An Ecofriendly and Stability-Indicating HPLCMethod for Determination of Permethrin Isomers : Application to Pharmaceutical Analysis

A green, simple, and stability-indicating RP-HPLC method was developed for simultaneous determination of permethrin isomers in pharmaceutical preparations.e separation was based on a C18 analytical column (150 × 4.6mm, i.d., 5 μμm).emobile phase consisted of ethanol: phosphoric acid solution (pH= 3) (67 : 33, v/v). e elution was carried out at 30C temperature with a �ow rate of 1.0mL/min. Quantitation was achieved with UV detection at 215 nm. In forced degradation studies, the drug was subjected to oxidation, hydrolysis, photolysis, and heat.emethodwas validated for speci�city, linearity, precision, accuracy, and robustness. e applied procedure was found to be linear in permethrin concentration range of 0.5–50 μμg/mL with correlation coefficients of 0.9996 for each isomer. Precision was evaluated by replicate analysis in which% relative standard deviation (RSD) values for areas were found below 2.0. e recoveries obtained (99.24%–100.72%) ensured the accuracy of the developed method. e peaks of permethrin isomers well resolved from various degradation products as well as the pharmaceutical excipients. Accordingly, the proposed validated and sustainable procedure was proved to be proper for routine analyzing and stability studies of permethrin in pharmaceutical preparations.


Introduction
Permethrin, (±)-3-phenoxybenzyl 3-(2,2-dichlorovinyl)-2,2dimethylcyclopropanecarboxylate (Figure 1), is a synthetic mixture of cis-and trans-isomers of pyrethrin, chemically altered to bestow light and heat stability [1].Permethrin isomers have different chemical, physical, and toxicological properties.It has been reported that the toxicity of permethrin with a cis-: trans-ratio of 25 : 75 is lower than that of permethrin with a cis-: trans-ratio of 40 : 60. is drug acts on the nervous system of insects and interferes with sodium channels to disrupt the function of neurons, and causes muscles to spasm, culminating in paralysis, and death [2][3][4].Permethrin is used predominantly for treatment of scabies, caused by head lice and mites [5,6].Recently, studies have demonstrated the efficacy of permethrin therapy in the treatment of acne rosacea for its mite eradicating ability [7][8][9][10].Moreover, it has been used as ectoparasiticides in agriculture, in the impregnation of clothing, and also in veterinary medicine, since pyrethroids development in the 1970s [11].
Concerning new therapeutic application of permethrin, validated and stability-indicating methods should be available to determine this drug in topical preparations.However, no method was compiled in British and United state pharmacopoeias for the analysis of this medicine in pharmaceutical preparations.Few methods reported the quantitation of permethrin in pharmaceutical dosage forms using spectrophotometric methods, [12] and LC methods with ultraviolet detection [13][14][15][16], most of which have not been validated according to International Conference on Harmonization (ICH) stability-indicating guidelines.
In all of the aforementioned LC techniques, acetonitrile or methanol has been used as a part of mobile phase or extraction procedure.It is worth mentioning that these solvents are ranked by US Environmental Protection Agency (EPA) as hazardous solvents [17] and because of their inherent toxicity [18], safe detoxi�cation of the waste solvents is essential, which may lead to high to very high disposal costs.
Due to scienti�c and public concern about the environment pollution, environmentally friendly practices have been introduced in different areas of society and research.In green analytical chemistry, sample preparation and LC analysis need special attention because hazardous solvents, are oen used.Possibilities toward green LC include reducing solvent use, switching to more benign solvents and/or eliminating organic solvents [19][20][21][22][23].
Taking ICH guidelines in consideration, the present study describes a simple, validated, and stability-indicating analytical method for determination of permethrin isomers, which meets the green aspects in analytical chemistry.e performances of the method were evaluated, and its potential for the determination of permethrin isomers in pharmaceutical preparations was investigated.

Experimental Section
2.1.Materials, Reagents, and Chemicals.Quali�ed permethrin standard (99.88%) was kindly provided by Gilaranco (Rasht, Iran).All solvents and reagents were of gradient and analytical grade, respectively, and were purchased from Merck (Darmstadt, Germany).HPLC grade water was obtained through a Milli-Q system (Millipore, Milford, MA, USA) and was used to prepare all solutions.e pharmaceutical formulations (cream 5%) and the corresponding placebos (mixture of all the excipients) were prepared in our laboratory.Permethrin cream 5% manufactured by Gilaranco (Rasht, Iran) was purchased from local pharmacy.

2.2.
Equipments. e HPLC method was carried out on a Shimadzu HPLC system (Shimadzu, Kyoto, Japan), set to recycle the mobile phase and was equipped with an SCL-10AVP system controller, LC-10 ADVP pump, DGU-14A degasser, and a SPD-M10AVP PDA detector.e peak areas were integrated automatically by computer using a Shimadzu Class VP soware program.A 20 L volume of sample was introduced into a Rheodyne model 7725i injector.

Chromatographic Conditions.
e elution was carried out on a C 18 column (150 mm × 4.6 mm, 5 m particle size) from Teknokroma (Barcelona, Spain).All analyses were performed at the column temperature of 30 ± 1 ∘ C under isocratic conditions with a mobile phase of ethanol: phosphoric acid solution (pH � 3.0) (67 : 33, v/v), and a �owrate of 1.0 mL/min, using PDA detection at 215 nm.

Preparation of Standard Solutions.
Stock standard solution of permethrin was prepared in ethanol 96% at a concentration of 10 mg/mL.is solution was found to be stable for at least 1 month, when stored at 2-8 ∘ C. Freshly prepared working standards at concentration levels of 0.5, 2, 5, 10, 20, and 50 g/mL were obtained from stock solution by the appropriate dilution in HPLC-grade water.

Preparation of Test Solutions.
A 1 gr portion of cream (equivalent to 50 mg of permethrin) was transferred into a 100 mL volumetric �ask.e volume was adjusted to the mark with ethanol 96% to provide a theoretical concentration of 500 g/mL of permethrin.e solution was serial diluted with HPLC-grade water to make �nal concentration of 9 g/mL.e experiment was performed in triplicate.ese samples were assayed using calibration curves of working standard solutions.e same procedure was applied to placebo to be sure about the selectivity of the method.

Method Development.
A variety of mobile phases were investigated in the development of a stability-indicating LC method for the analysis of permethrin isomers in pharmaceutical preparations.e suitability of mobile phase was decided on the basis of green analytical chemistry principles, selectivity, and sensitivity of the assay, stability studies, and separation of permethrin isomers from the impurities formed during forced degradation studies.Different wavelengths were also investigated.

Forced Degradation
Studies.e stability-indicating capability of the method was determined by subjecting reference solutions (50 g/mL) to accelerated degradation by acidic, basic, heat, oxidative, and photolytic conditions to evaluate the interferences in the quantitation of permethrin isomers.Sample solutions prepared in 1 M hydrochloric acid and 1 M sodium hydroxide were used for the acidic and basic hydrolysis, respectively.Both solutions were kept at ambient temperature for 12 h and then neutralized with basic or acidic solutions, as necessary.For evaluating the heat condition, the reference solution heated at 80 ∘ C for 12 h.For oxidative degradation, sample solutions were prepared in a solution of hydrogen peroxide (3%) and kept at ambient temperature for 4 h, protected from light.Photodegradation was induced by exposing the sample solution to UV-Lamp at a wavelength of 254 nm placed in a wooden cabinet for 1 hour.e solutions were diluted with HPLC-grade water to �nal concentration of 10 g/mL and were injected into chromatograph.
2.8.Method Validation.e developed method was validated as per the requirements of the ICH guidelines.Linearity was evaluated by determining six working standard solutions at a concentration range of 0.5-50 g/mL.Five sets of such solutions were prepared.Each set was analyzed to plot a calibration curve.Slope, intercept, and coefficient of determination ( 2 ) of the calibration curves were calculated to ascertain linearity of the method.e limit of quanti�cation (L��) was de�ned as the lowest concentrations with the RSDs lower than 5% and accuracies within ±5%, considering at least seven times the response compared to that of the blank.In order to check the robustness, the effect of small but deliberate variations in the chromatographic conditions was evaluated.e conditions studied were �ow rate (altered by ±0.2 mL/min), column temperature (altered by ±2 ∘ C), and pH of phosphoric acid solution (altered by ±0.2). ese chromatographic variations were evaluated for resolution between permethrin isomers and % assay of the drug.For method repeatability, assay of working standard solutions (0.5, 10, 20 and 50 g/mL) was repeatedly performed �ve times on the same day (intraday).For reproducibility, freshly prepared solutions at aforementioned concentration levels were analyzed at different days (interday), and results were statistically evaluated in terms of % RSD.For recovery studies, 0.5 gr portions of preassayed permethrin cream 5% were spiked with extra 1, 2, and 4 mL of stock standard solution.ese samples were handled as explain in Section 2.5, and the �nal target levels of 7, �, and 13 g/mL were prepared.e concentrations were calculated using calibration curves.Accuracy was calculated as the deviation of the mean from nominal concentration.To assess accuracy, freshly prepared placebo of the permethrin pharmaceutical formulations was spiked with various amounts of permethrin to obtain the concentration levels of 0.5, 10, 20, and 50 g/mL.Each solution was injected in triplicate.Selectivity of this method was indicated by the absence of any excipient interference at retention times of the peaks of permethrin isomers.e absence of interfering peak was evaluated by injecting a blank sample consisting of diluent and placebo.e double check of the lack of interferences of the resulting by-products with the elution of the peaks of permethrin isomers was done by calculating the F factor, meaning the ratio of the UV molar absorption coefficients of permethrin isomers at the 215 nm and 272 nm, respectively, using (1) where A (215) and A (272) are the permethrin peaks areas obtained at 215 nm and 272 nm, respectively.e resulting F factors were compared with those of the standards [24].
2.9.Estimation of the Uncertainty of the Measurements.An expanded uncertainty budget was constructed for permethrin isomers in pharmaceutical preparations by the RP-HPLC-DAD method according to previously reported procedures [25][26][27][28].Four individual sources were taken into account to assess the expanded uncertainty.
2.9.1.Uncertainty of the Measurement Standard.e uncertainty of the measurement standard is calculated by the quadratic addition of two terms� the uncertainty certi�ed by manufacturer ( stock ) and the uncertainty corresponding to its preparation by dilution or weighting ( preparation ) (2) [25].
e stock uncertainty ( stock ) is calculated from a value given by the manufacturer using where the purity is expressed as P%.When there are independent standard preparations at each concentration level, the  preparation term could be eliminated.In this case, the contribution of this term is included in the  precision term [25,26].
where  0 is the concentration calculated from the calibration curve and   0 is the standard deviation of the concentration, calculated from the calibration curve using where  is the slope of the line,  is the y-intercept of the line,  is the number of replicate unknowns,  is the number of the standards,  0 is the mean of  repeat measurements of  for the sample,  is the mean of the  values for the calibration standards,   are the concentrations of the standards,  is the average concentration of the standards.
2.9.3.Uncertainty Associated to Precision.is value is calculated using where  is standard deviation of the experimental data for precision and  is the number of assays [27].
2.9.4.Uncertainty Associated to Accuracy.is parameter is calculated using is the relative standard deviation of the recovery and  is the number of assays [27].e value of the expanded uncertainty was calculated according to ISO GUM guidelines using where  is expanded uncertainty,  the coverage factor (for con�dence interval 95%,  = 2),  is the concentration of the drug [28].

Results and Discussion
3.1.Optimization of the Chromatographic Conditions.Optimum conditions, which are necessary for the quantitative analysis of the permethrin isomers with maximum sensitivity, were established by a number of preliminary experiments.In this context, different detection wavelengths and compositions of mobile phase, considering the green chemistry principles [18] were explored.Optimum conditions were �xed by varying one parameter at a time while �xing other parameters constant and observing its effect on the peak resolution and also on the response.Initially, ethanol was replaced with acetonitrile in mobile phase previously reported containing acetonitrile: water (70 : 30, v/v) [29], in order to use a benign and green solvent.Although ethanol has some shared or similar characteristics to methanol and acetonitrile, some properties are different.By nature, ethanol has twice the viscosity of methanol, and almost 4 times the viscosity of acetonitrile.In addition, it has different strength of the solvents.us, the mobile phase composition as well as the operating conditions needed to be optimized.e aforementioned change in mobile phase resulted in unresolved permethrin isomers appearing before 12 minutes.In an attempt to improve separation, ethanol content of mobile phase were decreased, and the resolution values of higher than 2 was achieved with the ratio of 65 : 35.But in this situation, the peak shapes and retention times were not satisfactory.Decreasing the pH of water to 3.0 using phosphoric acid solution helped to reduce the retention times and to sharpen the peaks.e chromatograms related to the above procedures are shown in Figure 2. According these preliminary results, the detection wavelength of 215 nm and the mobile phase of ethanol: phosphoric acid solution (pH = 3) (67 : 33, v/v) were �nalized.Under such chromatographic condition, trans-and cis-isomers separated adequately and appeared at 16.2 and 20.3 min, respectively, (Figure 3(a)).Before fully implemented in the quantitative determination of drug substance and pharmaceutical preparation, this method was thoroughly validated according to ICH guidelines.

Forced Degradation Studies.
Stability-indicating method is de�ned as an analytical method that accurately quanti�es the active ingredients without interference from degradation products, process impurities, excipients, or other potential impurities [30].e forced degradation studies in photolytic condition, resulted in nonsigni�cant decrease of the area without any detectable eluting degradation product (Figure 3(f)).However, under the acidic condition, permethrin isomers exhibited a signi�cant decrease, in that only 68.08% of trans-and 64.57% of cis-isomers were recovered and four additional hydrophilic peaks were detected before 6 min (Figure 3(c)).e heat condition also exhibited decrease of the areas to 96.80% and 97.54% for trans-and cisisomers, respectively, and presumably only one peak was detected, which eluted in solvent front (Figure 3(b)).Under the oxidative condition, the trans-and cis-isomer contents decreased to 88.05% and 84.76%, respectively, without any additional peak (Figure 3(e)), indicating that the degradation products did not elute in the HPLC method or may have been degraded to nonchromophoric products.As reported previously [15], permethrin isomers were degraded thoroughly under alkaline condition, and three additional peaks were observed before 6 min (Figure 3(d)).Summary of all degradation studies is mentioned in Table 1.

Method Validation
3.3.1.Selecti�ity and Speci�city.e application of the whole procedure to placebo samples in order to verify the method selectivity demonstrates that no interferences were detected (Figure 4(b)).e speci�city of the method was illustrated by the complete separation of permethrin isomers from each other and from different degradation products as shown in Figure 3. Furthermore, the decreases observed in isomer contents in stability studies, when degradation products appeared, proved the speci�city of the method (Table 1     the LOQ, in the range of 0.5-50 (sum of isomers) g/mL.e peak area of each isomer against the respective concentration was used for plotting the graph considering the ratio of trans-: cis-as 3 : 1, and the linearity evaluated by the least square regression analysis.e linearity curves were de�ned by the following equation for trans-isomer: y = 225965x + 43623, and y = 220640x + 9149.9 for cisisomer.e value of the determination coefficient calculated ( 2 = 0.9996 for each isomer) indicated the linearity of the calibration curve for the method.Moreover, the relative  standard error of slope can be used as a parameter with respect to the precision of the regression, as a general acceptance criterion for the linearity performance of the analytical procedure [31].is parameter should be comparable to the calculated RSD in the evaluation of the precision.In this study, the results obtained for the RSDs of the slopes were 2.14% and 1.39% for trans-and cisisomers, respectively, which are comparable to those of the maximum respective precision values of 2.07% and 1.94% (Table 2).e results from the validation of method are summarized in Table 2. e method proved to be precise, as the intra-and interday precision ranged from 0.70%−1.90%and 1.30%−2.07%,respectively, for all analytes.ese values ful�ll the validation criteria of an analytical method designed for quality control of pharmaceutical preparations for which RSD values <2% are acceptable [31].
e LOQ is the lowest concentration that can be quan-ti�ed with acceptable precision and accuracy.e LOQ of permethrin was determined to be 0.5 g/mL, considering the mean accuracy value of 101.18% and maximum RSD value of 2.07% (Table 1).is value indicates that the proposed method is much more sensitive than what have been reported previously [12][13][14][15][16].

Recovery and Accuracy
. e accuracy was evaluated applying the proposed method to the analysis of the in-house mixture of the cream excipients with known amounts of the drug, to obtain solutions at concentrations of 0.5, 10, 20, and 50 g/mL.e accuracy was assessed from three replicate determinations and calculated as the percentage of the drug recovered from the formulation matrix.e means and RSD% obtained for permethrin isomers are shown in Table 2 with a range of 99.53%-101.28%and 99.58%-101.42%for trans-and cis-isomers, respectively, demonstrating that the method is accurate within the desired range.Also, the results obtained from the analysis of pre-assayed creams spiked with different amounts of permethrin stock solution revealed acceptable recoveries with the mean values of 99.79% and 100.04% for trans-and cis-isomers, respectively, and %RSDs < 0.81.ese values document a high recovery in this method.

Robustness.
Chromatographic parameters including % of assay and permethrin isomers resolution were not signi�cantly affected with the slight changes in the chromatographic conditions like alteration in �ow rates, p� of the aqueous solution of mobile phase, and column temperature.Analysis was carried out in triplicate and only one parameter was changed in the experiments at a time.e results of the experimental variables evaluated were within the acceptable deviation (RSD < 2%), and the resolution of permethrin isomers were more than 2, indicating that the proposed method is robust under the conditions tested.
3.3.5.e Uncertainty of the Method.e expanded uncertainty of the method for quanti�cation of permethrin isomers in pharmaceutical preparations was calculated.e results are shown in Table 3.
3.3.6.Application of the Method.e optimized and validated method was applied to the determination of permethrin isomers in marketed creams.e amounts of permethrin isomers in creams were calculated using calibration curve method.(Figure 4(a)) shows typical chromatogram obtained following the assay of Gilaranco creams.e results of the assay undertaken and the calculated uncertainties are shown in Table 3. e value of 99.77% of label claim indicates that the method is selective for the analysis of permethrin without interference from the excipients used to formulate and produce these creams.

Conclusion
e aim of this study was to develop a green and speci�c method for determination of permethrin isomers in pharmaceutical preparations.e method was designed to be speci�c, selective, sensitive, robust, reproducible, accurate, inexpensive, and easy to perform.e principal advantage of the method is the use of available environmentally friendly solvents and reagents for LC-analyzing and extractions to follow the �rst principle of green chemistry which emphasizes on waste prevention instead of remediation [18].To the best of our knowledge, this is the �rst method which is thoroughly green and reports the metrological parameters in quanti�cation of permethrin in topical preparations.In addition, recycling signi�cantly reduced the mobile phase consumption and made the method economic.Moreover, the method is much more sensitive than the previously reported procedures [12][13][14][15][16].
In conclusion, the newly developed method was successfully performed to simultaneous analysis of permethrin isomers in pharmaceutical preparations, and it can thus be used for routine analysis, quality control, and for studies of the stability of pharmaceutical formulations containing permethrin.

Con�ict of �nterests
e authors declared no con�ict of interests.

T 3 :
nm, 8 nm permethrin-f-blank.dat(b) F 4: A chromatogram obtained from analyzing of the commercially available cream.e solution contains the target permethrin concentration of 9 g/mL.(b) blank.Partial and expanded uncertainties associated to the analytical results (expressed as % relative standard deviation).
2: Precision, accuracy, and recovery data for the proposed method.