spectrophotometricandmultivariatecalibrationtechniquesfor...

Research ArticleSpectrophotometric and Multivariate Calibration Techniques forSimultaneous Determination of Different Drugs inPharmaceutical Formulations and Human Urine Evaluation ofGreenness Profile

Ahmed Mostafa

Department of Pharmaceutical Chemistry College of Clinical Pharmacy Imam Abdulrahman Bin Faisal UniversityKing Faisal Road PO Box 1982 Dammam 31441 Saudi Arabia

Correspondence should be addressed to Ahmed Mostafa ammostafuwaterlooca

Received 9 April 2020 Accepted 1 May 2020 Published 29 May 2020

Academic Editor Antonio V Herrera-Herrera

Copyright copy 2020 Ahmed Mostafa is 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

Eco-friendly rapid and cost-effective two spectrophotometric methods were developed and validated for the determination ofatenolol paracetamol hydrochlorothiazide and levofloxacin e first method is the newly developed extended derivative ratio(EDR) and the second method is multivariate curve resolutionmdashalternating least squares (MCR-ALS) In the EDR method theextended derivative ratio amplitudes at 2816 2376 2792 and 2828 nm were used for quantification of atenolol paracetamolhydrochlorothiazide and levofloxacin respectively In the MCR-ALS method calibration model was developed and correlationconstraint was employed External validation data set composed of sevenmixtures was used and different figures of merits such asroot mean square error of prediction standard error of prediction bias and relative error of prediction were calculated andsatisfactory results were obtained Both methods provided comparable results e methods were validated and applied for thedetermination of the target analytes in dosage forms spiked and real human urine ereafter the obtained results were sta-tistically compared to the published methods and revealed no significant difference regarding accuracy and precision Fur-thermore the greenness profile of the methods was evaluated using the National Environmental Methods Index ldquoNEMIrdquo andAnalytical Eco-Scale e developed methods can be used as a valid eco-friendly and simple cost-effective alternative to thecommonly used chromatographic methods for the routine analysis of the studied drugs in dosage forms and human urine

1 Introduction

Atenolol (AT) (Figure 1(a)) is a selective szlig1 receptor blockerand is used for the treatment of hypertension and coronaryheart disease AT undergoes little or no metabolism in theliver and is mainly excreted in urine (over 85 of theabsorbed drug is excreted in urine as unchanged drug) [1]Different analytical techniques have been reported for theanalysis of AT such as high performance liquid chroma-tography (HPLC) [2ndash4] spectrophotometric [2 5 6] cap-illary electrophoresis [7 8] gas chromatography (GC) [9]and spectrofluorimetric [10] methods

Paracetamol (PR) or acetaminophen (Figure 1(b)) is anover-the-counter (OTC) analgesic and antipyretic drug PR

is metabolized in liver and mainly excreted in urine Around1ndash4 of the administered dose is excreted unchanged [11]However 47ndash62 of the dose is excreted as paracetamolconjugated to glucuronide and 25ndash36 as sulphate conju-gates ([12 13] A small amount (8ndash10) is metabolizedthrough oxidation to form cysteine and mercapturic acidconjugates [13]

Various studies can be found in the literature for theanalysis of PR including HPLC [14ndash17] GC [18] spectro-photometry [19 20] voltammetry [21] and electrophoresis[22]

Hydrochlorothiazide (HZ) (Figure 1(c)) is a thiazidediuretic that is used to treat hypertension and edema as-sociated with certain disease conditions such as congestive

HindawiJournal of Analytical Methods in ChemistryVolume 2020 Article ID 8873003 15 pageshttpsdoiorg10115520208873003

heart failure HZ is excreted (gt95) as unchanged drug inurine [23 24] Different techniques have been reported forHZ quantitation such as HPLC [25ndash28] GC [29] spec-trophotometry [30 31] and voltammetry [32]

Levofloxacin (LV) (Figure 1(d)) is a fluoroquinoloneantibiotic that inhibits DND topoisomerase IV and gyrase inGram-positive and Gram-negative bacteria respectively Itis used for urinary respiratory skin and soft tissue infec-tions [33] Several reports are found in the literature for thedetermination of LV including different techniques such asHPLC [34 35] spectrophotometry [34 36] voltammetry[37] and capillary electrophoresis [38]

AT PR HZ and LV can be coadministered together bysome patients and they are excreted mainly in the urine Tothe best of our knowledge no method has been reported forthe simultaneous determination of the four drugs

Green analytical chemistry (GAC) which is concernedwith developing new methods that are sustainable and moreenvironmentally friendly has gained much interest amonganalytical chemists [39 40] However the challenge is to

achieve a compromise between increasing the quality of thedeveloped methods and improving the method greenness[39 41] UV-Vis spectroscopy is considered a greener an-alytical technique when compared to HPLC UV-Visspectroscopy is a fast technique that consumes low solventvolumes thus reducing the amount of generated waste Inaddition this technique is cost-effective and does not needexpensive solvents or sophisticated instruments [42 43]However the major challenge when using UV-Vis spec-troscopy is the analysis of multicomponent mixtures es-pecially when the analytes spectra are strongly overlapped[42 44] To overcome this challenge the author proposedthe use of nonconventional spectrophotometric techniquenamely extended derivative ratio (EDR) In addition the useof multivariate calibration helped in the mathematicalresolution of the multicomponent mixture

erefore this work aimed to develop green cost-ef-fective and precise methods for the analysis of AT PR HZand LV Two methods including a nonconventional uni-variate method (ie extended derivative ratio (EDR)) and a

O

NH2

O

OH

NH

(a)

OH

HN O

(b)

SNH

HN

OO

Cl

SO

OH2N

(c)

N

N F

O

N

O

OH

O

(d)

Figure 1 Chemical structures of atenolol (a) paracetamol (b) hydrochlorothiazide (c) and levofloxacin (d)

2 Journal of Analytical Methods in Chemistry

multivariate method (ie multivariate curve reso-lutionmdashalternating least squares (MCR-ALS)) were devel-oped and validated and then successfully applied foranalyzing the four analytes in different pharmaceuticaldosage forms spiked and real human urine In addition themethod greenness was assessed using the National Envi-ronmental Methods Index (NEMI) [45 46] and the ana-lytical Eco-Scale [47]

11 eoretical Background

111 Extended Derivative Ratio Technique (EDR) Letrsquosconsider mixtureM composed of four compounds (W X Yand Z) Once Beerrsquos law is obeyed over the whole wavelengthrange the absorption spectrum ofMwill be equal to the sumof the individual spectra of the four analytes (ie the additiveproperty of Beerrsquos law) It can be defined by the followingequation

AM λi εW λi

CW + εX λiCX + εY λi

CY + εZ λiCZ (1)

where AMλiis the absorbance of M at wavelength λi εWλi

εXλi

εYλiand εZλi

are the molar absorptivity of compoundsW X Y and Z at wavelength λi and CW CX CY and CZ arethe concentrations of W X Y and Z respectively

For compound W determination a mixture (m1) con-taining all other three compounds (X Y and Z) exceptcompound W is scanned and it can be represented by thefollowing equation

Aprimem1λi εXλi

CdegX + εYλiCdegY + εZλi

CdegZ (2)

For both equations (1) and (2) the optical path is con-sidered to be 1 cm

Dividing the mixture spectrumM bym1 will result in thefollowing ratio spectrum

AM λi

Aprimem1 λi

εWλi

CW + εXλiCX + εYλi

CY + εZλiCZ

εXλiCdegX + εYλi

CdegY + εZλiCdegZ

εWλi

CW


CdegY + εZλiCdegZ

+εXλi

CX + εYλiCY + εZλi

CZ


CdegY + εZλiCdegZ

εW λi

CW


CdegY + εZλiCdegZ

+ K

(3)

where K is constante derivative ratio spectra can be represented as

follows

d

dλAM λi

Alsquom1 λi

1113890 1113891 d

dλεW λi

CW

εX λiCdegX + εY λi

CdegY + εZ λiCdegZ

1113890 1113891

d

dλεW λi



1113890 1113891CW

(4)

Equation (4) indicates that the derivative ratio spectrumamplitude is dependent and directly proportional to theconcentration of the compound W erefore a calibrationcurve can be drawn by dividing the spectra of variousconcentrations of a pure compound W by the ternarymixture of the other three compounds and a regressionequation can be obtained Similarly the other three com-pounds can be determined using the same procedure as forcompound W

112 Multivariate Curve ResolutionmdashAlternating LeastSquares (MCR-ALS) MCR is a soft-modeling algorithmthat can extract relevant information of the pure compo-nents in multicomponent systems It can be performedthrough the bilinear decomposition of the data matrix D asfollows

D CST

+ E (5)

where C and ST are the pure concentration and spectralprofiles [48] E is the residuals matrix of the data notexplained by the bilinear model e C and STprofiles can beoptimized iteratively using Alternating Least Squares (ALS)until a certain convergence criterion is achieved e initialestimates of the componentsrsquo spectra were used to start theoptimization process and simplendashto-use interactive self-modeling mixture analysis (SIMPLISMA) [49] wasemployed Specific constraints such as nonnegativity clo-sure unimodality and correlation constraints [50 51] can beapplied during the optimization process In the presentedwork nonnegativity spectra constraint nonnegativity con-centration constraint and correlation constraint were ap-plied e latter constraint enhances building a calibrationmodel that allows the quantitative analysis of components inthe presence of unknown interferences [50 52]

eMCR-ALS modelrsquos consistency and reliability can bemeasured using the percentage of lack of fit (Equation (6))and the percentage of total explained variance (Equation(7)) e following equations help to calculate the twoparameters

lack of fit () 100

1113936ije2ij

1113936ijd2ij

11139741113972

(6)

S2

100

1113936ijd2ij minus 1113936ije

2ij

1113936ijd2ij

11139741113972

(7)

where dij is an element of the data matrix D and eij is theassociated residual (the difference between experimentaldata input and model reproduced data)

(1) Validation of the Model e performance of the de-veloped model will be evaluated employing a data setcomposed of 7 mixtures used as an external validation setDifferent figures of merit were calculated to evaluate theobtained results according to the following equations [50]

Journal of Analytical Methods in Chemistry 3

Root mean square error of prediction (RMSEP)

RMSEP

1113936ni1 ci minus 1113954ci( 1113857

2

n

1113971

(8)

Bias

bias 1113936

ni1 ci minus 1113954ci( 1113857

n (9)

Standard error of prediction (SEP)

SEP

1113936ni1 ci minus 1113954ci minus bias( 1113857

2

n minus 1

1113971

(10)

Relative percentage error in the concentration predic-tions (RE )

RE () 100

1113936ni1 ci minus 1113954ci( 1113857

2

1113936ni1 c2i

1113971

(11)

where ci and 1113954ci are the known and predicted analyte con-centration in sample i respectively and n is the total numberof validation samples

Furthermore a linear regression fit was performed be-tween the known and predicted concentrations and slopeintercept and determination coefficients were calculated

2 Material and Methods

21 Instrumentation and Software Shimadzu UVndash1800double-beam spectrophotometer (Tokyo Japan) with 1 cmquartz cells was used for data acquisition Scans wererecorded in the wavelength range of 200ndash330 nm at 02 nmintervals Spectra were acquired by Shimadzu UV-Probeversion 262 e MCR model was developed using MCR-ALS GUI 20 software for use with Matlab 2015a [53] esoftware is freely available at httpwwwmcralsinfo

22ChemicalsandReagents Atenolol (CAS no 29122-68-798 minimum purity) paracetamol (CAS no 103-90-298 minimum purity) and hydrochlorothiazide (CAS no58-93-5 98 minimum purity) were purchased fromSigmandashAldrich (Steinheim Germany) Levofloxacinhemihydrate (CAS no 138199-71-0 99 minimum purity)was purchased from Santa Cruz Biotechnology Inc (SantaCruz Canada) Helix pomatia β-glucuronidase enzymeused for hydrolysis was obtained from SigmandashAldrich (StLouis USA)

Ethanol (LiChrosolvreg HPLC grade) was obtained fromMerck (Darmstadt Germany) Ultrapure water (182MΩ)was obtained by Pure Lab Ultra water system (ELGA HighWycombe United Kingdom) and used for all samplepreparation

is study was approved by the Research and EthicsCommittee of the College of Clinical Pharmacy ImamAbdulrahman Bin Faisal University Saudi Arabia Uponreceiving informed written consent blank urine sampleswere collected from three healthy volunteers (23ndash25 yearsold) and were kept at minus20degC

Commercial pharmaceutical dosage forms of the studieddrugs were purchased from the local market and they were asfollows

(i) Tenorminreg tablets (batch PF742) produced byAstraZeneca UK Limited United Kingdom andlabeled to contain 100mg AT per tablet

(ii) Panadrexreg tablets (batch JT377) produced byKuwait Saudi Pharmaceutical Industries Co Kuwaitand labeled to contain 500mg PR per tablet

(iii) HCT Georetic 25reg tablets (batch 1832207) pro-duced by Marcyrl Pharmaceutical Industries Egyptand labeled to contain 25mg HZ per tablet

(iv) Tavanicreg tablets (batch 7PK7A) produced bySanofi Winthrop Industries France and labeled tocontain 500mg LV per tablet

23 Procedures

231 Standard Solutions Stock solutions were prepared bydissolving 10mg of AT PR HZ and LV separately in 10mLethanol and were used as stock standard solutions for theEDR and MCR-ALS methods All solutions were stored at4degC and were stable for at least three months Workingsolutions were obtained by appropriate dilutions using ul-trapure water to reach the calibration range of each method

232 Spectroscopic Characteristics e UV spectra of so-lutions comprising 10 μgmLminus1 of AT and 5 μgmLminus1 of PRHZ and LV were analyzed separately over the wavelengthrange of 200ndash330 nm

233 EDR Method Appropriate volumes of each stockstandard solution of AT PR HZ and LV were transferredinto four different sets of 10-mL volumetric flasks and di-luted with ultrapure water to reach the calibration range of5ndash40 1ndash25 1ndash15 and 1ndash15 μgmLminus1 for AT PR HZ and LVrespectively e absorption spectra were measured over therange of 200 to 330 nm using ultrapure water as blank

(1) Determination of AT A mixture composed of 7 2 and4 μgmLminus1 of PR HZ and LV respectively was scannedagainst ultrapure water as blank and spectra were stored (ATdivisor) e UV spectra of working standard solutions ofAT were divided by the stored divisor and the secondderivative of the resulting ratio spectra was calculatedemploying Δλ 4 nm and 10 as a scaling factor e am-plitudes at 2816 nm were directly proportional to ATconcentration e calibration curve was obtained and thecorresponding regression equation was calculated

(2) Determination of PR A mixture composed of 15 3 and5 μgmLminus1 of AT HZ and LV respectively was scannedagainst ultrapure water as blank and spectra were stored (PRdivisor) e UV spectra of working standard solutions ofPR were divided by the stored divisor and the first derivativeof the resulting ratio spectra was calculated employing


Δλ 4 nm and 10 as a scaling factor e amplitudes at2376 nm were directly proportional to PR concentratione calibration curve was obtained and the correspondingregression equation was calculated

(3) Determination of HZ A mixture composed of 15 10 and5 μgmLminus1 of AT PR and LV respectively was scannedagainst ultrapure water as blank and spectra were stored (HZdivisor) e UV spectra of working standard solutions ofHZ were divided by the stored divisor and the first de-rivative of the resulting ratio spectra was calculated usingΔλ 2 nm and 10 as a scaling factor e amplitudes at2792 nm were directly proportional to HZ concentratione calibration curve was obtained and the correspondingregression equation was calculated

(4) Determination of LV A mixture composed of 15 7 and2 μgmLminus1 of AT PR and HZ respectively was scannedagainst ultrapure water as blank and spectra were stored (LVdivisor) e UV spectra of working standard solutions ofLV were divided by the stored divisor and the secondderivative of the resulting ratio spectra was calculatedemploying Δλ 4 nm and 10 as a scaling factor e am-plitudes at 2828 nm were directly proportional to theconcentration of LV e calibration curve was obtainedand the corresponding regression equation was calculated

234 MCR-ALS Method Calibration and validation setscomposed of 25 samples were constructed using a five-levelfour-factors design [54] in which five different concentra-tion levels of AT PR HZ and LV were introduced elevels ranges were 5ndash25 1ndash10 1ndash10 and 1ndash7 μgmLminus1 for ATPR HZ and LV respectively e UV spectra of all sampleswere scanned from 220 to 330 nm at 02 nm intervalsEighteen samples were used to build the calibration modelwhile seven samples randomly selected were used for ex-ternal validation e absorption spectra were exported intoMatlab to build the MCR-ALS calibration model usingMCR-ALS GUI 20 software [53] Fast nonnegativity leastsquares (for spectral and concentration profiles) and cor-relation constraints were employed

24 Sample Preparation

241 Analysis of Pharmaceutical Preparations Ten tabletsof each commercial formulation were weighed and pul-verized A portion equivalent to 1000mg of AT PR HZand LV were accurately added to a 100mL conical flask andsonicated with 50mL ethanol for 30min e solution wasfiltered into a 100mL volumetric flask and completed tomark with ethanol e procedures for EDR and MCR-ALSmethods were followed as detailed under Sections 233 and234 respectively

242 Analysis of Spiked and Real Human Urine SamplesAll urine samples were syringe filtered through 045 μmnylon filters Aliquots of 100 μL of blank urine were

transferred into a set of 10mL volumetric flasks Differentvolumes of the stock standard solutions of AT PR HZ andLV were added vortex mixed and the volume was com-pleted to mark with ultrapure water e procedures forEDR and MCR-ALS methods were followed as detailedunder Sections 233 and 234 respectively Analytes con-centrations were calculated using the corresponding re-gression equations

For method application in the analysis of real urinesamples (A) urine was collected 0ndash12 h from a healthyvolunteer after the administration of a single oral dose of500mg PR and (B) urine was collected 0ndash12 h from ahealthy volunteer after the administration of 500mg of LVe collected urine volumes were accurately measured A100 μL aliquot was diluted into 10mL using ultrapure waterand used for the direct determination of PR and LV

3 Results and Discussion

Figure 2 shows the zero-order absorption spectra of AT PRHZ and LV ere is a severe overlap between the ab-sorption bands of the four analytes over the whole wave-length range (200ndash330 nm) us the simultaneousdetermination of these drugs is hindered using conventionalcalibration procedures without prior separation ereforesimple reliable and precise nonconventional univariate andmultivariate chemometrics-assisted spectrophotometricmethods were proposed for the simultaneous analysis of ATPR HZ and LV in pharmaceutical dosage forms spiked andreal human urine samples Furthermore the developedmethodsrsquo performance was evaluated and statisticallycompared with published methods [3 16 28 34]

31 EDR Method

311 Selection of Divisors and Derivative Parameterse severe spectral overlap of the analytes of interestdemonstrates the resolving power of the proposed methodEach analyte can be determined by using a divisor mixturecomposed of the other three analytes

Six synthetic mixtures were prepared containing dif-ferent concentration ratios of AT PR HZ and LV withintheir linear ranges e zero-order absorption spectra ofthese solutions were recorded and stored

For determination of AT the stored spectra of standardsolutions containing AT PR HZ and LV were divided bythe stored AT divisor then the second derivative of theobtained ratio spectra (2DD) was calculated using Δλ 4 nmand scaling factor 10 (Figure 3(a)) For the determination ofAT a reproducible amplitude was selected from the ob-tained derivative spectra e calculated amplitude at2816 nm was found to be proportional to the concentrationof AT

On the other hand for PR determination the sameprocedures were followed and the ratio spectra were ob-tained as described above using PR divisor en the firstderivative of the ratio spectra (1DD) was calculatedemploying Δλ 4 nm and 10 as a scaling factor (Figure 3(b))


0000

0200

0400

0600

Abs

orba

nce

3000025000 3300020300Wavelength (nm)

AT

HZ

PRLV

Figure 2 Zero-order absorption spectra of 10 μgmlminus1 atenolol (AT) 5 μgmlminus1 paracetamol (PR) 10 μgmlminus1 hydrochlorothiazide (HZ) and5 μgmlminus1 levofloxacin (LV)

0157

0100

0000

ndash0100

ndash0200

ndash0238

Am

plitu

de

26990 28000 29000 30170Wavelength (nm)

2816 nm

(a)

2376 nm

Am

plitu

de

Wavelength (nm)

5298

4000

2000

0000

ndash2000

ndash4000ndash4247

20495 22000 24000 26000 27715

(b)

Am

plitu

de

Wavelength (nm)

1758

1000

0000

ndash1000

ndash2000ndash2112

24152 26000 28000

2792 nm

30000 31702

(c)

Am

plitu

de

Wavelength (nm)

1760

1000

0000

ndash088525462 26000 27000 28000 29000 29622

2828 nm

(d)

Figure 3 Overlay of the derivative ratio spectra obtained (a) 2DD of AT (5ndash40 μgmLminus1) (b) 1DD of PR (1ndash25 μgmLminus1) (c) 1DD of HZ(1ndash15 μgmLminus1) and (d) 2DD of LV (1ndash15 μgmLminus1)


e calculated amplitude at 2376 nm was found to beproportional to the concentration of PR

Moreover HZ was determined using similar proceduresusing the HZ divisor and the 1DD was calculated employingΔλ 2 nm and 10 as a scaling factor (Figure 3(c)) eamplitude at 2792 nm was measured and found to beproportional to the concentration of HZ

Furthermore LV was determined by using LV divisorand the 2DD was calculated using Δλ 4 nm and scalingfactor of 10 (Figure 3(d)) e amplitude at 2828 nm wascalculated and found to be proportional to LVconcentration

e main parameters affecting the developed methodperformance were optimized for reliable and accurate de-termination of the analytes of interest is included opti-mization of the divisor selection which is a vital step in theproposed technique e concentration of each analyte inthe divisor was selected in a way that absorbances are low butmeanwhile spectral features are evident [55] Differentmixtures were prepared and tested as divisor and the op-timum one was selected First and second derivatives of theratio spectra were calculated and tested e first derivativeprovided reliable and accurate results in case of PR and HZHowever 2DD was preferred than 1DD in case of ATand LVbecause it provided better spectral resolution and moreaccurate and precise results Moreover the effect of the Δλfor 1DD and 2DD was optimized and the best results wereobtained using Δλ 2 for HZ and Δλ 4 for AT PR and LVFurthermore the scaling factor was optimized and a factorof 10 was found suitable for all analytes

312 Method Validation Method validation was performedaccording to the International Conference on Harmoniza-tion (ICH) recommendations [56]

(1) Linearity Seven concentrations were used in the con-centration range of 5ndash40 1ndash25 1ndash15 and 1ndash15 μgmLminus1 ofAT PR HZ and LV respectively e developed methodshowed a high correlation coefficient (rge 09994) and in-tercept value that was not statistically (plt 005) differentfrom zero Regression parameters are shown in Table 1

(2) Detection and Quantitation Limits Limit of detection(LOD) was calculated according to ICH as 33 (σS) andlimit of quantification was calculated as 10(σS) where ldquoσrdquo isthe standard deviation of the intercept and ldquoSrdquo is the slope ofthe calibration curve (Table 1)

(3) Accuracy and Precision Method accuracy was evaluatedusing three different concentrations of each drug analyzed intriplicate and percentage recovery of each analyte wascalculated Precision was evaluated by analyzing threedifferent concentrations of each drug in triplicate withinthe same day (Intraday) and for three consecutive days(Interday) and relative standard deviation (RSD) wascalculated e samples were analyzed according to theprocedures under ldquo233rdquo e results summed in Table 1shows excellent recoveries and RSD lower than 126

indicating the good accuracy and precision of the proposedmethod

(4) Selectivity Method selectivity was investigated usingseveral laboratory synthetic mixtures comprising the fouranalytes in different concentration ratios within the linearityrange e results shown in Table 2 indicate the high se-lectivity of the EDR method

(5) Statistical Analysis e obtained results of the developedmethods were compared with the published methods for thedetermination of AT [3] PR [16] HZ [28] and LV [34] eresults obtained in Table 3 revealed no significant differencebetween the proposed and the published methods withrespect to accuracy and precision

32 Multivariate Curve ResolutionmdashAlternating LeastSquares e severe spectral overlap between the fouranalytes and the presence of unknown sample interferencesin the urine matrix necessitate the use of multivariate cal-ibration models to resolve such kind of mixtures First-ordermultivariate calibration methods may be a good choice insuch case only if the interfering background is well repre-sented in the calibration phase However if such interfer-ences are not represented in the calibration first-ordermethods may not be a good choice In such instancessecond-order multivariate calibration models may workbetter due to their good prediction ability even in thepresence of unknown interferences [57] In this work MCR-ALS model was developed for the determination of the fouranalytes of interest in pharmaceutical dosage forms andspiked human urine samples without including urine in thecalibration samples

321 Calibration Set Multilevel multifactor experimentaldesign was employed to construct 25 mixtures of the fouranalytes thus providing mutual orthogonal factors [54] Fivedifferent concentration levels within the calibration rangewere used for each analyte A total of 18 mixtures were usedto build the calibration model and 7 mixtures were utilizedas an external validation set as shown in Table 4

322 Selection of the Wavelength Range e quality ofmultivariate calibration relies on the wavelength range se-lected to build up the calibration model erefore the datain the region 200ndash220 nm were discarded due to noiseMoreover the band of 290ndash330 nm was excluded as wellbecause AT had nonsignificant absorption in this regionus the region of 220ndash290 nm with 04 nm interval wasused in the developed MCR-ALS method

323 Developing the MCR-ALS Model No data pre-processing was conducted on the calibration matrix whiledeveloping the MCR-ALS model To obtain a reasonableresolution byMCR-ALSmodel initial estimation of the purespectra of the target analytes was conducted Five compo-nents were found to be responsible for the variations in


different samples Nonnegativity constraint (for spectral andconcentration profiles) and correlation constraints wereused in developing the model e model resolved fivespecies e first four curves were very similar to AT PRHZ and LV spectra with a correlation coefficient (r2 09996 09998 09997 and 09999 respectively) betweenthe real and calculated AT PR HZ and LV spectrae fifthspectrum was estimated as the interfering urine matrix eestimated spectral profiles are shown in Figure 4 e re-solved matrix for each analyte was used to find the con-centration profiles of each analyte and satisfactory resultswere obtained with a low lack of fit ( lof) of 01721 edeveloped model captured 9999 of variance in the ana-lyzed spectra Figure 5 shows the scatter plot of concen-tration values resolved by the MCR-ALS model versus the

true concentration values Good predictive ability of themodel was obtained with determination coefficients of notless than 09998 for the four analytes Table 5 shows thedifferent figures of merit of the developed model

324 Validation of the MCR-ALS Model e developedmodel was applied on a series of external validation data setcomposed of 7 different synthetic mixtures within the cal-ibration range of the analytes of interest (Table 4)e percentrecoveries summarized in Table 6 show satisfactory results Tofurther validate the model different figures of merit includingRMSEP SEP RE and r2 were calculated for the externalvalidation set Satisfactory results were obtained as shown inTable 6 Moreover accuracy intraday and interday precision

Table 1 Characteristic validation parameters of the proposed EDR method for the determination of AT PR HZ and LV

Parameters AT PR HZ LVCalibration range (μg mLminus1) 5ndash40 1ndash25 1ndash15 1ndash15Regression equationSlope (b) 00053 01830 01211 00998Intercept (a) minus143times10minus4 minus323times10minus4 minus610times10minus3 minus137times10minus3

Correlation coefficient (r2) 09995 09998 09994 09996Standard deviation of slope (Sb) 519times10minus5 124times10minus3 132times10minus3 850times10minus4

Standard deviation of intercept (Sa) 122times10minus3 175times10minus2 118times10minus2 766times10minus3

LODa (μg mLminus1) 076 031 032 025LOQb (μg mLminus1) 231 095 098 077Accuracy (meanplusmn SDc) 9982plusmn 088 9993plusmn 085 9998plusmn 069 10026plusmn 077PrecisionIntraday ( RSDd) 070 066 093 098Interday ( RSDe) 126 088 090 094aLimit of detection bLimit of quantification cStandard deviation of three different concentrations each analyzed in triplicate dRelative standard deviation ofthree different concentrations analyzed in triplicate within the same day eRelative standard deviation of three different concentrations analyzed in triplicateon three consecutive days

Table 2 Determination of AT PR HZ and LV in synthetic mixtures using the proposed EDR method

Mix NoConc (μg mLminus1) Recovery

AT PR HZ LV AT PR HZ LV1 15 10 3 6 9815 9970 10070 10152 30 2 5 5 9785 9915 9970 99403 25 6 8 4 10020 10120 9845 100004 25 5 7 4 9956 10091 10162 100255 10 1 4 1 10211 9870 10190 99106 15 4 5 3 9840 10000 9980 9980Mean 9938 9994 10036 10001plusmn SD 161 097 130 084

Table 3 Statistical comparison of the proposed EDR method with published methods for the determination of AT PR HZ and LV

AT PR HZ LVEDR Published method [3] EDR Published method [16] EDR Published method [28] EDR Published method [34]

Mean 9977 10005 10104 10011 10107 10030 9969 10015plusmn SD 110 106 116 105 071 096 122 112n 3 3 3 3 3 3 3 3ta 023 132 108 163Fa 108 121 186 119aCritical values of t and F are 430 and 1900 respectively at (p 005)


were calculated for the external validation set to furtherevaluate themodel Satisfactory results were obtained in termsof accuracy and precision (Table 6)

33 Analysis of Real Samples

331 Analysis of Pharmaceutical Formulations e devel-oped methods were successfully used for the quantificationof AT PR HZ and LV in different pharmaceutical

formulations and the results were statistically compared withthe reported methods using paired t-test and F ratio at 95confidence level Satisfactory results were obtained showingno significant difference with the reported methods in termsof accuracy and precision (Table 7)

332 Analysis of Spiked and Real Human Urine SamplesEDR and MCR-ALS methods were successfully employedfor the quantification of the four analytes of interest in

Table 4 e concentration matrix used for preparation of the calibration and validation sets for the MCR-ALS method

Mix no AT PR HZ LV1 15 55 55 42 15 1 1 73lowast 5 1 10 254lowast 5 10 35 75 25 35 10 46 10 10 55 257 25 55 35 258 15 35 35 559lowast 10 35 8 710 10 8 10 5511 20 10 8 412 25 8 55 713 20 55 10 714 15 10 10 115lowast 25 10 1 5516 25 1 8 117 5 8 1 418lowast 20 1 55 5519 5 55 8 5520lowast 15 8 8 2521 20 8 35 122 20 35 1 2523lowast 10 1 35 424 5 35 55 125 10 55 1 1lowastMixtures used as external validation set

012

01

008

006

004

002

0

Abso

rban

ce

2200 2300 2400 2500 2600 2700 2800 2900Wavelength (nm)

ATPRHZ

LVUrine matrix

Figure 4 MCR-ALS resolved spectral profiles of the four target analytes (AT PR HZ and LV) and the interfering urine matrix


spiked human urine samples and satisfactory results wereobtained (Table 8)

Pharmacokinetic studies on PR reported that it is excretedin urine mainly as conjugates [12 13] erefore PR urinesample was exposed to enzymatic hydrolysis to obtain thetotal PR (free + conjugated) An aliquot equivalent to 400units of β-glucuronidase enzyme with sulfatase activity wasadded to 1mL of the collected urine sample after oral ad-ministration of the PR dose e pH was adjusted to 5 usingacetate buffer and incubated at 37degC for 1 h [58] e de-velopedmethods were applied to determine PR concentrationfor the same sample with and without enzymatic hydrolysisbefore and after incubation No significant difference thatcould be attributed to the increase in PR concentration due to

enzymatic hydrolysis was obtained is result providedevidence that the developed methods can determine total PRdirectly in urine without prior hydrolysis step Furthermorethe published HPLCmethod [16] was employed to determinethe total PR in the urine sample after enzymatic hydrolysisand the concentration determined was very closed from theconcentration obtained using the developed methods with noenzymatic hydrolysis (Table 9)

Total PR excreted in urine was found in a concentrationof 4545 and 4597 μgmLminus1 and a cumulative (0ndash12 h) urineexcretion of 891 and 901 of the administered dose usingEDR and MCR-ALS respectively is is in agreement withprevious studies that reported similar PR urine excretionpercentage [16 20]

3000

2500

2000

1500

1000

500

000

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)

Actual concentration (μg mLndash1)0 5 10 15 20 25 30

AT

CalVal

(a)

Actual concentration (μg mLndash1)

000

200

400

600

800

1000

1200

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)

0 2 4 6 8 10 12

PR

CalVal

(b)

Actual concentrationn (μg mLndash1)0 2 4 6 8 10 12

000

200

400

600

800

1000

1200

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)

HZ

CalVal

(c)


000100200300400500600700800

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)0 1 2 3 4 5 6 7 8

LV

CalVal

(d)

Figure 5 Scatter plot of actual drugs concentrations versus MCR-ALS predicted values of AT PR HZ and LV

Table 5 Figures of merit of the MCR-ALS model for the calibration set of AT PR HZ and LV

Parameters AT PR HZ LVCalibration range (μg mLminus1) 5ndash25 1ndash10 1ndash10 1ndash7Slope 09998 10000 10001 10014Standard error of slope 328times10minus3 300times10minus3 280times10minus3 188times10minus3

Intercept 327times10minus3 496times10minus4 minus784times10minus5 minus329times10minus3

Standard error of intercept 567times10minus2 195times10minus2 181times 10minus2 800times10minus3

RMSEC 908times10minus2 334times10minus2 367times10minus2 166times10minus2

SEP 883times10minus2 325times10minus2 356times10minus2 162times10minus2

Bias minus715times10minus4 minus390times10minus4 minus300times10minus4 minus201times 10minus3

RE () 0526 0515 0567 0391Determination coefficient (r2) 09998 09999 09999 09999


Table 6 Figures of merit of the MCR-ALS model for the validation set of AT PR HZ and LV

Parameters AT NP HZ LVAccuracy (meanplusmn SD)a 9971plusmn 116 9971plusmn 122 9994plusmn 085 10001plusmn 079PrecisionIntraday ( RSD)b 157 132 135 086Interday ( RSD)c 170 124 165 072RMSEP 02033 00307 00461 00366SEP 01882 00284 00426 00339Bias 663times10minus2 minus834times10minus3 minus108times10minus3 260times10minus3

RE () 139 049 072 071r2 09994 09999 09999 09996aMean and standard deviation of 7 determinations bRelative standard deviation of three different concentrations analyzed in triplicate within the same daycRelative standard deviation of three different concentrations analyzed in triplicate on three consecutive days

Table 7 Determination results of AT PR HZ and LV in dosage forms by the proposed and reference methods

Parameter Conc Claimed(μg mLminus1)

EDR MCR-ALS Reference methodlowast

Conc Found(μg mLminus1) Recovery Conc Found

(μg mLminus1) Recovery Conc Found(μg mLminus1) Recovery

AT

Tenorminreg tablets 10 1005 10050 996 9960 1010 1010015 1485 9900 1505 10033 1508 1005320 1961 9805 1975 9875 199 9950

Mean 9918 9956 10034plusmn SDa 124 079 077tb 350 226Fb 259 107

PR

Panadrexreg tablets 3 296 9867 302 10067 298 99335 505 10100 493 9860 503 1006010 1004 10040 101 10100 98 9800


HZ

HCT georetic 25reg tablets 3 303 10100 301 10033 304 101335 492 9840 496 9920 498 996010 988 9880 1011 10110 992 9920


LV

Tavanicreg tablets 3 298 9933 301 10033 298 99334 403 10075 404 10100 402 100505 503 10060 501 10020 503 10060


aStandard deviation of the mean of percentage recovery from the label claim amount beoretical values for t and F are 430 and 1900 at (p 005)respectively lowastReference methods for AT [3] PR [16] HZ [28] and LV [34]

Table 8 Determination results of AT PR HZ and LV in spiked human urine samples using the proposed methods

Sample noConc (μg mLminus1) EDR MCR-ALS

AT PR HZ LV AT PR HZ LV AT PR HZ LV1 25 3 35 4 10410 10290 10315 1029 9988 9920 10183 102642 10 5 4 1 9781 10130 1047 10535 10141 9794 10086 102123 15 7 2 3 10215 10370 10371 9811 10277 9686 9869 10104Mean 10135 10263 10385 10212 10135 9800 10046 10193plusmn SD 322 122 078 368 145 117 161 082


On the other hand when urine sample B (LV dose) wasanalyzed using the developed methods LV was determinedwith cumulative (0ndash12 h) urinary excretion of 605 and624 of the administered dose using EDR and MCR-ALSrespectively e results are in agreement with previousstudies which reported similar LV excretion percentage [34]Furthermore the obtained results were compared to apublished HPLC method [34] and showed no significantdifference as shown in Table 9 e urinary excretion resultsof PR and LV are summarized in Table 9

34 Assessment of the Methods Greenness Greenness assess-ment of the developed methods was accomplished using twodifferent methods namely NEMI and Analytical Eco-Scale

341 National Environmental Methods Index (NEMI)is assessment method utilizes a greenness profile symbolcomposed of four quadrants representing method aspectsaccording to the following criteria any of the chemicals usedis persistent bioaccumulative or toxic (PBT) hazardouscorrosive (pH lt2 or gt12) and the amount of waste gen-erated (gt50 g) If the method is green all four quadrants willbe colored green However if the procedure does not meetany of these aspects the corresponding quadrant will be leftblank (ie uncolored)

In the proposed methods ethanol which is considered agreen solvent was used to prepare the stock solutions andwater was used as a solvent for all further dilutions andspectrophotometric measurements Neither ethanol norwater is listed as PBT or hazardous e pH is not corrosiveand the produced waste is lt50 g per sample erefore theproposed method passes the four criteria and the fourquadrants of the greenness profile are green

342 Analytical Eco-Scale In this method a more quanti-tative assessment is presented where a numerical score iscalculated to assess the method greenness and high Eco-Scale score (iegt 75) denotes the method greennessWhenever the method uses hazardous chemicals generateswaste has high energy consumption or has exposure riskpenalty points are deducted from the total 100 points [47]e high Eco-Scale score of the proposed methods (95)indicates the excellent greenness of the developed method(Table 10) us they can be employed for the routineanalysis of the studied drugs in pharmaceutical formulations

and urine samples with very few harmful effects on theenvironment

4 Conclusions

Rapid accurate and cost-effective green nonconventionalunivariate and multivariate chemometrics-assisted spec-trophotometric methods were developed for the determi-nation of AT PR HZ and LV in different pharmaceuticaldosage forms In addition the proposed methods weresuccessfully applied for the determination of the four ana-lytes in spiked and real human urine samples and bothmethods provided comparable results e assessment of thegreenness profile of the two methods showed they representan excellent green analysis of the studied drugs with very lowharmful effects on the environment Moreover the proposedmethods do not need any sample preparation sophisticatedHPLC instrumentation or expensive solvents Furthermorethe EDRmethod has the advantage of being simple and doesnot need any sophisticated mathematical algorithms neededfor the MCR method However the main challenge in de-veloping the EDR method is the selection of the optimumdivisor to get selective and reproducible results MCR-ALS

Table 9 Determination of urinary excretion of PR and LV after administration of a single 500mg oral dose of PR or LV by healthyvolunteers using EDR and MCR-ALS methods

Compound Parameter EDR MCR-ALS Reference methodlowast

PR Concentration (μg mLminus1) 4545 4597 4574Excretion (0ndash12 h) (mg) 4454 4505 4482 Excretion (0ndash12 h) 891 901 896

LV Concentration (μg mLminus1) 3153 3249 3185Excretion (0ndash12 h) (mg) 3027 3119 3058 Excretion (0ndash12 h) 605 624 612

lowastReference methods PR [16] and LV [34]

Table 10 Analytical Eco-Scale score for the proposed methods

Method items ValuePenaltypoints(PPs)

Reagents(Water) Reagent amount lt10mL 1Reagent hazardous None 0(Ethanol) Reagent amount lt10mL 1Reagent hazardous 2Total PPs (PPs of reagentamount timesPPs reagent hazard) 2

Instruments

Energy lt01 kWh persample 0

Occupational hazardsEmission ofvapors and

gases to the air0

Waste 1ndash10mL 3Total PPs (sum of instrument PPs) 3Total PPs (PPs reagent + PPsinstrument) 5

Analytical Eco-Scale total score 100minus 5 95 95


method has the advantage over other first-order multivariatecalibration methods of being able to produce good pre-diction even in the presence of inferences that are notrepresented in the calibration phase e proposed methodscan be used as a valid eco-friendly and simple cost-effectivealternative to the commonly used chromatographic methodsfor the routine analysis of the studied drugs in dosage formsand human urine

Data Availability

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

Conflicts of Interest

e author has no conflicts of interest to declare

Acknowledgments

is work was supported by the Deanship of ScientificResearch Imam Abdulrahman Bin Faisal UniversityKingdom of Saudi Arabia (Grant No Pharm-2017-250)

References

[1] J J Lilja L Juntti-Patinen and P J Neuvonen ldquoEffect ofrifampicin on the pharmacokinetics of atenololrdquo Basic ampClinical Pharmacology amp Toxicology vol 98 pp 555ndash5582006

[2] A El-Gindy S Emara and A Mostafa ldquoHPLC and che-mometric-assisted spectrophotometric methods for simulta-neous determination of atenolol amiloride hydrochloride andchlorthalidonerdquo Il Farmaco vol 60 pp 269ndash278 2005

[3] B Yilmaz S Arslan and A Asci ldquoHPLC method for de-termination of atenolol in human plasma and application to apharmacokinetic study in Turkeyrdquo Journal of Chromato-graphic Science vol 50 pp 914ndash919 2012

[4] N S Mohammed ldquoMethod development and validation ofatenolol using two hplc systemsrdquo International Journal ofPharmaceutical Sciences and Research vol 8 pp 2706ndash27112017

[5] D K Sharma and P Raj ldquoSimple and rapid spectropho-tometry determination of atenolol and esmolol beta-blockersin pharmaceutical formulations and spiked water samplesrdquoInternational Journal of Pharmaceutical Sciences and Re-search vol 8 p 5168 2017

[6] M N Khan J Ahmad M N Jan H Gulab and M IdreesldquoDevelopment and validation of a new spectrophotometricmethod for the determination of cephalexin monohydrate inpure form and pharmaceutical formulationsrdquo Journal of theBrazilian Chemical Society vol 27 pp 912ndash918 2016

[7] Y Y Chen and W P Yang ldquoPharmacokinetic study ofatenolol in rabbit blood by capillary electrophoresis withlaser-induced fluorescence detectionrdquo Asian Journal ofChemistry vol 23 pp 3958ndash3960 2011

[8] Y G Kuraeva A I Kamenskaya M V VasilrsquoevaA A Stupnikov and L A Onuchak ldquoCapabilities of capillaryelectrophoresis for the determination of atenolol and biso-prololrdquo Journal of Analytical Chemistry vol 71 pp 396ndash4012016

[9] B Yilmaz and V Akba ldquoGas chromatographymass spec-trometry method for determination of atenolol in rabbitplasmardquo Asian Journal of Chemistry vol 23 pp 54ndash58 2011

[10] E Bakir M Gouda A Alnajjar and W E Boraie ldquoSpec-trofluorimetric method for atenolol determination based ongold nanoparticlesrdquoActa Pharmaceutica vol 68 pp 243ndash2502018

[11] C D Oscier and Q J W Milner ldquoPeri-operative use ofparacetamolrdquo Anaesthesia vol 64 pp 65ndash72 2009

[12] A Kulo M Y Peeters K Allegaert et al ldquoPharmacokineticsof paracetamol and its metabolites in women at delivery andpost-partumrdquo British Journal of Clinical Pharmacologyvol 75 pp 850ndash860 2013

[13] J A H Forrest A H Forrest J A Clements J A ClementsL F Prescott and L F Prescott ldquoClinical pharmacokineticsof paracetamolrdquo Clinical Pharmacokinetics vol 7 pp 93ndash1071982

[14] S F Cook A D King J N van den Anker and D GWilkinsldquoSimultaneous quantification of acetaminophen and fiveacetaminophen metabolites in human plasma and urine byhigh-performance liquid chromatographyndashelectrospray ion-izationndashtandem mass spectrometry method validation andapplication to a neonatal pharmacokinetic studyrdquo Journal ofChromatography B vol 1007 pp 30ndash42 2015

[15] S Abbasi S A Haeri and S Sajjadifar ldquoBio-dispersive liquidliquid microextraction based on nano rhamnolipid aggregatescombined with molecularly imprinted-solid phase extractionfor selective determination of paracetamol in human urinesamples followed by HPLCrdquo Microchemical Journal vol 146pp 106ndash114 2019

[16] L S Jensen J Valentine R W Milne and A M Evans ldquoequantification of paracetamol paracetamol glucuronide andparacetamol sulphate in plasma and urine using a single high-performance liquid chromatography assayrdquo Journal ofPharmaceutical and Biomedical Analysis vol 34 pp 585ndash5932004

[17] Q-y Tan R-h Zhu H-d Li F Wang M Yan and L-b DaildquoSimultaneous quantitative determination of paracetamol andits glucuronide conjugate in human plasma and urine byliquid chromatography coupled to electrospray tandem massspectrometry application to a clinical pharmacokineticstudyrdquo Journal of Chromatography B vol 893-894 pp 162ndash167 2012

[18] A Trettin A A Zoerner A Bohmer et al ldquoQuantification ofacetaminophen (paracetamol) in human plasma and urine bystable isotope-dilution GCndashMS and GCndashMSMS as penta-fluorobenzyl ether derivativerdquo Journal of Chromatography Bvol 879 pp 2274ndash2280 2011

[19] J Parojcic K Karljikovic-Rajic Z Duric M Jovanovic andS Ibric ldquoDevelopment of the second-order derivative UVspectrophotometric method for direct determination ofparacetamol in urine intended for biopharmaceutical char-acterisation of drug productsrdquo Biopharmaceutics Drug Dis-position vol 24 pp 309ndash314 2003

[20] A R Khaskheli A Shah M I Bhanger A Niaz andS Mahesar ldquoSimpler spectrophotometric assay of para-cetamol in tablets and urine samplesrdquo Spectrochimica ActaPart A Molecular Biomolecular Spectroscopy vol 68pp 747ndash751 2007

[21] T Li J Xu L Zhao et al ldquoAu nanoparticlespoly (caffeic acid)composite modified glassy carbon electrode for voltammetricdetermination of acetaminophenrdquo Talanta vol 159pp 356ndash364 2016


[22] M Lecoeur G Rabenirina N Schifano et al ldquoDeterminationof acetaminophen and its main metabolites in urine bycapillary electrophoresis hyphenated to mass spectrometryrdquoTalanta vol 205 Article ID 120108 2019

[23] H Hasannejad M Takeda K Taki et al ldquoInteractions ofhuman organic anion transporters with diureticsrdquo Journal ofPharmacology amp Pharmacotherapeutics vol 308 pp 1021ndash1029 2004

[24] S A Van Wart S E Shoaf S Mallikaarjun and D E MagerldquoPopulation-based meta-analysis of hydrochlorothiazidepharmacokineticsrdquo Biopharmaceutics Drug Dispositionvol 34 pp 527ndash539 2013

[25] J De Vries and A Voss ldquoSimple determination of hydro-chlorothiazide in human plasma and urine by high perfor-mance liquid chromatographyrdquo Biomedical Chromatographyvol 7 pp 12ndash14 1993

[26] H Albishri D Abd El-Hady and R A Tayeb ldquoEco-friendlysimultaneous chromatographic determination of amiloridehydrochloride atenolol and hydrochlorothiazide in urinerdquoActa Chromatographica vol 27 pp 461ndash476 2015

[27] W Fang W Xie J K Hsieh and B Matuszewski ldquoDevel-opment and application of HPLC methods with tandem massspectrometric detection for the determination of hydro-chlorothiazide in human plasma and urine using 96-wellliquid-liquid extractionrdquo Journal of Liquid Chromatographyamp Related Technologies vol 28 pp 2681ndash2703 2005

[28] D Farthing I Fakhry E B D Ripley and D Sica ldquoSimplemethod for determination of hydrochlorothiazide in humanurine by high performance liquid chromatography utilizingnarrowbore chromatographyrdquo Journal of Pharmaceutical andBiomedical Analysis vol 17 pp 1455ndash1459 1998

[29] L Amendola C Colamonici M Mazzarino and F BotreldquoRapid determination of diuretics in human urine by gaschromatographyndashmass spectrometry following microwaveassisted derivatizationrdquo Analytica Chimica Acta vol 475pp 125ndash136 2003

[30] A Mostafa A El-Gindy and S Emara ldquoSimultaneousspectrophotometric estimation of bisoprolol fumarate andhydrochlorothiazide in tablet formulation using partial least-squares principal component regression multivariate cali-brations and RP-HPLC methodsrdquo Journal of Analytical ampPharmaceutical Research vol 4 Article ID 00124 2017

[31] F F Mohammed K Badr El-Din and S M Derayea ldquoTwosmart spectrophotometric methods for simultaneous deter-mination of Lisinopril and Hydrochlorothiazide in binarymixturesrdquo Journal of Advanced Biomedical PharmaceuticalSciences vol 2 pp 47ndash53 2019

[32] H Beitollahi and F Ghorbani ldquoBenzoylferrocene-modifiedcarbon nanotubes paste electrode as a voltammetric sensor fordetermination of hydrochlorothiazide in pharmaceutical andbiological samplesrdquo Ionics vol 19 pp 1673ndash1679 2013

[33] O Szerkus J Jacyna A Gibas et al ldquoRobust HPLCndashMSMSmethod for levofloxacin and ciprofloxacin determination inhuman prostate tissuerdquo Journal of Pharmaceutical BiomedicalAnalysis vol 132 pp 173ndash183 2017

[34] A El-Gindy S Emara and A Mostafa ldquoUV partial least-squares calibration and liquid chromatographic methods fordirect quantitation of levofloxacin in urinerdquo Journal of AOACInternational vol 90 pp 1258ndash1265 2007

[35] S Ghimire K van Hateren N Vrubleuskaya R KosterD Touw and J-W Alffenaar ldquoDetermination of levofloxacinin human serum using liquid chromatography tandem massspectrometryrdquo Journal of Applied Bioanalysis vol 4 p 31522018

[36] O Abdel-Aziz M F Abdel-Ghany R Nagi and L Abdel-Fattah ldquoApplication of SavitzkyndashGolay differentiation filtersand Fourier functions to simultaneous determination ofcefepime and the co-administered drug levofloxacin inspiked human plasmardquo Spectrochimica Acta Part A Molec-ular and Biomolecular Spectroscopy vol 139 pp 449ndash4552015

[37] M Rkik M B Brahim and Y Samet ldquoElectrochemical de-termination of levofloxacin antibiotic in biological samplesusing boron doped diamond electroderdquo Journal of Electro-analytical Chemistry vol 794 pp 175ndash181 2017

[38] A Rusu P Antonoaea A Ciurba M Birsan G Hancu andN Todoran ldquoDevelopment of a rapid capillary zone elec-trophoresis method to quantify levofloxacin and meloxicamfrom transdermal therapeutic systemsrdquo Studia UniversitatisBabes-Bolyai Chemia vol 64 2019

[39] A Gałuszka Z Migaszewski and J Namiesnik ldquoe 12principles of green analytical chemistry and the significancemnemonic of green analytical practicesrdquo TrAC Trends inAnalytical Chemistry vol 50 pp 78ndash84 2013

[40] H Shaaban and A Mostafa ldquoSustainable eco-friendly ultra-high-performance liquid chromatographic method for si-multaneous determination of caffeine and theobromine incommercial teas evaluation of greenness profile using NEMIand eco-scale Assessment toolsrdquo Journal of AOAC Interna-tional vol 101 pp 1781ndash1787 2018

[41] P T Anastas and J C Warner Green Chemistry eory andPractice TPB Oxford University Press New York NY USA2012

[42] M A Korany H Mahgoub R S Haggag M A Ragab andO Elmallah ldquoChemometrics-assisted spectrophotometricgreen method for correcting interferences in biowaiverstudies application to assay and dissolution profiling study ofdonepezil hydrochloride tabletsrdquo Spectrochimica Acta Part AMolecular Biomolecular Spectroscopy vol 199 pp 328ndash3392018

[43] A Mostafa H Shaaban M Almousa M Al Sheqawi andM Almousa ldquoEco-friendly pharmaceutical analysis of mul-ticomponent drugs coformulated in different dosage formsusing multivariate curve resolution and partial least squares acomparative studyrdquo Journal of AOAC International vol 102pp 465ndash472 2019

[44] A Mostafa and H Shaaban ldquoQuantitative analysis and res-olution of pharmaceuticals in the environment using multi-variate curve resolution-alternating least squares (MCR-ALS)rdquo Acta Pharm vol 69 pp 217ndash231 2019

[45] L Keith L Gron and J Young ldquoGreen analytical method-ologiesrdquo Chemical Reviews vol 107 pp 2695ndash2708 2007

[46] M Tobiszewski M Marc A Gałuszka and J NamiesnikldquoGreen chemistry metrics with special reference to greenanalytical chemistryrdquo Molecular Cells vol 20 pp 10928ndash10946 2015

[47] A Gałuszka Z M Migaszewski P Konieczka andJ Namiesnik ldquoAnalytical Eco-Scale for assessing the green-ness of analytical proceduresrdquo TrAC Trends in AnalyticalChemistry vol 37 pp 61ndash72 2012

[48] R Tauler ldquoMultivariate curve resolution applied to secondorder datardquo Chemometrics and Intelligent Laboratory Systemsvol 30 pp 133ndash146 1995

[49] W Windig and J Guilment ldquoInteractive self-modelingmixture analysisrdquo Analytical Chemistry vol 63 pp 1425ndash1432 1991

[50] T Azzouz and R Tauler ldquoApplication of multivariate curveresolution alternating least squares (MCR-ALS) to the


quantitative analysis of pharmaceutical and agriculturalsamplesrdquo Talanta vol 74 pp 1201ndash1210 2008

[51] A de Juan and R Tauler ldquoChemometrics applied to unravelmulticomponent processes and mixtures revisiting latesttrends in multivariate resolutionrdquo Analytica Chimica Actavol 500 pp 195ndash210 2003

[52] R R de Oliveira K M G de Lima R Tauler and A de JuanldquoApplication of correlation constrained multivariate curveresolution alternating least-squares methods for determina-tion of compounds of interest in biodiesel blends using NIRand UVndashvisible spectroscopic datardquo Talanta vol 125pp 233ndash241 2014

[53] J Jaumot A de Juan and R Tauler ldquoMCR-ALS GUI 20 newfeatures and applicationsrdquo Chemometrics and IntelligentLaboratory Systems vol 140 pp 1ndash12 2015

[54] R G Brereton ldquoMultilevel multifactor designs for multi-variatecalibrationrdquo Analyst vol 122 pp 1521ndash1529 1997

[55] O Abdel-Aziz A M El Kosasy and S M E Okeil ldquoCom-parative study for determination of some polycyclic aromatichydrocarbons ldquoPAHsrdquo by a new spectrophotometric methodand multivariate calibration coupled with dispersive liquid-liquid extractionrdquo Spectrochimica Acta Part A Molecular andBiomolecular Spectroscopy vol 133 pp 119ndash129 2014

[56] ICH Guideline ldquoValidation of analytical procedures text andmethodology Q2 (R1)rdquo in Proceedings of the InternationalConference on Harmonization Geneva Switzerland 2005

[57] K S Booksh and B R Kowalski ldquoeory of analyticalchemistryrdquo Analytical Chemistry vol 66 pp 782Andash791A1994

[58] J L Vila-Jato J Blanco and M J Alonso ldquoe effect of themolecular weight of polyethylene glycol on the bioavailabilityof paracetamol-polyethylene glycol solid dispersionsrdquo Journalof Pharmacy Pharmacology vol 38 pp 126ndash128 1986


heart failure HZ is excreted (gt95) as unchanged drug inurine [23 24] Different techniques have been reported forHZ quantitation such as HPLC [25ndash28] GC [29] spec-trophotometry [30 31] and voltammetry [32]

Levofloxacin (LV) (Figure 1(d)) is a fluoroquinoloneantibiotic that inhibits DND topoisomerase IV and gyrase inGram-positive and Gram-negative bacteria respectively Itis used for urinary respiratory skin and soft tissue infec-tions [33] Several reports are found in the literature for thedetermination of LV including different techniques such asHPLC [34 35] spectrophotometry [34 36] voltammetry[37] and capillary electrophoresis [38]

AT PR HZ and LV can be coadministered together bysome patients and they are excreted mainly in the urine Tothe best of our knowledge no method has been reported forthe simultaneous determination of the four drugs

Green analytical chemistry (GAC) which is concernedwith developing new methods that are sustainable and moreenvironmentally friendly has gained much interest amonganalytical chemists [39 40] However the challenge is to

achieve a compromise between increasing the quality of thedeveloped methods and improving the method greenness[39 41] UV-Vis spectroscopy is considered a greener an-alytical technique when compared to HPLC UV-Visspectroscopy is a fast technique that consumes low solventvolumes thus reducing the amount of generated waste Inaddition this technique is cost-effective and does not needexpensive solvents or sophisticated instruments [42 43]However the major challenge when using UV-Vis spec-troscopy is the analysis of multicomponent mixtures es-pecially when the analytes spectra are strongly overlapped[42 44] To overcome this challenge the author proposedthe use of nonconventional spectrophotometric techniquenamely extended derivative ratio (EDR) In addition the useof multivariate calibration helped in the mathematicalresolution of the multicomponent mixture

erefore this work aimed to develop green cost-ef-fective and precise methods for the analysis of AT PR HZand LV Two methods including a nonconventional uni-variate method (ie extended derivative ratio (EDR)) and a

O

NH2

O

OH

NH

(a)

OH

HN O

(b)

SNH

HN

OO

Cl

SO

OH2N

(c)

N

N F

O

N

O

OH

O

(d)

Figure 1 Chemical structures of atenolol (a) paracetamol (b) hydrochlorothiazide (c) and levofloxacin (d)





AM λi εW λi


CY + εZ λiCZ (1)


εXλi

εYλiand εZλi



Aprimem1λi εXλi


CdegZ (2)



AM λi

Aprimem1 λi

εWλi


CY + εZλiCZ


CdegY + εZλiCdegZ

εWλi

CW


CdegY + εZλiCdegZ

+εXλi


CZ


CdegY + εZλiCdegZ

εW λi

CW


CdegY + εZλiCdegZ

+ K

(3)


follows

d

dλAM λi

Alsquom1 λi

1113890 1113891 d

dλεW λi

CW



1113890 1113891

d

dλεW λi



1113890 1113891CW

(4)



D CST

+ E (5)



lack of fit () 100

1113936ije2ij

1113936ijd2ij

11139741113972

(6)

S2

100


2ij

1113936ijd2ij

11139741113972

(7)





RMSEP

1113936ni1 ci minus 1113954ci( 1113857

2

n

1113971

(8)

Bias

bias 1113936

ni1 ci minus 1113954ci( 1113857

n (9)


SEP


2

n minus 1

1113971

(10)


RE () 100

1113936ni1 ci minus 1113954ci( 1113857

2

1113936ni1 c2i

1113971

(11)













23 Procedures


















31 EDR Method






0000

0200

0400

0600

Abs

orba

nce

3000025000 3300020300Wavelength (nm)

AT

HZ

PRLV


0157

0100

0000

ndash0100

ndash0200

ndash0238

Am

plitu

de

26990 28000 29000 30170Wavelength (nm)

2816 nm

(a)

2376 nm

Am

plitu

de

Wavelength (nm)

5298

4000

2000

0000

ndash2000

ndash4000ndash4247

20495 22000 24000 26000 27715

(b)

Am

plitu

de

Wavelength (nm)

1758

1000

0000

ndash1000

ndash2000ndash2112

24152 26000 28000

2792 nm

30000 31702

(c)

Am

plitu

de

Wavelength (nm)

1760

1000

0000

ndash088525462 26000 27000 28000 29000 29622

2828 nm

(d)









































012

01

008

006

004

002

0

Abso

rban

ce

2200 2300 2400 2500 2600 2700 2800 2900Wavelength (nm)

ATPRHZ

LVUrine matrix







3000

2500

2000

1500

1000

500

000

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)


AT

CalVal

(a)


000

200

400

600

800

1000

1200

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)

0 2 4 6 8 10 12

PR

CalVal

(b)


000

200

400

600

800

1000

1200

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)

HZ

CalVal

(c)


000100200300400500600700800

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)0 1 2 3 4 5 6 7 8

LV

CalVal

(d)



















AT



PR



HZ



LV














4 Conclusions










Instruments



gases to the air0





Data Availability




Acknowledgments


References


































































AM λi εW λi


CY + εZ λiCZ (1)


εXλi

εYλiand εZλi



Aprimem1λi εXλi


CdegZ (2)



AM λi

Aprimem1 λi

εWλi


CY + εZλiCZ


CdegY + εZλiCdegZ

εWλi

CW


CdegY + εZλiCdegZ

+εXλi


CZ


CdegY + εZλiCdegZ

εW λi

CW


CdegY + εZλiCdegZ

+ K

(3)


follows

d

dλAM λi

Alsquom1 λi

1113890 1113891 d

dλεW λi

CW



1113890 1113891

d

dλεW λi



1113890 1113891CW

(4)



D CST

+ E (5)



lack of fit () 100

1113936ije2ij

1113936ijd2ij

11139741113972

(6)

S2

100


2ij

1113936ijd2ij

11139741113972

(7)





RMSEP

1113936ni1 ci minus 1113954ci( 1113857

2

n

1113971

(8)

Bias

bias 1113936

ni1 ci minus 1113954ci( 1113857

n (9)


SEP


2

n minus 1

1113971

(10)


RE () 100

1113936ni1 ci minus 1113954ci( 1113857

2

1113936ni1 c2i

1113971

(11)













23 Procedures


















31 EDR Method






0000

0200

0400

0600

Abs

orba

nce

3000025000 3300020300Wavelength (nm)

AT

HZ

PRLV


0157

0100

0000

ndash0100

ndash0200

ndash0238

Am

plitu

de

26990 28000 29000 30170Wavelength (nm)

2816 nm

(a)

2376 nm

Am

plitu

de

Wavelength (nm)

5298

4000

2000

0000

ndash2000

ndash4000ndash4247

20495 22000 24000 26000 27715

(b)

Am

plitu

de

Wavelength (nm)

1758

1000

0000

ndash1000

ndash2000ndash2112

24152 26000 28000

2792 nm

30000 31702

(c)

Am

plitu

de

Wavelength (nm)

1760

1000

0000

ndash088525462 26000 27000 28000 29000 29622

2828 nm

(d)









































012

01

008

006

004

002

0

Abso

rban

ce

2200 2300 2400 2500 2600 2700 2800 2900Wavelength (nm)

ATPRHZ

LVUrine matrix







3000

2500

2000

1500

1000

500

000

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)


AT

CalVal

(a)


000

200

400

600

800

1000

1200

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)

0 2 4 6 8 10 12

PR

CalVal

(b)


000

200

400

600

800

1000

1200

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)

HZ

CalVal

(c)


000100200300400500600700800

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)0 1 2 3 4 5 6 7 8

LV

CalVal

(d)



















AT



PR



HZ



LV














4 Conclusions










Instruments



gases to the air0





Data Availability




Acknowledgments


References
































































RMSEP

1113936ni1 ci minus 1113954ci( 1113857

2

n

1113971

(8)

Bias

bias 1113936

ni1 ci minus 1113954ci( 1113857

n (9)


SEP


2

n minus 1

1113971

(10)


RE () 100

1113936ni1 ci minus 1113954ci( 1113857

2

1113936ni1 c2i

1113971

(11)













23 Procedures


















31 EDR Method






0000

0200

0400

0600

Abs

orba

nce

3000025000 3300020300Wavelength (nm)

AT

HZ

PRLV


0157

0100

0000

ndash0100

ndash0200

ndash0238

Am

plitu

de

26990 28000 29000 30170Wavelength (nm)

2816 nm

(a)

2376 nm

Am

plitu

de

Wavelength (nm)

5298

4000

2000

0000

ndash2000

ndash4000ndash4247

20495 22000 24000 26000 27715

(b)

Am

plitu

de

Wavelength (nm)

1758

1000

0000

ndash1000

ndash2000ndash2112

24152 26000 28000

2792 nm

30000 31702

(c)

Am

plitu

de

Wavelength (nm)

1760

1000

0000

ndash088525462 26000 27000 28000 29000 29622

2828 nm

(d)









































012

01

008

006

004

002

0

Abso

rban

ce

2200 2300 2400 2500 2600 2700 2800 2900Wavelength (nm)

ATPRHZ

LVUrine matrix







3000

2500

2000

1500

1000

500

000

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)


AT

CalVal

(a)


000

200

400

600

800

1000

1200

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)

0 2 4 6 8 10 12

PR

CalVal

(b)


000

200

400

600

800

1000

1200

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)

HZ

CalVal

(c)


000100200300400500600700800

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)0 1 2 3 4 5 6 7 8

LV

CalVal

(d)



















AT



PR



HZ



LV














4 Conclusions










Instruments



gases to the air0





Data Availability




Acknowledgments


References










































































31 EDR Method






0000

0200

0400

0600

Abs

orba

nce

3000025000 3300020300Wavelength (nm)

AT

HZ

PRLV


0157

0100

0000

ndash0100

ndash0200

ndash0238

Am

plitu

de

26990 28000 29000 30170Wavelength (nm)

2816 nm

(a)

2376 nm

Am

plitu

de

Wavelength (nm)

5298

4000

2000

0000

ndash2000

ndash4000ndash4247

20495 22000 24000 26000 27715

(b)

Am

plitu

de

Wavelength (nm)

1758

1000

0000

ndash1000

ndash2000ndash2112

24152 26000 28000

2792 nm

30000 31702

(c)

Am

plitu

de

Wavelength (nm)

1760

1000

0000

ndash088525462 26000 27000 28000 29000 29622

2828 nm

(d)









































012

01

008

006

004

002

0

Abso

rban

ce

2200 2300 2400 2500 2600 2700 2800 2900Wavelength (nm)

ATPRHZ

LVUrine matrix







3000

2500

2000

1500

1000

500

000

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)


AT

CalVal

(a)


000

200

400

600

800

1000

1200

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)

0 2 4 6 8 10 12

PR

CalVal

(b)


000

200

400

600

800

1000

1200

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)

HZ

CalVal

(c)


000100200300400500600700800

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)0 1 2 3 4 5 6 7 8

LV

CalVal

(d)



















AT



PR



HZ



LV














4 Conclusions










Instruments



gases to the air0





Data Availability




Acknowledgments


References































































0000

0200

0400

0600

Abs

orba

nce

3000025000 3300020300Wavelength (nm)

AT

HZ

PRLV


0157

0100

0000

ndash0100

ndash0200

ndash0238

Am

plitu

de

26990 28000 29000 30170Wavelength (nm)

2816 nm

(a)

2376 nm

Am

plitu

de

Wavelength (nm)

5298

4000

2000

0000

ndash2000

ndash4000ndash4247

20495 22000 24000 26000 27715

(b)

Am

plitu

de

Wavelength (nm)

1758

1000

0000

ndash1000

ndash2000ndash2112

24152 26000 28000

2792 nm

30000 31702

(c)

Am

plitu

de

Wavelength (nm)

1760

1000

0000

ndash088525462 26000 27000 28000 29000 29622

2828 nm

(d)









































012

01

008

006

004

002

0

Abso

rban

ce

2200 2300 2400 2500 2600 2700 2800 2900Wavelength (nm)

ATPRHZ

LVUrine matrix







3000

2500

2000

1500

1000

500

000

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)


AT

CalVal

(a)


000

200

400

600

800

1000

1200

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)

0 2 4 6 8 10 12

PR

CalVal

(b)


000

200

400

600

800

1000

1200

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)

HZ

CalVal

(c)


000100200300400500600700800

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)0 1 2 3 4 5 6 7 8

LV

CalVal

(d)



















AT



PR



HZ



LV














4 Conclusions










Instruments



gases to the air0





Data Availability




Acknowledgments


References





































































































012

01

008

006

004

002

0

Abso

rban

ce

2200 2300 2400 2500 2600 2700 2800 2900Wavelength (nm)

ATPRHZ

LVUrine matrix







3000

2500

2000

1500

1000

500

000

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)


AT

CalVal

(a)


000

200

400

600

800

1000

1200

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)

0 2 4 6 8 10 12

PR

CalVal

(b)


000

200

400

600

800

1000

1200

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)

HZ

CalVal

(c)


000100200300400500600700800

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)0 1 2 3 4 5 6 7 8

LV

CalVal

(d)



















AT



PR



HZ



LV














4 Conclusions










Instruments



gases to the air0





Data Availability




Acknowledgments


References



































































3000

2500

2000

1500

1000

500

000

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)


AT

CalVal

(a)


000

200

400

600

800

1000

1200

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)

0 2 4 6 8 10 12

PR

CalVal

(b)


000

200

400

600

800

1000

1200

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)

HZ

CalVal

(c)


000100200300400500600700800

Pred

icte

d co

ncen

trat

ion

(μg

mLndash1

)0 1 2 3 4 5 6 7 8

LV

CalVal

(d)



















AT



PR



HZ



LV














4 Conclusions










Instruments



gases to the air0





Data Availability




Acknowledgments


References







































































AT



PR



HZ



LV














4 Conclusions










Instruments



gases to the air0





Data Availability




Acknowledgments


References





































































4 Conclusions










Instruments



gases to the air0





Data Availability




Acknowledgments


References
































































Data Availability




Acknowledgments


References































































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