Research ArticleDetermination of Atenolol and Trimetazidine inPharmaceutical Tablets and Human Urine Using a HighPerformance Liquid Chromatography-Photo Diode ArrayDetection Method

Walaa El-Alfy Omnia A Ismaiel Magda Y El-Mammli and Abdalla Shalaby

Department of Analytical Chemistry Faculty of Pharmacy Zagazig University Zagazig 44519 Egypt

Correspondence should be addressed to Walaa El-Alfy walaaalfy78gmailcom

Received 30 August 2018 Revised 19 October 2018 Accepted 28 October 2018 Published 3 January 2019

Academic Editor Samuel Carda-Broch

Copyright copy 2019 Walaa El-Alfy et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A simple RP-HPLC-PDA method for determination of atenolol (ATN) and trimetazidine (TMZ) in human urine and tabletshas been developed Analytes were separated on a Caltrex BI column (125times 40 mm 5 120583m) with 25mM potassium dihydrogenphosphate pH 33 methanol and acetonitrile mobile phases The PDA detector was operated at 210 nm for TMZ and 225 nm forATN and the flow rate was 10 mL min Linearity was obtained over a concentration range of (10-100 120583gmL) for both analytesin standard solutions and the method was successfully applied for determination of target analytes in their pharmaceutical tabletsExcellent linearity was also obtained over concentration ranges of (025-25 120583gmL) and (05-25 120583gmL) in human urine for TMZand ATN respectively A simple liquid-liquid extraction was applied for urine sample clean-up and a gradient method was used forchromatographic separationThe lower limit of quantitation (LOQ) was 099 and 060 120583gmL for ATN and TMZ respectivelyThelimit of detection (LOD) was 030 and 018 120583gmL for ATN and TMZ respectively Inter- and intraday precision and accuracy forATN were within plusmn189 in pure form and within plusmn285 in urine samples Inter- and intraday precision and accuracy for TMZwere within plusmn 399 in pure form and within plusmn 319 in urine samples

1 Introduction

Atenolol (ATN) (4-(2-hydroxy-3-isopropylamino propoxy)phenyl acetamide) (Figure 1) is a cardioselective 120573-blockercompound it is documented officially in BP [1] and USP[2] It has been reported that ATN suppresses the releaseof rennin and angiotensin-II and lowers the aldosteroneproduction [3] It has neither intrinsic sympathomimeticnor membrane stabilizing actions It is clinically used incontrolling of hypertension cardiac arrhythmias myocardialinfarction and angina pectoris It can also be used forpreventive treatment of migraine [4]

Trimetazidine dihydrochloride (TMZ) (1-[(234-trime-thoxyphenyl)methyl]-piperazine dihydrochloride) (Figure 1)is also an official drug in BP [1] TMZ is used as anantianginal drug acting mainly on ischemic cells [5] Themechanism of action is based on changing the energysubstrate preference from fatty acid oxidation to glucose

oxidation which adjusts the cellular energy processes and inthe main time it maintains appropriate energy metabolismduring ischemia condition It has no effect on the myocardialoxygen consumption or the coronary blood flow [6]

TMZ has no negative inotropic effects or vasodilatorycharacteristics and can be combined with other antianginaldrugs as a complementary therapy mostly for patients withcompanion diseases such as left ventricular dysfunction anddiabetes mellitus [7] A comparison between ATN-TMZcombined therapy and atenolol plus placebo has been estab-lished and has shown an enhancement in the total exercisetest duration and time to 1-mm ST segment depression Thiscombination was found to decrease the necessity of nitrateconsumption reduce the angina pain grade and also decreasethe overall number of angina attacks [7]

Coadministration of different medications for controllingchronic diseases especially in geriatric patients may causepolypharmacy problems such as adverse drug effects and

HindawiInternational Journal of Analytical ChemistryVolume 2019 Article ID 9625849 8 pageshttpsdoiorg10115520199625849

2 International Journal of Analytical Chemistry

NH

OHO

NH2

O

(a) Chemical structure of atenolol

HNN

O CH3

O CH3

O CH3

2 HCl

(b) Chemical structure of trimetazidinedihydrochloride

Figure 1

toxicity Changes in kidney and liver functions may affectdrug pharmacokinetic and pharmacodynamics and mayincrease the mortality rates Monitoring concomitant drugsin blood plasma and urine samples is mandatory in somecases [8] Atenolol and trimetazidine can be coadministereddue to their combined therapy benefits [7] and absence ofinteractions [9] Doses may be adjusted to avoid any adverseeffects with other drugs

Atenolol experiences low or no hepatic metabolism andis excreted unchanged mainly in urine [4] TMZ is weaklybound to plasma proteins It is metabolized at a low extentinto different metabolites andmajority of the drug is excretedintact in urine [7] Both analytes are considered good candi-dates for simple and rapid urine analysis therapeutic moni-toring polypharmacy issues investigation and toxicologicalstudies On the other hand urine samples are noninvasiveand easily accessible [10]

Different analytical methods have been applied for thedetermination of atenolol in biological fluids and pharmaceu-tical products such as HPLC [3 11] LC-MS [5 6] GC-MS[12] TLC [13] spectrophotometric and spectrofluorimetricmethods [14ndash16] titrimetry [17] voltammetry [18] chemilu-minescence [19] and capillary electrophoresis [20] Severalclean-up methods such as liquid-liquid extraction [3 11 12]solid phase extraction [21] and protein precipitation [22]have been used for extracting ATN from biological fluidsamples

Several techniques have been also reported for the deter-mination of trimetazidine including HPLC [23 24] in humanplasma and dosage forms respectively LC-MS [5 25] GC-MS [26 27] HPTLC [28 29] spectrophotometry [30 31]potentiometry [32] voltammetry [33] and chemilumines-cence [34] TMZ has been isolated from biological fluidsusing liquid-liquid extraction [5] solid phase extraction [35]and protein precipitation methods [6]

To our knowledge there are no published methods fordetermination of TMZ and ATN in human urine simulta-neously This paper describes simple HPLC-PDA methodsfor simultaneous determination of TMZ and ATN in humanurine and also for determination of TMZ and ATN in theircorresponding tablets

2 Experimental

21 Materials and Reagents All chemicals were of analyticalgrade and all solvents were of HPLC grade Trimetazidinewas kindly supplied from SIGMA Pharmaceutical Industries(Cairo Egypt) Atenolol was kindly supplied from Egyptian

Int Pharmaceutical Industries Co (10th of Ramadan cityEgypt) Metacardia film-coated tablets labeled to contain 20mg trimetazidine per tablet and Blokium film-coated tabletslabeled to contain 100 mg atenolol per tablet were obtainedfrom local pharmacy Acetonitrile HPLC grade orthophos-phoric acid and potassium orthophosphate monobasic wereobtained from Fischer Scientific UK (Bishop Meadow RoadUK)Methanol and water were obtained fromTEDIAChem-icals (USA) Sodium hydroxide chloroform and n-butanolwere purchased from El-Nasr Pharmaceutical ChemicalsCo (Abu-Zaabal Cairo Egypt) Human blank urine wascollected from healthy nonsmoker adult volunteers Urinesamples were stored at -20∘C

22 Instrumentation The HPLC system consists of AgilentTechnologies 1200 series chromatographic apparatus (Agi-lent Technologies Palo Alto CA USA) equipped with anautosampler injector 100 120583l injection loop Agilent quater-nary pumps and solvent cabinets Mobile phase was filteredthrough a 045 120583m membrane filter (Millipore Ireland)and degassed with vacuum degasser Separation was carriedout on Caltrex BI (125 times 40 mm 5 120583m) using UV lamp(Germany) and G1315D photodiode array detector (PDA)

23 Preparation of Stock Solutions Stock solutions of eithertrimetazidine or atenolol were prepared individually at 10mgmL in methanol Working solutions were prepared byfurther dilution of the stock solutions with water for spikingcalibration standards and quality control samples All stocksolutions were stored in amber glass containers at 2-8∘C

24 Preparation of Calibration Standards andQuality Control Samples

241 Neat Standard Solutions Calibration standards wereprepared in HPLC grade water at concentrations of 10 3050 10 25 50 75 and 100 120583gmL of both analytes Low medand high quality control (QC) samples were prepared at con-centrations of 3 50 and 75 120583gmL respectively 20 120583L of theprepared solutions were injected into the chromatographicsystem Standard calibration curves were constructed byplotting peak area against the corresponding concentrations

242 Human Urine Calibration standards were prepared inblank human urine at concentrations of 025 05 10 10 20and 25 120583gmL and of 05 10 10 20 and 25 120583gmL for TMZand ATN respectively Low med and high quality control

International Journal of Analytical Chemistry 3

Table 1 Analytical performance data for HPLC determination of atenolol and trimetazidine in pure form and urine samples

Parameter Atenolol TrimetazidinePure form Urine Pure form Urine

Linearity range (120583gml) 10ndash100 05-25 10ndash 100 025-25Correlation coefficient (r2) 09998 09999 09999 09995Regression equation Y=3915X-57394 Y=0037X-80947 Y=11806X-5805 Y=03464X+3183LOD (120583gml) 030 011 018 005LOQ (120583gml) 099 038 060 016

(QC) samples were prepared at concentrations of 05 10 and20 120583g mL of TMZ and 10 10 and 20 120583g mL of ATNSamples were stored at ndash20∘C extracted as described belowand calibration curves were constructed by plotting peak areaagainst concentrations

25 Preparation of Pharmaceutical Tablets Working Solutions

251 Metacardia Tablets Five tablets were weighedground and mixed well An accurately weighed amount ofpowdered tablets equivalent to 50 mg of trimetazidine wasextracted with 50 ml methanol and sonicated for 30 minutesThe solution was filtered and subsequent dilutions weremade in water for HPLC-PDA analysis

252 Blokium Tablets Five tablets were weighed groundand mixed together An accurately weighed amount of pow-dered tablets equivalent to 50 mg of atenolol was extractedwith 50 ml methanol and sonicated for 30 minutes Thesolution was filtered and the filtrate was subsequently dilutedwith water for HPLC analysis

26 Human Urine Sample Preparation Human urine sam-ples were thawed at room temperature and mixed well A20 mL aliquot of each sample was transferred into screw capculture tube 05 mL of 10 N NaOHwas added and the tubeswere vortex mixed for 30 seconds Samples were extracted byadding 60 ml of (41) chloroform n-butanol vv sampleswere vortex mixed for 10 minutes After centrifugation at3000 rpm for 10 min the organic layer was transferred intoclean tube and evaporated to dryness under a nitrogen streamat approximately 40∘C The residue was reconstituted with05 mL methanol vortex mixed briefly A 20 120583L of the finalextract was injected into the HPLC system

27 Chromatographic and Detection Conditions

271 Neat Standard Solutions and Pharmaceutical TabletsChromatographic separation was carried out on a Caltrex BI(125 times 40 mm 5 120583m) column Mobile phase A consisted of25mMpotassiumdihydrogen orthophosphate adjusted to pH33with orthophosphoric acidmobile phase B wasmethanoland mobile phase C was acetonitrile The autosampler rinsesolution was methanol A gradient method at a flow rate of10mLmin was used column temperature wasmaintained atRT and total run time was 10 0minutes The gradient elutionwas as follows 0-1 min 10 B and 0 C 11 min 20 B and

0 C 40 min 15 B and 15 C 41 min 20 B and 20C80 min 80B and 0 C and 81-100 min 10 B and 0 CPhotodiode array detector (PDA)was operated at 210 and 225nm for trimetazidine and atenolol respectively

272 Urine Samples Same conditions as described abovewere applied for the analysis of target analytes in urinesamples except the following the gradient was 0-20 min 10B and 0 C 21 min 20 B and 0 C 50 min 15 B and15 C 51 min 20 B and 20C 51-80 min 80B and 0C80-110min 90 B and 0C and 111-130min 10B and 0C Run time was 130 min

3 Results and Discussions

31 Method Development Target analytes exhibit differentabsorption characteristics so 210 and 225 nm were selectedfor detection of TMZ and ATN respectively All chromato-graphic parameters were fully optimized to achieve the bestchromatographic separation Both methanol and acetonitrilewere tested as organic modifiers in different proportionsusing both isocratic and gradient elution methanol showedhigher sensitivity but with asymmetric peak shape for bothanalytes A gradient elution using both methanol and ace-tonitrile (as described above) showed excellent symmetricpeak shape for both analytes Addition of buffer modifiers(using different mobile phase buffers) and effect of mobilephase pH (pH 3-5) have been studied in terms of peak shapeelution time and sensitivity Lower pHmobile phase showedrapid elution however pH 5 showed late elution for bothanalytes 25 mM phosphate buffer pH 33 was found to bethemost appropriate buffermodifier providing well-resolvedpeaks in reasonable elution time The flow rate range from05 to 10 mLmin was tested It was found that 10 mLminwas optimum for good chromatographic separation in areasonable time without interference from either pharma-ceutical preparation placebo components or biological fluidendogenous peaks Different gradient programs have beenapplied peak symmetry resolution selectivity and numberof theoretical plates were calculated in each case and summa-rized in Table 1 Figure 2 shows a typical chromatogram for alaboratory prepared mixture of the studied drugs under theoptimized chromatographic conditions The retention timesfor atenolol and trimetazidine were 372 and 43 minutesrespectively Due to the nature of biological fluid extracts andpresence of extracted endogenous components the gradienthas been slightly modified for urine sample analysis Longer

4 International Journal of Analytical Chemistry

Table 2 System suitability parameters for the determination of the atenolol and trimetazidine in pure formhuman urine

Parameters Atenolol Trimetazidine Reference value [39 40]Retention time (tR) 372432 431579 -----

Number of theoretical plates (N) 132112896 1393513668 gt2000increase with efficiency of separation

Tailing factor (T) 097185 071111 le2Capacity factor ldquoMass distribution ratiordquo (K1015840) 244232 298346 1-10 acceptableHeight equivalent to one theoretical plate (HETP) 001004 001001 The smaller the value the higher the column efficacy

Resolution (Rs) 162489 gt15Good separation between peaks of interest

Selectivity factor (120572) 111149 gt 1

1 2 3 4 5

mAU

0

20

40

60

80

10

86

37

19

43

07(A)

(B)

Figure 2 HPLC-PDA chromatogram of standard neat solution of(A) atenolol 10 120583gmL and (B) trimetazidine 10 120583gmL

0 2 4 6

mAU

050

100150200250300350400

09

53 1

049

11

08

15

52

26

97 43

18

52

36

57

95

63

36

(A)

(B)

Figure 3 HPLC-PDA chromatogram of (A) atenolol 25 120583gmL and(B) trimetazidine 25 120583gmL in urine sample

desalting step at high percentage of aqueous mobile phasewas applied to ensure complete removal of polar componentsbefore elution of target analytes Also the column wash stepusing high percentage organic mobile phase was extendedin order to elute any endogenous matrix components inthe final extracts and avoid any coelution of late elutingpeaks with target analytes in subsequent injections Figure 3represents the chromatogram of the analytes in urine samplesshowing that ATN and TMZ were eluted at 432 and 58minutes respectively and Figure 4 shows the blank urinechromatogram in which there was no interference at theelution times of our analytes

Liquid-liquid extraction using organic solvents has beenshown to provide clean extracts of biological fluid samplesit is also considered a cheaper sample treatment approachDifferent organic solvents (dichloromethane chloroform n-hexane ethyl acetate (11 vv) and chloroform n-butanol

2 4 6

mAU

0

100

200

300

400

09

56 1

052

11

15

15

70

27

65

45

13

52

78

57

18

63

92

Figure 4 HPLC-PDA chromatogram of blank urine

(41 vv)) have been evaluated for extraction recoveriesIt was found that 41 chloroform n-butanol vv not onlyprovided the highest extraction recoveries for both analytesbut also eliminated the interference of endogenous matrixcomponents pH of the sample is known to strongly affect theionization of different compounds and subsequently affectthe extracted portion of the analyte into the organic layeracidic and basic conditions using 10 N HCl and 10 NNaOH respectively and matrix samples without any pHoptimization (using water) have been compared in termsof extraction recoveries and cleanness of the final extractsBoth target drugs are basic compounds (of PKa 96 and 914for ATN [36] and TMZ [37] respectively) their extractionrecoveries were significantly improved by raising the pHof the sample with sodium hydroxide solutions Differentvolumes of 10 N NaOH were tested a 500 120583l aliquot of 10N NaOH provided the best extraction recoveries Extractionrecoveries were found to be 45 and 100 for ATN andTMZrespectively and were consistent against different levels forboth analytes

System suitability parameters such as resolution (Rs)tailing factor (T) capacity factor (K) and selectivity factor(120572) were calculated according to USP [2] as shown in Table 2The resolution was always more than 15 the selectivity wasmore than one and the symmetry factor had an acceptedvalue

Linearity Peak area responses were plotted against the cor-responding concentrations Linearity was obtained over aconcentration range of 10-100 120583gmL in pure form for bothanalytes and over concentration ranges of (025-25) 120583gmL

International Journal of Analytical Chemistry 5

Table 3 Statistical analysis of the proposed method in standard solution and the officialreported methods [1 38]

Atenolol TrimetazidineProposed Method Reported method [38]lowastlowast Proposed Method Official method [1]

Mean 10034 9966 10035 9839SD 117 087 106 077RSD 116 088 106 112Variance 136 076 113 059N 8 5 8 3F ndash test 18 (412)lowast - 109 (474)lowast -Studentrsquos t-test 111 (2201)lowast - 193 (2262)lowast -lowastFigures between parenthesis represent the corresponding tabulated values of t and F at P= 005lowastlowastSpectrophotometric method based on measurement of atenolol absorbance in 01 N HCl at 2246 nm

Table 4 Intra- and interday precision and accuracy calculated from quality control (QC) samples in pure form

QC conc (120583gmL) Intra-day Inter-dayMean (120583gmL) SD RSD DFN Mean (120583gmL) SD RSD DFN

ATN3 297 002 077 -112 301 005 17 02550 50 95 045 088 189 5017 041 082 03475 75004 012 017 0005 7636 029 039 182TMZ3 289 009 345 -367 288 009 302 -39950 5009 023 045 018 5007 026 052 01575 7714 131 17 285 7634 205 268 178

Table 5 Intra- and interday precision and accuracy calculated from quality control (QC) samples in human urine

Intra-day Inter-dayMean (120583gmL) SD RSD DFN Mean (120583gmL) SD RSD DFN

ATN10 100 002 176 008 099 003 285 -00110 992 007 073 -081 985 011 110 -15020 2002 025 126 011 2008 017 082 041TMZ05 050 0004 075 098 050 0004 069 09010 1019 001 098 19 1014 012 118 13720 1936 008 042 -319 1941 008 042 -294

and (05-25) 120583gmL in urine samples for trimetazidine andatenolol respectively The correlation coefficient was ge 0999as shown in Table 1

Limit of Detection and Limit of Quantitation The limit ofdetection (LOD) is the lowest concentration at which theanalyte can be detected The limit of quantitation (LOQ) isthe lowest concentration that can be quantitatively measuredwith acceptable precision and accuracy The LOD was estab-lished as the lowest concentration that provides a signal-to-noise (SN) ratio of 31 and the LOQ was established as thelowest concentration that provides SN ratio of 101 as givenin (Table 1)

Statistical Analysis for Pure Form Method The proposedmethod was compared to the official and reported methods

[1 38] statistically using Students t-test and variance ratio F-test at 95 confidence level The calculated t and F values didnot exceed the theoretical values No significant differencesbetween the proposed method and the official or reportedones have been observed as given in (Table 3)

Precision and Accuracy Inter- and intraday precision (mea-sured as percent relative standard deviation RSD) andinter- and intraday accuracy (measured as percent differencefrom the nominal concentration DFN) were measuredat low mid and high QC levels Excellent precision andaccuracy were obtained for both analytes in pure forms andurine samples (Tables 4 and 5)

Selectivity Selectivity is the ability of the analytical methodto determine the target compound in the presence of other

6 International Journal of Analytical Chemistry

Table 6 Application of the proposed method in standard solution on pharmaceutical tablets

Pharmaceutical Preparations Blokium tablets Metacardia tabletsTaken (120583gmL) Foundlowast (120583gmL) Recovery Taken (120583gmL) Foundlowast (120583gmL) Recovery

500 487 9744 500 473 94541000 960 9597 1000 974 97403000 2863 9542 3000 2850 95055000 4750 9501 5000 4915 98307500 7167 9556 7500 7124 949910000 9527 9527 10000 9536 9536

Mean 9578 9594SD 088 153SE 036 063RSD 091 160Variance 077 234lowastAverage of three determinations

components such as excipients degradation products coad-ministered drugs andor endogenous matrix componentswithout interferenceThe proposedmethod showed excellentchromatographic separation and quantitation of the citeddrugs in the presence of common tablet excipients and urineextracted matrix No significant peaks were detected at theexpected retention times of the two cited drugs and thereis no coelution with any of the other components that maypresent in the final extracts

Robustness Small variations in the experimental parameterswere applied and the effect of these variations on the analyti-cal performance was evaluated to ensure the robustness of theproposed method The studied parameters were as followsphosphate buffer pH (33plusmn 02) flow rate (10plusmn 001)mLminand wavelength 210 nm plusmn10 and 225 nm plusmn10 for TMZ andATN respectively Only one parameter was changed in atime and the others were kept unchanged The effects of theproposed changes on retention time peak area tailing factorand number of theoretical plates were evaluated It was foundthat all the studied minor changes have no significant effectson the method performance

Applications

Pharmaceutical Tablets The optimized HPLC method wassuccessfully applied for determination of the studied drugsin their corresponding tablets Good percentage recoverieswithout any excipients interference were obtained (Table 6)the proposed method can be applied for quality controlstudies

4 Conclusion

RP-HPLCmethod with PDA detection was developed for thedetermination of atenolol and trimetazidine in pure formpharmaceutical tablets and human urine The proposedmethod was found to be simple sensitive and accurate Theoptimized chromatographic conditions allowed separation ofthe studied drugs in reasonable time without any interferencefrom excipients andor extracted matrix components This

method can be used for therapeutic drug monitoring ofpatients treated with ATN and TMZ concomitantly and alsocan be applied for quality control analysis of the studied drugsin their pharmaceutical tablets

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

References

[1] The Biritish Pharmacopeia Her Majesty stationary office Lon-don UK 2017 II Appendix I D

[2] United States Pharmacopoeia (USP) 36 National Formulary 31US Pharmacoppieal Convention Rockville Md USA 2013

[3] M Zeeb H Farahani and M K Papan ldquoDetermination ofatenolol in human plasma using ionic-liquid-based ultrasound-assisted in situ solvent formation microextraction followed byhigh-performance liquid chromatographyrdquo Journal of Separa-tion Science vol 39 no 11 pp 2138ndash2145 2016

[4] S C Sweetman Martindale The Complete Drug Reference volAThe Pharmaceutical Press London UK 36th edition 2009

[5] L Ding B Gong X Chu J Hu and H Zheng ldquoSensi-tive and rapid LC-ESI-MS method for the determination oftrimetazidine in human plasmardquo Journal of Pharmaceutical andBiomedical Analysis vol 44 no 2 pp 526ndash531 2007

[6] Y Jiao M Su M Chen et al ldquoLCESI-MS method for thedetermination of trimetazidine in human plasma Applicationto a bioequivalence study on Chinese volunteersrdquo Journal ofPharmaceutical and Biomedical Analysis vol 43 no 5 pp 1804ndash1807 2007

[7] C A Dezsi ldquoTrimetazidine in practice Review of the clinicaland experimental evidencerdquo American Journal of Therapeuticsvol 23 no 3 pp e871ndashe879 2016

[8] R Bennis Nechba M El MrsquoBarki Kadiri M Bennani-ZiatniA A Zeggwagh and A Mesfioui ldquoDifficulty in managing

International Journal of Analytical Chemistry 7

polypharmacy in the elderly Case report and review of theliteraturerdquo Journal of Clinical Gerontology and Geriatrics vol 6no 1 pp 30ndash33 2015

[9] N Siddharth Shah and S Ambardekar ldquoAtenolol Interac-tion with Other Drugsrdquo Supplement Of JAPI vol 57 2009httpwwwjapiorgspecial issue 2009article 08pdf

[10] I Baranowska P Markowski and J Baranowski ldquoDevelopmentand validation of an HPLC method for the simultaneousanalysis of 23 selected drugs belonging to different therapeuticgroups in human urine samplesrdquoAnalytical Sciences vol 25 no11 pp 1307ndash1313 2009

[11] B Yilmaz and S Arslan ldquoDetermination of atenolol in humanurine by using HPLCrdquo Separation Science Plus vol 1 no 1 pp4ndash10 2018

[12] B Yilmaz and S Arslan ldquoDetermination of atenolol in humanurine by gas chromatography-mass spectrometry methodrdquoJournal of Chromatographic Science (JCS) vol 49 no 5 pp 365ndash369 2011

[13] N S Abdelwahab ldquoDetermination of atenolol chlorthalidoneand their degradation products by TLC-densitometric andchemometric methods with application of model updatingrdquoAnalytical Methods vol 2 no 12 pp 1994ndash2001 2010

[14] G C Hiremath R M Mulla and S T Nandibewoor ldquoKineticmethods of determination of atenolol in pure compound and inpharmaceutical formulationsrdquo Chemia Analityczna vol 50 no2 pp 449ndash455 2005

[15] A M El-didamony andM A Moustafa ldquoDirect spectrophoto-metric determination of atenolol and timolol anti-hypertensivedrugsrdquo International Journal of Pharmacy and PharmaceuticalSciences vol 9 no 3 pp 47ndash53 2017

[16] E Bakir M Gouda A Alnajjar and W E Boraie ldquoSpectroflu-orimetric method for atenolol determination based on goldnanoparticlesrdquo Acta Pharmaceutica vol 68 no 2 pp 243ndash2502018

[17] K N Prashanth K Basavaiah M S Raghu and K B VinayldquoDetermination of atenolol and its preparations by acid-basetitration in non-aqueous mediumrdquo Der Pharmacia Lettre vol4 no 5 pp 1534ndash1540 2012

[18] J Scremin and E R Sartori ldquoSimultaneous determination ofnifedipine and atenolol in combined dosage forms using aboron-doped diamond electrodewith differential pulse voltam-metryrdquo Canadian Journal of Chemistry vol 96 no 1 pp 1ndash72018

[19] D Li J Du and J Lu ldquoChemiluminescence determina-tion of atenolol in biological fluids by a europium-sensitizedpermanganate-sulfite systemrdquo Microchimica Acta vol 161 no1-2 pp 169ndash173 2008

[20] J Huang J Sun X Zhou and T You ldquoDetermination ofatenolol and metoprolol by capillary electrophoresis withtris(221015840-bipyridyl)ruthenium(II) electrochemiluminescencedetectionrdquo Analytical Sciences vol 23 no 2 pp 183ndash188 2007

[21] P M Harrison A M Tonkin and A J McLean ldquoSimple andrapid analysis of atenolol andmetoprolol in plasma using solid-phase extraction and high-performance liquid chromatogra-phyrdquo Journal of Chromatography B Biomedical Sciences andApplications vol 339 no C pp 429ndash433 1985

[22] E M Phyo Lwin C Gerber Y Song et al ldquoA new LC-MSMSbioanalytical method for atenolol in human plasma and milkrdquoBioanalysis vol 9 no 7 pp 517ndash530 2017

[23] M K Jeoung K S Kim C S Kim et al ldquoAn HPLC Determi-nation of Trimetazidine inHuman Plasma Using Liquid-Liquid

Extraction for Sample Clean-Uprdquo Journal of Liquid Chromatog-raphy ampRelated Technologies vol 28 no 9 pp 1299ndash1309 2005

[24] F Hasin S Noon H Al Hasan and T Jahan ldquoValidation ofhigh performance liquid chromatography (HPLC) method fordetermination of erlotinib related substance in pharmaceuticaldosage formrdquo European Journal of Biomedical and Pharmaceu-tical Sciences (EJBPS) vol 4 no 7 pp 66ndash73 2017

[25] Jefri A D Puspitasari J S Talpaneni and R R Tjandraw-inata ldquoIdentification and control of unspecified impurity intrimetazidine dihydrochloride tablet formulationrdquo IOP Confer-ence Series Materials Science and Engineering vol 349 ArticleID 012029 2018

[26] L Fay G Michel P Goupit C Harpey and M ProstldquoDetermination of trimetazidine in biological fluids by gaschromatography-mass spectrometryrdquo Journal of Chromatogra-phy B Biomedical Sciences and Applications vol 490 no C pp198ndash205 1989

[27] T S Belal T Awad and C R Clark ldquoStability-indicatingdetermination of trimetazidine dihydrochloride in the presenceof two of its related substances using a direct GCMS methodrdquoJournal of AOAC International vol 97 no 6 pp 1514ndash1518 2014

[28] S O Thoppil R M Cardoza and P D Amin ldquoStabilityindicating HPTLC determination of trimetazidine as bulk drugand in pharmaceutical formulationsrdquo Journal of Pharmaceuticaland Biomedical Analysis vol 25 no 1 pp 15ndash20 2001

[29] S Agrawal P Gurjar and B Katheriya ldquoAnalytical methoddevelopment and validation for simultaneous estimation ofTrimetazidine hydrochloride and Metoprolol succinate usingHPTLCrdquo Current Pharmaceutical Analysis vol 13 2017

[30] A A Chis F G Gligor G Cormos E Curea and MBojita ldquoSpectrophotometric method for the determination oftrimetazidine dihydrochloride from pharmaceutical formsrdquoFarmacia vol 58 no 5 pp 629ndash636 2010

[31] Md F Hasin Md H Mukhlasy Md I Hussain and MdM Hossain ldquoValidation of spectrophotometric dissolutionmethod for modified release Trimetazidine pharmaceuticaldosage formrdquo JIPBS vol 4 no 2 pp 68ndash73 2017

[32] M S Abd-Elmonem A S M Eltantawy and E Khaled ldquoAlphacyclodextrin-based trimetazidine potentiometric sensorsrdquo IndoAmerican Journal of Pharmaceutical Research vol 6 no 8 2016

[33] B Dogan-Topal A Sibel and B Uslu ldquoOzkan and Bengi UsluThe Analytical Applications of Square Wave Voltammetry onPharmaceutical Analysisrdquo The Open Chemical and BiomedicalMethods Journal vol 3 pp 56ndash73 2010

[34] J Li J Quan J Du and M Liu ldquoChemiluminescence deter-mination of trimetazidine via inducing the aggregation ofgold nanoparticlesrdquo Spectrochimica Acta Part A Molecular andBiomolecular Spectroscopy vol 114 pp 33ndash37 2013

[35] HNMistri AG Jangid andP Shrivastav ldquoSensitive and rapidmethod to determine trimetazidine in human plasma by liquidchromatographytandemmass spectrometryrdquo Journal of AOACInternational vol 91 no 3 pp 562ndash571 2008

[36] V Martınez M I Maguregui R M Jimenez and R MAlonso ldquoDetermination of the pK(a) values of 120573-blockers byautomatedpotentiometric titrationsrdquo Journal of Pharmaceuticaland Biomedical Analysis vol 23 no 2-3 pp 459ndash468 2000

[37] V V Mohylyuk and L L Davtian ldquoInteraction of weak basedrug trimetazidine and carbopol as further retardation in thematrix tabletrdquoActa Poloniae Pharmaceutica Drug Research vol72 no 6 pp 1259ndash1261 2015

[38] J H A Girdhari P Prabhakara K Pankaj K Annu and KMarina ldquoSimultaneous Estimation of Atenolol and Amlodipine

International Journal of Analytical Chemistry 3

Table 1 Analytical performance data for HPLC determination of atenolol and trimetazidine in pure form and urine samples

Parameter Atenolol TrimetazidinePure form Urine Pure form Urine

Linearity range (120583gml) 10ndash100 05-25 10ndash 100 025-25Correlation coefficient (r2) 09998 09999 09999 09995Regression equation Y=3915X-57394 Y=0037X-80947 Y=11806X-5805 Y=03464X+3183LOD (120583gml) 030 011 018 005LOQ (120583gml) 099 038 060 016

(QC) samples were prepared at concentrations of 05 10 and20 120583g mL of TMZ and 10 10 and 20 120583g mL of ATNSamples were stored at ndash20∘C extracted as described belowand calibration curves were constructed by plotting peak areaagainst concentrations

25 Preparation of Pharmaceutical Tablets Working Solutions

251 Metacardia Tablets Five tablets were weighedground and mixed well An accurately weighed amount ofpowdered tablets equivalent to 50 mg of trimetazidine wasextracted with 50 ml methanol and sonicated for 30 minutesThe solution was filtered and subsequent dilutions weremade in water for HPLC-PDA analysis

252 Blokium Tablets Five tablets were weighed groundand mixed together An accurately weighed amount of pow-dered tablets equivalent to 50 mg of atenolol was extractedwith 50 ml methanol and sonicated for 30 minutes Thesolution was filtered and the filtrate was subsequently dilutedwith water for HPLC analysis

26 Human Urine Sample Preparation Human urine sam-ples were thawed at room temperature and mixed well A20 mL aliquot of each sample was transferred into screw capculture tube 05 mL of 10 N NaOHwas added and the tubeswere vortex mixed for 30 seconds Samples were extracted byadding 60 ml of (41) chloroform n-butanol vv sampleswere vortex mixed for 10 minutes After centrifugation at3000 rpm for 10 min the organic layer was transferred intoclean tube and evaporated to dryness under a nitrogen streamat approximately 40∘C The residue was reconstituted with05 mL methanol vortex mixed briefly A 20 120583L of the finalextract was injected into the HPLC system

27 Chromatographic and Detection Conditions

271 Neat Standard Solutions and Pharmaceutical TabletsChromatographic separation was carried out on a Caltrex BI(125 times 40 mm 5 120583m) column Mobile phase A consisted of25mMpotassiumdihydrogen orthophosphate adjusted to pH33with orthophosphoric acidmobile phase B wasmethanoland mobile phase C was acetonitrile The autosampler rinsesolution was methanol A gradient method at a flow rate of10mLmin was used column temperature wasmaintained atRT and total run time was 10 0minutes The gradient elutionwas as follows 0-1 min 10 B and 0 C 11 min 20 B and

0 C 40 min 15 B and 15 C 41 min 20 B and 20C80 min 80B and 0 C and 81-100 min 10 B and 0 CPhotodiode array detector (PDA)was operated at 210 and 225nm for trimetazidine and atenolol respectively

272 Urine Samples Same conditions as described abovewere applied for the analysis of target analytes in urinesamples except the following the gradient was 0-20 min 10B and 0 C 21 min 20 B and 0 C 50 min 15 B and15 C 51 min 20 B and 20C 51-80 min 80B and 0C80-110min 90 B and 0C and 111-130min 10B and 0C Run time was 130 min

3 Results and Discussions

31 Method Development Target analytes exhibit differentabsorption characteristics so 210 and 225 nm were selectedfor detection of TMZ and ATN respectively All chromato-graphic parameters were fully optimized to achieve the bestchromatographic separation Both methanol and acetonitrilewere tested as organic modifiers in different proportionsusing both isocratic and gradient elution methanol showedhigher sensitivity but with asymmetric peak shape for bothanalytes A gradient elution using both methanol and ace-tonitrile (as described above) showed excellent symmetricpeak shape for both analytes Addition of buffer modifiers(using different mobile phase buffers) and effect of mobilephase pH (pH 3-5) have been studied in terms of peak shapeelution time and sensitivity Lower pHmobile phase showedrapid elution however pH 5 showed late elution for bothanalytes 25 mM phosphate buffer pH 33 was found to bethemost appropriate buffermodifier providing well-resolvedpeaks in reasonable elution time The flow rate range from05 to 10 mLmin was tested It was found that 10 mLminwas optimum for good chromatographic separation in areasonable time without interference from either pharma-ceutical preparation placebo components or biological fluidendogenous peaks Different gradient programs have beenapplied peak symmetry resolution selectivity and numberof theoretical plates were calculated in each case and summa-rized in Table 1 Figure 2 shows a typical chromatogram for alaboratory prepared mixture of the studied drugs under theoptimized chromatographic conditions The retention timesfor atenolol and trimetazidine were 372 and 43 minutesrespectively Due to the nature of biological fluid extracts andpresence of extracted endogenous components the gradienthas been slightly modified for urine sample analysis Longer

4 International Journal of Analytical Chemistry

Table 2 System suitability parameters for the determination of the atenolol and trimetazidine in pure formhuman urine

Parameters Atenolol Trimetazidine Reference value [39 40]Retention time (tR) 372432 431579 -----

Number of theoretical plates (N) 132112896 1393513668 gt2000increase with efficiency of separation

Tailing factor (T) 097185 071111 le2Capacity factor ldquoMass distribution ratiordquo (K1015840) 244232 298346 1-10 acceptableHeight equivalent to one theoretical plate (HETP) 001004 001001 The smaller the value the higher the column efficacy

Resolution (Rs) 162489 gt15Good separation between peaks of interest

Selectivity factor (120572) 111149 gt 1

1 2 3 4 5

mAU

0

20

40

60

80

10

86

37

19

43

07(A)

(B)

Figure 2 HPLC-PDA chromatogram of standard neat solution of(A) atenolol 10 120583gmL and (B) trimetazidine 10 120583gmL

0 2 4 6

mAU

050

100150200250300350400

09

53 1

049

11

08

15

52

26

97 43

18

52

36

57

95

63

36

(A)

(B)

Figure 3 HPLC-PDA chromatogram of (A) atenolol 25 120583gmL and(B) trimetazidine 25 120583gmL in urine sample

desalting step at high percentage of aqueous mobile phasewas applied to ensure complete removal of polar componentsbefore elution of target analytes Also the column wash stepusing high percentage organic mobile phase was extendedin order to elute any endogenous matrix components inthe final extracts and avoid any coelution of late elutingpeaks with target analytes in subsequent injections Figure 3represents the chromatogram of the analytes in urine samplesshowing that ATN and TMZ were eluted at 432 and 58minutes respectively and Figure 4 shows the blank urinechromatogram in which there was no interference at theelution times of our analytes

Liquid-liquid extraction using organic solvents has beenshown to provide clean extracts of biological fluid samplesit is also considered a cheaper sample treatment approachDifferent organic solvents (dichloromethane chloroform n-hexane ethyl acetate (11 vv) and chloroform n-butanol

2 4 6

mAU

0

100

200

300

400

09

56 1

052

11

15

15

70

27

65

45

13

52

78

57

18

63

92

Figure 4 HPLC-PDA chromatogram of blank urine

(41 vv)) have been evaluated for extraction recoveriesIt was found that 41 chloroform n-butanol vv not onlyprovided the highest extraction recoveries for both analytesbut also eliminated the interference of endogenous matrixcomponents pH of the sample is known to strongly affect theionization of different compounds and subsequently affectthe extracted portion of the analyte into the organic layeracidic and basic conditions using 10 N HCl and 10 NNaOH respectively and matrix samples without any pHoptimization (using water) have been compared in termsof extraction recoveries and cleanness of the final extractsBoth target drugs are basic compounds (of PKa 96 and 914for ATN [36] and TMZ [37] respectively) their extractionrecoveries were significantly improved by raising the pHof the sample with sodium hydroxide solutions Differentvolumes of 10 N NaOH were tested a 500 120583l aliquot of 10N NaOH provided the best extraction recoveries Extractionrecoveries were found to be 45 and 100 for ATN andTMZrespectively and were consistent against different levels forboth analytes

System suitability parameters such as resolution (Rs)tailing factor (T) capacity factor (K) and selectivity factor(120572) were calculated according to USP [2] as shown in Table 2The resolution was always more than 15 the selectivity wasmore than one and the symmetry factor had an acceptedvalue

Linearity Peak area responses were plotted against the cor-responding concentrations Linearity was obtained over aconcentration range of 10-100 120583gmL in pure form for bothanalytes and over concentration ranges of (025-25) 120583gmL

International Journal of Analytical Chemistry 5

Table 3 Statistical analysis of the proposed method in standard solution and the officialreported methods [1 38]

Atenolol TrimetazidineProposed Method Reported method [38]lowastlowast Proposed Method Official method [1]

Mean 10034 9966 10035 9839SD 117 087 106 077RSD 116 088 106 112Variance 136 076 113 059N 8 5 8 3F ndash test 18 (412)lowast - 109 (474)lowast -Studentrsquos t-test 111 (2201)lowast - 193 (2262)lowast -lowastFigures between parenthesis represent the corresponding tabulated values of t and F at P= 005lowastlowastSpectrophotometric method based on measurement of atenolol absorbance in 01 N HCl at 2246 nm

Table 4 Intra- and interday precision and accuracy calculated from quality control (QC) samples in pure form

QC conc (120583gmL) Intra-day Inter-dayMean (120583gmL) SD RSD DFN Mean (120583gmL) SD RSD DFN

ATN3 297 002 077 -112 301 005 17 02550 50 95 045 088 189 5017 041 082 03475 75004 012 017 0005 7636 029 039 182TMZ3 289 009 345 -367 288 009 302 -39950 5009 023 045 018 5007 026 052 01575 7714 131 17 285 7634 205 268 178

Table 5 Intra- and interday precision and accuracy calculated from quality control (QC) samples in human urine

Intra-day Inter-dayMean (120583gmL) SD RSD DFN Mean (120583gmL) SD RSD DFN

ATN10 100 002 176 008 099 003 285 -00110 992 007 073 -081 985 011 110 -15020 2002 025 126 011 2008 017 082 041TMZ05 050 0004 075 098 050 0004 069 09010 1019 001 098 19 1014 012 118 13720 1936 008 042 -319 1941 008 042 -294

and (05-25) 120583gmL in urine samples for trimetazidine andatenolol respectively The correlation coefficient was ge 0999as shown in Table 1

Limit of Detection and Limit of Quantitation The limit ofdetection (LOD) is the lowest concentration at which theanalyte can be detected The limit of quantitation (LOQ) isthe lowest concentration that can be quantitatively measuredwith acceptable precision and accuracy The LOD was estab-lished as the lowest concentration that provides a signal-to-noise (SN) ratio of 31 and the LOQ was established as thelowest concentration that provides SN ratio of 101 as givenin (Table 1)

Statistical Analysis for Pure Form Method The proposedmethod was compared to the official and reported methods

[1 38] statistically using Students t-test and variance ratio F-test at 95 confidence level The calculated t and F values didnot exceed the theoretical values No significant differencesbetween the proposed method and the official or reportedones have been observed as given in (Table 3)

Precision and Accuracy Inter- and intraday precision (mea-sured as percent relative standard deviation RSD) andinter- and intraday accuracy (measured as percent differencefrom the nominal concentration DFN) were measuredat low mid and high QC levels Excellent precision andaccuracy were obtained for both analytes in pure forms andurine samples (Tables 4 and 5)

Selectivity Selectivity is the ability of the analytical methodto determine the target compound in the presence of other

6 International Journal of Analytical Chemistry

Table 6 Application of the proposed method in standard solution on pharmaceutical tablets

Pharmaceutical Preparations Blokium tablets Metacardia tabletsTaken (120583gmL) Foundlowast (120583gmL) Recovery Taken (120583gmL) Foundlowast (120583gmL) Recovery

500 487 9744 500 473 94541000 960 9597 1000 974 97403000 2863 9542 3000 2850 95055000 4750 9501 5000 4915 98307500 7167 9556 7500 7124 949910000 9527 9527 10000 9536 9536

Mean 9578 9594SD 088 153SE 036 063RSD 091 160Variance 077 234lowastAverage of three determinations

components such as excipients degradation products coad-ministered drugs andor endogenous matrix componentswithout interferenceThe proposedmethod showed excellentchromatographic separation and quantitation of the citeddrugs in the presence of common tablet excipients and urineextracted matrix No significant peaks were detected at theexpected retention times of the two cited drugs and thereis no coelution with any of the other components that maypresent in the final extracts

Robustness Small variations in the experimental parameterswere applied and the effect of these variations on the analyti-cal performance was evaluated to ensure the robustness of theproposed method The studied parameters were as followsphosphate buffer pH (33plusmn 02) flow rate (10plusmn 001)mLminand wavelength 210 nm plusmn10 and 225 nm plusmn10 for TMZ andATN respectively Only one parameter was changed in atime and the others were kept unchanged The effects of theproposed changes on retention time peak area tailing factorand number of theoretical plates were evaluated It was foundthat all the studied minor changes have no significant effectson the method performance

Applications

Pharmaceutical Tablets The optimized HPLC method wassuccessfully applied for determination of the studied drugsin their corresponding tablets Good percentage recoverieswithout any excipients interference were obtained (Table 6)the proposed method can be applied for quality controlstudies

4 Conclusion

RP-HPLCmethod with PDA detection was developed for thedetermination of atenolol and trimetazidine in pure formpharmaceutical tablets and human urine The proposedmethod was found to be simple sensitive and accurate Theoptimized chromatographic conditions allowed separation ofthe studied drugs in reasonable time without any interferencefrom excipients andor extracted matrix components This

method can be used for therapeutic drug monitoring ofpatients treated with ATN and TMZ concomitantly and alsocan be applied for quality control analysis of the studied drugsin their pharmaceutical tablets

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

References

[1] The Biritish Pharmacopeia Her Majesty stationary office Lon-don UK 2017 II Appendix I D

[2] United States Pharmacopoeia (USP) 36 National Formulary 31US Pharmacoppieal Convention Rockville Md USA 2013

[3] M Zeeb H Farahani and M K Papan ldquoDetermination ofatenolol in human plasma using ionic-liquid-based ultrasound-assisted in situ solvent formation microextraction followed byhigh-performance liquid chromatographyrdquo Journal of Separa-tion Science vol 39 no 11 pp 2138ndash2145 2016

[4] S C Sweetman Martindale The Complete Drug Reference volAThe Pharmaceutical Press London UK 36th edition 2009

[5] L Ding B Gong X Chu J Hu and H Zheng ldquoSensi-tive and rapid LC-ESI-MS method for the determination oftrimetazidine in human plasmardquo Journal of Pharmaceutical andBiomedical Analysis vol 44 no 2 pp 526ndash531 2007

[6] Y Jiao M Su M Chen et al ldquoLCESI-MS method for thedetermination of trimetazidine in human plasma Applicationto a bioequivalence study on Chinese volunteersrdquo Journal ofPharmaceutical and Biomedical Analysis vol 43 no 5 pp 1804ndash1807 2007

[7] C A Dezsi ldquoTrimetazidine in practice Review of the clinicaland experimental evidencerdquo American Journal of Therapeuticsvol 23 no 3 pp e871ndashe879 2016

[8] R Bennis Nechba M El MrsquoBarki Kadiri M Bennani-ZiatniA A Zeggwagh and A Mesfioui ldquoDifficulty in managing

International Journal of Analytical Chemistry 7

polypharmacy in the elderly Case report and review of theliteraturerdquo Journal of Clinical Gerontology and Geriatrics vol 6no 1 pp 30ndash33 2015

[9] N Siddharth Shah and S Ambardekar ldquoAtenolol Interac-tion with Other Drugsrdquo Supplement Of JAPI vol 57 2009httpwwwjapiorgspecial issue 2009article 08pdf

[10] I Baranowska P Markowski and J Baranowski ldquoDevelopmentand validation of an HPLC method for the simultaneousanalysis of 23 selected drugs belonging to different therapeuticgroups in human urine samplesrdquoAnalytical Sciences vol 25 no11 pp 1307ndash1313 2009

[11] B Yilmaz and S Arslan ldquoDetermination of atenolol in humanurine by using HPLCrdquo Separation Science Plus vol 1 no 1 pp4ndash10 2018

[12] B Yilmaz and S Arslan ldquoDetermination of atenolol in humanurine by gas chromatography-mass spectrometry methodrdquoJournal of Chromatographic Science (JCS) vol 49 no 5 pp 365ndash369 2011

[13] N S Abdelwahab ldquoDetermination of atenolol chlorthalidoneand their degradation products by TLC-densitometric andchemometric methods with application of model updatingrdquoAnalytical Methods vol 2 no 12 pp 1994ndash2001 2010

[14] G C Hiremath R M Mulla and S T Nandibewoor ldquoKineticmethods of determination of atenolol in pure compound and inpharmaceutical formulationsrdquo Chemia Analityczna vol 50 no2 pp 449ndash455 2005

[15] A M El-didamony andM A Moustafa ldquoDirect spectrophoto-metric determination of atenolol and timolol anti-hypertensivedrugsrdquo International Journal of Pharmacy and PharmaceuticalSciences vol 9 no 3 pp 47ndash53 2017

[16] E Bakir M Gouda A Alnajjar and W E Boraie ldquoSpectroflu-orimetric method for atenolol determination based on goldnanoparticlesrdquo Acta Pharmaceutica vol 68 no 2 pp 243ndash2502018

[17] K N Prashanth K Basavaiah M S Raghu and K B VinayldquoDetermination of atenolol and its preparations by acid-basetitration in non-aqueous mediumrdquo Der Pharmacia Lettre vol4 no 5 pp 1534ndash1540 2012

[18] J Scremin and E R Sartori ldquoSimultaneous determination ofnifedipine and atenolol in combined dosage forms using aboron-doped diamond electrodewith differential pulse voltam-metryrdquo Canadian Journal of Chemistry vol 96 no 1 pp 1ndash72018

[19] D Li J Du and J Lu ldquoChemiluminescence determina-tion of atenolol in biological fluids by a europium-sensitizedpermanganate-sulfite systemrdquo Microchimica Acta vol 161 no1-2 pp 169ndash173 2008

[20] J Huang J Sun X Zhou and T You ldquoDetermination ofatenolol and metoprolol by capillary electrophoresis withtris(221015840-bipyridyl)ruthenium(II) electrochemiluminescencedetectionrdquo Analytical Sciences vol 23 no 2 pp 183ndash188 2007

[21] P M Harrison A M Tonkin and A J McLean ldquoSimple andrapid analysis of atenolol andmetoprolol in plasma using solid-phase extraction and high-performance liquid chromatogra-phyrdquo Journal of Chromatography B Biomedical Sciences andApplications vol 339 no C pp 429ndash433 1985

[22] E M Phyo Lwin C Gerber Y Song et al ldquoA new LC-MSMSbioanalytical method for atenolol in human plasma and milkrdquoBioanalysis vol 9 no 7 pp 517ndash530 2017

[23] M K Jeoung K S Kim C S Kim et al ldquoAn HPLC Determi-nation of Trimetazidine inHuman Plasma Using Liquid-Liquid

Extraction for Sample Clean-Uprdquo Journal of Liquid Chromatog-raphy ampRelated Technologies vol 28 no 9 pp 1299ndash1309 2005

[24] F Hasin S Noon H Al Hasan and T Jahan ldquoValidation ofhigh performance liquid chromatography (HPLC) method fordetermination of erlotinib related substance in pharmaceuticaldosage formrdquo European Journal of Biomedical and Pharmaceu-tical Sciences (EJBPS) vol 4 no 7 pp 66ndash73 2017

[25] Jefri A D Puspitasari J S Talpaneni and R R Tjandraw-inata ldquoIdentification and control of unspecified impurity intrimetazidine dihydrochloride tablet formulationrdquo IOP Confer-ence Series Materials Science and Engineering vol 349 ArticleID 012029 2018

[26] L Fay G Michel P Goupit C Harpey and M ProstldquoDetermination of trimetazidine in biological fluids by gaschromatography-mass spectrometryrdquo Journal of Chromatogra-phy B Biomedical Sciences and Applications vol 490 no C pp198ndash205 1989

[27] T S Belal T Awad and C R Clark ldquoStability-indicatingdetermination of trimetazidine dihydrochloride in the presenceof two of its related substances using a direct GCMS methodrdquoJournal of AOAC International vol 97 no 6 pp 1514ndash1518 2014

[28] S O Thoppil R M Cardoza and P D Amin ldquoStabilityindicating HPTLC determination of trimetazidine as bulk drugand in pharmaceutical formulationsrdquo Journal of Pharmaceuticaland Biomedical Analysis vol 25 no 1 pp 15ndash20 2001

[29] S Agrawal P Gurjar and B Katheriya ldquoAnalytical methoddevelopment and validation for simultaneous estimation ofTrimetazidine hydrochloride and Metoprolol succinate usingHPTLCrdquo Current Pharmaceutical Analysis vol 13 2017

[30] A A Chis F G Gligor G Cormos E Curea and MBojita ldquoSpectrophotometric method for the determination oftrimetazidine dihydrochloride from pharmaceutical formsrdquoFarmacia vol 58 no 5 pp 629ndash636 2010

[31] Md F Hasin Md H Mukhlasy Md I Hussain and MdM Hossain ldquoValidation of spectrophotometric dissolutionmethod for modified release Trimetazidine pharmaceuticaldosage formrdquo JIPBS vol 4 no 2 pp 68ndash73 2017

[32] M S Abd-Elmonem A S M Eltantawy and E Khaled ldquoAlphacyclodextrin-based trimetazidine potentiometric sensorsrdquo IndoAmerican Journal of Pharmaceutical Research vol 6 no 8 2016

[33] B Dogan-Topal A Sibel and B Uslu ldquoOzkan and Bengi UsluThe Analytical Applications of Square Wave Voltammetry onPharmaceutical Analysisrdquo The Open Chemical and BiomedicalMethods Journal vol 3 pp 56ndash73 2010

[34] J Li J Quan J Du and M Liu ldquoChemiluminescence deter-mination of trimetazidine via inducing the aggregation ofgold nanoparticlesrdquo Spectrochimica Acta Part A Molecular andBiomolecular Spectroscopy vol 114 pp 33ndash37 2013

[35] HNMistri AG Jangid andP Shrivastav ldquoSensitive and rapidmethod to determine trimetazidine in human plasma by liquidchromatographytandemmass spectrometryrdquo Journal of AOACInternational vol 91 no 3 pp 562ndash571 2008

[36] V Martınez M I Maguregui R M Jimenez and R MAlonso ldquoDetermination of the pK(a) values of 120573-blockers byautomatedpotentiometric titrationsrdquo Journal of Pharmaceuticaland Biomedical Analysis vol 23 no 2-3 pp 459ndash468 2000

[37] V V Mohylyuk and L L Davtian ldquoInteraction of weak basedrug trimetazidine and carbopol as further retardation in thematrix tabletrdquoActa Poloniae Pharmaceutica Drug Research vol72 no 6 pp 1259ndash1261 2015

[38] J H A Girdhari P Prabhakara K Pankaj K Annu and KMarina ldquoSimultaneous Estimation of Atenolol and Amlodipine

4 International Journal of Analytical Chemistry

Table 2 System suitability parameters for the determination of the atenolol and trimetazidine in pure formhuman urine

Parameters Atenolol Trimetazidine Reference value [39 40]Retention time (tR) 372432 431579 -----

Number of theoretical plates (N) 132112896 1393513668 gt2000increase with efficiency of separation

Tailing factor (T) 097185 071111 le2Capacity factor ldquoMass distribution ratiordquo (K1015840) 244232 298346 1-10 acceptableHeight equivalent to one theoretical plate (HETP) 001004 001001 The smaller the value the higher the column efficacy

Resolution (Rs) 162489 gt15Good separation between peaks of interest

Selectivity factor (120572) 111149 gt 1

1 2 3 4 5

mAU

0

20

40

60

80

10

86

37

19

43

07(A)

(B)

Figure 2 HPLC-PDA chromatogram of standard neat solution of(A) atenolol 10 120583gmL and (B) trimetazidine 10 120583gmL

0 2 4 6

mAU

050

100150200250300350400

09

53 1

049

11

08

15

52

26

97 43

18

52

36

57

95

63

36

(A)

(B)

Figure 3 HPLC-PDA chromatogram of (A) atenolol 25 120583gmL and(B) trimetazidine 25 120583gmL in urine sample

desalting step at high percentage of aqueous mobile phasewas applied to ensure complete removal of polar componentsbefore elution of target analytes Also the column wash stepusing high percentage organic mobile phase was extendedin order to elute any endogenous matrix components inthe final extracts and avoid any coelution of late elutingpeaks with target analytes in subsequent injections Figure 3represents the chromatogram of the analytes in urine samplesshowing that ATN and TMZ were eluted at 432 and 58minutes respectively and Figure 4 shows the blank urinechromatogram in which there was no interference at theelution times of our analytes

Liquid-liquid extraction using organic solvents has beenshown to provide clean extracts of biological fluid samplesit is also considered a cheaper sample treatment approachDifferent organic solvents (dichloromethane chloroform n-hexane ethyl acetate (11 vv) and chloroform n-butanol

2 4 6

mAU

0

100

200

300

400

09

56 1

052

11

15

15

70

27

65

45

13

52

78

57

18

63

92

Figure 4 HPLC-PDA chromatogram of blank urine

(41 vv)) have been evaluated for extraction recoveriesIt was found that 41 chloroform n-butanol vv not onlyprovided the highest extraction recoveries for both analytesbut also eliminated the interference of endogenous matrixcomponents pH of the sample is known to strongly affect theionization of different compounds and subsequently affectthe extracted portion of the analyte into the organic layeracidic and basic conditions using 10 N HCl and 10 NNaOH respectively and matrix samples without any pHoptimization (using water) have been compared in termsof extraction recoveries and cleanness of the final extractsBoth target drugs are basic compounds (of PKa 96 and 914for ATN [36] and TMZ [37] respectively) their extractionrecoveries were significantly improved by raising the pHof the sample with sodium hydroxide solutions Differentvolumes of 10 N NaOH were tested a 500 120583l aliquot of 10N NaOH provided the best extraction recoveries Extractionrecoveries were found to be 45 and 100 for ATN andTMZrespectively and were consistent against different levels forboth analytes

System suitability parameters such as resolution (Rs)tailing factor (T) capacity factor (K) and selectivity factor(120572) were calculated according to USP [2] as shown in Table 2The resolution was always more than 15 the selectivity wasmore than one and the symmetry factor had an acceptedvalue

Linearity Peak area responses were plotted against the cor-responding concentrations Linearity was obtained over aconcentration range of 10-100 120583gmL in pure form for bothanalytes and over concentration ranges of (025-25) 120583gmL

International Journal of Analytical Chemistry 5

Table 3 Statistical analysis of the proposed method in standard solution and the officialreported methods [1 38]

Atenolol TrimetazidineProposed Method Reported method [38]lowastlowast Proposed Method Official method [1]

Mean 10034 9966 10035 9839SD 117 087 106 077RSD 116 088 106 112Variance 136 076 113 059N 8 5 8 3F ndash test 18 (412)lowast - 109 (474)lowast -Studentrsquos t-test 111 (2201)lowast - 193 (2262)lowast -lowastFigures between parenthesis represent the corresponding tabulated values of t and F at P= 005lowastlowastSpectrophotometric method based on measurement of atenolol absorbance in 01 N HCl at 2246 nm

Table 4 Intra- and interday precision and accuracy calculated from quality control (QC) samples in pure form

QC conc (120583gmL) Intra-day Inter-dayMean (120583gmL) SD RSD DFN Mean (120583gmL) SD RSD DFN

ATN3 297 002 077 -112 301 005 17 02550 50 95 045 088 189 5017 041 082 03475 75004 012 017 0005 7636 029 039 182TMZ3 289 009 345 -367 288 009 302 -39950 5009 023 045 018 5007 026 052 01575 7714 131 17 285 7634 205 268 178

Table 5 Intra- and interday precision and accuracy calculated from quality control (QC) samples in human urine

Intra-day Inter-dayMean (120583gmL) SD RSD DFN Mean (120583gmL) SD RSD DFN

ATN10 100 002 176 008 099 003 285 -00110 992 007 073 -081 985 011 110 -15020 2002 025 126 011 2008 017 082 041TMZ05 050 0004 075 098 050 0004 069 09010 1019 001 098 19 1014 012 118 13720 1936 008 042 -319 1941 008 042 -294

and (05-25) 120583gmL in urine samples for trimetazidine andatenolol respectively The correlation coefficient was ge 0999as shown in Table 1

Limit of Detection and Limit of Quantitation The limit ofdetection (LOD) is the lowest concentration at which theanalyte can be detected The limit of quantitation (LOQ) isthe lowest concentration that can be quantitatively measuredwith acceptable precision and accuracy The LOD was estab-lished as the lowest concentration that provides a signal-to-noise (SN) ratio of 31 and the LOQ was established as thelowest concentration that provides SN ratio of 101 as givenin (Table 1)

Statistical Analysis for Pure Form Method The proposedmethod was compared to the official and reported methods

[1 38] statistically using Students t-test and variance ratio F-test at 95 confidence level The calculated t and F values didnot exceed the theoretical values No significant differencesbetween the proposed method and the official or reportedones have been observed as given in (Table 3)

Precision and Accuracy Inter- and intraday precision (mea-sured as percent relative standard deviation RSD) andinter- and intraday accuracy (measured as percent differencefrom the nominal concentration DFN) were measuredat low mid and high QC levels Excellent precision andaccuracy were obtained for both analytes in pure forms andurine samples (Tables 4 and 5)

Selectivity Selectivity is the ability of the analytical methodto determine the target compound in the presence of other

6 International Journal of Analytical Chemistry

Table 6 Application of the proposed method in standard solution on pharmaceutical tablets

Pharmaceutical Preparations Blokium tablets Metacardia tabletsTaken (120583gmL) Foundlowast (120583gmL) Recovery Taken (120583gmL) Foundlowast (120583gmL) Recovery

500 487 9744 500 473 94541000 960 9597 1000 974 97403000 2863 9542 3000 2850 95055000 4750 9501 5000 4915 98307500 7167 9556 7500 7124 949910000 9527 9527 10000 9536 9536

Mean 9578 9594SD 088 153SE 036 063RSD 091 160Variance 077 234lowastAverage of three determinations

components such as excipients degradation products coad-ministered drugs andor endogenous matrix componentswithout interferenceThe proposedmethod showed excellentchromatographic separation and quantitation of the citeddrugs in the presence of common tablet excipients and urineextracted matrix No significant peaks were detected at theexpected retention times of the two cited drugs and thereis no coelution with any of the other components that maypresent in the final extracts

Robustness Small variations in the experimental parameterswere applied and the effect of these variations on the analyti-cal performance was evaluated to ensure the robustness of theproposed method The studied parameters were as followsphosphate buffer pH (33plusmn 02) flow rate (10plusmn 001)mLminand wavelength 210 nm plusmn10 and 225 nm plusmn10 for TMZ andATN respectively Only one parameter was changed in atime and the others were kept unchanged The effects of theproposed changes on retention time peak area tailing factorand number of theoretical plates were evaluated It was foundthat all the studied minor changes have no significant effectson the method performance

Applications

Pharmaceutical Tablets The optimized HPLC method wassuccessfully applied for determination of the studied drugsin their corresponding tablets Good percentage recoverieswithout any excipients interference were obtained (Table 6)the proposed method can be applied for quality controlstudies

4 Conclusion

RP-HPLCmethod with PDA detection was developed for thedetermination of atenolol and trimetazidine in pure formpharmaceutical tablets and human urine The proposedmethod was found to be simple sensitive and accurate Theoptimized chromatographic conditions allowed separation ofthe studied drugs in reasonable time without any interferencefrom excipients andor extracted matrix components This

method can be used for therapeutic drug monitoring ofpatients treated with ATN and TMZ concomitantly and alsocan be applied for quality control analysis of the studied drugsin their pharmaceutical tablets

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

References

[1] The Biritish Pharmacopeia Her Majesty stationary office Lon-don UK 2017 II Appendix I D

[2] United States Pharmacopoeia (USP) 36 National Formulary 31US Pharmacoppieal Convention Rockville Md USA 2013

[3] M Zeeb H Farahani and M K Papan ldquoDetermination ofatenolol in human plasma using ionic-liquid-based ultrasound-assisted in situ solvent formation microextraction followed byhigh-performance liquid chromatographyrdquo Journal of Separa-tion Science vol 39 no 11 pp 2138ndash2145 2016

[4] S C Sweetman Martindale The Complete Drug Reference volAThe Pharmaceutical Press London UK 36th edition 2009

[5] L Ding B Gong X Chu J Hu and H Zheng ldquoSensi-tive and rapid LC-ESI-MS method for the determination oftrimetazidine in human plasmardquo Journal of Pharmaceutical andBiomedical Analysis vol 44 no 2 pp 526ndash531 2007

[6] Y Jiao M Su M Chen et al ldquoLCESI-MS method for thedetermination of trimetazidine in human plasma Applicationto a bioequivalence study on Chinese volunteersrdquo Journal ofPharmaceutical and Biomedical Analysis vol 43 no 5 pp 1804ndash1807 2007

[7] C A Dezsi ldquoTrimetazidine in practice Review of the clinicaland experimental evidencerdquo American Journal of Therapeuticsvol 23 no 3 pp e871ndashe879 2016

[8] R Bennis Nechba M El MrsquoBarki Kadiri M Bennani-ZiatniA A Zeggwagh and A Mesfioui ldquoDifficulty in managing

International Journal of Analytical Chemistry 7

polypharmacy in the elderly Case report and review of theliteraturerdquo Journal of Clinical Gerontology and Geriatrics vol 6no 1 pp 30ndash33 2015

[9] N Siddharth Shah and S Ambardekar ldquoAtenolol Interac-tion with Other Drugsrdquo Supplement Of JAPI vol 57 2009httpwwwjapiorgspecial issue 2009article 08pdf

[10] I Baranowska P Markowski and J Baranowski ldquoDevelopmentand validation of an HPLC method for the simultaneousanalysis of 23 selected drugs belonging to different therapeuticgroups in human urine samplesrdquoAnalytical Sciences vol 25 no11 pp 1307ndash1313 2009

[11] B Yilmaz and S Arslan ldquoDetermination of atenolol in humanurine by using HPLCrdquo Separation Science Plus vol 1 no 1 pp4ndash10 2018

[12] B Yilmaz and S Arslan ldquoDetermination of atenolol in humanurine by gas chromatography-mass spectrometry methodrdquoJournal of Chromatographic Science (JCS) vol 49 no 5 pp 365ndash369 2011

[13] N S Abdelwahab ldquoDetermination of atenolol chlorthalidoneand their degradation products by TLC-densitometric andchemometric methods with application of model updatingrdquoAnalytical Methods vol 2 no 12 pp 1994ndash2001 2010

[14] G C Hiremath R M Mulla and S T Nandibewoor ldquoKineticmethods of determination of atenolol in pure compound and inpharmaceutical formulationsrdquo Chemia Analityczna vol 50 no2 pp 449ndash455 2005

[15] A M El-didamony andM A Moustafa ldquoDirect spectrophoto-metric determination of atenolol and timolol anti-hypertensivedrugsrdquo International Journal of Pharmacy and PharmaceuticalSciences vol 9 no 3 pp 47ndash53 2017

[16] E Bakir M Gouda A Alnajjar and W E Boraie ldquoSpectroflu-orimetric method for atenolol determination based on goldnanoparticlesrdquo Acta Pharmaceutica vol 68 no 2 pp 243ndash2502018

[17] K N Prashanth K Basavaiah M S Raghu and K B VinayldquoDetermination of atenolol and its preparations by acid-basetitration in non-aqueous mediumrdquo Der Pharmacia Lettre vol4 no 5 pp 1534ndash1540 2012

[18] J Scremin and E R Sartori ldquoSimultaneous determination ofnifedipine and atenolol in combined dosage forms using aboron-doped diamond electrodewith differential pulse voltam-metryrdquo Canadian Journal of Chemistry vol 96 no 1 pp 1ndash72018

[19] D Li J Du and J Lu ldquoChemiluminescence determina-tion of atenolol in biological fluids by a europium-sensitizedpermanganate-sulfite systemrdquo Microchimica Acta vol 161 no1-2 pp 169ndash173 2008

[20] J Huang J Sun X Zhou and T You ldquoDetermination ofatenolol and metoprolol by capillary electrophoresis withtris(221015840-bipyridyl)ruthenium(II) electrochemiluminescencedetectionrdquo Analytical Sciences vol 23 no 2 pp 183ndash188 2007

[21] P M Harrison A M Tonkin and A J McLean ldquoSimple andrapid analysis of atenolol andmetoprolol in plasma using solid-phase extraction and high-performance liquid chromatogra-phyrdquo Journal of Chromatography B Biomedical Sciences andApplications vol 339 no C pp 429ndash433 1985

[22] E M Phyo Lwin C Gerber Y Song et al ldquoA new LC-MSMSbioanalytical method for atenolol in human plasma and milkrdquoBioanalysis vol 9 no 7 pp 517ndash530 2017

[23] M K Jeoung K S Kim C S Kim et al ldquoAn HPLC Determi-nation of Trimetazidine inHuman Plasma Using Liquid-Liquid

Extraction for Sample Clean-Uprdquo Journal of Liquid Chromatog-raphy ampRelated Technologies vol 28 no 9 pp 1299ndash1309 2005

[24] F Hasin S Noon H Al Hasan and T Jahan ldquoValidation ofhigh performance liquid chromatography (HPLC) method fordetermination of erlotinib related substance in pharmaceuticaldosage formrdquo European Journal of Biomedical and Pharmaceu-tical Sciences (EJBPS) vol 4 no 7 pp 66ndash73 2017

[25] Jefri A D Puspitasari J S Talpaneni and R R Tjandraw-inata ldquoIdentification and control of unspecified impurity intrimetazidine dihydrochloride tablet formulationrdquo IOP Confer-ence Series Materials Science and Engineering vol 349 ArticleID 012029 2018

[26] L Fay G Michel P Goupit C Harpey and M ProstldquoDetermination of trimetazidine in biological fluids by gaschromatography-mass spectrometryrdquo Journal of Chromatogra-phy B Biomedical Sciences and Applications vol 490 no C pp198ndash205 1989

[27] T S Belal T Awad and C R Clark ldquoStability-indicatingdetermination of trimetazidine dihydrochloride in the presenceof two of its related substances using a direct GCMS methodrdquoJournal of AOAC International vol 97 no 6 pp 1514ndash1518 2014

[28] S O Thoppil R M Cardoza and P D Amin ldquoStabilityindicating HPTLC determination of trimetazidine as bulk drugand in pharmaceutical formulationsrdquo Journal of Pharmaceuticaland Biomedical Analysis vol 25 no 1 pp 15ndash20 2001

[29] S Agrawal P Gurjar and B Katheriya ldquoAnalytical methoddevelopment and validation for simultaneous estimation ofTrimetazidine hydrochloride and Metoprolol succinate usingHPTLCrdquo Current Pharmaceutical Analysis vol 13 2017

[30] A A Chis F G Gligor G Cormos E Curea and MBojita ldquoSpectrophotometric method for the determination oftrimetazidine dihydrochloride from pharmaceutical formsrdquoFarmacia vol 58 no 5 pp 629ndash636 2010

[31] Md F Hasin Md H Mukhlasy Md I Hussain and MdM Hossain ldquoValidation of spectrophotometric dissolutionmethod for modified release Trimetazidine pharmaceuticaldosage formrdquo JIPBS vol 4 no 2 pp 68ndash73 2017

[32] M S Abd-Elmonem A S M Eltantawy and E Khaled ldquoAlphacyclodextrin-based trimetazidine potentiometric sensorsrdquo IndoAmerican Journal of Pharmaceutical Research vol 6 no 8 2016

[33] B Dogan-Topal A Sibel and B Uslu ldquoOzkan and Bengi UsluThe Analytical Applications of Square Wave Voltammetry onPharmaceutical Analysisrdquo The Open Chemical and BiomedicalMethods Journal vol 3 pp 56ndash73 2010

[34] J Li J Quan J Du and M Liu ldquoChemiluminescence deter-mination of trimetazidine via inducing the aggregation ofgold nanoparticlesrdquo Spectrochimica Acta Part A Molecular andBiomolecular Spectroscopy vol 114 pp 33ndash37 2013

[35] HNMistri AG Jangid andP Shrivastav ldquoSensitive and rapidmethod to determine trimetazidine in human plasma by liquidchromatographytandemmass spectrometryrdquo Journal of AOACInternational vol 91 no 3 pp 562ndash571 2008

[36] V Martınez M I Maguregui R M Jimenez and R MAlonso ldquoDetermination of the pK(a) values of 120573-blockers byautomatedpotentiometric titrationsrdquo Journal of Pharmaceuticaland Biomedical Analysis vol 23 no 2-3 pp 459ndash468 2000

[37] V V Mohylyuk and L L Davtian ldquoInteraction of weak basedrug trimetazidine and carbopol as further retardation in thematrix tabletrdquoActa Poloniae Pharmaceutica Drug Research vol72 no 6 pp 1259ndash1261 2015

[38] J H A Girdhari P Prabhakara K Pankaj K Annu and KMarina ldquoSimultaneous Estimation of Atenolol and Amlodipine

International Journal of Analytical Chemistry 5

Table 3 Statistical analysis of the proposed method in standard solution and the officialreported methods [1 38]

Atenolol TrimetazidineProposed Method Reported method [38]lowastlowast Proposed Method Official method [1]

Mean 10034 9966 10035 9839SD 117 087 106 077RSD 116 088 106 112Variance 136 076 113 059N 8 5 8 3F ndash test 18 (412)lowast - 109 (474)lowast -Studentrsquos t-test 111 (2201)lowast - 193 (2262)lowast -lowastFigures between parenthesis represent the corresponding tabulated values of t and F at P= 005lowastlowastSpectrophotometric method based on measurement of atenolol absorbance in 01 N HCl at 2246 nm

Table 4 Intra- and interday precision and accuracy calculated from quality control (QC) samples in pure form

QC conc (120583gmL) Intra-day Inter-dayMean (120583gmL) SD RSD DFN Mean (120583gmL) SD RSD DFN

ATN3 297 002 077 -112 301 005 17 02550 50 95 045 088 189 5017 041 082 03475 75004 012 017 0005 7636 029 039 182TMZ3 289 009 345 -367 288 009 302 -39950 5009 023 045 018 5007 026 052 01575 7714 131 17 285 7634 205 268 178

Table 5 Intra- and interday precision and accuracy calculated from quality control (QC) samples in human urine

Intra-day Inter-dayMean (120583gmL) SD RSD DFN Mean (120583gmL) SD RSD DFN

ATN10 100 002 176 008 099 003 285 -00110 992 007 073 -081 985 011 110 -15020 2002 025 126 011 2008 017 082 041TMZ05 050 0004 075 098 050 0004 069 09010 1019 001 098 19 1014 012 118 13720 1936 008 042 -319 1941 008 042 -294

and (05-25) 120583gmL in urine samples for trimetazidine andatenolol respectively The correlation coefficient was ge 0999as shown in Table 1

Limit of Detection and Limit of Quantitation The limit ofdetection (LOD) is the lowest concentration at which theanalyte can be detected The limit of quantitation (LOQ) isthe lowest concentration that can be quantitatively measuredwith acceptable precision and accuracy The LOD was estab-lished as the lowest concentration that provides a signal-to-noise (SN) ratio of 31 and the LOQ was established as thelowest concentration that provides SN ratio of 101 as givenin (Table 1)

Statistical Analysis for Pure Form Method The proposedmethod was compared to the official and reported methods

[1 38] statistically using Students t-test and variance ratio F-test at 95 confidence level The calculated t and F values didnot exceed the theoretical values No significant differencesbetween the proposed method and the official or reportedones have been observed as given in (Table 3)

Precision and Accuracy Inter- and intraday precision (mea-sured as percent relative standard deviation RSD) andinter- and intraday accuracy (measured as percent differencefrom the nominal concentration DFN) were measuredat low mid and high QC levels Excellent precision andaccuracy were obtained for both analytes in pure forms andurine samples (Tables 4 and 5)

Selectivity Selectivity is the ability of the analytical methodto determine the target compound in the presence of other

6 International Journal of Analytical Chemistry

Table 6 Application of the proposed method in standard solution on pharmaceutical tablets

Pharmaceutical Preparations Blokium tablets Metacardia tabletsTaken (120583gmL) Foundlowast (120583gmL) Recovery Taken (120583gmL) Foundlowast (120583gmL) Recovery

500 487 9744 500 473 94541000 960 9597 1000 974 97403000 2863 9542 3000 2850 95055000 4750 9501 5000 4915 98307500 7167 9556 7500 7124 949910000 9527 9527 10000 9536 9536

Mean 9578 9594SD 088 153SE 036 063RSD 091 160Variance 077 234lowastAverage of three determinations

components such as excipients degradation products coad-ministered drugs andor endogenous matrix componentswithout interferenceThe proposedmethod showed excellentchromatographic separation and quantitation of the citeddrugs in the presence of common tablet excipients and urineextracted matrix No significant peaks were detected at theexpected retention times of the two cited drugs and thereis no coelution with any of the other components that maypresent in the final extracts

Robustness Small variations in the experimental parameterswere applied and the effect of these variations on the analyti-cal performance was evaluated to ensure the robustness of theproposed method The studied parameters were as followsphosphate buffer pH (33plusmn 02) flow rate (10plusmn 001)mLminand wavelength 210 nm plusmn10 and 225 nm plusmn10 for TMZ andATN respectively Only one parameter was changed in atime and the others were kept unchanged The effects of theproposed changes on retention time peak area tailing factorand number of theoretical plates were evaluated It was foundthat all the studied minor changes have no significant effectson the method performance

Applications

Pharmaceutical Tablets The optimized HPLC method wassuccessfully applied for determination of the studied drugsin their corresponding tablets Good percentage recoverieswithout any excipients interference were obtained (Table 6)the proposed method can be applied for quality controlstudies

4 Conclusion

RP-HPLCmethod with PDA detection was developed for thedetermination of atenolol and trimetazidine in pure formpharmaceutical tablets and human urine The proposedmethod was found to be simple sensitive and accurate Theoptimized chromatographic conditions allowed separation ofthe studied drugs in reasonable time without any interferencefrom excipients andor extracted matrix components This

method can be used for therapeutic drug monitoring ofpatients treated with ATN and TMZ concomitantly and alsocan be applied for quality control analysis of the studied drugsin their pharmaceutical tablets

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

References

[1] The Biritish Pharmacopeia Her Majesty stationary office Lon-don UK 2017 II Appendix I D

[2] United States Pharmacopoeia (USP) 36 National Formulary 31US Pharmacoppieal Convention Rockville Md USA 2013

[3] M Zeeb H Farahani and M K Papan ldquoDetermination ofatenolol in human plasma using ionic-liquid-based ultrasound-assisted in situ solvent formation microextraction followed byhigh-performance liquid chromatographyrdquo Journal of Separa-tion Science vol 39 no 11 pp 2138ndash2145 2016

[4] S C Sweetman Martindale The Complete Drug Reference volAThe Pharmaceutical Press London UK 36th edition 2009

[5] L Ding B Gong X Chu J Hu and H Zheng ldquoSensi-tive and rapid LC-ESI-MS method for the determination oftrimetazidine in human plasmardquo Journal of Pharmaceutical andBiomedical Analysis vol 44 no 2 pp 526ndash531 2007

[6] Y Jiao M Su M Chen et al ldquoLCESI-MS method for thedetermination of trimetazidine in human plasma Applicationto a bioequivalence study on Chinese volunteersrdquo Journal ofPharmaceutical and Biomedical Analysis vol 43 no 5 pp 1804ndash1807 2007

[7] C A Dezsi ldquoTrimetazidine in practice Review of the clinicaland experimental evidencerdquo American Journal of Therapeuticsvol 23 no 3 pp e871ndashe879 2016

[8] R Bennis Nechba M El MrsquoBarki Kadiri M Bennani-ZiatniA A Zeggwagh and A Mesfioui ldquoDifficulty in managing

International Journal of Analytical Chemistry 7

polypharmacy in the elderly Case report and review of theliteraturerdquo Journal of Clinical Gerontology and Geriatrics vol 6no 1 pp 30ndash33 2015

[9] N Siddharth Shah and S Ambardekar ldquoAtenolol Interac-tion with Other Drugsrdquo Supplement Of JAPI vol 57 2009httpwwwjapiorgspecial issue 2009article 08pdf

[10] I Baranowska P Markowski and J Baranowski ldquoDevelopmentand validation of an HPLC method for the simultaneousanalysis of 23 selected drugs belonging to different therapeuticgroups in human urine samplesrdquoAnalytical Sciences vol 25 no11 pp 1307ndash1313 2009

[11] B Yilmaz and S Arslan ldquoDetermination of atenolol in humanurine by using HPLCrdquo Separation Science Plus vol 1 no 1 pp4ndash10 2018

[12] B Yilmaz and S Arslan ldquoDetermination of atenolol in humanurine by gas chromatography-mass spectrometry methodrdquoJournal of Chromatographic Science (JCS) vol 49 no 5 pp 365ndash369 2011

[13] N S Abdelwahab ldquoDetermination of atenolol chlorthalidoneand their degradation products by TLC-densitometric andchemometric methods with application of model updatingrdquoAnalytical Methods vol 2 no 12 pp 1994ndash2001 2010

[14] G C Hiremath R M Mulla and S T Nandibewoor ldquoKineticmethods of determination of atenolol in pure compound and inpharmaceutical formulationsrdquo Chemia Analityczna vol 50 no2 pp 449ndash455 2005

[15] A M El-didamony andM A Moustafa ldquoDirect spectrophoto-metric determination of atenolol and timolol anti-hypertensivedrugsrdquo International Journal of Pharmacy and PharmaceuticalSciences vol 9 no 3 pp 47ndash53 2017

[16] E Bakir M Gouda A Alnajjar and W E Boraie ldquoSpectroflu-orimetric method for atenolol determination based on goldnanoparticlesrdquo Acta Pharmaceutica vol 68 no 2 pp 243ndash2502018

[17] K N Prashanth K Basavaiah M S Raghu and K B VinayldquoDetermination of atenolol and its preparations by acid-basetitration in non-aqueous mediumrdquo Der Pharmacia Lettre vol4 no 5 pp 1534ndash1540 2012

[18] J Scremin and E R Sartori ldquoSimultaneous determination ofnifedipine and atenolol in combined dosage forms using aboron-doped diamond electrodewith differential pulse voltam-metryrdquo Canadian Journal of Chemistry vol 96 no 1 pp 1ndash72018

[19] D Li J Du and J Lu ldquoChemiluminescence determina-tion of atenolol in biological fluids by a europium-sensitizedpermanganate-sulfite systemrdquo Microchimica Acta vol 161 no1-2 pp 169ndash173 2008

[20] J Huang J Sun X Zhou and T You ldquoDetermination ofatenolol and metoprolol by capillary electrophoresis withtris(221015840-bipyridyl)ruthenium(II) electrochemiluminescencedetectionrdquo Analytical Sciences vol 23 no 2 pp 183ndash188 2007

[21] P M Harrison A M Tonkin and A J McLean ldquoSimple andrapid analysis of atenolol andmetoprolol in plasma using solid-phase extraction and high-performance liquid chromatogra-phyrdquo Journal of Chromatography B Biomedical Sciences andApplications vol 339 no C pp 429ndash433 1985

[22] E M Phyo Lwin C Gerber Y Song et al ldquoA new LC-MSMSbioanalytical method for atenolol in human plasma and milkrdquoBioanalysis vol 9 no 7 pp 517ndash530 2017

[23] M K Jeoung K S Kim C S Kim et al ldquoAn HPLC Determi-nation of Trimetazidine inHuman Plasma Using Liquid-Liquid

Extraction for Sample Clean-Uprdquo Journal of Liquid Chromatog-raphy ampRelated Technologies vol 28 no 9 pp 1299ndash1309 2005

[24] F Hasin S Noon H Al Hasan and T Jahan ldquoValidation ofhigh performance liquid chromatography (HPLC) method fordetermination of erlotinib related substance in pharmaceuticaldosage formrdquo European Journal of Biomedical and Pharmaceu-tical Sciences (EJBPS) vol 4 no 7 pp 66ndash73 2017

[25] Jefri A D Puspitasari J S Talpaneni and R R Tjandraw-inata ldquoIdentification and control of unspecified impurity intrimetazidine dihydrochloride tablet formulationrdquo IOP Confer-ence Series Materials Science and Engineering vol 349 ArticleID 012029 2018

[26] L Fay G Michel P Goupit C Harpey and M ProstldquoDetermination of trimetazidine in biological fluids by gaschromatography-mass spectrometryrdquo Journal of Chromatogra-phy B Biomedical Sciences and Applications vol 490 no C pp198ndash205 1989

[27] T S Belal T Awad and C R Clark ldquoStability-indicatingdetermination of trimetazidine dihydrochloride in the presenceof two of its related substances using a direct GCMS methodrdquoJournal of AOAC International vol 97 no 6 pp 1514ndash1518 2014

[28] S O Thoppil R M Cardoza and P D Amin ldquoStabilityindicating HPTLC determination of trimetazidine as bulk drugand in pharmaceutical formulationsrdquo Journal of Pharmaceuticaland Biomedical Analysis vol 25 no 1 pp 15ndash20 2001

[29] S Agrawal P Gurjar and B Katheriya ldquoAnalytical methoddevelopment and validation for simultaneous estimation ofTrimetazidine hydrochloride and Metoprolol succinate usingHPTLCrdquo Current Pharmaceutical Analysis vol 13 2017

[30] A A Chis F G Gligor G Cormos E Curea and MBojita ldquoSpectrophotometric method for the determination oftrimetazidine dihydrochloride from pharmaceutical formsrdquoFarmacia vol 58 no 5 pp 629ndash636 2010

[31] Md F Hasin Md H Mukhlasy Md I Hussain and MdM Hossain ldquoValidation of spectrophotometric dissolutionmethod for modified release Trimetazidine pharmaceuticaldosage formrdquo JIPBS vol 4 no 2 pp 68ndash73 2017

[32] M S Abd-Elmonem A S M Eltantawy and E Khaled ldquoAlphacyclodextrin-based trimetazidine potentiometric sensorsrdquo IndoAmerican Journal of Pharmaceutical Research vol 6 no 8 2016

[33] B Dogan-Topal A Sibel and B Uslu ldquoOzkan and Bengi UsluThe Analytical Applications of Square Wave Voltammetry onPharmaceutical Analysisrdquo The Open Chemical and BiomedicalMethods Journal vol 3 pp 56ndash73 2010

[34] J Li J Quan J Du and M Liu ldquoChemiluminescence deter-mination of trimetazidine via inducing the aggregation ofgold nanoparticlesrdquo Spectrochimica Acta Part A Molecular andBiomolecular Spectroscopy vol 114 pp 33ndash37 2013

[35] HNMistri AG Jangid andP Shrivastav ldquoSensitive and rapidmethod to determine trimetazidine in human plasma by liquidchromatographytandemmass spectrometryrdquo Journal of AOACInternational vol 91 no 3 pp 562ndash571 2008

[36] V Martınez M I Maguregui R M Jimenez and R MAlonso ldquoDetermination of the pK(a) values of 120573-blockers byautomatedpotentiometric titrationsrdquo Journal of Pharmaceuticaland Biomedical Analysis vol 23 no 2-3 pp 459ndash468 2000

[37] V V Mohylyuk and L L Davtian ldquoInteraction of weak basedrug trimetazidine and carbopol as further retardation in thematrix tabletrdquoActa Poloniae Pharmaceutica Drug Research vol72 no 6 pp 1259ndash1261 2015

[38] J H A Girdhari P Prabhakara K Pankaj K Annu and KMarina ldquoSimultaneous Estimation of Atenolol and Amlodipine

6 International Journal of Analytical Chemistry

Table 6 Application of the proposed method in standard solution on pharmaceutical tablets

Pharmaceutical Preparations Blokium tablets Metacardia tabletsTaken (120583gmL) Foundlowast (120583gmL) Recovery Taken (120583gmL) Foundlowast (120583gmL) Recovery

500 487 9744 500 473 94541000 960 9597 1000 974 97403000 2863 9542 3000 2850 95055000 4750 9501 5000 4915 98307500 7167 9556 7500 7124 949910000 9527 9527 10000 9536 9536

Mean 9578 9594SD 088 153SE 036 063RSD 091 160Variance 077 234lowastAverage of three determinations

components such as excipients degradation products coad-ministered drugs andor endogenous matrix componentswithout interferenceThe proposedmethod showed excellentchromatographic separation and quantitation of the citeddrugs in the presence of common tablet excipients and urineextracted matrix No significant peaks were detected at theexpected retention times of the two cited drugs and thereis no coelution with any of the other components that maypresent in the final extracts

Robustness Small variations in the experimental parameterswere applied and the effect of these variations on the analyti-cal performance was evaluated to ensure the robustness of theproposed method The studied parameters were as followsphosphate buffer pH (33plusmn 02) flow rate (10plusmn 001)mLminand wavelength 210 nm plusmn10 and 225 nm plusmn10 for TMZ andATN respectively Only one parameter was changed in atime and the others were kept unchanged The effects of theproposed changes on retention time peak area tailing factorand number of theoretical plates were evaluated It was foundthat all the studied minor changes have no significant effectson the method performance

Applications

Pharmaceutical Tablets The optimized HPLC method wassuccessfully applied for determination of the studied drugsin their corresponding tablets Good percentage recoverieswithout any excipients interference were obtained (Table 6)the proposed method can be applied for quality controlstudies

4 Conclusion

RP-HPLCmethod with PDA detection was developed for thedetermination of atenolol and trimetazidine in pure formpharmaceutical tablets and human urine The proposedmethod was found to be simple sensitive and accurate Theoptimized chromatographic conditions allowed separation ofthe studied drugs in reasonable time without any interferencefrom excipients andor extracted matrix components This

method can be used for therapeutic drug monitoring ofpatients treated with ATN and TMZ concomitantly and alsocan be applied for quality control analysis of the studied drugsin their pharmaceutical tablets

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

References

[1] The Biritish Pharmacopeia Her Majesty stationary office Lon-don UK 2017 II Appendix I D

[2] United States Pharmacopoeia (USP) 36 National Formulary 31US Pharmacoppieal Convention Rockville Md USA 2013

[3] M Zeeb H Farahani and M K Papan ldquoDetermination ofatenolol in human plasma using ionic-liquid-based ultrasound-assisted in situ solvent formation microextraction followed byhigh-performance liquid chromatographyrdquo Journal of Separa-tion Science vol 39 no 11 pp 2138ndash2145 2016

[4] S C Sweetman Martindale The Complete Drug Reference volAThe Pharmaceutical Press London UK 36th edition 2009

[5] L Ding B Gong X Chu J Hu and H Zheng ldquoSensi-tive and rapid LC-ESI-MS method for the determination oftrimetazidine in human plasmardquo Journal of Pharmaceutical andBiomedical Analysis vol 44 no 2 pp 526ndash531 2007

[6] Y Jiao M Su M Chen et al ldquoLCESI-MS method for thedetermination of trimetazidine in human plasma Applicationto a bioequivalence study on Chinese volunteersrdquo Journal ofPharmaceutical and Biomedical Analysis vol 43 no 5 pp 1804ndash1807 2007

[7] C A Dezsi ldquoTrimetazidine in practice Review of the clinicaland experimental evidencerdquo American Journal of Therapeuticsvol 23 no 3 pp e871ndashe879 2016

[8] R Bennis Nechba M El MrsquoBarki Kadiri M Bennani-ZiatniA A Zeggwagh and A Mesfioui ldquoDifficulty in managing

International Journal of Analytical Chemistry 7

polypharmacy in the elderly Case report and review of theliteraturerdquo Journal of Clinical Gerontology and Geriatrics vol 6no 1 pp 30ndash33 2015

[9] N Siddharth Shah and S Ambardekar ldquoAtenolol Interac-tion with Other Drugsrdquo Supplement Of JAPI vol 57 2009httpwwwjapiorgspecial issue 2009article 08pdf

[10] I Baranowska P Markowski and J Baranowski ldquoDevelopmentand validation of an HPLC method for the simultaneousanalysis of 23 selected drugs belonging to different therapeuticgroups in human urine samplesrdquoAnalytical Sciences vol 25 no11 pp 1307ndash1313 2009

[11] B Yilmaz and S Arslan ldquoDetermination of atenolol in humanurine by using HPLCrdquo Separation Science Plus vol 1 no 1 pp4ndash10 2018

[12] B Yilmaz and S Arslan ldquoDetermination of atenolol in humanurine by gas chromatography-mass spectrometry methodrdquoJournal of Chromatographic Science (JCS) vol 49 no 5 pp 365ndash369 2011

[13] N S Abdelwahab ldquoDetermination of atenolol chlorthalidoneand their degradation products by TLC-densitometric andchemometric methods with application of model updatingrdquoAnalytical Methods vol 2 no 12 pp 1994ndash2001 2010

[14] G C Hiremath R M Mulla and S T Nandibewoor ldquoKineticmethods of determination of atenolol in pure compound and inpharmaceutical formulationsrdquo Chemia Analityczna vol 50 no2 pp 449ndash455 2005

[15] A M El-didamony andM A Moustafa ldquoDirect spectrophoto-metric determination of atenolol and timolol anti-hypertensivedrugsrdquo International Journal of Pharmacy and PharmaceuticalSciences vol 9 no 3 pp 47ndash53 2017

[16] E Bakir M Gouda A Alnajjar and W E Boraie ldquoSpectroflu-orimetric method for atenolol determination based on goldnanoparticlesrdquo Acta Pharmaceutica vol 68 no 2 pp 243ndash2502018

[17] K N Prashanth K Basavaiah M S Raghu and K B VinayldquoDetermination of atenolol and its preparations by acid-basetitration in non-aqueous mediumrdquo Der Pharmacia Lettre vol4 no 5 pp 1534ndash1540 2012

[18] J Scremin and E R Sartori ldquoSimultaneous determination ofnifedipine and atenolol in combined dosage forms using aboron-doped diamond electrodewith differential pulse voltam-metryrdquo Canadian Journal of Chemistry vol 96 no 1 pp 1ndash72018

[19] D Li J Du and J Lu ldquoChemiluminescence determina-tion of atenolol in biological fluids by a europium-sensitizedpermanganate-sulfite systemrdquo Microchimica Acta vol 161 no1-2 pp 169ndash173 2008

[20] J Huang J Sun X Zhou and T You ldquoDetermination ofatenolol and metoprolol by capillary electrophoresis withtris(221015840-bipyridyl)ruthenium(II) electrochemiluminescencedetectionrdquo Analytical Sciences vol 23 no 2 pp 183ndash188 2007

[21] P M Harrison A M Tonkin and A J McLean ldquoSimple andrapid analysis of atenolol andmetoprolol in plasma using solid-phase extraction and high-performance liquid chromatogra-phyrdquo Journal of Chromatography B Biomedical Sciences andApplications vol 339 no C pp 429ndash433 1985

[22] E M Phyo Lwin C Gerber Y Song et al ldquoA new LC-MSMSbioanalytical method for atenolol in human plasma and milkrdquoBioanalysis vol 9 no 7 pp 517ndash530 2017

[23] M K Jeoung K S Kim C S Kim et al ldquoAn HPLC Determi-nation of Trimetazidine inHuman Plasma Using Liquid-Liquid

Extraction for Sample Clean-Uprdquo Journal of Liquid Chromatog-raphy ampRelated Technologies vol 28 no 9 pp 1299ndash1309 2005

[24] F Hasin S Noon H Al Hasan and T Jahan ldquoValidation ofhigh performance liquid chromatography (HPLC) method fordetermination of erlotinib related substance in pharmaceuticaldosage formrdquo European Journal of Biomedical and Pharmaceu-tical Sciences (EJBPS) vol 4 no 7 pp 66ndash73 2017

[25] Jefri A D Puspitasari J S Talpaneni and R R Tjandraw-inata ldquoIdentification and control of unspecified impurity intrimetazidine dihydrochloride tablet formulationrdquo IOP Confer-ence Series Materials Science and Engineering vol 349 ArticleID 012029 2018

[26] L Fay G Michel P Goupit C Harpey and M ProstldquoDetermination of trimetazidine in biological fluids by gaschromatography-mass spectrometryrdquo Journal of Chromatogra-phy B Biomedical Sciences and Applications vol 490 no C pp198ndash205 1989

[27] T S Belal T Awad and C R Clark ldquoStability-indicatingdetermination of trimetazidine dihydrochloride in the presenceof two of its related substances using a direct GCMS methodrdquoJournal of AOAC International vol 97 no 6 pp 1514ndash1518 2014

[28] S O Thoppil R M Cardoza and P D Amin ldquoStabilityindicating HPTLC determination of trimetazidine as bulk drugand in pharmaceutical formulationsrdquo Journal of Pharmaceuticaland Biomedical Analysis vol 25 no 1 pp 15ndash20 2001

[29] S Agrawal P Gurjar and B Katheriya ldquoAnalytical methoddevelopment and validation for simultaneous estimation ofTrimetazidine hydrochloride and Metoprolol succinate usingHPTLCrdquo Current Pharmaceutical Analysis vol 13 2017

[30] A A Chis F G Gligor G Cormos E Curea and MBojita ldquoSpectrophotometric method for the determination oftrimetazidine dihydrochloride from pharmaceutical formsrdquoFarmacia vol 58 no 5 pp 629ndash636 2010

[31] Md F Hasin Md H Mukhlasy Md I Hussain and MdM Hossain ldquoValidation of spectrophotometric dissolutionmethod for modified release Trimetazidine pharmaceuticaldosage formrdquo JIPBS vol 4 no 2 pp 68ndash73 2017

[32] M S Abd-Elmonem A S M Eltantawy and E Khaled ldquoAlphacyclodextrin-based trimetazidine potentiometric sensorsrdquo IndoAmerican Journal of Pharmaceutical Research vol 6 no 8 2016

[33] B Dogan-Topal A Sibel and B Uslu ldquoOzkan and Bengi UsluThe Analytical Applications of Square Wave Voltammetry onPharmaceutical Analysisrdquo The Open Chemical and BiomedicalMethods Journal vol 3 pp 56ndash73 2010

[34] J Li J Quan J Du and M Liu ldquoChemiluminescence deter-mination of trimetazidine via inducing the aggregation ofgold nanoparticlesrdquo Spectrochimica Acta Part A Molecular andBiomolecular Spectroscopy vol 114 pp 33ndash37 2013

[35] HNMistri AG Jangid andP Shrivastav ldquoSensitive and rapidmethod to determine trimetazidine in human plasma by liquidchromatographytandemmass spectrometryrdquo Journal of AOACInternational vol 91 no 3 pp 562ndash571 2008

[36] V Martınez M I Maguregui R M Jimenez and R MAlonso ldquoDetermination of the pK(a) values of 120573-blockers byautomatedpotentiometric titrationsrdquo Journal of Pharmaceuticaland Biomedical Analysis vol 23 no 2-3 pp 459ndash468 2000

[37] V V Mohylyuk and L L Davtian ldquoInteraction of weak basedrug trimetazidine and carbopol as further retardation in thematrix tabletrdquoActa Poloniae Pharmaceutica Drug Research vol72 no 6 pp 1259ndash1261 2015

[38] J H A Girdhari P Prabhakara K Pankaj K Annu and KMarina ldquoSimultaneous Estimation of Atenolol and Amlodipine

International Journal of Analytical Chemistry 7

polypharmacy in the elderly Case report and review of theliteraturerdquo Journal of Clinical Gerontology and Geriatrics vol 6no 1 pp 30ndash33 2015

[9] N Siddharth Shah and S Ambardekar ldquoAtenolol Interac-tion with Other Drugsrdquo Supplement Of JAPI vol 57 2009httpwwwjapiorgspecial issue 2009article 08pdf

[10] I Baranowska P Markowski and J Baranowski ldquoDevelopmentand validation of an HPLC method for the simultaneousanalysis of 23 selected drugs belonging to different therapeuticgroups in human urine samplesrdquoAnalytical Sciences vol 25 no11 pp 1307ndash1313 2009

[11] B Yilmaz and S Arslan ldquoDetermination of atenolol in humanurine by using HPLCrdquo Separation Science Plus vol 1 no 1 pp4ndash10 2018

[12] B Yilmaz and S Arslan ldquoDetermination of atenolol in humanurine by gas chromatography-mass spectrometry methodrdquoJournal of Chromatographic Science (JCS) vol 49 no 5 pp 365ndash369 2011

[13] N S Abdelwahab ldquoDetermination of atenolol chlorthalidoneand their degradation products by TLC-densitometric andchemometric methods with application of model updatingrdquoAnalytical Methods vol 2 no 12 pp 1994ndash2001 2010

[14] G C Hiremath R M Mulla and S T Nandibewoor ldquoKineticmethods of determination of atenolol in pure compound and inpharmaceutical formulationsrdquo Chemia Analityczna vol 50 no2 pp 449ndash455 2005

[15] A M El-didamony andM A Moustafa ldquoDirect spectrophoto-metric determination of atenolol and timolol anti-hypertensivedrugsrdquo International Journal of Pharmacy and PharmaceuticalSciences vol 9 no 3 pp 47ndash53 2017

[16] E Bakir M Gouda A Alnajjar and W E Boraie ldquoSpectroflu-orimetric method for atenolol determination based on goldnanoparticlesrdquo Acta Pharmaceutica vol 68 no 2 pp 243ndash2502018

[17] K N Prashanth K Basavaiah M S Raghu and K B VinayldquoDetermination of atenolol and its preparations by acid-basetitration in non-aqueous mediumrdquo Der Pharmacia Lettre vol4 no 5 pp 1534ndash1540 2012

[18] J Scremin and E R Sartori ldquoSimultaneous determination ofnifedipine and atenolol in combined dosage forms using aboron-doped diamond electrodewith differential pulse voltam-metryrdquo Canadian Journal of Chemistry vol 96 no 1 pp 1ndash72018

[19] D Li J Du and J Lu ldquoChemiluminescence determina-tion of atenolol in biological fluids by a europium-sensitizedpermanganate-sulfite systemrdquo Microchimica Acta vol 161 no1-2 pp 169ndash173 2008

[20] J Huang J Sun X Zhou and T You ldquoDetermination ofatenolol and metoprolol by capillary electrophoresis withtris(221015840-bipyridyl)ruthenium(II) electrochemiluminescencedetectionrdquo Analytical Sciences vol 23 no 2 pp 183ndash188 2007

[21] P M Harrison A M Tonkin and A J McLean ldquoSimple andrapid analysis of atenolol andmetoprolol in plasma using solid-phase extraction and high-performance liquid chromatogra-phyrdquo Journal of Chromatography B Biomedical Sciences andApplications vol 339 no C pp 429ndash433 1985

[22] E M Phyo Lwin C Gerber Y Song et al ldquoA new LC-MSMSbioanalytical method for atenolol in human plasma and milkrdquoBioanalysis vol 9 no 7 pp 517ndash530 2017

[23] M K Jeoung K S Kim C S Kim et al ldquoAn HPLC Determi-nation of Trimetazidine inHuman Plasma Using Liquid-Liquid

Extraction for Sample Clean-Uprdquo Journal of Liquid Chromatog-raphy ampRelated Technologies vol 28 no 9 pp 1299ndash1309 2005

[24] F Hasin S Noon H Al Hasan and T Jahan ldquoValidation ofhigh performance liquid chromatography (HPLC) method fordetermination of erlotinib related substance in pharmaceuticaldosage formrdquo European Journal of Biomedical and Pharmaceu-tical Sciences (EJBPS) vol 4 no 7 pp 66ndash73 2017

[25] Jefri A D Puspitasari J S Talpaneni and R R Tjandraw-inata ldquoIdentification and control of unspecified impurity intrimetazidine dihydrochloride tablet formulationrdquo IOP Confer-ence Series Materials Science and Engineering vol 349 ArticleID 012029 2018

[26] L Fay G Michel P Goupit C Harpey and M ProstldquoDetermination of trimetazidine in biological fluids by gaschromatography-mass spectrometryrdquo Journal of Chromatogra-phy B Biomedical Sciences and Applications vol 490 no C pp198ndash205 1989

[27] T S Belal T Awad and C R Clark ldquoStability-indicatingdetermination of trimetazidine dihydrochloride in the presenceof two of its related substances using a direct GCMS methodrdquoJournal of AOAC International vol 97 no 6 pp 1514ndash1518 2014

[28] S O Thoppil R M Cardoza and P D Amin ldquoStabilityindicating HPTLC determination of trimetazidine as bulk drugand in pharmaceutical formulationsrdquo Journal of Pharmaceuticaland Biomedical Analysis vol 25 no 1 pp 15ndash20 2001

[29] S Agrawal P Gurjar and B Katheriya ldquoAnalytical methoddevelopment and validation for simultaneous estimation ofTrimetazidine hydrochloride and Metoprolol succinate usingHPTLCrdquo Current Pharmaceutical Analysis vol 13 2017

[30] A A Chis F G Gligor G Cormos E Curea and MBojita ldquoSpectrophotometric method for the determination oftrimetazidine dihydrochloride from pharmaceutical formsrdquoFarmacia vol 58 no 5 pp 629ndash636 2010

[31] Md F Hasin Md H Mukhlasy Md I Hussain and MdM Hossain ldquoValidation of spectrophotometric dissolutionmethod for modified release Trimetazidine pharmaceuticaldosage formrdquo JIPBS vol 4 no 2 pp 68ndash73 2017

[32] M S Abd-Elmonem A S M Eltantawy and E Khaled ldquoAlphacyclodextrin-based trimetazidine potentiometric sensorsrdquo IndoAmerican Journal of Pharmaceutical Research vol 6 no 8 2016

[33] B Dogan-Topal A Sibel and B Uslu ldquoOzkan and Bengi UsluThe Analytical Applications of Square Wave Voltammetry onPharmaceutical Analysisrdquo The Open Chemical and BiomedicalMethods Journal vol 3 pp 56ndash73 2010

[34] J Li J Quan J Du and M Liu ldquoChemiluminescence deter-mination of trimetazidine via inducing the aggregation ofgold nanoparticlesrdquo Spectrochimica Acta Part A Molecular andBiomolecular Spectroscopy vol 114 pp 33ndash37 2013

[35] HNMistri AG Jangid andP Shrivastav ldquoSensitive and rapidmethod to determine trimetazidine in human plasma by liquidchromatographytandemmass spectrometryrdquo Journal of AOACInternational vol 91 no 3 pp 562ndash571 2008

[36] V Martınez M I Maguregui R M Jimenez and R MAlonso ldquoDetermination of the pK(a) values of 120573-blockers byautomatedpotentiometric titrationsrdquo Journal of Pharmaceuticaland Biomedical Analysis vol 23 no 2-3 pp 459ndash468 2000

[37] V V Mohylyuk and L L Davtian ldquoInteraction of weak basedrug trimetazidine and carbopol as further retardation in thematrix tabletrdquoActa Poloniae Pharmaceutica Drug Research vol72 no 6 pp 1259ndash1261 2015

[38] J H A Girdhari P Prabhakara K Pankaj K Annu and KMarina ldquoSimultaneous Estimation of Atenolol and Amlodipine

8 International Journal of Analytical Chemistry

Besylate in tablet Formulations by VierodtS Method UsingUVSpectrophotometryrdquo IRJP vol 3 no 2 pp 248ndash250 2012

[39] M Swartz and I Krull ldquoValidation of chromatographic Meth-odsrdquo Pharmaceutical Technology vol 22 pp 104ndash119 1998

[40] M A Hegazya N Yahiaa Y Z Sewilamb and S A WeshahyaldquoStability Indicating High Performance Liquid Chromato-graphic Method for The Determination of Bromazepam inthe presence of its degradation productsrdquo Asian Journal ofBiomedical and Pharmaceutical Sciences vol 5 no 51 pp 13ndash20 2015

TribologyAdvances in

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

International Journal ofInternational Journal ofPhotoenergy

Hindawiwwwhindawicom Volume 2018

Journal of

Chemistry

Hindawiwwwhindawicom Volume 2018

Advances inPhysical Chemistry

Hindawiwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2018

Bioinorganic Chemistry and ApplicationsHindawiwwwhindawicom Volume 2018

SpectroscopyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Medicinal ChemistryInternational Journal of

Hindawiwwwhindawicom Volume 2018

NanotechnologyHindawiwwwhindawicom Volume 2018

Journal of

Applied ChemistryJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

Journal of

SpectroscopyAnalytical ChemistryInternational Journal of

Hindawiwwwhindawicom Volume 2018

MaterialsJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

BioMed Research International Electrochemistry

International Journal of

Hindawiwwwhindawicom Volume 2018

Na

nom

ate

ria

ls

Hindawiwwwhindawicom Volume 2018

Journal ofNanomaterials

Submit your manuscripts atwwwhindawicom

TribologyAdvances in

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

International Journal ofInternational Journal ofPhotoenergy

Hindawiwwwhindawicom Volume 2018

Journal of

Chemistry

Hindawiwwwhindawicom Volume 2018

Advances inPhysical Chemistry

Hindawiwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2018

Bioinorganic Chemistry and ApplicationsHindawiwwwhindawicom Volume 2018

SpectroscopyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Medicinal ChemistryInternational Journal of

Hindawiwwwhindawicom Volume 2018

NanotechnologyHindawiwwwhindawicom Volume 2018

Journal of

Applied ChemistryJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

Journal of

SpectroscopyAnalytical ChemistryInternational Journal of

Hindawiwwwhindawicom Volume 2018

MaterialsJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

BioMed Research International Electrochemistry

International Journal of

Hindawiwwwhindawicom Volume 2018

Na

nom

ate

ria

ls

Hindawiwwwhindawicom Volume 2018

Journal ofNanomaterials

Submit your manuscripts atwwwhindawicom