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Research ArticleSimultaneous HPLC Determination of Chlordiazepoxide andMebeverine HCl in the Presence of Their Degradation Productsand Impurities

Rania N El-Shaheny and Fathalla F Belal

Analytical Chemistry Department Faculty of Pharmacy Mansoura University Mansoura 35516 Egypt

Correspondence should be addressed to Rania N El-Shaheny rania n2010yahoocom

Received 8 December 2014 Revised 10 February 2015 Accepted 10 February 2015

Academic Editor Victor David

Copyright copy 2015 R N El-Shaheny and F F Belal This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

A simple rapid and sensitive RP-HPLC method was developed and validated for the simultaneous determination ofchlordiazepoxide (CDO) and mebeverine HCl (MBV) in the presence of CDO impurity (2-amino-5-chlorobenzophenone ACB)and MBV degradation product (veratric acid VER) Separation was achieved within 9min on a BDS Hypersil phenyl column(45mmtimes 250mm 5 120583m particle size) using a mobile phase consisting of acetonitrile 01M potassium dihydrogen phosphatetriethylamine (35 65 02 vvv) in an isocratic mode at a flow rate of 1mLmin The pH of the mobile phase was adjusted to 45with orthophosphoric acid and UV detection was set at 260 nm A complete validation procedure was conducted The proposedmethod exhibited excellent linearity over the concentration ranges of 10ndash1000 100ndash2000 20ndash400 and 20ndash400 120583gmL for CDOMBV VER and ACB respectively The proposed method was applied for the simultaneous determination of CDO and MBV intheir coformulated tablets withmean percentage recoveries of 9975plusmn 062 and 9861plusmn 038 respectivelyThe results of the proposedmethod were favorably compared with those of a comparison HPLCmethod using Student t-test and the variance ratio F-test Thechemical structure of MBV degradation product was ascertained by mass spectrometry and IR studies

1 Introduction

Chlordiazepoxide (CDO Figure 1(a)) 7-chloro-2-methyl-amino-5-phenyl-3H-14-benzodiazepine 4-oxide is a ben-zodiazepine with general properties similar to those ofdiazepam It is used for the short-term treatment of anx-iety disorders and insomnia CDO is also used in musclespasm and in alcohol withdrawal syndrome [1] Mebever-ine hydrochloride (MBV Figure 1(b)) 4-[ethyl(4-methoxy-120572-methylphenethyl)amino]butyl veratrate hydrochloride isan antispasmodic with a direct action on the smooth musclesof the gastrointestinal tract It is used in conditions suchas irritable bowel syndrome [1] The combined therapy ofMBV as an antispasmodic agent with the anxiolytic drugCDO provides effective relief of spastic colon and associatedsymptoms of anxiety and tension in the gastrointestinal tract

CDO is the subject of amonograph in theBritish Pharma-copoeia (BP) [2] and the United States Pharmacopoeia (USP)[3] The BP [2] recommends a potentiometric non-aqueoustitration method with 01M perchloric acid as a titrant forits determination in pure form and a spectrophotometricmethod for its determination in capsules The USP [3]describes HPLC methods for its determination in pure formand single or coformulated tablets with either clidiniumbromide or amitriptyline HCl Literature survey revealedmany analytical methods for the determination of CDOeither alone or in combination with other drugs Some exam-ples of these methods include ion-selective electrode [4]spectrophotometry [5ndash8] and HPLC [9ndash11] Some methodswere also applied to separate CDO from its photodegradationproducts [12] or impurities [13]

MBV is also an official drug in the BP [2] that recom-mends a potentiometric non-aqueous titration method with

Hindawi Publishing CorporationJournal of ChemistryVolume 2015 Article ID 293719 9 pageshttpdxdoiorg1011552015293719

2 Journal of Chemistry

NHN

N+

OminusCl

(a)

NO

O

O

O

OHCl

(b)

O NH2

Cl

(c)

OH

O

O

O

(d)

Figure 1 Structural formulas of (a) chlordiazepoxide (b) mebeverine HCl (c) 2-amino-5-chlorobenzophenone and (d) veratric acid

01M perchloric acid and a spectrophotometric method forits determination in pure form and tablets respectivelyManyanalytical methods were applied for determination of MBVsuch as spectrophotometry [14ndash17] TLC [15] HPLC [14 1518 19] and spectrofluorimetry [20]

Reviewing the scientific literature revealed an HPLCmethod [21] and two spectrophotometric methods based onthe simultaneous equations and absorbance ratio method[22] for the simultaneous estimation of CDO and MBV intheir coformulated tablets These methods are not stability-indicating moreover the pharmaceutical ratio of the twodrugs (1 CDO 27 MBV) was not achieved in the two spec-trophotometricmethods [22] which are linear over the rangesof 10ndash100 and 20ndash120 120583gmL for CDO and MBV respec-tively so they are not practically applicable to coformulateddosage forms Only one HPLC method [23] was publishedas a stability-indicating assay for the two drugsrsquo combinationNevertheless this method suffers from some drawbacks suchas poor sensitivity narrow linearity range and complicatedgradient elution program Hence our main target was todevelop a simple sensitive and rapid isocratic stability-indicatingHPLCmethod for the simultaneous determinationof the two drugs in their combined tablets in the presence oftheir degradation products or synthesis impurities

The BP [2] and the USP [3] define 2-amino-5-chlorobenzophenone (ACB Figure 1(c)) as an impurityin CDO pure and dosage forms ACB is the starting materialfor synthesis of many diazepams including CDO [24]On the other hand MBV being an ester-containing drugis easily susceptible to hydrolytic degradation giving thecorresponding acid degradation product veratric acid (34-dimethoxy benzoic acid VER Figure 1(d)) [25] Moreoverthe BP [2] defines VER as a related substance in MBV pureform and tablets

In this study we developed a stability-indicating HPLCmethod for the analysis of CDO and MBV in combinationand in the presence of MBV degradation product (VER) andCDO synthetic precursor and main impurity (ACB) Theidentification of the degradation product of MBV was ascer-tained by mass spectrometry and IR studies The developedmethod was validated according to ICH Guidelines [26]Theproposed procedure is suitable for the quality control analysisand purity assessment of dosage forms containing the twostudied compounds

2 Experimental

21 Instruments Separation was performed using a Shi-madzu HPLC system (Shimadzu Corporation Japan) con-sisting of LC-20AD liquid chromatograph equipped with aRheodyne injector valve and a 20120583L sample loop a SPD20A UV-Vis detector DGU-20A5 online solvent degasserand CBM-20A communication busmodule A Consort P-901pH-meter (Turnhout Belgium) was used for pH adjustment

22 Materials Gift samples of chlordiazepoxide and mebev-erine HCl were kindly provided by EVA Pharma Co (CairoEgypt) Their purities are 10010 and 10012 respectivelyas determined by the reference method [21] 2-Amino-5-chlorobenzophenone was purchased from Sigma Aldrich Co(Steinheim am Albuch Germany) Coloverin A film coatedtablets labeled to contain 5mg CDO + 135mg MBVtablet(batch number 120960A) product of Chemipharm Pharma-ceutical Industries SAE 6th of October city Egypt werepurchased from a local pharmacy

23 Reagents All solvents used were of HPLC grade and allchemicals were of analytical reagent grade High purity water

Journal of Chemistry 3

obtained by filtration of distilled water through a 045 120583mmembrane filter was used throughout the study Acetonitrileand methanol (HPLC grade) were obtained from SigmaAldrich Co (Steinheim am Albuch Germany) Orthophos-phoric acid (85 wv) and triethylamine (TEA) wereobtained from Riedel-de Haen (Seelze Germany) Potassiumdihydrogen phosphate sodiumhydroxide hydrochloric acidethanol methylene chloride and diethyl ether were obtainedfrom ADWIC Co (Cairo Egypt)

24 Preparation and Isolation of Veratric Acid Veratric acidwas prepared in our laboratory according to the method ofNaguib and Abdelkawy [27] 05 gm of MBV was dissolvedin 25mL of 1M NaOH methanolic solution The solutionwas heated at 45∘C for 12 h till complete degradation ofMBV as confirmed by the disappearance of MBV spot uponapplying qualitative TLC on silica gel F

254plates using

absolute ethanol methylene chloride TEA (7 3 02 vvv)as a mobile phase [27] The solution was evaporated to aresidue The residue was dissolved in 20mL distilled waterand extracted with diethyl ether (3 times 15mL) The aqueouslayer containing VER was separated and acidified with 5NHCl till acidic to litmus paper where a heavywhite precipitateof VER was produced which was then filtered and washedwith distilled water (3 times 15mL) The white needles of VERwere dried in an oven at 90∘C for 1 hThe identity of VER wasconfirmed by mass spectrometry and IR studies

25 Chromatographic Conditions Separation was performedon a BDS Hypersil phenyl column (45mm times 250mm 5 120583mparticle size) from Thermo Electron Corporation (RuncornUK) using a mobile phase consisted of a ternary mixtureof acetonitrile 01M potassium dihydrogen phosphate TEA(35 65 02 vvv) The final pH of the mixed mobile phasewas adjusted to 45 with orthophosphoric acid and it wasdelivered at a flow rate of 1mLminThe detector wavelengthwas set at 260 nm

26 Standard Stock Solutions Standard stock solutionsof CDO (4000120583gmL) MBV (4000 120583gmL) and VER(2000120583gmL) were prepared individually in methanol ACBstandard stock solution (2000 120583gmL) was prepared in ace-tonitrileThe standard stock solutions were found to be stablefor 2 weeks when kept in a refrigerator at 4∘C

27 Recommended Procedures

271 Construction of Calibration Graphs Seven-point cali-bration graphs were constructed by quantitatively transfer-ring increasing volumes of the standard stock solutions ofCDO (25120583L-25mL)MBV (025ndash25mL) ACB (01ndash20mL)and VER (01ndash20mL) to four sets of 10mL volumetric flasksThe solutions were made up to the volume with the mobilephase and mixed well to obtain final concentration rangesof 10ndash1000 100ndash2000 20ndash400 and 20ndash400120583gmL forCDO MBV ACB and VER respectively Twenty 120583L aliquotswere injected in triplicate and eluted under the optimumchromatographic conditions Average peak areas of each

compound were plotted versus the corresponding concen-tration (120583gmL) to obtain the calibration graphs and thecorresponding regression equations were derived

272 Analysis of Laboratory-PreparedMixtures of CDOMBVFor the analysis of binarymixtures of the two studied drugs intheir pharmaceutical ratio (1 CDO 27 MBV) a mixed stan-dard stock solution containing both CDO and MBV (1000and 27000120583gmL resp) was prepared in methanol Fromthis solution aliquots of 03 05 and 07mL were transferredinto a series of 10mL volumetric flasks The solutions weremade up to volume with the mobile phase to obtain mixtureswith final concentrations of 30 CDO + 810MBV 50 CDO+ 1350MBV and 70 CDO + 1890MBV respectively Thesolutions were mixed well and 20 120583L aliquots were injectedin triplicate and eluted under the optimum chromatographicconditions The average percentage recovery of each com-pound was determined from the corresponding regressionequations

273 Analysis of Laboratory-Prepared Mixtures of CDOACBand MBVVER Aliquots of 25mL of the standard solutionof CDO were transferred into a series of 10mL volumetricflasks together with increasing volumes (01 02 04 and05mL) of its impurity (ACB) Similarly aliquots of 50mLof MBV standard solution were transferred into a series of10mL volumetric flasks together with increasing volumes(01 02 04 and 05mL) of its degradation product andimpurity (VER) The volumes were completed to the markwith the mobile phase Solutions were mixed well and 20120583Lwere injected (triplicate) Samples were eluted under theoptimum chromatographic conditions The average percent-age recoveries of CDO and MBV were calculated from thecorresponding regression equations

274 Analysis of Tablets Ten tablets were accurately weighedand finely powdered An amount of the powder equivalent to5mg CDO + 135mg MBV was weighed and transferred into50mL volumetric flask About 40mL of methanol was addedand the solution was sonicated for about 30minThe volumewas made up to the mark with the same solvent solutionwas mixed well and filtered through cellulose acetate-syringefiltration disks The obtained tablet extract contains CDOand MBV in the concentrations of 1000 and 27000120583gmLrespectively Different volumes of this solution (03 05 and07mL) were transferred into a series of 10mL volumetricflasks the volumes were made up to the mark with themobile phase and mixed well 20 120583L aliquots were injectedin triplicate and eluted under the optimum chromatographicconditions The average percentage recoveries of CDO andMBV were calculated from the previously derived regressionequations

3 Results and Discussion

A fast reliable and selective HPLC method was developedand validated for the simultaneous analysis of CDO andMBV in the presence of their impurities and degradation


100

80

60

404000 4003000 2000 1000

12 3

4

56

7

8

910

11

12

13

1415

161718

19

20

21

22

23

24

Tran

smitt

ance

()

Wave number (cmminus1)

(a)

70

60

50

40

30

20

10

060 80 100 120 140 160 180

56

6496

110120

165

182

Rela

tive a

bund

ance

Mass to charge ration (mz)

Veratric acid(MW = 18217)

O

OO

OH

(b)

Figure 2 (a) IR spectrum and (b) mass spectrum of veratric acid

products The degradation product of MBV was identifiedby mass spectrometry exhibiting a molecular ion peak atmass to charge ration (119898119911) of 182 which is consistent with34-dimethoxybenzoic acid (veratric acid VER) formed asa result of the hydrolysis of the ester linkage of MBV TheIR spectrum of the degradation product showed an intenseband at 1705 cmminus1 corresponding to the carbonyl stretchC=O of a carboxylic acid and a strong broad band centeredaround 3000 cmminus1 corresponding to the carboxylic acid OndashHstretch [28] thus confirming the structure of VER Figure 2illustrates the mass and IR spectra of VER

Hence the separation of the two drugs and their cor-responding degradation products and impurities was opti-mized as illustrated below

31 Optimization of Chromatographic Conditions Severalmobile phases were tried using various portions of organicmodifier and buffer of different molarities at different pHvalues The ratio of acetonitrile has a marked effect on theretention of the four compounds A ratio of 35 (vv) ace-tonitrile was selected as the optimum yielding well separatedpeaks for the four compounds with sufficient sensitivity andsuitable peak symmetry (peaks tailing factor lt16) in a shortrun time (lt9min) At a ratio of 40 (vv) acetonitrile thechromatographic peak of VER strongly overlapped with thatof CDO and that of MBV overlapped with that of ACB Onthe other hand decreasing the ratio of acetonitrile (le30vv) led to longer retention times especially for MBV andACB

Phosphate buffer of different ionic strengths (0025ndash01M) was investigated and 01Mwas chosen as the optimumconcentration allowing the simultaneous determination ofthe four compounds within a reasonable run time At lowerionic strengths the retention of MBV and ACB increased

with asymmetric distorted peaks which are completely over-lapped when using buffers at concentrations of 0025 to005M with no significant effect on CDO and VER peaks

Increasing the pH of the mobile phase resulted in anincrease of the retention of CDO MBV and ACB withouta significant change in the retention of VER At pH 45optimum resolution with reasonable retention times wasachieved At higher pH values unacceptable long retentiontimes of MBV and ACB were observed where at lower pHvalues the resolution between VERCDO and MBVACBpairs was adversely affected

TEA was added to the mobile phase in order to improvepeak symmetry and sharpness It prevents the interactionof the basic compounds (MBV and ACB) with the phenyl-bonded stationary phase where TEA associates with thesilanol sites blocking ion-exchange processes When usingmobile phase deprived of this ion suppressing agent theretention times of both MBV and ACB increased probablydue to interaction of these basic drugs with the silanolgroups of silica and they were coeluted at 12min Theconcentration of TEA was kept at 02mL for achieving bestpeak symmetry where at lower concentrations broadeningas well as distortion of the peaks was observed

For selection of the optimum detection wavelength theabsorption spectra of the four analytes were scanned inthe mobile phase MBV exhibited two absorption maximaat 260 and 290 nm and VER at 256 and 288 nm CDOshows maximum absorption at 250 nm while ACB hashighest absorption intensity at 260 nm For the simultaneousdetection and determination of the four components withhigh sensitivity the UV detection was carried out at 260 nmsince all compounds show appreciable absorption at thiswavelength

A study of the influence of the flow rate of the mobilephase on the separation process was also carried out over therange of 08ndash12mLmin At a flow rate of 08mLmin the


VER

CDO

MBV

ACB38

46

69

77

Time (min)

Det

ecto

r res

pons

e (Ab

s)

(a)

400

300

200

100

0

00 25 50 75 100 125

Figure 3 Representative chromatogram showing good separa-tion of CDO (500 120583gmL) MBV (1000120583gmL) veratric acid(100120583gmL) and ACB (100 120583gmL) where (a) is the solvent front

analysis time increased and broad peakswere obtainedwhileat a flow rate of 12mLmin the resolution of VERCDOand MBVACB pairs was adversely affected A flow rate of10mLmin was chosen as the optimum flow rate allowinggood resolution of the four peaks of the studied compoundswithin a reasonable run time (lt9min)

After this thorough experimental study good separationbetween the two drugs and their degradation products andimpurities was achieved Figure 3 shows a typical chro-matogram for the separation of the four compounds atretention times (119905

119877) of 38 46 69 and 77min forVERCDO

MBV and ACB respectively

32 Method Validation Method validation was performedaccording to ICH guidelines [26] to evaluate the suitabilityof the method for separation and quantitative determinationof CDO MBV and their degradation products A summaryof the validation results is illustrated below

321 Linearity and Range The linearity of the proposedmethod was evaluated by processing 7-point calibrationgraph for each compound The peak area of each drug wasplotted versus its nominal concentration (120583gmL) and alinear regression analysis was conducted to demonstrate thelinearity of the method [29]The small values of the standarddeviations of the residuals (119878

119910119909) slope (119878

119887) and intercept

(119878119886) and the relative error indicate good linearity of the

proposed method (Table 1)

322 Limit of Quantification (LOQ) and Limit of Detection(LOD) Both LOQ and LOD were calculated according toICH guidelines using the following equations [26]

LOQ =10119878119886

119887

(1)

LOD =33119878119886

119887

(2)

where 119878119886is the standard deviation of the intercept of regres-

sion line and 119887 is the slope of the regression line The resultsare summarized in Table 1

323 Accuracy The accuracy was studied by applying theproposed method for the determination of pure samplesof CDO and MBV over the working concentration rangesThe mean percentage recovery plusmn SD was calculated and theresults are presented in Table 2 The obtained results werestatistically compared with those obtained by a comparisonHPLC method [21] using Student 119905-test and the varianceration 119865-test revealing no significant difference regarding theaccuracy and precision respectively [29]

324 Precision The intraday precision was assessed fromthe results of the analyses of quality control samples of threedifferent concentrations at three successive times in the sameday The interday precision was also determined from threequality control samples analyzed on 3 consecutive days [26]The results of intraday and interday precision illustrated inTable 3 indicate the high precision of the proposed method

325 Selectivity The selectivity of the proposed method wasproven by its ability to separate the two drugs from theirdegradation products and impurities Different mixtures ofCDOMBV CDOACB and MBVVER were prepared andanalyzed according to the previously mentioned procedureThe recovery RSD and error values shown in Table 4were satisfactory thus proving the selectivity and accuracyof the proposed method and its ability to separate thedrugs from each other and from degradation products andimpurities Moreover no interference with the peaks ofinterest was observed from common tablet excipients

326 System Suitability Resolution (119877119904) selectivity factor

(120572) (both were calculated between each two adjacent peaks)retention factor (1198961015840) number of theoretical plates (119873) andtailing factor (119879) were calculated as per USP guidelines ascriteria for system suitability test [3] (Table 5)

327 Robustness Thereliability of the proposedmethodwithrespect to deliberate variations in method parameters wasstudiedThe influences of variation of pH of themobile phase(45 plusmn 02) ratio of acetonitrile in the mobile phase (35 plusmn 1vv) and molar strength of phosphate buffer (01 plusmn 0005M)were examined These minor deliberate variations in themethod parameters did not significantly affect the resolutionor peak area of the four test compounds

33 Application of the Proposed Method for Quality Controlof Coformulated Tablets Containing CDO and MBV MixtureThe applicability of the validated HPLC method to thesimultaneous determination of MBV and CDO was verifiedby the analysis of their coformulated Coloverin A tabletsThe obtained results were statistically compared with thoseof the comparison method [21] by Student 119905-test and thevariance ratio F-test revealing no significant differences[29] The results in Table 2 demonstrate the quality of theanalyzed pharmaceutical samples and the applicability of themethod for quality control analysisThe obtained results werein accordance with the labeled claim amount The resultsof the assay indicate that the method is selective for the


Table 1 Analytical performance data for determination of CDO MBV VER and ACB by the proposed method

Parameter ResultCDO MBV VER ACB

Concentration range (120583gmL) 10ndash1000 100ndash2000 20ndash400 20ndash400Limit of detection (LOD) (120583gmL) 023 067 005 015Limit of quantification (LOQ) (120583gmL) 071 204 015 045Correlation coefficient (119903) 09999 09999 09999 09999Slope 105 times 105 292 times 104 693 times 104 687 times 104

Intercept 215 times 104 108 times 104 minus176 times 104 734 times 104

Standard deviation of the residuals (119878119910119909

) 150 times 104 998 times 103 381 times 103 885 times 103

Standard deviation of the intercept (119878119886


Standard deviation of the slope (119878119887


RSD 091 099 094 094 error ( RSDradic119899) 034 037 035 038

Table 2 Application of the proposed and comparison methods for the determination of CDO and MBV in pure form and coformulatedtablets

MatrixProposed method Comparison method [21]

CDO MBV CDO MBVConc taken (120583gmL) founda Conc taken (120583gmL) founda founda

Pure form

10 9800 100 9817 10140 1015325 9860 200 9825 9830 981650 10000 400 10010 10060 10066150 10006 600 10010250 9952 800 10039500 10055 1000 100461000 9989 2000 9983

Mean plusmn SD 9952 plusmn 090 9961 plusmn 098 10010 plusmn 161 10012 plusmn 175119905-value 0753 (2306)b 0597 (2306)b

119865-value 3187 (514)b 3176 (514)b

Coloverin Atablets (5mgCDO + 135mgMBVtablet)

30 10022 810 9825 10120 1008850 9998 1350 9901 9858 989570 9905 1890 9858 10051 10038


119865-value 4829 (1900)b 691 (1900)baEach result is the average of three separate determinationsbValues between parentheses are the tabulated 119905- and 119865-values at 119875 = 005 [29]

Table 3 Precision data for the determination of CDO and MBV by the proposed method

Compound Conc (120583gmL) Intraday precision Interday precisionMean found plusmn SD RSD Mean found plusmn SD RSD

CDO10 10010 plusmn 091 091 9945 plusmn 107 108500 10077 plusmn 144 143 9915 plusmn 095 0951000 10044 plusmn 055 055 10050 plusmn 064 063

MBV100 10083 plusmn 063 062 9909 plusmn 081 0821000 10041 plusmn 067 067 9949 plusmn 133 1332000 9975 plusmn 053 053 10042 plusmn 109 109


Table 4 Application of the proposed method for the determination of CDO MBV and their degradation products in binary mixtures

Parameter Conc taken (120583gmL) founda

CDO MBV CDO MBV

Binary mixture of CDOMBV30 810 9982 998750 1350 9852 1005870 1890 9865 9908

Mean plusmn SD 9899 plusmn 072 9984 plusmn 075 RSD 072 075 error 041 043

Conc taken (120583gmL) found of CDOa

CDO ACB

Binary mixture of CDOACB

1000 20 99081000 40 98701000 80 99121000 100 10001

Mean plusmn SD 9922 plusmn 056 RSD 056 error 032

Conc taken (120583gmL) found of MBVa

MBV VER

Binary mixture of MBVVER

2000 20 100202000 40 99682000 80 100032000 100 9998

Mean plusmn SD 9997 plusmn 022 RSD 022 error 008aEach result is the average of three separate determinations

Table 5 Final system suitability test parameters for the proposed method

Compound Number of theoretical plates (119873) Retention factor (1198961015840) Tailing factor (119879)CDO 2778 042 144MBV 2542 113 155VER 2433 015 152ACB 2326 140 148Compoundsa Resolution (119877

119904

) Selectivity factor (120572)CDOVER 264 280CDOMBV 504 269MBVACB 145 124aResolution and selectivity factor were calculated for each two adjacent peaksCalculations were done according to USP guidelines [3]

assay of MBV and CDO without interference from commontablet excipients A typical chromatogram obtained from theanalysis of tablets is shown in Figure 4 As can be seenno degradation of the sample was observed indicating thestability of tablets under storage conditions

4 Conclusion

A fast simple and accurate stability-indicating HPLCmethod has been developed for the simultaneous determina-tion of MBV and CDO and their impurities and degradation

products that are VER and ACB respectively The methodallows identification quantitative analysis and purity assess-ment to be performed simultaneouslyThe proposed methodappears to be suitable for quality control laboratories whereeconomy and time-saving are essential

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


Time (min)

Det

ecto

r res

pons

e (Ab

s)

CDO46

MBV69

(a)

200

150

100

50

0

00 25 50 75 100 125 150

Figure 4 Representative chromatogram obtained for simultaneousdetermination of CDO (50120583gmL) and MBV (1350 120583gmL) incoformulated Coloverin A tablets where (a) is the solvent front

Authorsrsquo Contribution

Rania N El-Shaheny proposed the method and carried outthe experimental work and statistical analysis of the dataFathalla F Belal supervised the work Both authors approvedthe final paper

References

[1] S C SweetmanMartindale The Complete Drug Reference ThePharmaceutical Press London UK 36th edition 2009

[2] The British Pharmacopoeia Her Majestyrsquos Stationary OfficeLondon UK 2012

[3] The United States Pharmacopoeia 35th andThe National formu-lary 30th United Book Press Rockville Md USA 2012

[4] I P Gorelov S S Ryasenskii S V Kartamyshev and MV Fedorova ldquoA solid-state ionic-selective electrode with anionic-electronic transducer for determining chlordiazepoxiderdquoJournal of Analytical Chemistry vol 60 no 1 pp 65ndash69 2005

[5] M R Khoshayand H Abdollahi A Moeini A Shamsaie AGhaffari and S Abbasian ldquoSimultaneous spectrophotometricdetermination of chlordiazepoxide and clidinium using multi-variate calibration techniquesrdquo Drug Testing and Analysis vol2 no 9 pp 430ndash435 2010

[6] M I Toral P Richter N Lara P Jaque C Soto and MSaavedra ldquoSimultaneous determination of chlordiazepoxideand clidinium bromide in pharmaceutical formulations byderivative spectrophotometryrdquo International Journal of Phar-maceutics vol 189 no 1 pp 67ndash74 1999

[7] A H M Sarrafi Z Khodakarami and M Karkeabadi ldquoSimul-taneous spectrophotometric determination of amitriptylinehydrochloride and chlordiazepoxide in pharmaceutical tabletsbymultivariate calibrationmethodrdquoE-Journal of Chemistry vol6 supplement 1 pp S111ndashS116 2009

[8] S Patel N J Patel and S A Patel ldquoSimultaneous spec-trophotometric estimation of imipramine hydrochloride andchlordiazepoxide in tabletsrdquo Indian Journal of PharmaceuticalSciences vol 71 no 4 pp 468ndash472 2009

[9] K B Borges E F Freire I Martins and M E P Bde Siqueira ldquoSimultaneous determination of multibenzodi-azepines by HPLCUV investigation of liquid-liquid and solid-phase extractions in human plasmardquo Talanta vol 78 no 1 pp233ndash241 2009

[10] A Pathak P Rai and S J Rajput ldquoStability-indicating HPLCmethod for simultaneous determination of clidinium bromide

and chlordiazepoxide in combined dosage formsrdquo Journal ofChromatographic Science vol 48 no 3 pp 235ndash239 2010

[11] S Khodadoust and M Ghaedi ldquoOptimization of dispersiveliquid-liquidmicroextraction with central composite design forpreconcentration of chlordiazepoxide drug and its determina-tion by HPLC-UVrdquo Journal of Separation Science vol 36 no 11pp 1734ndash1742 2013

[12] V Soentjens-Werts J G Dubois G Atassi and M HanocqldquoChlordiazepoxide photoisomerization kinetics into oxaziri-dine A HPLC studyrdquo Talanta vol 42 no 4 pp 581ndash589 1995

[13] S E Roberts and M F Delaney ldquoDetermination of chlor-diazepoxide its hydrochloride and related impurities in phar-maceutical formulations by reversed-phase high-performanceliquid chromatographyrdquo Journal of Chromatography vol 283pp 265ndash272 1984

[14] E A Abdelaleem and N S Abdelwahab ldquoValidated chro-matographic and spectrophotometric methods for analysis ofsome amoebicide drugs in their combined pharmaceuticalpreparationrdquo Pakistan Journal of Pharmaceutical Sciences vol26 no 1 pp 175ndash183 2013

[15] A F M ElWalily A El Gindy andM F Bedair ldquoApplication offirst-derivative UV-spectrophotometry TLC-densitometry andliquid chromatography for the simultaneous determination ofmebeverine hydrochloride and sulpiriderdquo Journal of Pharma-ceutical andBiomedical Analysis vol 21 no 3 pp 535ndash548 1999

[16] A M El-Didamony ldquoSpectrophotometric determination ofbenzydamine HCl levamisole HCl and mebeverine HClthrough ion-pair complex formation with methyl orangerdquoSpectrochimica ActamdashPart A Molecular and Biomolecular Spec-troscopy vol 69 no 3 pp 770ndash775 2008

[17] S A Shama and A S Amin ldquoSpectrophotometric microde-termination of nefopam mebevrine and phenylpropanolaminehydrochloride in pharmaceutical formulations using alizarinsrdquoSpectrochimica Acta Part A Molecular and Biomolecular Spec-troscopy vol 60 no 8-9 pp 1769ndash1774 2004

[18] M S Arayne N Sultana and F A Siddiqui ldquoA new RP-HPLC method for analysis of mebeverine hydrochloride inraw materials and tabletsrdquo Pakistan Journal of PharmaceuticalSciences vol 18 no 2 pp 11ndash14 2005

[19] M A Radwan H H Abdine and H Y Aboul-Enein ldquoA vali-dated chiralHPLCmethod for the determination ofmebeverineHCI enantiomers in pharmaceutical dosage forms and spikedrat plasmardquo Biomedical Chromatography vol 20 no 2 pp 211ndash216 2006

[20] MWalash M Sharaf El-Din N El-Enany M Eid and Sh Sha-lan ldquoFirst derivative synchronous fluorescence spectroscopy forthe simultaneous determination of sulpiride and mebeverinehydrochloride in their combined tablets and application to realhuman plasmardquo Journal of Fluorescence vol 20 no 6 pp 1275ndash1285 2010

[21] R S Haggag R A Shaalan and T S Belal ldquoValidated HPLCdetermination of the two fixed dose combinations (chlor-diazepoxide hydrochloride and mebeverine hydrochloridecarvedilol and hydrochlorothiazide) in their tabletsrdquo Journal ofAOAC International vol 93 no 4 pp 1192ndash1200 2010

[22] J Patel J K Patel and V P Patel ldquoSimultaneousspectrophotometric estimation of mebeverine hydrochlorideand chlordiazepoxide in tablet dosage formrdquo Inventi RapidPharm Analysis amp Quality Assurance vol 78 no 11 2011httpinventiinjournalarticlerapid49277pharm-analysis-quality-assurancepi


[23] H M Heneedak I Salama S Mostafa and M El-Sadek ldquoAstability-indicating HPLC method for the simultaneous deter-mination ofmebeverine hydrochloride and chlordiazepoxide incommercial tabletsrdquoCurrent Analytical Chemistry vol 10 no 4pp 565ndash573 2014

[24] R Vardanyan and V Hruby Synthesis of Essential DrugsElsevier Amsterdam The Netherlands 1st edition 2006

[25] J A de Schutter F de Croo G van der Weken W van denBossche and P de Moerloose ldquoStability study and quantitativedetermination ofmebeverine hydrochloride in tablets bymeansof reversed-phase high-performance liquid chromatographyrdquoChromatographia vol 20 no 3 pp 185ndash192 1985

[26] ldquoICH Harmonized Tripartite Guidelines Validation ofAnalytical Procedures Text and Methodology Q2(R1)International Conference on Harmonization 2005rdquo May 2013httpwwwichorgproductsguidelinesqualityarticlequal-ity-guidelineshtml

[27] I A Naguib and M Abdelkawy ldquoDevelopment and valida-tion of stability indicating HPLC and HPTLC methods fordetermination of sulpiride and mebeverine hydrochloride incombinationrdquo European Journal ofMedicinal Chemistry vol 45no 9 pp 3719ndash3725 2010

[28] B C Smith Infrared Spectral Interpretation A SystematicApproach CRC Press LLC Boca Raton Fla USA 1999

[29] J N Miller and J C Miller Statistics and Chemometrics forAnalytical Chemistry Pearson Education Harlow UK 5thedition 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

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International Journal ofPhotoenergy


Carbohydrate Chemistry

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Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


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Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


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Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


NHN

N+

OminusCl

(a)

NO

O

O

O

OHCl

(b)

O NH2

Cl

(c)

OH

O

O

O

(d)

Figure 1 Structural formulas of (a) chlordiazepoxide (b) mebeverine HCl (c) 2-amino-5-chlorobenzophenone and (d) veratric acid

01M perchloric acid and a spectrophotometric method forits determination in pure form and tablets respectivelyManyanalytical methods were applied for determination of MBVsuch as spectrophotometry [14ndash17] TLC [15] HPLC [14 1518 19] and spectrofluorimetry [20]

Reviewing the scientific literature revealed an HPLCmethod [21] and two spectrophotometric methods based onthe simultaneous equations and absorbance ratio method[22] for the simultaneous estimation of CDO and MBV intheir coformulated tablets These methods are not stability-indicating moreover the pharmaceutical ratio of the twodrugs (1 CDO 27 MBV) was not achieved in the two spec-trophotometricmethods [22] which are linear over the rangesof 10ndash100 and 20ndash120 120583gmL for CDO and MBV respec-tively so they are not practically applicable to coformulateddosage forms Only one HPLC method [23] was publishedas a stability-indicating assay for the two drugsrsquo combinationNevertheless this method suffers from some drawbacks suchas poor sensitivity narrow linearity range and complicatedgradient elution program Hence our main target was todevelop a simple sensitive and rapid isocratic stability-indicatingHPLCmethod for the simultaneous determinationof the two drugs in their combined tablets in the presence oftheir degradation products or synthesis impurities

The BP [2] and the USP [3] define 2-amino-5-chlorobenzophenone (ACB Figure 1(c)) as an impurityin CDO pure and dosage forms ACB is the starting materialfor synthesis of many diazepams including CDO [24]On the other hand MBV being an ester-containing drugis easily susceptible to hydrolytic degradation giving thecorresponding acid degradation product veratric acid (34-dimethoxy benzoic acid VER Figure 1(d)) [25] Moreoverthe BP [2] defines VER as a related substance in MBV pureform and tablets

In this study we developed a stability-indicating HPLCmethod for the analysis of CDO and MBV in combinationand in the presence of MBV degradation product (VER) andCDO synthetic precursor and main impurity (ACB) Theidentification of the degradation product of MBV was ascer-tained by mass spectrometry and IR studies The developedmethod was validated according to ICH Guidelines [26]Theproposed procedure is suitable for the quality control analysisand purity assessment of dosage forms containing the twostudied compounds

2 Experimental

21 Instruments Separation was performed using a Shi-madzu HPLC system (Shimadzu Corporation Japan) con-sisting of LC-20AD liquid chromatograph equipped with aRheodyne injector valve and a 20120583L sample loop a SPD20A UV-Vis detector DGU-20A5 online solvent degasserand CBM-20A communication busmodule A Consort P-901pH-meter (Turnhout Belgium) was used for pH adjustment

22 Materials Gift samples of chlordiazepoxide and mebev-erine HCl were kindly provided by EVA Pharma Co (CairoEgypt) Their purities are 10010 and 10012 respectivelyas determined by the reference method [21] 2-Amino-5-chlorobenzophenone was purchased from Sigma Aldrich Co(Steinheim am Albuch Germany) Coloverin A film coatedtablets labeled to contain 5mg CDO + 135mg MBVtablet(batch number 120960A) product of Chemipharm Pharma-ceutical Industries SAE 6th of October city Egypt werepurchased from a local pharmacy

23 Reagents All solvents used were of HPLC grade and allchemicals were of analytical reagent grade High purity water




254plates using













100

80

60

404000 4003000 2000 1000

12 3

4

56

7

8

910

11

12

13

1415

161718

19

20

21

22

23

24

Tran

smitt

ance

()


(a)

70

60

50

40

30

20

10

060 80 100 120 140 160 180

56

6496

110120

165

182

Rela

tive a

bund

ance



O

OO

OH

(b)












VER

CDO

MBV

ACB38

46

69

77

Time (min)

Det

ecto

r res

pons

e (Ab

s)

(a)

400

300

200

100

0

00 25 50 75 100 125








119910119909) slope (119878





LOQ =10119878119886

119887

(1)

LOD =33119878119886

119887

(2)

























Pure form

10 9800 100 9817 10140 1015325 9860 200 9825 9830 981650 10000 400 10010 10060 10066150 10006 600 10010250 9952 800 10039500 10055 1000 100461000 9989 2000 9983


119865-value 3187 (514)b 3176 (514)b


30 10022 810 9825 10120 1008850 9998 1350 9901 9858 989570 9905 1890 9858 10051 10038










CDO MBV CDO MBV




CDO ACB


1000 20 99081000 40 98701000 80 99121000 100 10001



MBV VER


2000 20 100202000 40 99682000 80 100032000 100 9998




119904



4 Conclusion






Time (min)

Det

ecto

r res

pons

e (Ab

s)

CDO46

MBV69

(a)

200

150

100

50

0

00 25 50 75 100 125 150




References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




254plates using













100

80

60

404000 4003000 2000 1000

12 3

4

56

7

8

910

11

12

13

1415

161718

19

20

21

22

23

24

Tran

smitt

ance

()


(a)

70

60

50

40

30

20

10

060 80 100 120 140 160 180

56

6496

110120

165

182

Rela

tive a

bund

ance



O

OO

OH

(b)












VER

CDO

MBV

ACB38

46

69

77

Time (min)

Det

ecto

r res

pons

e (Ab

s)

(a)

400

300

200

100

0

00 25 50 75 100 125








119910119909) slope (119878





LOQ =10119878119886

119887

(1)

LOD =33119878119886

119887

(2)

























Pure form

10 9800 100 9817 10140 1015325 9860 200 9825 9830 981650 10000 400 10010 10060 10066150 10006 600 10010250 9952 800 10039500 10055 1000 100461000 9989 2000 9983


119865-value 3187 (514)b 3176 (514)b


30 10022 810 9825 10120 1008850 9998 1350 9901 9858 989570 9905 1890 9858 10051 10038










CDO MBV CDO MBV




CDO ACB


1000 20 99081000 40 98701000 80 99121000 100 10001



MBV VER


2000 20 100202000 40 99682000 80 100032000 100 9998




119904



4 Conclusion






Time (min)

Det

ecto

r res

pons

e (Ab

s)

CDO46

MBV69

(a)

200

150

100

50

0

00 25 50 75 100 125 150




References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


100

80

60

404000 4003000 2000 1000

12 3

4

56

7

8

910

11

12

13

1415

161718

19

20

21

22

23

24

Tran

smitt

ance

()


(a)

70

60

50

40

30

20

10

060 80 100 120 140 160 180

56

6496

110120

165

182

Rela

tive a

bund

ance



O

OO

OH

(b)












VER

CDO

MBV

ACB38

46

69

77

Time (min)

Det

ecto

r res

pons

e (Ab

s)

(a)

400

300

200

100

0

00 25 50 75 100 125








119910119909) slope (119878





LOQ =10119878119886

119887

(1)

LOD =33119878119886

119887

(2)

























Pure form

10 9800 100 9817 10140 1015325 9860 200 9825 9830 981650 10000 400 10010 10060 10066150 10006 600 10010250 9952 800 10039500 10055 1000 100461000 9989 2000 9983


119865-value 3187 (514)b 3176 (514)b


30 10022 810 9825 10120 1008850 9998 1350 9901 9858 989570 9905 1890 9858 10051 10038










CDO MBV CDO MBV




CDO ACB


1000 20 99081000 40 98701000 80 99121000 100 10001



MBV VER


2000 20 100202000 40 99682000 80 100032000 100 9998




119904



4 Conclusion






Time (min)

Det

ecto

r res

pons

e (Ab

s)

CDO46

MBV69

(a)

200

150

100

50

0

00 25 50 75 100 125 150




References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


VER

CDO

MBV

ACB38

46

69

77

Time (min)

Det

ecto

r res

pons

e (Ab

s)

(a)

400

300

200

100

0

00 25 50 75 100 125








119910119909) slope (119878





LOQ =10119878119886

119887

(1)

LOD =33119878119886

119887

(2)

























Pure form

10 9800 100 9817 10140 1015325 9860 200 9825 9830 981650 10000 400 10010 10060 10066150 10006 600 10010250 9952 800 10039500 10055 1000 100461000 9989 2000 9983


119865-value 3187 (514)b 3176 (514)b


30 10022 810 9825 10120 1008850 9998 1350 9901 9858 989570 9905 1890 9858 10051 10038










CDO MBV CDO MBV




CDO ACB


1000 20 99081000 40 98701000 80 99121000 100 10001



MBV VER


2000 20 100202000 40 99682000 80 100032000 100 9998




119904



4 Conclusion






Time (min)

Det

ecto

r res

pons

e (Ab

s)

CDO46

MBV69

(a)

200

150

100

50

0

00 25 50 75 100 125 150




References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of
















Pure form

10 9800 100 9817 10140 1015325 9860 200 9825 9830 981650 10000 400 10010 10060 10066150 10006 600 10010250 9952 800 10039500 10055 1000 100461000 9989 2000 9983


119865-value 3187 (514)b 3176 (514)b


30 10022 810 9825 10120 1008850 9998 1350 9901 9858 989570 9905 1890 9858 10051 10038










CDO MBV CDO MBV




CDO ACB


1000 20 99081000 40 98701000 80 99121000 100 10001



MBV VER


2000 20 100202000 40 99682000 80 100032000 100 9998




119904



4 Conclusion






Time (min)

Det

ecto

r res

pons

e (Ab

s)

CDO46

MBV69

(a)

200

150

100

50

0

00 25 50 75 100 125 150




References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




CDO MBV CDO MBV




CDO ACB


1000 20 99081000 40 98701000 80 99121000 100 10001



MBV VER


2000 20 100202000 40 99682000 80 100032000 100 9998




119904



4 Conclusion






Time (min)

Det

ecto

r res

pons

e (Ab

s)

CDO46

MBV69

(a)

200

150

100

50

0

00 25 50 75 100 125 150




References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Time (min)

Det

ecto

r res

pons

e (Ab

s)

CDO46

MBV69

(a)

200

150

100

50

0

00 25 50 75 100 125 150




References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

research article simultaneous hplc …downloads.hindawi.com/journals/jchem/2015/293719.pdfresearch...

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