development and validation of first order derivative method for simultaneous estimation of quinapril...

Int. J. Pharm. Res. Sci., 013, 01(1), 16-25. ISSN: 2348 –0882

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Development And Validation Of First Order Derivative MethodFor Simultaneous Estimation Of Quinapril Hcl And

Hydrochlorothiazide In Combined Pharmaceutical Formulation

Reema Jaiswal*1, Pinak patel1

1Department of Pharmaceutical Analysis, Indubhai Patel College of Pharmacy and ResearchCentre, Dharmaj, Gujarat-388430, India

2Department of Quality Assurance, Indubhai Patel College of Pharmacy and Research Centre,Dharmaj, Gujarat-388430, India

====================================================================ABSTRACTA new simple, sensitive, rapid, accurate,precise and economical DerivativeSpectrophotometric method for thesimultaneous determination of QuinaprilHCl (QUI) and Hydrochlorothiazide(HCTZ) in their combined pharmaceuticaldosage form was developed. The derivativeSpectrophotometric method was based theabsorbance of the solutions were measuredat 242.45 nm (λ1), and 257.17 nm (λ2) forthe estimation of both the drugs. Thelinearity was obtained in the concentrationrange of 80-240 μg/ml for QUI and 10-50μg/ml for HSCfl e mean recovery was99.93 – 100.33 % and 99.06-101.25% forQUI and HCTZ respectively. The results ofanalysis have been validated statistically asper ICH guidelines.KEYWORDSQuinapril Hydrochloride,Hydrochlorthiazide, Derivative method,Methanol.

Corresponding AuthorReema JaiswalEmail address: [email protected]: 18.12.2013Revised: 26.12.2013Accepted: 29.12.2013

INTRODUCTIONQuinapril Hydrochloride (QUI), a 3-Isoquinolinecarboxylic acid,2-[2-[[1-(ethoxycarbonyl)-3-phenylpropyl]amino]-1-oxopropyl]-1,2,3,4-tetrahydro-,monohydrochloride [Figure 1a]. QUI isofficial in United State Pharmacopoeia(USP) but it is listed in Merck Index1,Martindale and Complete Drug Reference2-7.It is a ACE inhibitor and indicated intreatment of symptomatic treatment of highblood pressure and used with some otherdrugs in combination therapy. Literaturesurvey revealed that various8-12 , Capillaryelectrophoresis, Ion-pair HPLC and HPTLCmethods have been reported for quantitativeestimation of QUI in pharmaceutical dosageforms and biological fluids individually or incombination with other drugs.Hydrochlorothiazide (HCTZ) is chemically6-Chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide[Figure 1b]. HCTZ is a thiazide diureticinhibits water reabsorption in the nephron byinhibiting the sodium-chloride symporter(SLC12A3) in the distal convoluted tubule,which is responsible for 5% of total sodiumreabsorption. It is used for the treatment ofthe treatment of blood pressure and in


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management of oedema.13-15 HCTZ is is no published RP-HPLC method for thisofficial in IP,BP16,17,USP18 and JP19 describeHPLC method for its estimation. The reviewof literature revealed that various analyticalmethods involving Spectrophotometry,

combination. So, the present paper describesa simple, accurate and precise method forsimultaneous estimation of QUI and HCTZin combined Pharmaceutical Formulation by

HPLC20-26and HPTLC27-29 have been RP-HPLC method. The veloped methodreported for HCTZ in pharmaceuticaldosage forms and biological fluids

was validated in accordance with ICHGuidelines30and successfully employed for

individually or in combination with other the assay of QUI and HCTZ in theirdrugs.

To the best of our knowledge, there.

combined dosage form

Figure 1: Chemical structure of (a) QUI and (b) HCTZMATERIALS AND METHODS An accurately weighed quantity ofReagents and Chemicals QUI (100 mg) and HCTZ (100 mg) were

Analytically pure QUIwere kindly provided by

and HCTZAurobindo

transferred to a separate 100 ml volumetricflask and 50 ml of methanol was added to

Pharmaceuticals Ltd.,Hyderabad and both volumetric flask and sonicated for 5Chemyes Corporation Vadodara, Gujarat minutes. Volume was adjusted up to theIndia as gratis samples. Methanol was used mark with methanol to obtain standardas solvent. Tablet of QUI and HCTZ in solution having concentration of QUI (1000combined dosage form, i.e. Q-PRIL H-10 μg/ml) and HCTZ (1000 μg/ml). 12.5 mlwas procured from local market.11-13 solutions of QUI (1000 μg/ml) and 10 mlINSTRUMENTS HCTZ (1000 μg/ml) were transferred to a

A Shimadzu UV/Vis 1800 doublebeam spectrophotometer with a wavelengthaccuracy (± 0.3 nm), 1 cm matched quartzcells and UV probe 2.32 software was usedfor all the spectral measurements and

separate 25 ml volumetric flask and 100 mlvolumetric flask respectively and diluted upto concentration of QUI (500 μg/ml) andHCTZ (50μg/ml) with methanol.First order Derivative method

Shimadzu UV/Vis 1601 double beam 80-240 µ g/ml solutions of QUI and 10-50spectrophotometer with a wavelength µ g/ml solutions of HCTZ were prepared inaccuracy (± 0.3 nm) and 1 cm matchedquartz cells was used for reproducibilityStudy. Calibrated analytical balance K-EA210 (K-Roy Instrument Pvt. Ltd) was usedfor weighing purpose.Preparation of standard stock solutions

methanol by appropriate dilution andspectrum was recorded between 200-400nm. The absorbance of the solutions weremeasured at 242.45 nm (λ1), and 257.17 nm(λ2), for the estimation of both the drugs byproposed method. The quantitative


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manner, at 257.17nm different Accuracyconcentrations of QUI possess zero D1 Accuracy was assessed by

determination of QUI is carried out bymeasuring absorbance difference between242.45 nm and 257.17 nm where HCTZ hassame absorbance at both the wavelengths.For estimation of QUI and HCTZ usingderivative spectroscopy, zero crossingmethod was decided to be used. In thismethod two wavelengths are required. Onewavelength is selected at which QUI showszero absorbance while other drug HCTZshows considerable absorbance. The secondwavelength is selected such that HCTZshows zero absorbance while QUI showsconsiderable absorbance.The overlain derivative spectrum (firstorder) of QUI and HCTZ at differentconcentrations revealed that at 242.45 nmdifferent concentration of HCTZ possesseszero D1 absorbance whereas QUI possessessignificant D1 absorbance. In a similar

stearate) were spiked into a pre weighedquantity of drugs. The fundamental ornormal spectrum was recorded byappropriate dilutions and the quantities ofdrugs were determined.Linearity

Appropriate volume of aliquot fromQUI and HCTZ standard stock solution wastransferred to volumetric flask of 10 mlcapacity. The volume was adjusted to themark with methanol to give solutionscontaining 80-240 µ g/ml QUI and 10-50µ g/ml HCTZ. All D0 Spectrum wererecorded for QUI and HCTZ respectively(n=5).Calibration curves were constructed byplotting average absorbance versusconcentrations for both drugs. Straight lineequations were obtained from thesecalibration curves.

absorbance whereas HCTZ possessessignificant D1 absorbance. Consideringabove facts, wavelength 242.45 nm and257.17nm were selected for the estimationof QUI and HCTZ respectivelyMethod validationThe proposed method has been extensivelyvalidated in terms of specificity, linearity,accuracy, precision, limits of detection(LOD) and quantification (LOQ), robustnessand reproducibility. The accuracy wasexpressed in terms of percent recovery ofthe known amount of the standard drugsadded to the known amount of thepharmaceutical dosage forms. The precision(Coefficient of Variation - C.V.) wasexpressed with respect to the repeatability,intra-day and inter-day variation in theexpected drug concentrations. Aftervalidation, the developed methods have beenapplied to pharmaceutical dosage form.Specificity

Commonly used excipients (starch,microcrystalline cellulose and magnesium

determination of the recovery of the methodby addition of standard drug to the pre-quantified placebo preparation at 3 differentconcentration levels 80, 100 and 120 %,taking into consideration percentage purityof added bulk drug samples. Eachconcentration was analyzed 3 times andaverage recoveries were measured.Precision

The repeatability was evaluated byassaying 6 times of sample solution preparedfor assay determination. The intraday andinterday precision study of QUI and HCTZwas carried out by estimating differentconcentrations of QUI (120, 160, 200µ g/ml) and HCTZ (20, 30, 40 µ g/ml), 3times on the same day and on 3 differentdays (second, third, fourth) and the resultsare reported in terms of C.V.Detection limit and Quantitation limit

ICH guideline describes severalapproaches to determine the detection andquantitation limits. These include visualevaluation, signal-to-noise ratio and the use


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of standard deviation of the response and theslope of the calibration curve. In the presentstudy, the LOD and LOQ were based on thethird approach and were calculatedaccording to the 3.3σ/S and 10σ/S criterions,respectively; where σ is the standarddeviation of y-intercepts of regression linesand s is the slope of the calibration curve.Robustness

The sample solution was preparedand then analyzed with change in the typicalanalytical conditions like stability ofanalytical solution.Reproducibility

The absorbance readings weremeasured at different laboratory for samplesolution using another spectrophotometer byanalyst and the values obtained wereevaluated using t- test to verify theirreproducibility.Determination of QUI and HCTZ in theirCombined DosageTwenty tablets were weighed and powdered.A powder quantity equivalent to 10 QUI and12.5 mg HCTZ was accurately weighed andtransferred to volumetric flask of 50 mlcapacity. 50 ml of methanol was transferredto this volumetric flask and sonicated for 15min. The flask was shaken and volume wasmade up to the mark with methanol. Theabove solution was filtered throughwhatman filter paper (0.45µ ).The flask wasshaken and volume was made up to the markwith methanol. From this solution 4 ml wastransferred to volumetric flask of 10 mlcapacity. Volume was made up to the markto give a solution containing 80 µ g/ml ofQUI using methanol(Solution A). Thissolution was used for the estimation of QUI,from (solution A) withdraw 1ml and diluteto 10 ml to give 8 μg/ml QUI and 10 μg/mlHCTZ and solution was used for estimationof HCTZ(solution B). The resulting solutionwas analyzed by proposed methods. Thequantitation was carried out by keeping

these values to the straight line equation ofcalibration curve.RESULTS AND DISCUSSIONIn First order Derivative wavelengthmethod, For estimation of QUI and HCTZusing derivative spectroscopy, zero crossingmethod was decided to be used. In thismethod two wavelengths are required. Onewavelength is selected at which QUI showszero absorbance while other drug HCTZshows considerable absorbance. The secondwavelength is selected such that HCTZshows zero absorbance while QUI showsconsiderable absorbance. The overlainderivative spectrum (first order) of QUI andHCTZ at different concentrations revealedthat at 242.45 nm different concentration ofHCTZ possesses zero D1 absorbancewhereas QUI possesses significant D1absorbance. In a similar manner, at257.17nm different concentrations of QUIpossess zero D1 absorbance whereas HCTZpossesses significant D1 absorbance.Considering above facts, wavelength 242.45nm and 257.17nm were selected for theestimation of QUI and HCTZ respectively .Calibration data for QUI and HCTZ areshown in Table 2 and 3 respectively.Calibration curves for QUI and HCTZ wereplotted between D1 absorbance andconcentration. The following equations forstraight line were obtained for QUI andHCTZ.Linear equation for QUI, y = 0.002x -0.011

Linear equation for HCTZ, y = 0.030x -0.203

The linearity of the calibration curve wasvalidated by the high values of correlationcoefficient of regression. The C.V values forQUI and HCTZ for repeatability was foundto be 0.54 and 0.51 %, respectively [Table1]. The Coefficient of Variance(C.V) (lessthan 2 %) indicates that the proposedmethod is repeatable. The C.V values ofIntraday (0.52 – 0.89% and 0.10 – 0.48%)


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and Interday (0.25 – 0.43 % and 0.11 – 0.23%) for QUI and HCTZ, respectively, revealthat the proposed method is precise. LODvalues for QUI and HCTZ were found to be1.88 and 0.18 μg/ml, respectively and LOQvalues for QUI and HCTZ were found to be6.20 and 0.54 μg/ml, respectively [Table 1].These data show that proposed method issensitive and precise for the determinationof QUI and HCTZ, respectively [Table 3, 4].

The recovery experiment wasperformed by the standard addition method.The percentage recovery was 99.93 –100.33% and 99.06 – 101.25% for QUI and

HCTZ, respectively [Table 5, 6]. The resultsof recovery studies indicate that theproposed method is accurate. The proposedvalidated method was successfully appliedto determine QUI and HCTZ in theircombined dosage form. The results obtainedfor QUI and HCTZ was comparable with thecorresponding labelled amount. Nointerference of the excipients with theabsorbance of interest appeared; hence theproposed method is applicable for theroutine simultaneous estimation of QUI andHCTZ in pharmaceutical dosage forms.

Figure 2-Calibration Curve for QUI Figure 3-Calibration Curve for HCTZ

Figure 4- Overlain D0 spectra of QUI (80-240)µg/ml in methanol


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Parameter QUI HCTZAnalytical wavelength 242.45 nm 257.17 nmRange (µ g/ml) 80– 240 10-50

Slope -0.002 0.030Intercept -0.010 -0.202Regression Coefficient (r2) 0.997 0.998Standard deviation of intercept 0.00114 0.00164

Figure 5- Overlain D0 spectra of HCTZ(10-50) µg/ml in methanol

Figure 6-QUI& HCTZ- first order ( D1) derivative overlain

Table 1: Summary of Validation Parameters of First order Derivative methodParameters QUI HCTZ% Recovery 99.70 – 101.10 99.80 – 101.62Repeatability(RSD, n=6) 0.54 0.51Precision(%RSD)Intra-day (n=3)Inter-day (n=3)

0.52-0.890.25-0.43

0.10-0.480.11-0.23

LOD 1.88 0.18LOQ 6.20 0.54Specificity specific specificSolvent Stability Stable for 24 hours Stable for 24 hours

Table 2: Statistical data QUI and HCTZ by First order Derivative method


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Table 3: Precision data for QUI

Concentration(μg/ml)

Intraday C.V. Interday C.V.

120 -0.2333±0.0020 0.89 -0.2321±0.0010 0.43

160 -0.3133±0.0024 0.64 -0.3120±0.0012 0.30

200 -0.3934±0.0022 0.52 -0.3921±0.0011 0.25

Table 4: Precision data for HCTZConcentration(μg/ml)

Intraday C.V. Interday C.V.

20 0.4333±0.0020 0.48 0.4320±0.0012 0.23

30 0.6836±0.0025 0.36 0.6820±0.0016 0.14

40 0.9960±0.0010 0.10 0.9981±0.0018 0.10

Table 5 :Reproducibility data for QUI (80 µg/ml)

Instrument1Mean ± S.D.


Result ofttest *

Value fromt-distributiontable

Inference

0.3153 ±0.0030

0.3166 ±0.0025

0.5783 4.30 There is no significancedifference

Table 6 :Reproducibility data for HCTZ( 10 µg/ml)



Result ofttest *

Value fromt-distributiontable

Inference

0.2956 ±0.0025

0.2936 ±0.0015 0.4380

4.30 There is no significancedifference

Table 7: Specificity and Selectivity study

Study QUI HCTZ

Specificity Specific Specific

Selectivity Selective Selective


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Table 8: Accuracy data for QUI and HCTZ

Level Amountof drugpresent

Amount Stddrug added(µg/ml)

AmountRecovered in(µg/ml) % Recovery ± S.D

US 80+10 QUI HCTZ QUI HCTZ QUI HCTZ

80 80+10 80 16 79.95 15.85 99.93±0.58 99.06± 0.41

100 80+10 100 20 100.20 20.10 100.20±0.36 100.50±0.52

120 80+10 120 24 120.40 24.30 100.33±0.47 101.25±0.68

Table 9: Assay Results of Marketed FormulationTablet Drug Labeled

claim(mg)

Amounttaken(µg/ml)

Amountfound(µg/ml)

% Label claim±S.D

Q-PRIL H-10TAB

QUI 10 80 79.60 98.50±0.0152HCTZ 12.5 10 9.97 99.70±0.0156

CONCLUSIONThe proposed dual wavelength methodprovides simple, specific, precise, accurateand reproducible quantitative analysis forsimultaneous determination of QUI andHCTZ in combined dosage form. Themethods were validated as per ICHguidelines in terms of specificity, linearity,accuracy, precision, limits of detection(LOD) and quantification (LOQ), robustnessand reproducibility. The proposed methodscan be used for routine analysis and qualitycontrol assay of QUI and HCTZ incombined dosage form.ACKNOWLEDGEMENT

The author is thankful to IndubhaiPatel College of Pharmacy and ResearchCentre (Dharmaj, India) for providing thenecessary facilities for research work and toall the staff members and friends for theirguidance and help throughout the researchwork.

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development and validation of first order derivative method for simultaneous estimation of quinapril...

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