Aspirin JLCRT


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Aspirin JLCRT

  1. 1. This article was downloaded by: [Chinmoy Ghosh]On: 22 November 2011, At: 20:31Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UKJournal of Liquid Chromatography &Related TechnologiesPublication details, including instructions for authors andsubscription information: DETERMINATION OFASPIRIN AND ITS METABOLITE FROMHUMAN PLASMA BY UPLC-UV DETECTION:APPLICATION TO PHARMACOKINETICSTUDYChinmoy Ghosha, Anita Upadhayaya, Ajay Singha, SaumyaBahadura, Priya Jaina& Bhaswat S. ChakrabortyaaCadila Pharmaceuticals Limited, Gujarat, IndiaAvailable online: 18 Nov 2011To cite this article: Chinmoy Ghosh, Anita Upadhayay, Ajay Singh, Saumya Bahadur, Priya Jain &Bhaswat S. Chakraborty (2011): SIMULTANEOUS DETERMINATION OF ASPIRIN AND ITS METABOLITE FROMHUMAN PLASMA BY UPLC-UV DETECTION: APPLICATION TO PHARMACOKINETIC STUDY, Journal of LiquidChromatography & Related Technologies, 34:19, 2326-2338To link to this article: SCROLL DOWN FOR ARTICLEFull terms and conditions of use: article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden.The publisher does not give any warranty express or implied or make any representationthat the contents will be complete or accurate or up to date. The accuracy of anyinstructions, formulae, and drug doses should be independently verified with primarysources. The publisher shall not be liable for any loss, actions, claims, proceedings,demand, or costs or damages whatsoever or howsoever caused arising directly orindirectly in connection with or arising out of the use of this material.
  2. 2. SIMULTANEOUS DETERMINATION OF ASPIRIN AND ITSMETABOLITE FROM HUMAN PLASMA BY UPLC-UV DETECTION:APPLICATION TO PHARMACOKINETIC STUDYChinmoy Ghosh, Anita Upadhayay, Ajay Singh, Saumya Bahadur,Priya Jain, and Bhaswat S. ChakrabortyCadila Pharmaceuticals Limited, Gujarat, India& A rapid and sensitive liquid chromatography method with UV detection for simultaneousmeasurement of aspirin (ASA) and salicylic acid (SA) was developed and validated completelyin human plasma. ASA, SA, and Benzoic acid (BA) as internal standard were extracted via pro-tein precipitation with Perchloric acid. An isocratic elution with binary mode was used to separateinterference peaks using a C18 Acquity column with only three minutes of analysis time. The lin-earity range was 15 to 6000 n g mLÀ1. Calibration functions, LOQ, stability, intra- and inter-dayreproducibility, and accuracy were estimated. The inter- and intra-day % CV for ASA and SA areÆ15% and the percentage change of all stability samples with comparison samples were within10%. The in vitro conversion of ASA to SA was also studied and it was controlled to keep at aminimum (<10%). With respect to other published methods this method is most sensitive in UVdetection, as well as its sensitivity and throughput is comparable or even better than publishedLC-MS=MS methods. This method was successfully applied to a single dose Bioequivalence (BE)study of following administration of 81 mg enteric coated Aspirin tablets.Keywords aspirin, Benzoic acid, Bioequivalence=protein precipitation, salicylic acid,UPLC-UVINTRODUCTIONAspirin, also known as acetylsalicylic acid (ASA)[1](CAS number:50-78-2; Figure 1), is often used as an analgesic to relieve minor achesand pains, as an antipyretic to reduce fever, and as an anti-inflammatorymedication. Aspirin also has an antiplatelet or ‘‘anti-clotting’’ effect andis used in low doses to prevent long-term heart attacks, strokes and bloodclot formation in people at high risk.[2]Furthermore, it has beenAddress correspondence to Chinmoy Ghosh, Research Scientist, Contract Research Organization,Cadila Pharmaceuticals Limited, 1389, Trasad Road, Dholka-387 810, Ahmedabad, Gujarat, India.E-mail: chinmoy_ghosh@yahoo.comJournal of Liquid Chromatography & Related Technologies, 34:2326–2338, 2011Copyright # Taylor & Francis Group, LLCISSN: 1082-6076 print/1520-572X onlineDOI: 10.1080/10826076.2011.589092Downloadedby[ChinmoyGhosh]at20:3122November2011
  3. 3. established that low doses of aspirin may be given immediately after a heartattack to reduce the risk of another heart attack or of the death of cardiactissue.[3,4]The main undesirable side effects of aspirin are gastrointestinalulcers, stomach bleeding, and tinnitus, especially in higher doses. In chil-dren under 19 years of age, aspirin is no longer used to control flu-likesymptoms or the symptoms of chickenpox, due to the risk of Reye’ssyndrome.[5]Salicylic acid (SA)[6](CAS number: 69-72-7; Figure 2), is a colorlesscrystalline organic acid and is widely used in organic synthesis and func-tions as a plant hormone. It is derived from the metabolism of salicin. Inaddition to being a compound that is chemically similar to but not identicalto the active component of aspirin (acetylsalicylic acid), it is probably bestknown for its use in anti-acne treatments. It is poorly soluble in water(0.2 g=100 ml H2O at 20C).[7]Several LC methods with UV detection[8–16]have been reported forestimation of ASA alone or with its metabolite, that is, SA. Although theother reported LC-UV methods were not sensitive enough, the reportedlower limit of quantitation (LLOQ) for simultaneous determination ofboth ASA and SA is around 100 ng mLÀ1with long analysis time. On theother hand, there were some LC-MS=MS methods[17–19]that are compara-ble with this LC-UV method; as in the present method, the limit of quanti-fication (LLOQ) is 15 ng mLÀ1for both ASA and SA, which is sufficientwith low doses of ASA with only 3 min. of analysis time. In the presentmethod, a simple precipitation method was used as the extraction tech-nique instead of other complex extraction techniques reported in otherFIGURE 2 SA (Salicylic Acid).FIGURE 1 ASA (Acetyl Salicylic Acid).Determination of Aspirin and Its Major Metabolite by UPLC-UV 2327Downloadedby[ChinmoyGhosh]at20:3122November2011
  4. 4. methods.[12,13]In the present method, benzoic acid (BA) was used as inter-nal standard (IS), whereas some other method used isotope labeled IS.[14]More over there is no UPLC method available, so it is the only UPLC-UVmethod. The novelty of this method is that, its sensitivity and throughputis equivalent or better to reported LC-MS=MS methods.[17–19]Additionally,this manuscript also studies the degradation of aspirin in human plasma. Asa result, this present method can be very useful for those laboratories wherecostly instruments such as LC-MS=MS or GC-MS are not available; however,by applying this method, the equivalent sensitivity and throughput can beachieved.The aim of the present research work was to develop an accurate andsensitive UPLC-UV method with a dynamic linearity range that can coverthe plasma concentrations following a single oral dose of aspirin. Themethod has been validated by evaluating the precision, accuracy, and othervalidation parameters for human plasma samples, as mentioned in regulat-ory guidelines.[20]EXPERIMENTALChemicals and ReagentsASA and the IS were procured from the Cadila Pharmaceuticals Ltd.,Ahmedabad. SA was obtained from the Chemisynth Laboratory Limited,Mumbai, India. Potassium dihydrogen phosphate, Ortho phosphoric Acid(OPA), and Hydrochloric acid used during analysis were procured fromS.D. Fine Chemicals Ltd., Ankleshwar, Gujarat, India and were of AR grade.Perchloric Acid (about 70% Purity) was procured from Merck Pharmaceu-ticals Limited, Mumbai, India. Water used was collected from water purifi-cation systems (Milli Q, Milli Pore, USA) installed in our Laboratory wherethis study was conducted. Methanol and acetonitrile were of HPLC gradeand were supplied by J. T. Baker, USA. Fresh frozen Human Plasma(K2-EDTA as anticoagulant) was used during validation, which was suppliedby Prathma Blood Centre, Ahmedabad, India. Plasma was stored atÀ70 Æ 5C.Apparatus and SoftwareWaters Acquity Ultra Performance Liquid Chromatography (UPLC) sys-tem with UV-PDA detector (Waters Corporation, USA) for detection andquantitation of samples was utilized. Chromatographic acquisition andintegration were performed using Empower 2 software developed and inte-grated with the UPLC by Waters Corporation, USA. Other instruments2328 C. Ghosh et al.Downloadedby[ChinmoyGhosh]at20:3122November2011
  5. 5. used were Analytical Balance (Mettler Toledo, Switzerland), RefrigeratedCentrifuge (Heraeus Multifuge 3S–Rþ, Thermo Electron Corporation,Germany), Vortexer (Remi, Mumbai, India), Deep freezer (Sanyo, Japan),and Refrigerator (Samsung, Ahmedabad, India).Standards and Working SolutionsA standard stock solution of ASA containing 1 mg mLÀ1was preparedby dissolving pure compound in acetonitrile, where separate stock standardsolutions of SA and BA (each 1 mg mLÀ1) were prepared by dissolving purecompounds in methanol. Intermediate and working solutions of ASA wereprepared from corresponding stock solutions by diluting with acetonitrile,whereas intermediate and working solutions of SA and BA were preparedfrom corresponding stock solutions by diluting with water:methanol(50:50 v=v). Calibration standards were established between 15 to6000 ng mLÀ1of ASA and SA both, using nine concentration levels. Qualitycontrol standards at three different levels low (45 ng mLÀ1), medium(2480 ng mLÀ1), and high (5000 ng mLÀ1) were also prepared. All thesestock solutions, calibration standards, and quality control samples werestored at 4 Æ 2C. These solutions were found to be stable and used forthe complete method validation.Chromatographic ConditionsChromatographic separation was performed on an Acquity UPLC BEHC18 column (100 Â 2.1 mm i.d., particle size 1.7 mm). The mobile phaseused was a mixture of Potassium dihydrogen phosphate (pH: 2.1 Æ 0.05,20 mM) in Milli-Q water:methanol:acetonitrile (70:15:15, v=v=v). The flowrate was 0.350 mL minÀ1. Total analysis time of single injection was threemin. Injection volume was 30 mL. Auto sampler rinsing volume was400 mL. Strong Needle Wash was a mixture of acetonitrile:water at a ratioof 50:50 v=v. Weak needle wash was 100% Milli-Q water.Sample PreparationProtein precipitation extraction technique was used to extract the ASAand SA from human plasma samples. Hydrochloric acid (1M) was used forproviding an acidic medium to prevent conversion of ASA to SA. Perchloricacid was used as extracting solvent. Plasma samples (500 mL) were trans-ferred to a centrifuge tube for analysis. An amount of 20 mL of IS(20 mg mLÀ1) followed by 50 mL of 1.0 M hydrochloric acid was added intoDetermination of Aspirin and Its Major Metabolite by UPLC-UV 2329Downloadedby[ChinmoyGhosh]at20:3122November2011
  6. 6. it and the samples were vortexed for 15 sec. Perchloric acid solution(200 mL of 20% v=v) was added to precipitate the plasma samples and vor-texed for 1 min and centrifuged at 4500 rpm for 5 min at 5C. The super-natant was centrifuged at 10000 rpm for 5 min at 5C and the wholesupernatant was injected onto UPLC.Degradation Study for Bioanalytical PurposeIt has been reported[21]that ASA converts to SA by hydrolysis over timeunder normal conditions. Therefore, tests were performed to monitor per-centage degradation of ASA into SA in different conditions of pH (1 MHCl, 0.1 M HCl, 1% Formic Acid, 1% Acetic Acid), Base (1 M KOH), andwithout addition of any acid or base at different time intervals.Method ValidationThe validation parameters were specifically, linearity, sensitivity,accuracy, precision, and hemolysis effects of the assay and the recoveryand stability in human plasma, according to the US Food and Drug Admin-istration (FDA) guidance for the validation of bioanalytical methods.[20]Selectivity was studied by comparing the chromatograms of six differentlots of plasma obtained from six subjects, with the plasma samples havingbeen spiked with SA, ASA, and the IS. Calibration curves were preparedby assaying standard plasma samples at SA and ASA concentrations, ran-ging from 15 to 6000 ng=mL for both SA and ASA.The linearity of each method matched calibration curve was deter-mined by plotting the peak area ratio (y) of SA or ASA to the IS versusthe nominal concentration (x) of SA or ASA, respectively. The calibrationcurves were constructed by weighing (1=x2) least-squares linear regression.The LLOQ for SA or ASA in human plasma was defined as the lowestconcentration giving acceptable accuracy (80–120%), and sufficientprecision (within 20%); this was verified by analysis of six replicates.Intra- and inter-day accuracy and precision for this method were deter-mined at three different concentration levels on three different days, andon each day, six replicates were analyzed with independently prepared cali-bration curves. The percentage accuracy was express as (mean observedconcentration)=(nominal concentration) Â 100, and the precision was therelative standard deviation [RSD (%)].For recovery calculation, the peak areas obtained by direct injection ofsolvent (or neat) standard solutions spiked after extraction into plasmaextracts as A and the peak areas for solvent (or neat) standard solutionsspiked before plasma extraction as B, the extraction recovery value can2330 C. Ghosh et al.Downloadedby[ChinmoyGhosh]at20:3122November2011
  7. 7. be calculated as follows:Extraction recovery ð%Þ ¼ B=A Â 100The stability of SA or ASA in human plasma was assessed by analyzing sixreplicate samples at LQC and HQC levels for SA and ASA, respectively,under six conditions: after long term storage of 6 months at À70C; afterfour freeze-thaw cycles; after sample preparation for 6 hr on bench topand after 49 hr within the auto sampler. The concentrations obtained werecompared with the nominal values of the QC samples.Clinical ProtocolThe Independent Medical Ethics Committee (IEC) of Cadila ContractResearch Organization, Ahmedabad, Gujarat, India, approved the bio-equivalence study protocol and bioanalytical method presented in thispaper. The study was a randomized, open-label, two-treatment, two-period,two-sequence, two-way crossover study, during which subjects were adminis-tered two different formulations of a single dose of Aspirin 81 mgtablet along with 240 mL of drinking water. Dose were administered afteran overnight fasting for at least 10 hr in each period with at least 14 d ofthe washout period between each administration. Volunteers were healthy,adult males from India. In each period, a total of 16 blood samples werecollected including a predose sample prior to drug administration andafter drug administration at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0,8.0, 10.0, 12.0, 14.0, and 16.0 hr. These samples were assayed by thedeveloped and validated UPLC method described in this paper.RESULTS AND DISCUSSIONSMethod DevelopmentMethod development was started with UPLC BEH C8 column(50 Â 2.1 mm i.d., particle size 1.7 mm), but there was almost no resolutionbetween SA and ASA with poor chromatography. As a result, the columnlength was increased to 100 mm keeping all other specifications the same,but poor chromatography was observed for both SA and ASA with little res-olution between SA and ASA. Therefore, BEH C18 column (50 Â 2.1 mmi.d., particle size 1.7 mm), was tried, which showed good chromatographyfor SA, ASA, and BA with comparatively better resolution. Initially, potass-ium dihydrogen phosphate (pH: 2.1 Æ 0.05, 20 mM) in Milli-Q water:aceto-nitrile (75:25, v=v) was tried as the mobile phase. Later, to improve theDetermination of Aspirin and Its Major Metabolite by UPLC-UV 2331Downloadedby[ChinmoyGhosh]at20:3122November2011
  8. 8. resolution between the peaks, methanol was introduced along with acetoni-trile as the organic phase. Finally, the mobile phase used was a mixture ofpotassium dihydrogen phosphate (pH: 2.1 Æ 0.05, 20 mM) in Milli-Q water:-methanol:acetonitrile (70:15:15, v=v=v). The flow rate was 0.350 mL minÀ1.Total analysis time of a single injection was three min. Injection volume was30 mL. Wave length was selected from the available literatures.Method ValidationThe method was validated in terms of linearity, specificity, LLOQ, recov-ery, accuracy, precision, dilution integrity, hemolysis effect, and stability stu-dies (e.g., short term stock solution stability, freeze thaw stability, bench topstability, and auto sampler stability). The accuracy and precision determi-nation were carried out in three different d with six replicates of LLOQ,low, medium, and high quality control samples.LinearityLinearity of calibration standards was assessed by subjecting the spikedconcentrations and the respective peak areas using 1=X2linearleast-squares regression analysis. Linearity of the assay ranged from 15 to6000 ng mLÀ1(r 0.9900). In aqueous solution linearity test, all calibrationstandards showed an accuracy within 85–115%, except LLOQ where it wasbetween 80–120%.Specificity and SelectivitySix different lots of plasma were analyzed to ensure that no endogenousinterference with the retention time chosen for ASA, SA, and the IS. SixLLOQ level samples along with the plasma blank from the respectiveplasma lot were prepared from six different lots of plasma and analyzed.In all six plasma blanks, the response at the retention time of ASA andSA was less than 20% of LLOQ response and at the retention time of IS,the response was less than 5% of mean IS response in LLOQ. Figure 3shows a typical chromatogram of plasma blank and Figure 4 representsthe chromatogram at LLOQ.Accuracy and PrecisionFor the validation of the assay, QC samples were prepared with threeconcentration levels of low, medium, and high. Six replicates of each QCsamples and LLOQ level were analyzed together with a set of calibration2332 C. Ghosh et al.Downloadedby[ChinmoyGhosh]at20:3122November2011
  9. 9. standards. The accuracy of each sample preparation was determined byinjection of calibration samples and LLOQ, LQC, MQC, and HQC samplesin six replicate for 3 d. The obtained accuracy and precision (inter- andintra-day) are presented in Table 1 for ASA and SA. The results showed thatthe analytical method is accurate, as the accuracy is within the acceptancelimits of Æ20% of the theoretical value at LLOQ and Æ15% at all other con-centration levels. The precision around the mean value was always within15% at any of the nominal concentration studied.Recovery StudyA recovery study was performed by comparing processed QC samples ofthree different levels in six replicate with aqueous samples of same level.The mean recovery of ASA was 58.11% and the CV (%) of mean recoveryFIGURE 4 Representative chromatogram of LLOQ (15.0 ng mLÀ1).FIGURE 3 Representative chromatogram of plasma blank.Determination of Aspirin and Its Major Metabolite by UPLC-UV 2333Downloadedby[ChinmoyGhosh]at20:3122November2011
  10. 10. of at all three QCs was 1.98, whereas the mean recovery of SA was 70.64. CV(%) of mean recovery of at all three QCs was 6.09. Recovery of the IS was73.22%.Hemolysis EffectsTo determine hemolysis effects on the estimation of plasma concen-tration, six hemolyzed plasma blank and QC samples were prepared inhemolyzed plasma with two concentration levels of low and high. Six repli-cates of each QC samples were analyzed together with a set of calibrationstandards prepared in normal plasma. The accuracy of each sample prep-aration was determined by injection of calibration samples and two QCsamples in six replicates. The average % accuracy of LQC and HQC levelwas 104.61 and 100.75, respectively, for ASA and % accuracy of LQC andHQC level was 98.32 and 101.71, respectively, for SA. The CV (%) ofLQC was 5.86 and for HQC was 9.27 for ASA and CV (%) of LQC was10.11 and for HQC was 9.51 for SA.Stability StudiesThe stability of ASA, SA, and IS were investigated in the stock and work-ing solutions, in plasma during storage, during processing (i.e., bench topTABLE 1 Inter and Intra-day Accuracy and Precision of ASA and SAASA SAQC LevelsMeanAccuracyMeanPrecision (% CV)MeanAccuracyMeanPrecision (% CV)Day 1 LLOQ 98.73 4.66 104.63 3.40LQC 91.44 9.14 97.08 2.27MQC 95.33 1.25 112.13 1.59HQC 101.53 4.33 113.59 2.30Day 2 LLOQ 103.83 4.05 111.25 1.20LQC 93.75 2.03 92.51 1.50MQC 98.21 3.24 103.52 4.75HQC 99.63 10.24 110.49 3.33Day 3 LLOQ 100.33 12.75 98.79 3.84LQC 100.79 4.11 85.69 2.55MQC 93.71 3.93 100.74 3.65HQC 100.93 2.91 108.05 1.32Inter day LLOQ 100.96 7.94 104.88 5.71LQC 95.42 7.09 92.12 5.48MQC 95.75 3.47 105.46 5.76HQC 100.69 6.24 110.71 3.13Each mean and % CV of intra-day accuracy and precision represent six observations (n ¼ 6). Theinter-day accuracy and precision are averages and % CV of three intra-day observations.2334 C. Ghosh et al.Downloadedby[ChinmoyGhosh]at20:3122November2011
  11. 11. stability), after four freeze-thaw cycles, and in the final extract (i.e., autosampler stability). Stability samples were compared with freshly processedcalibration standards and QC samples for comparison. Analyte, metabolite,and IS were considered stable when the change of concentration was Æ10%with respect to initial concentration. Summary of stability data is presentedin Table 2 for ASA and SA.Calibration Curve ParameterThe summaries of calibration curve parameters were as follows.The mean y-intercepts and slope for ASA were À0.0086 (range: À0.0149to À0.0032) and 0.00033 (range: 0.00027 to 0.00037) respectively. Themean correlation coefficient, r was 0.9968 (range: 0.9942 to 0.9987).For SA, the mean y-intercepts and slope were À0.0050 (Range: À0.0201to À0.0224) and 0.00063 (range: 0.000497 to 0.00072), respectively. Themean correlation coefficient, r was 0.9994 (range: 0.9935 to 0.9978).Degradation StudySummary of data after conversion of ASA to SA is summarized inFigure 5. The maximum conversion was observed in basic condition whichis suitable for the studies where only ASA is to be analyzed in terms of SA.The minimum conversion is obtained under acidic conditions. Hence, thiscondition was selected for simultaneous estimation of ASA and SA. Amongall the trials it has been observed that after six hr almost 70% of ASA wasconverted to SA in 1 M potassium hydroxide solution at room temperature,whereas approximately 10% of ASA is converted to SA in 1 M hydrochloricacid solution at room temperature after six hr.TABLE 2 Summary of Stability Data of ASAASA SAExperimentName QC LevelMeanAccuracyMeanPrecision(%CV)PercentChangeMeanAccuracyMeanPrecision(%CV)PercentChangeStabilityDurationBench top LQC 94.88 8.30 1.21 94.03 4.13 9.73 06 hrHQC 94.46 1.09 À5.19 99.06 4.65 À8.32Freeze thaw LQC 91.44 4.74 4.61 95.72 12.77 4.02 4 cyclesHQC 102.75 1.50 À1.90 106.80 0.73 À3.96Auto sampler LQC 97.54 13.88 4.04 93.70 6.36 9.35 60 hrHQC 95.34 1.48 À4.30 110.10 0.91 1.89Each mean accuracy, % CV and % change of each stability represents six observations (n ¼ 6) ofcorresponding QC levels.Determination of Aspirin and Its Major Metabolite by UPLC-UV 2335Downloadedby[ChinmoyGhosh]at20:3122November2011
  12. 12. Pharmacokinetic StudyThe sensitivity and specificity of the assay were found to be sufficient foraccurately characterizing the plasma pharmacokinetics of SA and ASA inhealthy volunteers. Profile of the mean plasma concentration and timeare shown in Figure 6. Maximum plasma concentration (Cmax of SA is800 Æ 100 ng=mL and ASA is 5500 Æ 500 ng=mL) was achieved at 2.50 hrfor SA and 6.00 hr for ASA. The higher sensitivity of this method comparedwith the currently existing methods in literature facilitates the quantitationof SA and ASA at lower concentration. Figure 7 represents the chromato-gram of real sample.FIGURE 5 Representative graph of % conversion of ASA to SA.FIGURE 6 Pharmacokinetic profile of plasma concentration and time of single volunteer.2336 C. Ghosh et al.Downloadedby[ChinmoyGhosh]at20:3122November2011
  13. 13. CONCLUSIONA simple, sensitive, selective, precise, and accurate UPLC-UV methodfor the simultaneous determination of ASA and its metabolite, SA, inhuman plasma, over a range of 15–6000 ng=mL for ASA and SA, wasdeveloped and validated. This is the only method for ASA and SA, whichwas developed and validated using UPLC with UV detection. This methodrequires only 0.500 mL of biological samples, owing to simple sample prep-aration and short run time (3 min), it allows high sample throughput. Themethod was successfully applied to a single dose 81 mg enteric coated tabletBio equivalence study of ASA and its major metabolite, SA.REFERENCES1. PubChem Public Chemical Database. (accessed on 19th October, 2011).2. Lewis, H. D.; Davis, J. W.; Archibald, D. G.; Steinke, W. E.; Smitherman, T. C.; Doherty, J. E.;Schnaper, H. W.; Le Winter, M. M.; Linares, E.; Pouget, J. M.; Sabharwal, S. C.; Chesler, E.; DeMots,H. Protective Effects of Aspirin Against Acute Myocardial Infarction and Death in Men withUnstable Angina. Results of a Veterans Administration Cooperative Study. N. Engl. J. Med. 1983,309, 396–403.3. Julian, D. G.; Chamberlain, D. A.; Pocock, S. J. A Comparison of Aspirin and AnticoagulationFollowing Thrombolysis for Myocardial Infarction (the AFTER Study): A Multicentre UnblindedRandomised Clinical Trial. Br. Med. J. 1996, 313, 429–1431.4. Krumholz, H. M.; Radford, M. J.; Ellerbeck, E. F.; Hennen, J.; Meehan, T. P.; Petrillo, M.; Wang, Y.;Kresowik, T. F.; Jencks, S. F. Aspirin in the Treatment of Acute Myocardial Infarction in ElderlyMedicare Beneficiaries. Patterns of Use and Outcomes. Circulation. 1995, 92, 2841–2847.5. Macdonald, S. Aspirin Use to be Banned in Under 16 Year Olds. Br. Med. J. 2002, 325, 988.6. PubChem Public Chemical Database. (accessed on 19th October, 2011).7. Salicilyc Acid. International Programme on Chemical Safety and the Commission of the EuropeanCommunities, Great Britain, 2001. (Retrieved on 13 October 2008).FIGURE 7 Representative chromatogram of real sample at 6:00 hrs.Determination of Aspirin and Its Major Metabolite by UPLC-UV 2337Downloadedby[ChinmoyGhosh]at20:3122November2011
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