is similar and found at the samem/z value as obtained from the analysis of standard solution(Fig. 2a and b).

Conclusions

An improved UPLC–MS/MS method for the rapid quantificationof AA and ASA in pharmaceutical formulation has been validated tobe a fast and appropriate method compared to previous HPLC–PDAmethod. This improved method supplies a rapid quantitative pro-cedure of AA and ASA with a run time of less than 2 min and showshigh sensitivity, good linearity over a wide range and good repro-ducibility. The results of the quality parameters, analysis time andresolution achieved with this system, confirm that they are ade-quate to propose it as a new methodology for the fast and reliableanalysis of AA and ASA in commercial pharmaceutical effervescenttablet formulations.

Acknowledgment

This research was supported by King Saud University, Deanshipof Scientific Research, College of Science Research Center.

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