Development And Validation Of UV Spectrophotometric
Original ArticleDevelopment and Validation of UVSpectrophotometric Method for Estimation of TeaTree Oil in Bulk and Cosmeceutical CreamsBasappa Palur Manjula, Vijaya Gopalachar Joshi, Ramachandra Setty Siddam Setty,Muniyappa GeethaDepartment of Pharmaceutics, Government College of Pharmacy, Rajiv Gandhi University of Health Sciences,Bengaluru, Karnataka, INDIA.ABSTRACTBackground: Tea tree oil, also known as Melaleuca alternifolia essential oil is widely used inskin care cosmeceuticals for its antibacterial, antifungal, analgesic and anti-inflammatoryeffect. The complex and variable composition of the oil poses many challenges to theanalytical chemist and a recent survey of analytical methods indicated only a few simpleand validated methods for estimation of the oil. Aim: A quality by design approach hastherefore been adopted to develop a simple and novel UV spectrophotometric methodfor estimation of tea tree oil in bulk and cosmeceutical creams. Materials and Methods:UV spectrophotometric method was developed for estimation of Tea tree oil usingdichloromethane- methanol (54 % v/v) solvent system. The method was then validatedunder optimized conditions for accuracy, precision and ruggedness, limit of detection andlimit of quantification as per ICH: Q2 (R1) guidelines. Results: A characteristic spectralpeak was observed at 267 nm and linearity was established over a concentration rangeof 10-160 mcg/ml with R2 value of 0.9961. Accuracy based on recovery studies,100.5-113.6 %, precision and ruggedness based on % RSD values less than 2 %,LOD, 0.1277mcg/ml and LOQ and 4.195 mcg/ml indicated that the method is sensitiveenough to be used for routine estimation. Conclusion: The method is robust and has beenused to determine the essential oil content of two cosmeceutical creams.Key words: Tea tree oil, UV spectrophotometry, Box Behnken design, Optimization,Routine determination, Cosmeceutical cream.INTRODUCTIONTTO, an essential oil obtained by steamdistillation of aerial parts of Melaleucaalternifolia is widely used in skin carecosmeceuticals for its antibacterial,antifungal, analgesic and anti-inflammatoryeffect.1 TTO is included in EuropeanPharmacopoeia, Merck Index and in IndianPharmacopoeia Addendum 2016. Thechemical composition of tea tree oil isdefined by international standard ISO 4730:2017 and the identical Australian standardAS 2782-2017 which specifies levels for 15of more than 113 components found in thepure oil including physical parameters likerelative density, refractive index and opticalrotation. Terpinen-4-ol is the principalconstituent of the oil (35%- 48%) followedby γ-terpinene (14% -28%), α-terpinene(6%-12%) and 1, 8-cineole also knownas eucalyptol ( 15%), all of which havepotential antimicrobial activity.2 The physicaland chemical properties specified in thedocument published by the Health CanadaPest Management Regulatory Agencystates that the oil is not expected to showa λmax beyond 300nm. Hence the obtainedλmax at 267nm cannot be attributed to anyparticular constituent and the oil used in thestudy conforms to the limits mentioned inISO4730:2017 and AS 2782:2017 standards.The complex and variable compositionof TTO poses many challenges to theanalytical chemist and as indicated from aIndian Journal of Pharmaceutical Education and Research Vol 55 Issue 1 [Suppl] Jan-Mar, 2021 Submission Date: 23-06-2020;Revision Date: 08-10-2020;Accepted Date: 03-01-2021DOI: 10.5530/ijper.55.1s.61Correspondence:Mrs. Basappa PalurManjulaDepartment ofPharmaceutics, GovernmentCollege of Pharmacy,Rajiv Gandhi Universityof Health Sciences,Bengaluru-560027,Karnataka, INDIA.Phone no: 91-0809449046511Email id: karthikamogh@gmail.comwww.ijper.orgS285
Manjula, et al.: UV Spectrophotometric Estimation of Tea Tree Oil in Bulk and Cosmeceutical Creamsliterature survey, only a few simple validated methods forestimation of the oil have been reported. However, UVspectrophotometric method for analysis of TTO has notbeen reported till date. Biju SS et al. 2005 has reporteda validated HPTLC method for determination of TTOcontent of cosmeceutical formulations by estimatingthe principal constituent, terpinen-4-ol.3 Likewise SoniaK and Anupama D. 2011 have determined the terpinen4-ol content for 5% TTO micro emulsion.4 Gulati Net al. 2012 developed a gas chromatographic methodusing flame ionization detector for assay of α-pinenein TTO formulations.5 Venugopal V. 2016 has analysedethosomes loaded with TTO for oil content by HPTLCmethod using terpinen-4-ol as reference standard.6 Anin–house validated reverse phase HPLC method wasdeveloped to determine the eucalyptol content of TTOin bulk and cosmeceutical formulations since higherlevels of eucalyptol, a putative skin irritant is generallyassociated with lower levels of terpinen-4-ol, the mainantimicrobial component of the oil.2,7 Estimation ofeucalyptol content may throw some light on the safetyand dermal irritancy of the pure oil as well as TTObased cosmeceuticals.8In spite of all these developments, there is still an absoluteneed for a simple and robust UV spectrophotometricmethod suitable for routine determination of TTOin bulk and cosmeceutical formulations. The presentresearch is an attempt in this direction. As describedearlier, the method has as an advantage that there isno need for the use of a standard or a specific markercompound since TTO complies with the specifications.MATERIALSTea tree oil was purchased from Messrs. Falcon EssentialOils, Bengaluru. Dichloromethane (SD Fine Chem)extra pure and methanol (Qualigens) HPLC grade wereused for method development. DXN Tea Tree Cream(DXN Cream) and Healthvit Bath and Body Tea TreeCream (HVT Cream) were purchased online fromAmazon.in. Product information of the cosmeceuticalcreams are given in Table 1.InstrumentsShimadzu UV 1800 Spectrophotometer and Shimadzuanalytical balance were used for the study.METHODSScreening StudyPreliminary studies were carried out to identify a suitablesolvent system for preparing standard solution of TTO.S286 The solubility of TTO was tested using various solventssuch as n-hexane, cyclohexane, toluene, DCM and MeOH; binary solvent systems like chloroform-methanol(2:1v/v) and DCM-Me OH (3:2v/v) were also chosenfor the study. DCM-Me OH (3:2v/v) was found to besuitable for carrying out analytical work.Preparation of standard solution of TTO0.025g TTO was weighed accurately into a 25mlvolumetric flask, dissolved using DCM-Me OH (3:2v/v)solvent system and volume made up to the mark usingthe same. The concentration of the resulting solutionwas 1.0mg/ml of TTO.UV spectrum of TTO standard solutionStandard solution of TTO was used for a spectral scanof TTO between 400-250nm using the solvent systemas blank.Preparation of calibration curve of TTOAliquots of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5 and 1.6 ml of standard TTOsolution were transferred into separate 10 ml volumetricflasks and the volume made up with the solvent system.The absorbance of the resulting solutions was read at267 nm against a solvent blank.Analytical method development based on designof experimentsDesign expert software trial version 11 was chosen foranalytical method development. Box-Behnken designwas most appropriate for the study. Factors chosen werepercentage of dichloromethane, time of measurementof absorbance and concentration range correspondingto A, B and C respectively. Factors and their coded levelsare summarized in Table 2.Table 1: Description of cosmeceutical creams.DXN Tea Tree CreamHealthvit Bath and Body TeaTree CreamNet weight:30gNet weight:50gIngredients: Tea treeoil, camphor, menthol,cetomacrogol, emulsifyingwax, waterIngredients: Tea tree oil, lightliquid paraffin, white softparaffin, cetostearyl alcohol,stearic acid, glycerin, phenoxyethanol, perfume, tartrazine,sunset yellow, brilliant blue, lightgreen, amaranth, waterBatch No:71C009Batch No:WCHV0132Best before:02/2020Expiry date:01/2019Registration CertificateNo:COS-778/15Mfg Lic No: GC/974Mfg by: DXNPharmaceutical, Malaysia.Mfg by: West CoastPharmaceutical Works Ltd,Gota, Ahmedabad.Indian Journal of Pharmaceutical Education and Research Vol 55 Issue 1 [Suppl] Jan-Mar, 2021
Manjula, et al.: UV Spectrophotometric Estimation of Tea Tree Oil in Bulk and Cosmeceutical CreamsTable 2: Factors and their coded ded HighMeanStd.Dev.ADCM%Numeric50.060.0-1 50.0 1 60.055.03.78BTIMEminNumeric0.060.0-1 0.0 1 1 10.0 1 200.0105.071.81The design suggested 15 runs and the response chosenwas measurement of absorbance at 267nm, details ofwhich are summarized in Table 3.All the runs were performed in triplicate and averagevalues of the response was analysed by ANOVA at0.05 level. F-test was used to assess each parameter andresponse by subjecting it to multiple regression analysis togenerate polynomial equations. Numerical optimizationas well as graphical optimization was carried out toarrive at the selected solution. The analysis of the oilwas carried out for the selected solution and observedresponses were compared with predicted responses.Table 3: Runs suggested by Box -Behnken design.StdRunFactor1A: DCM%Factor 2B:Time (min)Factor 0The method was then validated as per ICH guidelinesfor accuracy, precision, linearity, LOD, LOQ for themethod under optimized conditions.Accuracy of the method was carried out by addingknown amount of standard solution, 80%, 100%and 120% to a fixed concentration of oil and thendetermining the percent recovery.The precision of the method was established attwo levels- repeatability and intermediate precisionor ruggedness. Repeatability was based on intradaymeasurements at different times of the day andruggedness by using different instruments and inter-daymeasurements for three consecutive days respectively.Linearity represents the concentration range for whicha linear relation exists between concentration and theresponse. This was confirmed from the equation of bestfit and R2 value, a statistical measure of goodness of fit.An R2 value 0.99 is desirable for establishing linearity.LOD and LOQ were determined fromstandard deviation of the response at low concentrationand slope of the calibration curve.LOD 3.3 σ/SlopeLOQ 10 σ/SlopeWhere: σ the standard deviation of theresponse at low concentration.Robustness is a measure of the resistance of accuracyand precision of the method to small variations inthe method. A robust method has been developed bycarrying out optimization by which deliberate changes1095560106106030103115060105Validation of the method underconditions as per ICH 5are done for the three independent variables (factors)viz. percentage of dichloromethane and time ofmeasurement and concentration of TTO.Determination of TTO content in cosmeceuticalcream0.5g of the cosmeceutical cream was weighed accuratelyinto a 50 ml volumetric flask and dissolved in thesolvent system comprising of 54% v/v DCM in MeOH. 1.0ml of the solution was transferred into a 10mlvolumetric flask and diluted to the mark with thesame solvent system. The absorbance of the resultingsolution was read at 267 nm. All determinations weredone in triplicate and average absorbance was used forcalculating the content of TTO.TTO content of the cream was calculated usingthe following formula:TTO Content %w/v Absorbance x dilution factorx100 / (0.5 x 106)Where dilution factor 500Indian Journal of Pharmaceutical Education and Research Vol 55 Issue 1 [Suppl] Jan-Mar, 2021 S287
Manjula, et al.: UV Spectrophotometric Estimation of Tea Tree Oil in Bulk and Cosmeceutical CreamsRESULTS AND DISCUSSIONThe composition of TTO is governed by ISO4730:2017. TTO is known to contain more than 113components of which minimum and maximum levels ofoccurrence in the pure oil are specified for 15 differentcomponents. Few important constituents and theirminimum and maximum concentrations are indicated inthe chromatographic profile of the oil. Terpinen-4-ol isthe principal constituent of the oil (35%- 48%) followedby γ-Terpinene (14% -28%) and α-Terpinene (6%-12%)and 1, 8-Cineole or eucalyptol (traces -15%).9 Hence anattempt has been made to develop a simple and robustUV spectrophotometric method for determination ofTTO, based on sound principles of statistical designof experiments and validate the method as per ICHguidelines.Preliminary studies were carried out to identify a suitablesolvent system for preparing standard solution ofTTO. The oil was found to be miscible with less thantwo volumes of 85 % v/v ethanol at 20 C as per thespecifications provided by the supplier. However thesolubility of TTO was tested using various solvents suchas n-hexane, cyclohexane, toluene, dichloromethaneand methanol; binary solvent systems like chloroformmethanol (2:1v/v) and dichloromethane–methanol(3:2v/v) were also chosen for the study. This was donewith an intention of finding a common solvent for bothTTO and Neem seed oil since simultaneous estimationof both oils by UV spectrophotometry was planned aspart of an extended work. Dichloromethane - methanol(3:2v/v) was found to be suitable for carrying outanalytical work since both the oils were miscible withthis solvent system. A spectral scan from 400-250 nmwas obtained for standard solution of TTO whichexhibited a sharp peak at 267nm (Figure 1).Preliminary screening studies were carried out to identifythe factors which could affect the method and also toascertain the concentration range for establishing thelinearity. Accordingly three factors which could affectthe response viz. absorbance at 267 nm were identified aspercentage composition of DCM in the solvent system,time of measurement of absorbance and concentrationof TTO.The next stage of development began with selection ofa design using Design Expert software, Version 11. BoxBehnken design, a response surface design was chosenfor the study since it is most suitable for determiningthe best factor settings or operating conditions foroptimizing the response and to develop a polynomialmodel with less number of runs or experiments.Box Behnken design was generated using three factorsA, B and C corresponding to percentage of DCM, timeof measurement and concentration of TTO respectivelyand their minimum and maximum levels were fixed asshown in the Table 2. Absorbance at 267 nm was chosenas the response. The design generated 15 runs whichwere performed and the response was measured intriplicate. The response was then analysed and optimizedby numerical and graphical method (Figure 2-4).Optimization of response (absorbance at 267 nm)A linear factorial model was used for the analysis ofresponse, the model F-value (p-value) of 38.92(0.0001)implies the model is significant. There is only a 0.01%chance that an F-value this large could occur due tonoise. P-values less than 0.0500 indicate model terms aresignificant. In this case C (concentration) is a significantmodel term. Values greater than 0.1000 indicate themodel terms are not significant. The lack of fit F-valueof 0.0386 implies the lack of fit is not significant relativeto the pure error (Table 4). Non-significant lack of fitis good - we want the model to fit. The predicted R² of0.8247 is in reasonable agreement with the adjusted R²of 0.8904; i.e. the difference is less than 0.2. AdequatePrecision measures the signal to noise ratio. A ratiogreater than 4 is desirable. A ratio of 15.675 indicatesTable 4: ANOVA for Linear model.SourceSum of SquaresdfMean SquareF-valuep-valueModel3.6931.2338.92 0.0001*A-DCM0.013910.01390.43900.5212 NSB-TIME0.000210.00020.00600.9395 NS116.31 316Lack of Fit0.347390.0386Pure Error0.000020.0000Cor Total4.0314*-significant NS-not significantS288 Indian Journal of Pharmaceutical Education and Research Vol 55 Issue 1 [Suppl] Jan-Mar, 2021
Manjula, et al.: UV Spectrophotometric Estimation of Tea Tree Oil in Bulk and Cosmeceutical CreamsFigure 3: Contour plot of absorbance.Figure 1: UV spectrum of TTO.Figure 4: Predicted vs Actual values of response.Figure 2: Response surface graph of absorbance.Table 5: Optimized conditions for estimation.ResponseDCM %Time(min)Concentrationmcg/ml)Absorbance at 267nm545 min60an adequate signal. Hence the model can be used tonavigate the design space.Absorbance 0.472566 0.008325* DCM 0.000163*Time 0.007132 *Concentration. (Eqn5)The above equation clearly indicates that all the threefactors viz. DCM, time and concentration have apositive impact on absorbance.Analysis of TTO under optimized conditionsThe predicted versus actual value of the response wasfound to be in close agreement with each other whichTable 6: Predicted and actual values of response for60 mcg/ml of TTO.ResponsePredicted valueActual valueAbsorbance at 267 nm0.4090.404indicated the suitability of the method for estimationof TTODesirability approach was used to optimize the response,desirability value (di) for all factors and responses wasfound to be 1. The optimized conditions of the methodhave a desirability of 1, which indicates that the responseis also optimum (Figure 5) (Table 5 and 6).The optimized conditions indicated for analysis ofresponse are 54% DCM and time of measurement 5min. These conditions were maintained for validatingthe method as per ICH guidelines for accuracy,precision (repeatability, intermediate precision andruggedness), concentration range and linearity, LODand LOQ. The method showed linearity in the range of10-160mcg/ml with R2 value of 0.9961 (Figure 6). LODand LOQ values of 0.1277 mcg/ml and 4.195 mcg/mlIndian Journal of Pharmaceutical Education and Research Vol 55 Issue 1 [Suppl] Jan-Mar, 2021 S289
Manjula, et al.: UV Spectrophotometric Estimation of Tea Tree Oil in Bulk and Cosmeceutical CreamsTable 7: Calibration curve of Tea Tree Oil.Concentrationmcg/mlTrial ITrial IITrial IIIAverageabsorbance SD10.00.0860.0890.0850.087 0.00220.00.1340.1310.1310.132 0.00230.00.1530.1530.1560.154 0.00240.00.2560.2630.2610.260 0.00250.00.3370.3370.3360.337 0.00460.00.4010.4020.4040.402 0.00170.00.4530.4610.4650.460 0.00180.00.5430.5480.5460.546 0.00690.00.6280.6290.6320.630 0.003100.00.6540.6590.6560.656 0.002110.00.7020.7010.7040.702 0.003120.00.7820.7850.7900.786 0.002130.00.8510.8560.8550.854 0.004140.00.9270.9270.9310.928 0.003150.00.9480.9620.9620.957 0.008160.01.0381.0421.0671.049 0.016Figure 5: Desirability graph.AbsorbanceTable 8: Linearity parameters for estimation of TeaTree Oil, LOD and LOQ.S290 ValueEquationy 0.0065x 0.0046R2 value0.996LOD mcg/ml0.1277LOQ mcg/ml4.195Table 9: Results of accuracy determination.Figure 6: Calibration curve of Tea Tree Oil.were low indicating that the method is quite sensitivefor estimation of TTO (Table 7 and 8). Accuracy ofthe method was established from % recovered beingmore than 100 for all the levels (Table 9) and precisionof the method confirmed by % RSD values 2% forrepeatability, intermediate precision and ruggedness(Table 10 and 11).The content of TTO in two marketed creams wasdetermined by the developed method viz., DXNCream, 0.34%w/v and HVT Cream, 0.45%w/vrespectively (Table 12). Though, it is not possible toverify the label claim owing to the current labellingpractices adopted for cosmeceuticals, still it is possibleto conclude that the content of TTO in the creams iswell within the acceptable limit of 1-5%; however thecomposition of TTO is not mentioned on the label Itis our observation, based on a market survey of variouscosmetic and personal care products that 1-5% Tea mlAverageamountrecovered SD mcg/mlPercentagerecoveredAbsorbanceat 267 nm804042.6 1.19106.51006060.3 0.50100.51208090.9 0.47113.6*n 3Table 10: Results of method precision.Resp
analytical chemist and a recent survey of analytical methods indicated only a few simple and validated methods for estimation of the oil. Aim: A quality by design approach has therefore been adopted to develop a simple and novel UV spectrophotometric method for estimation of tea tree oil in bulk and cosm
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