Download - Research Article Validation Thin Layer Chromatography for ...downloads.hindawi.com/journals/bmri/2013/545703.pdf · with preparative thin layer chromatography. e separation of propyphenazone,

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 545703 10 pageshttpdxdoiorg1011552013545703

Research ArticleValidation Thin Layer Chromatography for theDetermination of Acetaminophen in Tablets and Comparisonwith a Pharmacopeial Method

Alina Pyka Marika Budzisz and MaBgorzata DoBowy

Department of Analytical Chemistry Faculty of Pharmacy Medical University of Silesia PL-4 Jagiellonska Street41-200 Sosnowiec Poland

Correspondence should be addressed to Alina Pyka apykasumedupl

Received 17 April 2013 Accepted 19 July 2013

Academic Editor Deliang Zhou

Copyright copy 2013 Alina Pyka et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Adsorption thin layer chromatography (NP-TLC) with densitometry has been established for the identification and thequantification of acetaminophen in three leading commercial products of pharmaceutical tablets coded as brand P1 (Productno 1) P2 (Product no 2) and P3 (Product no 3) Applied chromatographic conditions have separated acetaminophen from itsrelated substances namely 4-aminophenol and and 41015840-chloroacetanilide UV densitometry was performed in absorbance mode at248 nmThe presented method was validated by specificity range linearity accuracy precision detection limit quantitative limitand robustnessThe TLC-densitometric method was also compared with a pharmacopeial UV-spectrophotometric method for theassay of acetaminophen and the results confirmed statistically that the NP-TLC-densitometric method can be used as a substitutemethod It could be said that the validated NP-TLC-densitometric method is suitable for the routine analysis of acetaminophen inquantity control laboratories

1 Introduction

Acetaminophen (paracetamol) has pharmacological andpharmaceutical significance It is a nonsteroidal anti-inflam-matory drug and is used for the reduction of pain and feverAcetaminophen is commonly used for the relief of headachesand other minor aches and is a major ingredient in numerouscold and flu remedies

Acetaminophen was quantitatively determined in dif-ferent biological fluids namely plasma [1ndash6] urine [7ndash12]serum [11 13] and tissue [14] Moreover acetaminophen wasalso determined in different pharmaceutical preparations insingle and in combined dosage forms [15ndash30]

As was reported in the literature several methods likeliquid chromatography [1 4 5 21] high performance liquidchromatography [3 8 10 12 17 18] reversed-phase sequen-tial injection chromatography (SIC) [20] spectrophotometric[7 9 13 16] spectrophotometric with spectrodensitometric[22] spectrofluorimetric [15] capillary electrophoresis [1119] colorimetric [2 6] chronoamperometric [14] thin layerchromatography with spectrophotometric [23] and thin

layer chromatography with a fluorescence plate reader [24]and thin layer chromatography (HPTLC) with a densitome-try were successfully applied in qualitative and quantitativeacetaminophen analysis [25ndash30] Generally HPLC and UV-spectrophotometric methods have been reported in theUnited States and Polish Pharmacopeias for the analysis ofacetaminophen in pharmaceutical preparations [31 32]

Dimitrovska et al [23] described the conditions forthe determination of propyphenazone acetaminophen caf-feine and codeine phosphate in commercial tablet dosagewith preparative thin layer chromatography The separationof propyphenazone acetaminophen and caffeine was per-formed by use of a mobile phase chloroform + acetone +ammonium hydroxide (25) in volume rations 8 2 01Codeine phosphate was separated from the other compo-nents with chloroform + ethanol in volume ration 8 2 asa mobile phase UV-spectrophotometric determinations ofpropyphenazone acetaminophen caffeine and codeine aftertheir separation on thin layer and elution from the adsorbentwere performed Tavallali et al [24] developed a methodto determine caffeine and acetaminophen concentrations in

2 BioMed Research International

pharmaceutical formulations using TLC with a fluorescenceplate reader Separation of acetaminophen and caffeine wasperformed using the mobile phase n-hexane + ethyl acetate +ethanol (25 15 04 vv)

Many papers described the determination of acet-aminophen in single and in combined tablet dosage formsusing TLC with densitometry [24ndash30] However authorsdid not separate the related substances (4-aminophenol and41015840-choroacetanilide) to acetaminophen This fact indicatesthat the previous TLC-densitometric methods presentedin cited previous papers were not validated in term ofspecificity accordance to validation guidelines [33 34]Ferenczi-Fodor and coworkers described in their papersthat besides the typical validation characteristics suchas accuracy precision repeatability specificity detectionlimit quantification limit linearity and range in the caseof liquid chromatography the evaluation of robustnessshould be considered [34 35] It is a critical parameterhaving significant effect for final results of chromatographicanalysis It was stated that when this parameter is notconstant the analytical process should be improved [34 35]According to ICH guideline the robustness test includesthe influence of the variations of the following parameterslike variations of pH in a mobile phase variations in mobilephase composition different column and temperature onfinal results of liquid chromatographic analysis [33]

Presented work is a continuation of our previous TLCacetaminophen study In earlier paper we described thepreliminary investigations for the analysis of acetaminophenbyTLCwith densitometry including the choice of the optimalchromatographic conditions enabled to complete separationof examined acetaminophen from its related substances (4-aminophenol and 41015840-choroacetanilide) such as silica gel60F254

plates and a mixture of chloroform + acetone +ammonia (25) in volume compositions 8 2 01 as a mobilephase [36]

The aims of this work were to

(i) elaborate the conditions for quantitative determi-nation of acetaminophen in tablets by a TLC-densitometric method with regard to obligatory val-idation presented in validation guidelines and inFerenczi-Fodor and also Nagy-Turak reports includ-ing robustness test [33ndash35]

(ii) apply the spectrophotometric method recommendedby Polish Pharmacopoeia to the quantitative determi-nation of acetaminophen in tablets [31]

(iii) compare the results of quantitative determinationof acetaminophen in tablets obtained by TLC-densitometric and spectrophotometric methods

Based on the obtained results the usefulness of the vali-dated TLC-densitometric method for quantitative analysis ofacetaminophen in combined dosage form in comparison tospectrophotometric method (recommended by Polish Phar-macopoeia) was estimated Moreover the use of the changesof the following chromatographic conditions such as sorbenttype the chamber type extraction time the temperature ofplate activation the distance of development the wavelength

and the analyst as the new factors in robustness test of TLC-densitometric method was discussed

2 Materials and Methods

21 Apparatus Densitometer Camag (Muttenz Switzer-land) equipped with TLC Scanner

Spectrophotometer Specord 205 (Analytik Jena Ger-many)

IKAUltra-Turrax Tube DriveWorkstation with BMT-20-S Tube for grinding with balls of stainless steel

NP-TLC plates 10 times 20 cm glass plates precoated with020mm layers of silica gel 60F

254(EMerck 105570) 20 times

20 cm aluminium plates precoated with 020mm layers ofsilica gel 60F

254(EMerck 105554)

The 5 120583L Camag micropipettes were used to apply thesolutions to the plates

Chromatographic chambers twin-trough chamber for20 times 20 cm plates (02225255 Camag Muttenz Switzer-land) and twin-trough chamber for 20 times 10 cm plates(02225221 Camag Muttenz Switzerland)

22 Chemicals Acetaminophen (Sigma-Aldrich St LouisMO USA) testing and handling conforms to United StatesPharmacopeia 4-aminophenol (gt99 Sigma-Aldrich StLouis MO USA) and 41015840-chloroacetanilide (gt98 Sigma-Aldrich St Louis MO USA) were used as standards Allchemicals and reagents used for TLC were analytical gradeand were purchased from POCh Gliwice Poland

23 Pharmaceutical Preparation Pharmaceutical prepara-tions of three different pharmaceutical manufactures con-taining 500mg of acetaminophen per tablet (Product 1) andalso 500mg per dragged tablet (Product 2 andProduct 3) wereinvestigated

24 Preparation Sample of Tablets Ten tablets were groundfor 20min with a speed equal to 4000 rpm using anIKA Ultra-Turrax Tube Drive Workstation with a BMT-20-S tube for grinding with balls of stainless steel Theobtained powders of acetaminophen tablets (equivalent to25mg acetaminophen by weighing the powder to an accu-racy of 01mg) were shaken with ethanol (998 10mL)for 5min with a speed equal to 4000 rpm After shak-ing the solutions were filtered through a medium-densityfilter to volumetric flasks (25mL) and replenished usingethanol (998) to demanded volume From these solu-tions were next prepared the solutions with about theconcentrations of active substances (acetaminophen) equalto 030mg sdotmLminus1 020mg sdotmLminus1 and 010mg sdotmLminus1 Thesesolutions (5120583L)were used for theTLC-densitometric analysisand for quantitative determination of acetaminophen in cer-tain pharmaceutical preparations The equivalent of 100mgacetaminophen by weight of the powder was also used forUV-spectrophotometric analysis

25 Preparation of Standard Solution of Acetaminophen andIts Related Substances Standard solutions of acetaminophen

BioMed Research International 3

and standard solutions of 4-aminophenol and 41015840-chloro-acetanilide were prepared by dissolving the solutes in ethanol(998)

26 Thin Layer Chromatography The plates were prewashedwith methanol and dried for 24 h at room temperatureBefore use the plates used in NP-TLC were activated at 120∘Cfor 10min TLC-densitometric method for determination ofacetaminophen was performed on aluminium plates pre-coated with 020mm layers of silica gel 60F

254(EMerck

105570) Additionally 20 times 20 cm aluminum plates pre-coated with 020mm layers of silica gel 60F

254(EMerck

105554) were used to test robustness The robustness of ananalytical procedure is a measure of its capacity to remainunaffected by small but deliberate variations in methodparameters

The solutions of acetaminophen samples and acetam-inophen standards (5 120583L) were spotted manually on thechromatographic plates

Themixture of chloroform+acetone+ ammonia (25) involume compositions 8 2 01 was used as mobile phase Ofthemobile phase 50mLwas used in all cases After saturationof the twin-trough chamber (20 cmtimes 20 cm)with themobile-phase vapor for 15min the plates were developed verticallyat room temperature (20∘C) to a distance of 75 cmThe plateswere then dried for 20 h at room temperature (20∘C) in a fumecupboard

Additionally a twin-trough chamber of 20 times 10 cm(02225221 Camag) was used to test robustness

27 Densitometric and Spectrodensitometric InvestigationsDensitometric and spectrodensitometric investigations weredone using a TLC Scanner 3 operated in the absorbancemode The radiation source was a deuterium lamp emittinga continuous UV spectrum between 190 and 450 nm Den-sitometric scanning was then performed at multiwavelengthin the range of 200 to 400 nm at wavelength intervalsof 50 nm at each step Finally densitometric scanning forquantitative determination of acetaminophen was then per-formed at absorption maximum of acetaminophen equalto 248 nm The chromatographic bands obtained on thedensitograms were also investigated by spectrodensitometricanalysis under the following conditions The slit dimen-sions were 1000 times 040mm Macro the optimal opticalsystem was light the scanning speeds were 20mm sdot sminus1 and20 nm sdot sminus1 respectively for densitometric and spectrodensit-ometric analysis the data resolution was 100 120583m sdot stepminus1 and1 nm sdot stepminus1 respectively for densitometric and spectroden-sitometric analysis themeasurement typewas remission andthe measurement mode was absorption

28 Validation of the TLCMethod Theproposedmethodwasvalidated by specificity range linearity accuracy precisiondetection limit quantitative limit and robustness accordingto the ICH guidelines [33] and according to the guidelinesdescribed by Ferenczi-Fodor et al [34]

281 Specificity The specificity of the method was checkedby chromatography of working standard (acetaminophen)and related substances (4-aminophenol and 41015840-chloro-acetanilide) and sample solution of acetaminophen extractedfrom tablets

282 Linearity of Detector Response and Range The linearityof the TLCmethodwas evaluated by analysis of nine standardsolutions of acetaminophen of concentrations 008 010 012015 020 025 030 035 and 040mg sdotmLminus1 The solutions(5 120583L) were applied to the same plate The plates weredeveloped using above-mentioned mobile phases (in thinlayer chromatography section) and scannedThe experimentswere performed in six different analyses

283 Accuracy The accuracy of the TLC method was eval-uated by measurement of recovery Known amounts ofacetaminophen standards in the low medium and highlevels of the calibration plot were added to powdered tabletsof known acetaminophen content and the tablets wereextracted and analyzed under the optimized conditions Theexperiments were performed in six different analyses

284 Precision Repeatability (intraday precision) of themethod was determined by analysis of three replicates ofthree sample solutions (ethanol extracts of acetaminophen)of different concentrations (010 020 and 030mg sdotmLminus1)under the same operating conditions over a short intervalof time (the same day) Intermediate (interday) precisionwas obtained for three sample solutions of different concen-trations (010 020 and 030mg sdotmLminus1) by an analyst whoperformed the analysis over a period of two weeks To deter-mine the precision of the procedure the concentrations wereprepared independently and experiments were performed inthree different analyses The precision was evaluated as therelative standard deviation (coefficient of variation CV [])

285 Detection Limit and Quantitative Limit Based on theCalibration Curve A specific calibration curve was studiedusing samples containing acetaminophen in the range of thedetection limit namely 0125 0250 and 0400 120583g sdot spotminus1The experiments were performed in six different analyses

The detection limit (DL) was calculated as

DL = 33120590119878

(1)

The quantitative limit (QL) was calculated as

QL = 10120590119878

(2)

where 120590 is the standard deviation of the response and 119878 is theslope of the calibration curve

286 Robustness The robustness of the method was testedaccording to guidelines described in the papers by Nagy-Turak and Ferenczi-Fodor et al [34 35 37] The robustnessof the method was checked by spotting sample solution


Table 1 The factors and their levels investigated in robustness test

Symbol Factors Method condition Levels+ minus

1198831

Sorbent type(Merck )

Al sheet(105554)

Al sheet(105554)

Al sheet(105570)

1198832

Chamber type Twin trough20 cm times 20 cm

Twin trough20 cm times 20 cm


1198833

Temperature of plate activation (∘C) 120 130 1101198834

Extraction time (min) 5 6 41198835

Distance of development (cm) 75 80 701198836

Wavelength (nm) 248 250 2461198837

Analyst A A B

Table 2 Experimental design matrix (23) for robustness test

Experiment no 1198831

1198832

1198833

1198834

1198835

1198836

1198837

Acetaminophen content (119910119894)

[mg sdot tabletminus1] in tabletsa

P1 P2 P31 + + + + + + + 4990 4945 50122 + + minus + minus minus minus 4542 4516 45983 + minus + minus minus + minus 4964 4941 49674 + minus minus minus + minus + 4851 4814 48825 minus + + minus + minus minus 5011 5013 50346 minus + minus minus minus + + 4958 4915 49257 minus minus + + minus minus + 4780 4779 48568 minus minus minus + + + minus 4859 4772 4835Effect of the drug

P1 minus6525 1175 13375 minus15325 11675 14675 5075P2 minus6579 2078 16521 minus16777 9804 11297 5278P3 minus4775 0725 15725 minus12675 10425 9225 6025

aProduct 1 (P1) Product 2 (P2) Product 3 (P3)

(100 120583g sdot spotminus1) on the plate and developing the plate afteraltering the conditions (Table 1) The conditions changedwere the sorbent type the chamber type extraction time thetemperature of plate activation the distance of developmentthe wavelength and the analyst The method conditions andthe selected factors with the values of their (+) and (minus)levels are summarized in Table 1 A high level is representedby ldquo+rdquo and a low level by ldquominusrdquo The main effects of sevenfactors were tested on two levels in eight experiments [3537] The levels of factors investigated and the experimentaldesign matrix (23) are shown in Tables 1 and 2 respectivelyThe ways of calculation of the effects (119864) characterizing theparticular individual factors and rank probabilities [38] wereearly presented [35 37 39ndash43]

29 Comparison with Pharmacopeial Method When devel-oping a new analytical method it is desirable to com-pare the results from the new method with those froman accepted method Sample solutions were analyzed byTLC-densitometric method and by the method recom-mended in the Polish Pharmacopeia namely the UV-spectrophotometric method [31] The equivalent of 100mg

of acetaminophen with 15mL of hydrochloric acid (36 gL)was shaken for 30 minutes and then sufficient hydrochloricacid (36 gL) was added to produce 100mL and filtered The10mL of filtrate was diluted to 1000mL with hydrochloricacid (36 gL) and the absorbance of the resulting solutionwas measured at the maximum of 245 nm using Specord 205(Analytik JenaGermany)The content of acetaminophenwascalculated taking 668 as the value of absorbance (1 1 cm) atthe maximum of 245 nm

The comparison of TLC-densitometric method with UV-spectrophotometric method to determine acetaminophenin pharmaceutical preparations was studied by use of tenindependently repeated different analyses

3 Results and Discussion

31 Validation of TLC Method Summarized results of themethod of validation are presented in Figures 1 2 3 and 4as well as in Tables 1 2 and 3

311 Specificity As was reported in Introduction partour literature survey shows that many papers described


Table 3 Method-validation data for the quantitative determination of acetaminophen by NP-TLC with densitometrya

Method characteristic ResultsSpecificity SpecificRange [120583g sdot spotminus1] 040 divide 175

Linearity [120583g sdot spotminus1]A = 24444(plusmn683) + 72498(plusmn637) sdot x

119899 = 8 119903 = 09998 119904 = 830 119865 = 12952 119875 lt 00001Detection limit (DL) [120583g sdot spotminus1] 009Quantitation limit (QL) [120583g sdot spotminus1] 027

For tabletsProduct 1 Product 2 Product 3

AccuracyFor 50 acetaminophen added (119899 = 6) 119877 = 10072 CV = 113 119877 = 9969 CV = 091 119877 = 10134 CV = 129For 100 acetaminophen added (119899 = 6) 119877 = 10058 CV = 121 119877 = 9963 CV = 092 119877 = 10234 CV = 133For 150 acetaminophen added (119899 = 6) 119877 = 10123 CV = 086 119877 = 9927 CV = 112 119877 = 10087 CV = 108

Precision (CV [])Repeatability

For 050 120583g sdot spotminus1 (119899 = 3) 057 085 099For 100 120583g sdot spotminus1 (119899 = 3) 066 076 078For 150 120583g sdot spotminus1 (119899 = 3) 100 073 111

IntermediateFor 050 120583g sdot spotminus1 (119899 = 3) 085 141 111For 100 120583g sdot spotminus1 (119899 = 3) 120 157 099For 150 120583g sdot spotminus1 (119899 = 3) 113 136 156

Robustness (CV []) Robust Robust Robusta119860 peak area [AU] x amount [120583g sdot spotminus1] of drug analyzed r correlation coefficient R recovery [] CV coefficient of variation []

1000

900

800

700

600

500

400

300

200

100

0

(AU

)

minus006 014 034 054 074 094

Rf

P

PA

CA

Figure 1 Densitogram obtained from acetaminophen standard (P)spiked with related substances namely p-aminophenol (PA) and 41015840-chloroacetanilide (CA)

the determination of acetaminophen in commercial pro-ducts with the use of TLC and TLC-densitometry [24ndash30]Various chromatographic conditions such as mobile phasecomposition and the kind of TLC plates were studied toobtain satisfactory results of acetaminophen qualitative andquantitative analysis Based on the literature reports our

1000

(AU

)

800900

700600500400300200100

00

1000

(AU

)

800900

70060050040030020010000

2000 2500 3000 3500(nm)

4000

CA

PPA

Figure 2 Spectrodensitograms of acetaminophen standard (P) p-aminophenol (PA) and 41015840-chloroacetanilide (CA)

TLC-densitometry of acetaminophen was performed onaluminium plates precoated with silica gel 60F

254(recom-

mended in literature for acetaminophen TLC analysis) Inorder to choose the optimal mobile phase which allowsseparating acetaminophen and its related substances (4-aminophenol and 41015840-chloroacetanilide) from commercialproduct different compositions and ratios of ammoniatoluene ethyl acetate chloroform cyclohexane methanol n-hexane acetone and glacial acetic acid were examined Of all


300

250

200

150

100

50

0

(AU

)

minus006 014 034 054 074 094

Rf

P

Figure 3 Densitogram of acetaminophen (P) coming from Product2 sample

16000

14000

12000

10000

8000

6000

4000

2000

0000 025 050 075 100 125 150 175 200

A = 24444(plusmn683) + 72498(plusmn637)middotxn = 8 r = 09998 s = 830 F = 12952 P lt 00001

Peak

area

A(A

U)

x (120583gspot)

(a)

040 050

060

075

100125

150

175

300

200

100

0

minus100

minus200

minus300

Resid

ual (

AU)

(120583gspot)

(b)

Figure 4 Calibration plot (a) and plot of residuals (b) foracetaminophen in the linear working range

mobile phases used in experiment themixture of chloroform+ acetone + ammonia (25) in volume compositions 8 2 01as mobile phase resulted in compact bands and sharp andsymmetric peaks of acetaminophen (P) related substancesnamely p-aminophenol (PA) and 41015840-chloroacetanilide(CA) The mixture of chloroform + acetone + ammonia(25) (8 2 01 vvv) used as mobile phase resulted inoptimum migration acetaminophen (119877

119865(P) = 014 plusmn 001)

201510

50

minus5

minus10

minus15

minus20

minus25

X1

X2 X3

X4

X5 X6 X7Effec

t

(a)

Effect

1090807060504030201

00000 2000 4000 6000 8000 10000 12000140001600018000

Pi

R2 = 09742

(b)

Figure 5 Robustness test the effects of factors (a) and half-normalprobability plot of effects (b) for determination of acetaminophen inProduct 2

and resolution of the drug from its related substancesnamely p-aminophenol (119877

119865(PA) = 028 plusmn 002) and 41015840-chlo-roacetanilide (119877

119865(CA) = 059 plusmn 002) without interferencefrom other components of the formulations matrix Whenexcess p-aminophenol and 41015840-chloroacetanilide wereadded to the sample solution to check the specificity ofthe method the chromatogram presented the resolutionof the acetaminophen peak from the related substance(p-aminophenol and 41015840-chloroacetanilide) peaks wereequal to 119877

119878(PPA) = 125 119877119878(PACA) = 276 119877119878(PCA) = 373(Figure 1) Spectrodensitograms of acetaminophen (P)p-aminophenol (PA) and 41015840-chloroacetanilide (CA)are presented in Figure 2 The absorption maximum ofacetaminophen p-aminophenol and 41015840-chloroacetanilideare equal to 248 200 and 249 nm respectively Typicaldensitogram of acetaminophen coming from the extract ofProduct 2 is presented in Figure 3The identical densitogramswere also obtained for the extract of Products 1 and 3 It wasobserved that excipients present in the formulation did notinterfere with the acetaminophen peak It was also statedthat the analyte was not decomposed during development ofthe chromatogram and was stabled in solution and on thesorbent at room temperature These facts indicate that newmethod is specificity

312 Accuracy The accuracy of themethodwas evaluated bymeasurement of recovery (Table 3) When known amounts


of acetaminophen were added to powdered tablets of knownacetaminophen content quantitative recoveries of 9927 divide10234 (mean 10058) were obtained (Table 3) The lowcoefficient of variation values (CV lt 2) are indicative of theaccuracy of the method

313 Calibration and Range The statistical data shownin Table 3 and Figure 4(a) indicate that linear relationshipexists between area of peaks [AU] and concentration ofacetaminophen standard [120583g sdot spotminus1] The plot (119899 = 8)was linear in the range 040 to 175120583g sdot spotminus1 for NP-TLCanalysis The graphs of residuals against the concentrationof acetaminophen were also plotted (Figure 4(b)) It wasobserved that the residuals were distributed both above andbelow the zero residuals line

314 Precision The precision of the method was studiedas repeatability and intermediate of the system at three dif-ferent concentrations of tablet extractions The results fromthese experiments expressed as the coefficients of variation(CV ) of the respectively response factors (a relationshipbetween the peak area and concentration of acetaminophen)are presented in Table 3 Because CV for repeatability andintermediate were lt3 the method was precise

315 Detection Limit (DL) and Quantitative Limit (QL) Basedon theCalibrationCurve The limits of detection and the limitof quantification were 009120583g sdot spotminus1 and 027 120583g sdot spotminus1respectively

316 Robustness The main effects of seven factors weretested on two levels in eight experiments (Tables 1 and 2)for determination of robustness applied NP-TLC methodTable 2 shows the results obtained for acetaminophen content(119910119894) in three pharmaceutical preparations coded P1 (Product

1) P2 (Product 2) and P3 (Product 3) The main effects of thefactors calculated from these results (119910

119894) are also presented

in Table 2 These results show that no factor has significanteffect on the results These results were also evaluated byhalf-normal probability plotting of rank probabilities (119901

119894) as a

function of the absolute values of themain effects [35 38 39]For example the effects of factors andhalf-normal probabilityplot of effects for the determination of acetaminophen inProduct 2 are presented in Figure 5 Similarly results wereobtained for determination of acetaminophen in Product 1and Product 3 The points of all factors lie near the straightline which indicates that their effect is negligible Basedon the results of the robustness test (Table 2 experimentaldesign matrix (23) for robustness) which were determinedaccording to the guidelines described in the papers byNagy-Turak and Ferenczi-Fodor et al it was stated that theproposed TLC-densitometric method can be regarded asrobust Moreover it can be suggested that the modification ofrobustness test procedure in comparison to the methodologypresented by Nagy-Turak and Ferenczi-Fodor et al [3537] and to our previous investigations [39ndash43] is suitablefor robustness study of TLC-densitometric method used in

acetaminophen determination in combined pharmaceuticaldosage form

It was stated that besides the robustness factors pre-sented in our previous validation studies concerning TLC-densitometric investigations of different bioactive sub-stances in their pharmaceutical preparations like hydrocor-tisone naproxen tocopherol acetate and acetylsalicylic acid(eg volume of n-hexane saturation time of the chamberetc) the changes of the following chromatographic conditionsuch as sorbent type the chamber type extraction time thetemperature of plate activation the distance of developmentthe wavelength and the analyst used in this work can besuccessfully applied as the new factors in robustness test ofTLC-densitometric method which is successfully applied indrug analysis

317 Analysis of Acetaminophen in Commercial Tablets Asingle spot at an average 119877

119865equal 014 was observed in the

chromatograms obtained from the extract of acetaminophentablets There was no interference from excipients presentin the tablets It was also concluded that no degradationof acetaminophen had occurred in the formulation ana-lyzed by the TLC method In each case the 119877

119865values of

acetaminophen standard and acetaminophen from Product1 Product 2 and Product 3 were equal to 014 divide 001 forTLC-densitometric analysisThe identities of acetaminophenstandard with acetaminophen from the commercial sampleswere investigated on the basis of the comparison of theirspectra The very good correspondence between spectroden-sitograms was stated In all cases the absorption maximum(120582max) is equal to 248 nm The purities of acetaminophenpeaks from the samples of Product 1 Product 2 and Product3 were also assessed by comparing the spectra obtainedfrom a acetaminophen standard at the peak start peak apexand peak end of spot It was found that 119903(119878119872) gt 0999and 119903(119872 119864) gt 0999 for all of the analyses performed bythe TLC-densitometric technique Statistical data concerningthe results of quantitative determination of acetaminophenin the ten repeated different analyses of pharmaceuticalpreparations are presented in Table 4 It was stated thatacetaminophen amounts in pharmaceutical preparationsdetermined by TLC-densitometric method are equal to993 998 and 1008 respectively for Product 1 Product2 and Product 3 in relation to the amounts of acetaminophendeclared by the manufacturers

32 Comparison with Pharmacopeial Method To verify theresults obtained by the TLC-densitometric method com-parison was made with a previous report using the PolishPharmacopeia UV-spectrophotometric method [31] Whencompared with the pharmacopeial method recommendedfor acetaminophen tablets similar results were obtainedfor ten repeated different analyses (Table 4) The averageassays of acetaminophen were 4967 plusmn 64mg sdot tabletminus1 and4957plusmn70mg sdot tabletminus1 Product 1 4991 plusmn 56mg sdot tabletminus1 and4975 plusmn 48mg sdot tabletminus1 Product 2 5038 plusmn 51mg sdot tabletminus1and 5014plusmn79mg sdot tabletminus1 Product 3 for TLC-densitometric


Table 4 Acetaminophen assay [mg sdot tabletminus1] obtained from ten repeated different analyses by TLC-densitometric and UV-spectro-densitometric methods for tablets of three different pharmaceutical manufactures

No

Product 1 Product 2 Product 3Assay

by TLC-densitometric

method

Assayby UV-

spectrophotometricmethod

Assayby TLC-

densitometricmethod

Assayby UV-


Assayby TLC-

densitometricmethod

Assayby UV-


1 5003 5023 5090 5045 5024 50572 5035 4913 4881 4975 4968 48983 4883 4865 4996 4947 5096 51124 4952 4944 5044 4986 4978 49235 5005 4935 4943 5036 5027 50896 4918 4906 4996 4899 5058 50677 4891 4945 4992 4973 4976 49128 5042 5062 5015 5019 5098 50599 5031 5069 4969 4929 5067 497910 4912 4908 4988 4939 5089 5046Average assay 4967 4957 4991 4975 5038 5014Label claimed 5000 5000 5000 5000 5000 5000Standard deviation (SD) 64 70 56 48 51 79Coefficient of variation[CV ] 127 140 112 096 101 158

TLC-densitometric method compared with UV-spectrophotometric methodt-test

t calculated 034 069 081119905(9518) tabulated 210 210 210

F-testF calculated 124 136 239119865(951198911=1198912=9) tabulated 318 318 318

andUV-spectrophotometricmethods respectivelyThe coef-ficients of variance were smaller than 2 in each case Highreproducibility and insignificant differences between the twocomparedmethodswere obtained at the 95probability levelfor 119905-test and 119865-test of significance of 034 lt 210 and 124lt 318 069 lt 210 and 136 lt 318 081 lt 210 and 239 lt318 respectively for Product 1 Product 2 and Product 3These results confirmed statistically that TLC-densitometricmethod is accurate and can be used as a substitute method

Acetaminophen content in investigated pharmaceuticalpreparations is consistent with that reported by the PolishPharmacopoeia [31] acetaminophen content in the prepara-tion should not be smaller than 95 and larger than the 105of the declared value

The results of this study and those presented in ourprevious papers [39ndash43] indicate that the validated TLC-densitometric method can be successfully applied to thedetermination of biological active compounds in selectedpharmaceutical formulations

4 Conclusion

Our TLC-densitometric study of acetaminophen indicatesthat of all applied chromatographic conditions the most

suitable are aluminium plates precoated with silica gel 60F254

as stationary phase and a mixture of chloroform + ace-tone + ammonia (25) in volume compositions 8 2 01as mobile phase Above-mentioned chromatographic con-ditions resulted in optimum migration of acetaminophenand complete resolution of this compound from its relatedsubstances namely 4-aminophenol and 41015840-chloroacetanilidefrom commercial products in combined dosage form Thisfact confirmed the specificity of proposedmethod Accordingto the results of the experiments performed using the TLC-densitometric method it was determined that the procedureused in this study is reliable with specificity accuracyprecision and robustness The TLC-densitometric methodalso realizes the criterion of the linearity in the required rangeof acetaminophen concentrations The results of validationof developed TLC-densitometric method for acetaminophenanalysis indicate that the changes of the following chro-matographic conditions such as sorbent type the chambertype extraction time the temperature of plate activationthe distance of development the wavelength and the analystused in our work can be successfully used as the new factorsin robustness test of TLC-densitometric method which iswidely applied in drug analysis


Comparison of the acetaminophen content in tabletsobtained from the TLC-densitometry to those determinedby the use of the official UV-spectrophotometric methodshows that acetaminophen content in investigated pharma-ceutical preparations is consistent with that reported by thePolish Pharmacopoeia (is not smaller than 95 and notlarger than the 105 of the declared value) It could besaid that the TLC-densitometric andUV-spectrophotometricmethods mentioned in this study are suitable for the routineanalysis of acetaminophen in quantity control laboratoriesMoreover the results in our work confirmed statisticallythat TLC-densitometric method is accurate and can beused as a substitute method for the accurate assay of theacetaminophen in pharmaceutical dosage forms for examplein situationwhenUV-spectrophotometer orHPLC-UV is notaffordable in laboratory

Disclosure

The usage of this trade mark symbol or company name isfor proving the genuineness of the work and not for anyanother purposeThe authors submitted this paper in amuchelaborate manner by mentioning the trademarks or companynames in order to make each and every point or sentenceclear or transparentThe authors of the paper do not have anyfinancial relation with the commercial identity mentioned intheir paper

Conflict of Interests

The authors have no conflict of interests or no financial gainsin mentioning the company names or trademarks

Acknowledgment

This research was financed by the Medical University of Sile-sia as part of statutory research projects KNW-1-004P10and KNW-1-013N30

References

[1] A Wang J Sun H Feng S Gao and Z He ldquoSimultaneousdetermination of paracetamol and caffeine in human plasmaby LC-ESI-MSrdquo Chromatographia vol 67 no 3-4 pp 281ndash2852008

[2] C T Archer and R A Richardson ldquoAn improved colorimetricmethod for the determination of plasma paracetamolrdquo Annalsof Clinical Biochemistry vol 17 no 1 pp 45ndash46 1980

[3] A Esteban M Graells J Satorre and M Perez-Mateo ldquoDeter-mination of paracetamol and its four major metabolites inmouse plasma by reversed-phase ion-pair high-performanceliquid chromatographyrdquo Journal of Chromatography B vol 573no 1 pp 121ndash126 1992

[4] O Q P Yin S S L Lam and M S S Chow ldquoSimultane-ous determination of paracetamol and dextropropoxyphenein human plasma by liquid chromatographytandem massspectrometry application to clinical bioequivalence studiesrdquoRapid Communications in Mass Spectrometry vol 19 no 6 pp767ndash774 2005

[5] C Celma J A Allue J Prunonosa C Peraire and R ObachldquoSimultaneous determination of paracetamol and chlorpheni-ramine in human plasma by liquid chromatography-tandemmass spectrometryrdquo Journal of Chromatography A vol 870 no1-2 pp 77ndash86 2000

[6] F Shihana D Dissanayake P Dargan and A DawsonldquoA modified low-cost colorimetric method for paracetamol(acetaminophen) measurement in plasmardquo Clinical Toxicologyvol 48 no 1 pp 42ndash46 2010

[7] J Parojcic K Karljikovic-Rajic Z Duric M Jovanovic and SIbric ldquoDevelopment of the second-order derivative UV spec-trophotometric method for direct determination of paraceta-mol in urine intended for biopharmaceutical characterisationof drug productsrdquo Biopharmarmaceutics and Drug Dispositionvol 24 no 7 pp 309ndash314 2003

[8] R Krishnamurthy M K Malve and B M Shinde ldquoSimulta-neous determination of morphine caffeine and paracetamol inthe urine of addicts by HPTLC and HPLCrdquo Journal of PlanarChromatographymdashModern TLC vol 13 no 3 pp 171ndash175 2000

[9] H Filik I Sener S D Cekic E Kilic andRApak ldquoSpectropho-tometric determination of paracetamol in urine with tetrahy-droxycalix[4]arene as a coupling reagent and preconcentrationwith Triton X-114 using cloud point extractionrdquo Chemical andPharmaceutical Bulletin vol 54 no 6 pp 891ndash896 2006

[10] J Meyer and U Karst ldquoDetermination of paracetamol (acet-aminophen) by HPLC with post-column enzymatic derivatiza-tion and fluorescence detectionrdquo Chromatographia vol 54 no3-4 pp 163ndash167 2001

[11] S Heitmeier and G Blaschke ldquoDirect determination of parac-etamol and its metabolites in urine and serum by capillary elec-trophoresis with ultraviolet and mass spectrometric detectionrdquoJournal of Chromatography B vol 721 no 1 pp 93ndash108 1999

[12] I Baranowska and A Wilczek ldquoSimultaneous RP-HPLC deter-mination of sotalol metoprolol 120572-hydroxymetoprolol parac-etamol and its glucuronide and sulfate metabolites in humanurinerdquo Analytical Sciences vol 25 no 6 pp 769ndash772 2009

[13] A Afkhami N Sarlak and A R Zarei ldquoSpectrophotometricdetermination of salicylamide and paracetamol in biologicalsamples and pharmaceutical formulations by a differentialkinetic methodrdquo Acta Chimica Slovenica vol 53 no 3 pp 357ndash362 2006

[14] O Fatibello-Filho K O Lupetti and I C Vieira ldquoChronoam-perometric determination of paracetamol using an avocadotissue (Persea americana) biosensorrdquo Talanta vol 55 no 4 pp685ndash692 2001

[15] M A Oliva R A Olsina and A N Masi ldquoSelective spec-trofluorimetricmethod for paracetamol determination throughcoumarinic compound formationrdquo Talanta vol 66 no 1 pp229ndash235 2005

[16] S D Cekic H Filik and R Apak ldquoSimultaneous spectropho-tometric determination of paracetamol and p-aminophenol inpharmaceutical products with tiron using dissolved oxygen asoxidantrdquo Journal of Analytical Chemistry vol 60 no 11 pp1019ndash1023 2005

[17] K Milenkova A Dimitrovska L Ugrinova and S Trajkovic-Jolevska ldquoSimultaneous determination of paracetamol pseu-doephedrine hydrochloride and dextromethorphan hydrobro-mide in tablets by HPLCrdquo Bulletin of Chemical TechnologyMacedonia vol 22 pp 33ndash37 2003


[18] U D Pawar A V Naik A V Sulebhavikar T A Datar andKVMangaonkar ldquoSimultaneous determination of aceclofenacparacetamol and chlorzoxazone by HPLC in tablet dose formrdquoE-Journal of Chemistry vol 6 no 1 pp 289ndash294 2009

[19] S Zhao W Bai H Yuan and D Xiao ldquoDetection of parac-etamol by capillary electrophoresis with chemiluminescencedetectionrdquo Analytica Chimica Acta vol 559 no 2 pp 195ndash1992006

[20] D Satınsky I Neto P Solich et al ldquoSequential injectionchromatographic determination of paracetamol caffeine andacetylsalicylic acid in pharmaceutical tabletsrdquo Journal of Sepa-ration Science vol 27 no 7-8 pp 529ndash536 2004

[21] L Monser and F Darghouth ldquoSimultaneous LC determinationof paracetamol and related compounds in pharmaceuticalformulations using a carbon-based columnrdquo Journal of Phar-maceutical and Biomedical Analysis vol 27 no 6 pp 851ndash8602002

[22] H E Abdellatef M M Ayad S M Soliman and N F YoussefldquoSpectrophotometric and spectrodensitometric determinationof paracetamol and drotaverine HCl in combinationrdquo Spec-trochimica Acta A vol 66 no 4-5 pp 1147ndash1151 2007

[23] A Dimitrovska S Trajkovic-Jolevska A Nancovska and MIlievska ldquoDetermination of propyphenazone paracetamol caf-feine and codeine phosphate with thin layer chromatographyrdquoBulletin of Chemical Technology Macedonia vol 14 pp 39ndash411995

[24] H Tavallali S F Zareiyan and M Naghian ldquoAn efficientand simultaneous analysis of caffeine and paracetamol inpharmaceutical formulations using TLC with a fluorescenceplate readerrdquo Journal of AOAC International vol 94 no 4 pp1094ndash1099 2011

[25] C Sullivan and J Sherma ldquoDevelopment and validation ofan HPTLC-densitometry method for assay of caffeine andacetaminophen in multicomponent extra strength analgesictabletsrdquo Journal of Liquid Chromatography and Related Tech-nologies vol 26 no 20 pp 3453ndash3462 2003

[26] H J Shah I S Rathod S A Shah S S Savale and C JShishoo ldquoSensitive HPTLC method for monitoring dissolu-tion profiles of diclofenac from different tablets containingcombiined diclofenac and acetaminophenrdquo Journal of PlanarChromatographymdashModern TLC vol 16 no 1 pp 36ndash44 2003

[27] V K Mahajan S B Bari A A Shirkhedkar and S J SuranaldquoSimultaneous densitometric TLC analysis of aceclofenacparacetamol and chlorzoxazone in tabletsrdquo Acta Chromato-graphica vol 20 no 4 pp 625ndash636 2008

[28] S V Gandhi N S Barhate B R Patel D D Panchal and KG Bothara ldquoA validated densitometric method for analysis ofaceclofenac and paracetamol as the bulk drugs and in combinedtablet dosage formsrdquo Acta Chromatographica vol 20 no 2 pp175ndash182 2008

[29] C OrsquoSullivan and J Sherma ldquoModel procedure for the transferof TLC pharmaceutical product screening methods designedfor use in developing countries to quantitative HPTLC-densitometry methodsrdquo Acta Chromatographica vol 24 no 2pp 241ndash252 2012

[30] A Yadav R M Singh S C Mathur P K Saini and G NSingh ldquoA simple and sensitiveHPTLCmethod for simultaneousanalysis of domperidone and paracetamol in tablet dosageformsrdquo Journal of Planar ChromatographymdashModern TLC vol22 no 6 pp 421ndash424 2009

[31] Polish Pharmacopoeia Polish Pharmaceutical Society WarsawPoland 2002

[32] The United States Pharmacopoeia Twinbrook ParkwayRockville Md USA 2011

[33] ICH Harmonised Tripartite Guideline Validation of Analyti-cal Procedures Text and Methodology Q2(R1) ICH GenevaSwitzerland 2005 httpwwwichorg

[34] K Ferenczi-Fodor B Renger and Z Vegh ldquoThe frustratedreviewermdashrecurrent failures in manuscripts describing valida-tion of quantitative TLCHPTLC procedures for analysis ofpharmaceuticalsrdquo Journal of Planar ChromatographymdashModernTLC vol 23 no 3 pp 173ndash179 2010

[35] K Ferenczi-Fodor A Nagy-Turak and Z Vegh ldquoValidationand monitoring of quantitative thin layer chromatographicpurity tests for bulk drug substancesrdquo Journal of PlanarChromatographymdashModern TLC vol 8 no 5 pp 349ndash356 1995

[36] A Pyka M Budzisz and M Dołowy ldquoZastosowanie TLCi densytometrii do ilosciowego oznaczania paracetamolu wwybranych preparatach farmaceutycznychrdquo in Chromatografiaw Praktyce A Voelkel and W Wasiak Eds pp 137ndash147Wydawnictwo Politechniki Poznanskiej 2011

[37] A Nagy-Turak Z Vegh and K Ferenczi-Fodor ldquoValidation ofthe quantitative planar chromatographic analysis of drug sub-stances III Robustness testing in OPLCrdquo Journal of PlanarChromatographymdashModern TLC vol 8 no 3 pp 188ndash193 1995

[38] C D Hendix ldquoWhat every technologist should know aboutexperiment designrdquo Chemical Technology vol 9 pp 167ndash1741979

[39] A Pyka E Wiatr K Kwiska and D Gurak ldquoValidation thinlayer chromatography for the determination of naproxen intablets and comparison with a pharmacopeil methodrdquo Journalof Liquid Chromatography and Related Technologies vol 34 no10-11 pp 829ndash847 2011

[40] A Pyka D Nabiałkowska K Bober andM Dołowy ldquoCompar-ison of NP-TLC and RP-TLC with densitometry to quantitativeanalysis of tocopherol acetate in pharmaceutical preparationrdquoJournal of Liquid Chromatography and Related Technologies vol34 no 20 pp 2548ndash2564 2011

[41] A Pyka M Babuska-Roczniak and P Bochenska ldquoDetermi-nation of hydrocortisone in pharmaceutical drug by TLC withdensitometric detection in UVrdquo Journal of Liquid Chromatogra-phy and Related Technologies vol 34 no 9 pp 753ndash769 2011

[42] P Bochenska and A Pyka ldquoDetermination of acetylsalicylicacid in pharmaceutical drugs by TLC with densitometricdetection in UVrdquo Journal of Liquid Chromatography and RelatedTechnology vol 35 pp 1346ndash1363 2012

[43] P Bochenska and A Pyka ldquoUse of TLC for the quantitativedetermination of acetylsalicylic acid caffeine and ethoxyben-zamide in combined tabletsrdquo Journal of Liquid Chromatographyand Related Technology vol 36 pp 2405ndash2421 2013

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


pharmaceutical formulations using TLC with a fluorescenceplate reader Separation of acetaminophen and caffeine wasperformed using the mobile phase n-hexane + ethyl acetate +ethanol (25 15 04 vv)

Many papers described the determination of acet-aminophen in single and in combined tablet dosage formsusing TLC with densitometry [24ndash30] However authorsdid not separate the related substances (4-aminophenol and41015840-choroacetanilide) to acetaminophen This fact indicatesthat the previous TLC-densitometric methods presentedin cited previous papers were not validated in term ofspecificity accordance to validation guidelines [33 34]Ferenczi-Fodor and coworkers described in their papersthat besides the typical validation characteristics suchas accuracy precision repeatability specificity detectionlimit quantification limit linearity and range in the caseof liquid chromatography the evaluation of robustnessshould be considered [34 35] It is a critical parameterhaving significant effect for final results of chromatographicanalysis It was stated that when this parameter is notconstant the analytical process should be improved [34 35]According to ICH guideline the robustness test includesthe influence of the variations of the following parameterslike variations of pH in a mobile phase variations in mobilephase composition different column and temperature onfinal results of liquid chromatographic analysis [33]

Presented work is a continuation of our previous TLCacetaminophen study In earlier paper we described thepreliminary investigations for the analysis of acetaminophenbyTLCwith densitometry including the choice of the optimalchromatographic conditions enabled to complete separationof examined acetaminophen from its related substances (4-aminophenol and 41015840-choroacetanilide) such as silica gel60F254

plates and a mixture of chloroform + acetone +ammonia (25) in volume compositions 8 2 01 as a mobilephase [36]

The aims of this work were to

(i) elaborate the conditions for quantitative determi-nation of acetaminophen in tablets by a TLC-densitometric method with regard to obligatory val-idation presented in validation guidelines and inFerenczi-Fodor and also Nagy-Turak reports includ-ing robustness test [33ndash35]

(ii) apply the spectrophotometric method recommendedby Polish Pharmacopoeia to the quantitative determi-nation of acetaminophen in tablets [31]

(iii) compare the results of quantitative determinationof acetaminophen in tablets obtained by TLC-densitometric and spectrophotometric methods

Based on the obtained results the usefulness of the vali-dated TLC-densitometric method for quantitative analysis ofacetaminophen in combined dosage form in comparison tospectrophotometric method (recommended by Polish Phar-macopoeia) was estimated Moreover the use of the changesof the following chromatographic conditions such as sorbenttype the chamber type extraction time the temperature ofplate activation the distance of development the wavelength

and the analyst as the new factors in robustness test of TLC-densitometric method was discussed

2 Materials and Methods

21 Apparatus Densitometer Camag (Muttenz Switzer-land) equipped with TLC Scanner

Spectrophotometer Specord 205 (Analytik Jena Ger-many)

IKAUltra-Turrax Tube DriveWorkstation with BMT-20-S Tube for grinding with balls of stainless steel

NP-TLC plates 10 times 20 cm glass plates precoated with020mm layers of silica gel 60F

254(EMerck 105570) 20 times

20 cm aluminium plates precoated with 020mm layers ofsilica gel 60F

254(EMerck 105554)

The 5 120583L Camag micropipettes were used to apply thesolutions to the plates

Chromatographic chambers twin-trough chamber for20 times 20 cm plates (02225255 Camag Muttenz Switzer-land) and twin-trough chamber for 20 times 10 cm plates(02225221 Camag Muttenz Switzerland)

22 Chemicals Acetaminophen (Sigma-Aldrich St LouisMO USA) testing and handling conforms to United StatesPharmacopeia 4-aminophenol (gt99 Sigma-Aldrich StLouis MO USA) and 41015840-chloroacetanilide (gt98 Sigma-Aldrich St Louis MO USA) were used as standards Allchemicals and reagents used for TLC were analytical gradeand were purchased from POCh Gliwice Poland

23 Pharmaceutical Preparation Pharmaceutical prepara-tions of three different pharmaceutical manufactures con-taining 500mg of acetaminophen per tablet (Product 1) andalso 500mg per dragged tablet (Product 2 andProduct 3) wereinvestigated

24 Preparation Sample of Tablets Ten tablets were groundfor 20min with a speed equal to 4000 rpm using anIKA Ultra-Turrax Tube Drive Workstation with a BMT-20-S tube for grinding with balls of stainless steel Theobtained powders of acetaminophen tablets (equivalent to25mg acetaminophen by weighing the powder to an accu-racy of 01mg) were shaken with ethanol (998 10mL)for 5min with a speed equal to 4000 rpm After shak-ing the solutions were filtered through a medium-densityfilter to volumetric flasks (25mL) and replenished usingethanol (998) to demanded volume From these solu-tions were next prepared the solutions with about theconcentrations of active substances (acetaminophen) equalto 030mg sdotmLminus1 020mg sdotmLminus1 and 010mg sdotmLminus1 Thesesolutions (5120583L)were used for theTLC-densitometric analysisand for quantitative determination of acetaminophen in cer-tain pharmaceutical preparations The equivalent of 100mgacetaminophen by weight of the powder was also used forUV-spectrophotometric analysis

25 Preparation of Standard Solution of Acetaminophen andIts Related Substances Standard solutions of acetaminophen




254(EMerck


254(EMerck













DL = 33120590119878

(1)


QL = 10120590119878

(2)






1198831


Al sheet(105554)

Al sheet(105554)

Al sheet(105570)

1198832




1198833




Wavelength (nm) 248 250 2461198837

Analyst A A B



1198832

1198833

1198834

1198835

1198836

1198837
























1000

900

800

700

600

500

400

300

200

100

0

(AU

)

minus006 014 034 054 074 094

Rf

P

PA

CA



1000

(AU

)

800900

700600500400300200100

00

1000

(AU

)

800900

70060050040030020010000

2000 2500 3000 3500(nm)

4000

CA

PPA



254(recom-



300

250

200

150

100

50

0

(AU

)

minus006 014 034 054 074 094

Rf

P


16000

14000

12000

10000

8000

6000

4000

2000

0000 025 050 075 100 125 150 175 200


Peak

area

A(A

U)

x (120583gspot)

(a)

040 050

060

075

100125

150

175

300

200

100

0

minus100

minus200

minus300

Resid

ual (

AU)

(120583gspot)

(b)



119865(P) = 014 plusmn 001)

201510

50

minus5

minus10

minus15

minus20

minus25

X1

X2 X3

X4

X5 X6 X7Effec

t

(a)

Effect

1090807060504030201

00000 2000 4000 6000 8000 10000 12000140001600018000

Pi

R2 = 09742

(b)
















119894) as a







119865values of





No



method

Assayby UV-


Assayby TLC-

densitometricmethod

Assayby UV-


Assayby TLC-

densitometricmethod

Assayby UV-









4 Conclusion






Disclosure




Acknowledgment


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




254(EMerck


254(EMerck













DL = 33120590119878

(1)


QL = 10120590119878

(2)






1198831


Al sheet(105554)

Al sheet(105554)

Al sheet(105570)

1198832




1198833




Wavelength (nm) 248 250 2461198837

Analyst A A B



1198832

1198833

1198834

1198835

1198836

1198837
























1000

900

800

700

600

500

400

300

200

100

0

(AU

)

minus006 014 034 054 074 094

Rf

P

PA

CA



1000

(AU

)

800900

700600500400300200100

00

1000

(AU

)

800900

70060050040030020010000

2000 2500 3000 3500(nm)

4000

CA

PPA



254(recom-



300

250

200

150

100

50

0

(AU

)

minus006 014 034 054 074 094

Rf

P


16000

14000

12000

10000

8000

6000

4000

2000

0000 025 050 075 100 125 150 175 200


Peak

area

A(A

U)

x (120583gspot)

(a)

040 050

060

075

100125

150

175

300

200

100

0

minus100

minus200

minus300

Resid

ual (

AU)

(120583gspot)

(b)



119865(P) = 014 plusmn 001)

201510

50

minus5

minus10

minus15

minus20

minus25

X1

X2 X3

X4

X5 X6 X7Effec

t

(a)

Effect

1090807060504030201

00000 2000 4000 6000 8000 10000 12000140001600018000

Pi

R2 = 09742

(b)
















119894) as a







119865values of





No



method

Assayby UV-


Assayby TLC-

densitometricmethod

Assayby UV-


Assayby TLC-

densitometricmethod

Assayby UV-









4 Conclusion






Disclosure




Acknowledgment


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




1198831


Al sheet(105554)

Al sheet(105554)

Al sheet(105570)

1198832




1198833




Wavelength (nm) 248 250 2461198837

Analyst A A B



1198832

1198833

1198834

1198835

1198836

1198837
























1000

900

800

700

600

500

400

300

200

100

0

(AU

)

minus006 014 034 054 074 094

Rf

P

PA

CA



1000

(AU

)

800900

700600500400300200100

00

1000

(AU

)

800900

70060050040030020010000

2000 2500 3000 3500(nm)

4000

CA

PPA



254(recom-



300

250

200

150

100

50

0

(AU

)

minus006 014 034 054 074 094

Rf

P


16000

14000

12000

10000

8000

6000

4000

2000

0000 025 050 075 100 125 150 175 200


Peak

area

A(A

U)

x (120583gspot)

(a)

040 050

060

075

100125

150

175

300

200

100

0

minus100

minus200

minus300

Resid

ual (

AU)

(120583gspot)

(b)



119865(P) = 014 plusmn 001)

201510

50

minus5

minus10

minus15

minus20

minus25

X1

X2 X3

X4

X5 X6 X7Effec

t

(a)

Effect

1090807060504030201

00000 2000 4000 6000 8000 10000 12000140001600018000

Pi

R2 = 09742

(b)
















119894) as a







119865values of





No



method

Assayby UV-


Assayby TLC-

densitometricmethod

Assayby UV-


Assayby TLC-

densitometricmethod

Assayby UV-









4 Conclusion






Disclosure




Acknowledgment


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of












1000

900

800

700

600

500

400

300

200

100

0

(AU

)

minus006 014 034 054 074 094

Rf

P

PA

CA



1000

(AU

)

800900

700600500400300200100

00

1000

(AU

)

800900

70060050040030020010000

2000 2500 3000 3500(nm)

4000

CA

PPA



254(recom-



300

250

200

150

100

50

0

(AU

)

minus006 014 034 054 074 094

Rf

P


16000

14000

12000

10000

8000

6000

4000

2000

0000 025 050 075 100 125 150 175 200


Peak

area

A(A

U)

x (120583gspot)

(a)

040 050

060

075

100125

150

175

300

200

100

0

minus100

minus200

minus300

Resid

ual (

AU)

(120583gspot)

(b)



119865(P) = 014 plusmn 001)

201510

50

minus5

minus10

minus15

minus20

minus25

X1

X2 X3

X4

X5 X6 X7Effec

t

(a)

Effect

1090807060504030201

00000 2000 4000 6000 8000 10000 12000140001600018000

Pi

R2 = 09742

(b)
















119894) as a







119865values of





No



method

Assayby UV-


Assayby TLC-

densitometricmethod

Assayby UV-


Assayby TLC-

densitometricmethod

Assayby UV-









4 Conclusion






Disclosure




Acknowledgment


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


300

250

200

150

100

50

0

(AU

)

minus006 014 034 054 074 094

Rf

P


16000

14000

12000

10000

8000

6000

4000

2000

0000 025 050 075 100 125 150 175 200


Peak

area

A(A

U)

x (120583gspot)

(a)

040 050

060

075

100125

150

175

300

200

100

0

minus100

minus200

minus300

Resid

ual (

AU)

(120583gspot)

(b)



119865(P) = 014 plusmn 001)

201510

50

minus5

minus10

minus15

minus20

minus25

X1

X2 X3

X4

X5 X6 X7Effec

t

(a)

Effect

1090807060504030201

00000 2000 4000 6000 8000 10000 12000140001600018000

Pi

R2 = 09742

(b)
















119894) as a







119865values of





No



method

Assayby UV-


Assayby TLC-

densitometricmethod

Assayby UV-


Assayby TLC-

densitometricmethod

Assayby UV-









4 Conclusion






Disclosure




Acknowledgment


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of










119894) as a







119865values of





No



method

Assayby UV-


Assayby TLC-

densitometricmethod

Assayby UV-


Assayby TLC-

densitometricmethod

Assayby UV-









4 Conclusion






Disclosure




Acknowledgment


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



No



method

Assayby UV-


Assayby TLC-

densitometricmethod

Assayby UV-


Assayby TLC-

densitometricmethod

Assayby UV-









4 Conclusion






Disclosure




Acknowledgment


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



Disclosure




Acknowledgment


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of
































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of