UV-Visible Spectrophotometric Method Development And .

American Journal of Pharmacy and Pharmacology 2015; 2(1): 1-8Repeatability is also termed intra-assay precision.Repeatability is usually demonstrated by repeatedmeasurements of a single sample (e.g. use of the analyticalprocedure within a laboratory over a short period of timeusing the same analyst with the same equipment).Repeatability is determined by replicate measurements ofstandard and/or sample solutions. A minimum of threedeterminations at each of three concentrations across theintended range, or a minimum of six determinations at thetest concentration is recommended (BP, 2009, Swartz, 2009and ICH, 2005).(ii) Intermediate PrecisionIntermediate precision expresses within-laboratoryvariations: different days, different analysts or equipment,etc. Intermediate precision is usually demonstrated byrepeated measurements of the sample used in therepeatability experiment within the same laboratory. Usuallythe repeatability experiment is repeated on the same sampleby a different analyst, on a different day, using differentequipment if possible (BP, 2009).(iii) ReproducibilityReproducibility expresses the precision betweenlaboratories (collaborative studies, usually applied tostandardization of methodology). Reproducibility is usuallydemonstrated by means of an inter-laboratory trial. It isusually expressed as the concentration of analyte (e.g.,percentage, parts per billion) in the sample (BP, 2009)1.1.4. AccuracyThe accuracy of an analytical procedure expresses thecloseness of agreement between the value which is acceptedeither as a conventional true value or an accepted referencevalue and the value found. This is sometimes termedtrueness. Accuracy is usually demonstrated by adding knownamounts of analyte(s) to the sample matrix and determiningthe measured result using the analytical procedure. Therecovery of measured against actual amounts is thencalculated. Usually a minimum of three determinations ateach of three concentrations across the intended range isrecommended. The measured recovery is typically 98% to102% of the amount added (BP, 2009 and ICH, 2005).1.1.5. RangeThe range of an analytical method is the interval betweenthe upper and lower concentration (amounts) of analyte(including these concentrations) for which it has beendemonstrated that the analytical procedure has a suitablelevel of precision, accuracy and linearity. For assays therange is usually not less than 80 to 120% of the testconcentration. For determination of content uniformity therange is usually not less than 70 to 130% of the testconcentration. For dissolution testing the range is usually /20% over the expected concentrations. Range is usuallydemonstrated by confirming that the analytical procedureprovides an acceptable degree of linearity, accuracy andprecision when applied to samples containing amounts of3analyte within or at the extremes of the specified range (BP,2009).1.1.6. RobustnessThe robustness of an analytical procedure is a measure ofits capacity to remain unaffected by small but deliberatevariations in method parameters and provides an indicationof its reliability during normal usage (BP, 2009)The evaluation of robustness should be considered duringdevelopment of the analytical procedure. If measurements aresusceptible to variations in analytical conditions, theanalytical conditions should be suitably controlled or aprecautionary statement should be included in the procedure(Chung et al., 2004).Robustness is usually demonstrated by making smalldeliberate changes to one of the operating parameters of themethod, analyzing samples and comparing the results tothose obtained using the prescribed method (BP, 2009).1.2. Reported Analytical Methods forQuantitative Determination ofCiprofloxaciline in PharmaceuticalDosage FormNájla Mohamad et.al proposed HPLC methods forquantitative determination of ciprofloxacin and norfloxacinin pharmaceutical preparations by high performance liquidchromatography. The method uses water: acetonitrile:triethylamine (80:20:0.3 v/v/v) as a mobile phase where theorganic solvelts are not environmentally friendship. Pandey,et al. also proposes quantitative analysis of Ciprofloxacinusing FTIR Spectroscopy method. A Validated Method forthe Quantitation of Ciprofloxacin Hydrochloride UsingDiffuse Reflectance Infrared Fourier Transform Spectroscopymethod by Bhoomendra et al., Spectrophotometric andtitrimetric Determination of Ciprofloxacin Based on Reactionwith Cerium (IV) Sulphate by Kanakapura Basavaiah et al.,has been proposed.1.3. Official Method of Ciprofloxacin TabletThere are two common ways of analyzing assay of drugproducts, spectrophotometeric (UV) determinations and hotometeric determinations are usually faster,simpler, and require less solvent than HPLC (Swartz, 2009).HPLC is the official method for many of pharmacopoeialdrug products. However, it is not simple, and is timeconsuming and expensive since it uses mostly organicsolvents which are rarely available and costly. Among thosemany products Ciprofloxacin tablet is the one whosepharmacopoeial assay method is HPLC (BP, 2009 and USP,2008). The mobile phase is the mixture of phosphoric acidand acetonitrile composition which are expensive,environmental polutant and not easily available. Hence, itwas deemed necessary to provide a simple, quick,inexpensive and readily applicable method for assaying ofCiprofloxacin tablet, which is one of the essential drugsbeing produced in Ethiopia.

4Tadesse Haile Fereja et al.: UV-Visible Spectrophotometric Method Development and Quantification of Ciprofloxaciline inTablets Dosage Form2. Objectives3. Experimental2.1. General Objective3.1. InstrumentsThe study mainly aims to develop and validatespectrophotometeric assay method for Ciprofloxacintablet.2.2. Specific ObjectivesTo identify maximum wave length and quantifyCiprofloxacin in 500 mg Ciprofloxacin tablet.To studies the effect of excipients on assaydetermination of Ciprofloxacin tablet,To evaluate the linearity, accuracy, precision of themethod developed, and to determine the range of theCiprofloxacin concentration in which the method isreliablePrecision balance (Mettler Toledo, AL204-IC, rporation, UV-2401PC, China), sonicator, mortar andpestle, different glass wares were used throughout theexperimental work.3.2. Materials3.2.1. Test SamplesCiprofloxacin 500 mg film coated tablets (Table 3.2.1.)were obtained from RX Africa (Ethiopia), Ethio-Americanpharmaceuticals, Bishoftu, Ethiopia.Table 1. Description of sample investigatedProduct nameBatch numberMfg dateExp dateMode of packagingCiprofloxacin 500 mg film coated tabletCP301208/201208/201510 tabs/blister10 blisters/box3.2.2. Chemicals and ReagentsPharmaceutical grade (reference standards) CiprofloxacinHCl chemical were obtained from RX Africa (Ethiopia),Ethio-American pharmaceuticals, Bishoftu, Ethiopia andDistilled water was used as solvent.3.3. Methods3.3.1. Maximum Wavelength SelectionCiprofloxacin HCl reference standard equivalent to 0.1 gCiprofloxacin was weighed using precision balance (MettlerToledo, AL204-IC, and Switzerland) and dissolved in 100 mlof water and this solution was reserved as stock solution. 10ml of the resulting solution was diluted to 100 ml with water.Then 5 ml of the final solution was diluted to 100 ml with thesame solvent (5 µg/ml). Scanning was carried out over thewave length range of 200 – 600 nm using UV-Visspectrophotometer (Shimadzu Corporation, UV-2401PC,China). The maximum wavelength was taken at which theRS solution showed maximum absorption/peak.3.3.2. Quantification of Ciprofloxacin inCiprofloxacin Tablet (500 mg)Ten Ciprofloxacin tablets were taken and finely powderedafter each was weighed separately. Then, a weight of thefinely powdered tablets equivalent to 0.1g Ciprofloxacin wastaken and dissolved in 100 ml of water by shaking for 5 min.The solution was filtered using Whatman filter paper no. 42,and 10 ml of the filtrate was diluted to 100 ml with water.Then 5 ml of the final solution was diluted to 100 ml with thesame solvent (5 µg/ml). Finally, the absorbance of the samplesolution and the controlled reference standard (CRS) solutionof the same concentration was measured concurrently at theselected maximum wave length. The amount (in percent) ofCiprofloxacin in the Ciprofloxacin tablet was calculatedusing the following formula:As/Ars x 100,Where As and Ars are the absorbances of sample solutionand standard solution, respectively.3.3.3. SpecificityA placebo, consisting of all the excipients and coatingmaterial in the same amounts as in the formulation except theactive ingredient, was prepared. Then a solution wasprepared from the placebo by the same procedure as thatunder 3.3.2. Finally, the absorbance of the placebo solutionand the CRS solution (5µg/ml) was measured concurrently atthe selected maximum wave length. The percent placebointerference was calculated using the following formula:Ab/Ars x 100,Where Ab and Ars are the absorbances of placebo solutionand standard solution, respectively. It should not exceed 2%.3.3.4. LinearityA stock solution of 100µg/ml was prepared by dissolving aweight of Ciprofloxacin Hydrochloride RS equivalent to 0.1g of Ciprofloxacin in 100 ml of water and then diluting 10 mlof the resulting solution to 100 ml with water. Fiveconcentration levels were prepared by diluting 3, 4, 5, 6, and7 ml of the stock solution to 100 ml with water to get 3, 4, 5,6, and 7µg/ml, respectively. The respective absorbancereadings of the five concentration levels were taken at theselected maximum wavelength. Then, the concentrationversus the absorbance was plotted to obtain the Beer-Lambertcalibration curve.3.3.5. Repeatability5 µg/ml, a concentration mid point to the linearity range of

American Journal of Pharmacy and Pharmacology 2015; 2(1): 1-8a sample solution, was prepared six times over a shortinterval of time within one day by one analyst from finelypowdered tablet, by the same procedure as that under section3.3.2. And the absorbance of the solution was taken at eachtime. Finally, six assay results (n 6), indicating the amountof Ciprofloxacin in Ciprofloxacin tablet, were obtained andthe standard deviation (SD) was calculated using MicrosoftExcel 2003 and the corresponding relative standard deviation(RSD) was calculated by the following formula:RSD SD/X x100,Where X is the mean of the six assay results. It should notexceed 2 %.3.3.6. Intermediate PrecisionThe same procedure was followed as that under 3.3.2except that three assay results were obtained on one day byone analyst. And the other three assay results were obtainedon the second day (after 24 hrs of the fist test) by anotheranalyst from newly prepared5 µg/ml sample solution. Finally,the standard deviation (SD) of the six assay results (n 6)was calculated using Microsoft Excel 2003 and thecorresponding relative standard deviation (RSD) wascalculated by the following formula:RSD SD/X x100,Where X is the mean of the six assay results. It should notexceed 2 %.3.3.7. AccuracyA sample was prepared by blending the activepharmaceutical ingredient (Ciprofloxacin HCl) and theexcipients and the coating material in the same amount andthe same type as present in the formulation (Ciprofloxacin500 mg film coated tablet). A stock solution of 100µg/ml wasprepared by dissolving the sample equivalent to 0.1 g ofCiprofloxacin in 100 ml of water and then diluting 10 ml offiltered solution to 100 ml with water. Three concentrationlevels ranging from below (3 µg/ml) to above (7 µg/ml) and5 µg/ml as target concentration were prepared by diluting 3,7, and 5 ml of the stock solution to 100 ml with water,respectively. The respective absorbance readings of the threeconcentration levels and that of the correspondingconcentration levels of CRS were taken at the selectedmaximum wavelength. It was repeated three times to getthree replicates for each concentration by proceeding asdescribed above by preparing stock solution from the sample.Finally, nine test results were obtained and the percentrecovery was determined. It should be between 98 to 102%.3.3.8. Robustness(i) Solution StabilityThe same procedure was followed to prepare the solutionsas described under 3.2.2. The standard and the samplesolutions stability was evaluated initially and after 24 hrs and48 hrs preserved under ambient temperature and light5condition by determining the concentration against freshstandard solution. The test result should be between 98.0 and102.0% of the initial value.(ii) Wavelength ChangeThe effect of a small change in wavelength (λmax 1nm) onthe test result was studied. The assay results obtained at λmax 1nm and at λmax 1nm were compared with that obtained atλmax.3.3.9. Limit of Detection and Limit ofQuantificationThe limit of detection and limit of quantification weredetermined from the calibration curve data obtained by(Instant ) version 3.33 programs using the followingformulae:LOD 3.3 σ/SLOQ 10 σ/SWhere, σ is the standard deviation of the response and S isthe slope of the calibration curve.

6Tadesse Haile Fereja et al.: UV-Visible Spectrophotometric Method Development and Quantification of Ciprofloxaciline inTablets Dosage Form4.3.2. LinearityThe five different concentrations of Ciprofloxacin CRSand the corresponding absorbance readings are indicated inTable 3.Table 3. Linearity data indicating concentrations (n 5) and thecorresponding absorbance valuesFigure 4.1. Absorbance spectrum of Ciprofloxacin scanning from 200.00 to600.00nmConcentration(µg/ml)Absorbance (au)03456700.3090.4070.5230.6360.7284. Results and Discussion4.1. Maximum Wavelength IdentificationDistilled water was selected as a solvent as it is easilyavailable and cheaper than other solvents as well asCiprofloxacin HCl is soluble in it. As shown in Figure 4.1,the maximum wavelength (λ max) was obtained at 275 nmsince Ciprofloxacin Hydrochloride CRS solution showedmaximum absorption peak at 275 nm upon scanning over thewave length range of 200 – 600 nm using distilled water as ablank.The absorbance was plotted versus the concentration(µg/ml) to obtain the Beer-Lambert calibration curve (Figure4.2). The equation for the calibration curve was Y 0.105X 0.0035, where Y is the absorbance and X is the concentrationin µg/ml; r2 0.9994. The value of correlation coefficient (r)was 0.9997. The result reveals that there is a strong linearrelationship between the concentration of the test sample andthe absorbance values over the concentration range 3 to 7µg/ml Ciprofloxacin.0.80.74.2. Assay of CiprofloxacinAbsorbance0.6Ciprofloxacin 500 mg film coated tablet was then analyzedfor the quantification of Ciprofloxacin at the selectedmaximum wave length and found to be 97.93% of the labeledclaim. Ciprofloxacin tablet should contain not less than 90%and not more than 110% of the labeled claim ofCiprofloxacin (USP, 2008). The result shows that the assayvalue lies within the limit specified in the United Statespharmacopoeia (USP). Thus, Ciprofloxacin tablet sample metthe requirement for assay as specified in the on (ug/ml)4.3. Validation of the Proposed MethodsThe proposed method was validated for specificity,linearity, accuracy, repeatability, intermediate precision androbustness in accordance with ICH guidelines.4.3.1. SpecificityThe absorbance values of the placebo solution and theCRS solution (5 µg/ml) at 275 nm and the percent placebointerference are depicted below (Table 2).Table 2. Absorbance values placebo and test solutionTest solutionAbsorbancePlacebo0.009CRS0.567% Interference1.59Limit in %Not more than 2Figure 4.2. Beer-Lambert calibration curve for Ciprofloxacin HydrochlorideCRS in water and maximum wave length of 275 nm over the range of 3 to 7µg/ml Ciprofloxacin4.3.3. AccuracyThe percent mean recovery obtained from three differentconcentration levels, 60, 100, and 140% of the testconcentration, three replicates each, was 101.57% (Table 4).It should be between 98 - 102% (ICH, 2005). Therefore, theproposed method is accurate as the percent mean recoverylies within the recommended limit.Table 4. Assay results (n 9) and % Mean recovery%Nominal concentration*60100As can be seen from Table 2, the interference of theexcipients is found to be 1.59% which is less than 2%, i.e.absorption of the placebo solution is not significant which inturn indicates the insignificance of excipients interferenceand hence the specificity of the proposed method.140%Mean recoveryReplicate123123123101.57Assay 1100.84* target concentration at which any sample is to be analyzed

American Journal of Pharmacy and Pharmacology 2015; 2(1): 1-84.3.4. RepeatabilityThe repeatability data indicating assay results (n 6) ofCiprofloxacin tablet, determined at 100% test concentration,and the percent relative standard deviation (RSD) are shownbelow in Table 5.Table 5. Repeatability at 100% test concentrationReplicate123456% RSDAssay (%)101.65101.28100.37102.14101.96102.150.67The relative standard deviation value (%RSD) was lessthan 2.0%, the maximum limit recommended by ICHguidelines, indicating good repeatability of the test resultsobtained by the proposed method. In other wards, there is nosignificant variation among the test results generated by themethod under development within 12 hrs by one analyst.4.3.5. Intermediate PrecisionThe intermediate precision data indicating assay results (n 6) of Ciprofloxacin tablet, determined at 100% testconcentration (5 µg/ml), and the percent relative standarddeviation (RSD) are shown below in Table 6.Table 6. Intermediate precision (by two analysts on two different days) at100% test concentrationReplicate123%RSDAssay (%)Analyst 1 (on day 1)99.6598.9498.80.90Analyst 2 (on day 2)97.6998.99100.34The relative standard deviation value (%RSD) was 0.90,which is less than 2.0% (the maximum limit recommendedby ICH guidelines). This means that the variation betweenthe results obtained by two different analysts on two differentdays is insignificant and therefore the changes in analyst andday (24 hrs) did not affect the consistency of assay resultsobtained, indicating good precision of the proposed method.4.3.6. RangeThe proposed UV-Vis spectrophotometeric assay methodfor Ciprofloxacin tablet is accurate, precise and linear withinthe concentration range of 3 µg/ml (60%) to7 µg/ml (140%)of Ciprofloxacin. Therefore the range of the method isbetween 60% and 140% of the target concentration (5µg/ml).4.3.7. Robustness(i) Solution StabilityTable 7a shows sample and standard solutions stabilityperformed on first, second and third day after preparation.7Table 7a. Stability of sample and standard solutionsDay123% Initial sample100.0096.8491.70% Initial Standard100.0098.6295.81As can be seen from table 7a, the amount of Ciprofloxacindeclines as the solutions stay for long time for both thesample and the standard. For example, 96.84 and 91.70% ofthe initial amount remains, after reserving the samplesolution for 24 hrs and 48 hrs, respectively. The standardsolution also declines to 95.81% of the initial amount after 48hrs. This suggests that the solutions are not stable after 24 hrsand hence any analysis carried out to determine assay ofCiprofloxacin using this proposed method should be donewithin 12 hrs.(ii) Wavelength ChangeTable 7 b shows the assay results obtained at λ max 1 nmto investigate the effect of small change in wave length.Table 7 b. Assay results obtained at λ max - 1 nm, λ max, and λ max 1 nmWave length (nm)Assay (%)274100.1227599.3127699.87The assay results obtained at λ max – 1 nm (274 nm) and atλ max 1 nm (276 nm) are almost similar to that obtained at λmax (275 nm), indicating the consistency of test results in spiteof small change in wave length.4.3.8. Limit of Detection and Limit ofQuantificationTable 8 shows the calibration curve data used for thedetermination of LOD and LOQ.Table 8. Data obtained from calibration curveParameterSlope (S) of the calibration curveStandard deviation (σ) of the responseValue79.810.0029LOD 3.3 σ/S 3.3X0.0029/79.81 0.00012LOQ 10 σ/S 10X0.0029/79.81 0.000365. ConclusionIn this study, a simple UV-Vis spectrophotometeric assaymethod for Ciprofloxacin film coated tablet was developedand validated. Distilled water, which is easily available andcheaper than other solvents, was selected as a solvent sinceCiprofloxacin HCl is soluble in it. The maximum wavelength(λ max) for Ciprofloxacin HCl was found to be 275 nm. Themethod was then validated for specificity, linearity, accuracy,repeatability, intermediate precision and robustness inaccordance with ICH guidelines.The interference of the excipients in the formulation ofCiprofloxacin tablet investigated was not significant therebyshowing the specificity of the method. The method alsoindicated good linearity relationship between theconcentrations of the test sample and the absorbance values.

8Tadesse Haile Fereja et al.: UV-Visible Spectrophotometric Method Development and Quantification of Ciprofloxaciline inTablets Dosage FormIt is accurate as the percent mean recovery obtained(101.57%) lies within the recommended limit (98 - 102%).The RSD of assay results (n 6) of Ciprofloxacin tablet,determined at 100% test concentratio

analyte within or at the extremes of the specified range (BP, 2009). 1.1.6. Robustness The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its re