MATERIALS AND METHODS

Chapter 5

MATERIALS AND METHODS

5.1Material
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5.1.1 Reagents The Model drug artemether and lumefantrine was procured as a gift sample from IPCA Pharmaceuticals ltd Mumbai India. Methanol and acetonitrile were purchased from CDH Pvt. Ltd., New Delhi, India. Potassium bromide (spectroscopy grade) was procured from Sigma Aldrich, Chennai, India. All the other chemicals and solvents were of analytical grade and were used without any further purification. Deionized double distilled water was used throughout the study. 5.1.2 Instrumentation Capillary melting point apparatus, 1700 double beam UV Visible spectrophotometer, UV PC software (UV probe) version 2.31, Shimadzu, Japan and 380 FTIR spectrometer, Thermo Nicolet Corp., USA. 5.2 Methods 5.2.1 Physical appearance Physical appearance of the drug was noted by visual observation. 5.2.2 Melting Point A capillary melting point apparatus was used to determine melting point of the drug. Small amount of ART and LUME was filled in the capillary and melting point was determined. 5.2.3 Solubility Solubility was determined using pharmacopoeial method. One part of ART and LUME was added to different part of the solvents such as methanol, acetonitrile and water and shaken reciprocally at 37 C for 5 min. 5.2.4 Fourier transform Infra Red Spectroscopy The FTIR study was performed for identification of the procured (ART and LUME) & to confirm that it is in pure form. FTIR transmission spectrum of a pure ART and LUME was obtained using a Fourier Transform infrared spectrophotometer. A total of 2 % (w/w) of sample (with respect to the potassium bromide-KBr) was mixed with dry KBr. The mixture was ground into a fine powder using an agate mortar before compressing into KBr disc under a hydraulic press at 10,000 psi. Each KBr disc was scanned 16 times at 4 mm s -1 at a
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resolution of 2 cm-1 over a wave number region of 5004000 cm-1. The characteristic peaks were recorded.

5.3 HPTLC METHOD DEVELOPMENT 5.3.1 Equipments A Camag HPTLC system equipped with a sample applicator Linomat 5 TLC Scanner III, Reprostar and Wincats 4.02, integration software (Switzerland), twin trough glass development chamber and Ultra sonicator. 5.3.2 Chemicals Analytical grade methanol, hexane, and ethyl acetate were obtained from CDH (New Delhi, India).. Pre-coated silica gel 60 F254 TLC aluminium plates (20 x 20 cm, 0.2 mm thick) were purchased from E. Merck. Germany. 5.3.3 Drug The Model drug artemether was procured as a gift sample from IPCA Pharmaceuticals ltd Mumbai, India. 5.3.4 Instrumentation and Chromatographic Conditions

HPTLC System : CAMAG, Switzerland. Stationary phase : Pre-coated silica gel 60F254 TLC plate, 0.2mm thick Mobile phase : Hexane: Ethyl Acetate: (8: 2 ) v/v

Saturation time : 20 min Wavelength Lamp : 565 nm : Deuterium, Tungsten

Slit dimension : 6 x 0.30 mm Application position : 15 mm Position of Solvent front :75 mm

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MATERIALS AND METHODS

5.3.5 Sample application The samples were applied in the form of bands of width 8 mm with a Camag Linomat V sample applicator (Switzerland) on pre-coated silica gel aluminium plate 60F-254 with 200 m thickness (E.Merck, Germany). Application rate of 80 nL s1 was employed and space between the bands were 10 mm. Linear ascending development was carried out in a twin trough glass chamber saturated with mobile phase. Densitometric scanning was performed on Camag TLC scanner III in the absorbance/reflectance mode 5.3.6 Preparation of stock solution of Artemether A stock solution of 0.1g/ml was prepared by dissolving accurately weighed 10 mg of standard Artemether in 10 mL of methanol. This solution was termed as stock solution. 5.3.7 Preparation of market formulation extract
Dissolve the required quantity of crushed powder of 20 tablets to make a concentration of approx1g/mL and transfer into 10ml volumetric flask containing 10ml methanol. After this the suspension formed by excipients in market formulation was centrifuged and the supernatant was collected. Test solution of market formulation was prepared by diluting required quantity of supernatant stock solution of with methanol.

5.3.8 Method Validation

Once the chromatographic method had been developed and optimized, it must be validated. The validation of an analytical method verifies that the characteristics of the method satisfy the requirements of the application domain. The proposed method was validated in the light of ICH Guidelines for linearity, precision, sensitivity, and recovery. Consequently, the following were performed.

5.3.8.1 Experimental 5.3.8.2 Linearity:The linearity range of ART was obtained by plotting the peak area of artemether against the concentration over a range (100-700 ng band1). Then calibration curve was plotted at the range of (100-700 ng band1) and the correlation coefficient was determined. 5.3.8.3 Calibration curve of Artemether:A stock solution of ART (0.1 gmL1) was prepared in methanol. Different volumes of stock solution 1, 2, 3,4, 5, 6, 7L, were spotted on the TLC plate to obtain concentrations of 100, 200, 300, 400, 500, 600, 700 ng band1 of ART, respectively. The data of peak areas
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plotted against the corresponding concentrations were treated by least-square regression analysis. 5.3.8.4 Precision Repeatability of the sample application and measurement of peak area were carried out using six replicates of the same band (500 ng band1 of artemether) and was expressed in terms of percent relative standard deviation (% R.S.D.) and standard error (S.E.). The intraday and inter-day variation for the determination of ART was carried at three different concentration levels of 200, 500 and 700 ng band1. 5.3.8.5 Specificity The specificity of the method was ascertained by analyzing the standard drug and samples. The band for artemether in the sample was confirmed by comparing the Rf values and spectra of the band with that of the standard. The peak purity of the artemether was assessed by comparing the spectra at three different levels, viz. peak start (S), peak apex (M) and peak end (E) positions of the band. 5.3.8.6 Robustness of the method By introducing small changes in the mobile phase composition, mobile phase volume, duration of mobile phase saturation and different analyst; the effects on the results were examined. Robustness study of the method was done in triplicate at a concentration level of 500 ng band1 and the % R.S.D and S.E. of peak areas were calculated. Following are the changes introduced in the method: (1) Change in mobile phase composition Hexane: Ethyl acetate a) 9 : 1 b) 7 : 2.5 c) 7 : 3

(2) Change in mobile phase volume a) 8 ml b) 10 ml c) 12 ml

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(3) Change in time of chamber saturation a) 10 minutes b) 20 minutes c) 30 minutes

(4) Different analyst a) Analyst 1 b) Analyst 2 c) Analyst 3

5.3.8.7 Limit of detection and limit of quantification LOD was experimentally verified by diluting the known concentrations of ART until the peak is detectable. LOQ was calculated from the densitometric estimation of minimum content of standard ART.

5.3.8.8 Recovery studies The pre-analyzed samples were spiked with extra 80, 100 and 120 % of the standard ART and the mixtures were reanalyzed by the proposed method. The experiment was conducted six times. This was done to check the recovery of the ART at different levels in the formulation.

5.3.8.9 Ruggedness of the method A solution of concentration 700 ng band1 was prepared and analyzed on day 0 and after 3, 6, 24, 48 and 72 h. Data were treated for % R.S.D. to assess the ruggedness of the developed method.

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5.4 Analytical Method Development RP-HPLC DAD Method Method development involves evaluation and optimization of the various stages of sample preparation, chromatographic separation, detection and quantification. Optimization of various parameters was performed in order to develop a selective and sensitive method for analysis of ART and LUME on reverse phase high performance liquid chromatography (RPHPLC) using diode array detector (DAD). 5.4.1Material 5.4.1.1 Reagents Acetonitrile, water and Potassium dihydrogen and 20 % ortho-phosphoric acid (H3PO4) were purchased from Rankem, New Delhi, India. All these chemicals and solvents were of HPLC grade and were used without any further purification. All the solutions were prepared in HPLC grade water. Market formulation of Lumitroy (Batch NumberA100016, Troikaa Pharmaceuticals Ltd., Gujarat, India) was procured from the local drug store. All mobile phase solutions were degassed ultrasonically by Steryl 40050 bath sonicator before prior to use. 5.4.1.2 Instrumentation The analysis was performed on HPLC system of WATERS (Milford, USA) composed of 515 solvent delivery system equipped with a auto sampler, DAD detector set at wavelength range 190-800 nm. Separation was performed on a NUCLEOSIC-CN Ciano (1504.6mm i.d.; 5_m particle size).Chromatographic data were recorded and processed using EMPOWER-2 software 5.4.2 Methods 5.4.2.1 Sample Preparation Stock solution of ART (10g/ml) and LUME (2g/ml) was prepared in ACN. Aliquots were diluted stepwise with the ACN to obtain 1000g/ml of artemether and 200g/ml of lumefantrine. This solution was used for the optimization of the proposed method. Marketed formulation was prepared as similar to pure ART and LUME, then diluted accordingly after proper separation of excipients. 5.4.2.2 Optimization of chromatographic conditions
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The effects of different chromatographic conditions on the instrument response create a situation where one has to compromise between different experimental variables in order to achieve the best chromatographic separation. Chromatographic separations are significantly affected by the mobile phase conditions, such as the type and composition of the organic modifiers [42] and therefore before selecting the conditions for the optimization, a number of preliminary trials were conducted with different combinations of different organic solvents and buffers at various pH, compositions, and flow rate to check the retention time, shape, resolution, and other chromatographic parameters. From those experiments the mobile phase combination of ACN and PHOSPAHATE BUFFER in the acidic pH range was found to be most suitable. In order to achieve an optimum separation, following conditions were studied: (i) Mobile phase pH varied at 4.5, 3 and 2.6 keeping the composition of ACN: Phospahte Buffer 50:50 and flow rate of 1.0 ml/min fixed. (ii) Mobile phase composition varied at 60:40, 70:30, 80:20 and 90:10 with pH and flow rate kept constant at1.0 ml/min. (iii) Flow rate was varied (0.8, 1.0, and 1.5 ml/min) with mobile phase composition . Moreover, the effects of different level of all these three factors were systematically addressed on system suitability parameters such as resolution, theoretical plates, retention time, capacity factor, separation factor, asymmetry, and HETP etc. After applying the different chromatographic parameters during the method development, the recommended mobile phase consisted of ACN: Phosphate Buffer of 60:40 v/v adjusted to pH 2.6. The best resolution and sensitivity of the method was obtained at 215 nm or 234nm and mobile phase flow rate of 1.0 ml/min. typical chromatogram at the optimized condition give sharp and symmetric peak with retention time of 3.3 min or 4.9 min. After chromatographic method development and optimization it was validated. The validation of an analytical method verifies that the characteristics of the method satisfy the requirements of the application domain. The proposed method was validated according to ICH guidelines for linearity, precision, accuracy sensitivity, and recovery.

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5.4.2.3 Linearity For linearity studies, Stock solution of 10 mg of artemether reference standard or 2mg of Lumefantrine reference standard were accurately weighed and transferred to 10ml volumetric flask and made upto mark with ACN, to obtain a solution of 1000g/ml of artemether and 200g/ml of lumefantrine, and resultant solution was ultra sonicated of ART and LUME. 5.4.2.4 Precision and Accuracy Intra-day precision and inter-day precision for the developed method was measured in terms of % R.S.D. The experiments were repeated three times a day for intra-day precision and on three different days for inter-day precision. The concentration values for both intra-day precision and inter-day precision were calculated six times separately and percent relative standard deviation were calculated. Finally, the mean of % R.S.D. (% R.S.D. = [S/X] 100, where S is standard deviation and X is mean of the sample analysed) were taken for conclusion. For studying the accuracy of the proposed method, and for checking the interference from excipients used in the dosage form, recovery experiments were carried out by the standard addition method. This study was performed by addition of known amounts of ART and LUME to a known concentration of the commercial tablets. The amounts of standard recovered were calculated in terms of mean recovery with the upper and lower limits of percent relative standard deviation. So for the determination of accuracy of the analytical procedure three same dilution of market formulation were spiked with different concentrations of standard solution i.e. 80 %, 100 % and 120 %. Area of these solutions was observed and this observed area was plotted into calibration curve. Concentration and accuracies were calculated. 5.4.2.5 Detection and quantitation limits (LOD & LOQ) The limits of detection and quantitation were calculated by the method based on standard deviation () and slope (S) of the calibration plot using the formula LOD = 3.3/S and LOQ = 10/S. for 5min. Calibration graph was prepared by plotting the mean peak area versus concentration

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5.4.2.6 Percentage Purity Twenty tablets were weighed and powdered equivalent to 10 mg of ART and 2mg of LUME was accurately weighed and diluted up to 10 ml with ACN.

5.4.2.7 System Suitability Parameter System suitability tests are an integral part HPLC method. They are used to verify that the resolution and reproducibility of the chromatographic system are adequate for the analysis to be done.(94)

5.4.2.8 Plate number or number of theoretical plates (n) This is a measurement of the sharpness of the peaks and therefore the efficiency of the column. n = 16 (t / wb )2 (Where t is retention time of peak, Wb is peak width at base)

5.4.2.9 Capacity factor (K) This value gives an indication of how long each component is retained on the column (i.e. how many times longer the component is retarded by the stationary phase than it spends in the mobile phase). K = tR - tM / tM (Where tM is unrestrained peak retention time, tR is retention time of peak of interest.)

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5.4.2.10 Separation Factor () This describes the relative position of two adjacent peaks. Ideally, it is calculated using the capacity factor because the peaks' separation depends on the components' interaction with the stationary phase. = KB / KA (Where KB is capacity factor of peak B, KA is capacity factor peak A).

5.4.2.11 Resolution Factor (RS) This is not only a measure of the separation between two peaks, but also the efficiency of the column. It is expressed as the ratio of the distance between the two peak maxima to the mean value of the peak width at base (Wb). RS = 2(tR2 - tR1) / wb1 + wb2 (Where tR1 and tR2 are retention time of two adjacent peak, wb1 and wb2 are peak width of respective peak).

5.4.2.12 Peak Asymmetric Factor (Af ) and Peak Tailing Factor (T) The deviation from symmetry is measured by the Asymmetry Factor, Af or Tailing Factor T. Asymmetric factor, Af is described by the following equation: Af = A10% h / B10%h (Where A and B are sections in the horizontal line parallel to the baseline, drawn at 10% of the peak height) Tailing factor, T is described by following equation: T = (A5%h + B5%h) / 2A
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5.5 Bio-Analytical Method Development in Human Plasma Optimized analytical method was developed and validated which was selective and sensitive method of analysis for ART and LUME determination using RP-HPLC with DAD detector. The same method was applied for determination of ART and LUME in human plasma. The ART and LUME was spiked in externally obtained human plasma and method was fully validated. 5.5.1 Material 5.5.1.1 Reagents Acetonitrile, DMSO, Chloroform, di-hydrogen ortho phosphate (KH2PO4) and 85 % orthophosphoric acid (H3PO4) were purchased from Rankem, New Delhi, India. All these chemicals and solvents were of HPLC grade and were used without any further purification. All the solutions were prepared in HPLC grade water. Itraconazole (ITZ) was used as internal standard and was procured from Intas Bio Pharmaceuticals Industries Ltd., Mumbai, India. Frozen human plasma was procured from local blood bank & was stored at -20 C until analysis. 5.5.1.2 Instrumentations and Chromatographic Conditions The analysis was performed on HPLC system of WATERS (Milford, USA) composed of 515 solvent delivery system equipped with a auto sampler, DAD detector set at wavelength range 190-800 nm. Separation was performed on a NUCLEOSIC-CN Ciano (1504.6mm i.d.; 5_m particle size).Chromatographic data were recorded and processed using EMPOWER-2 software. Analysis was isocratic at 1.0 ml/min flow rate with ACN: Buffer (adjusted to pH 2.5 with H3PO4 0.2 %) (37:63, v/v) as mobile phase. The mobile phase was prepared freshly every day and degassed by sonication before use. The elution was detected at 238 nm. The substance was quantified using its peak area ratio of ART and LUME to IS (Internal Standard). Prior to injecting solutions, the column was equilibrated for at least 30 min with the mobile phase flowing through the system. Each solution was injected in triplicate, and the relative standard deviation (R.S.D.) was measured.

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5.5.1.3 Standard and test solutions of ART and LUME Stock solution of ART (100g/ml) and LUME (1g/ml) was freshly prepared in ACN(add 1ml of DMSO to increase solubility of lumefantrine). Test solutions of ART and LUME were prepared by diluting required quantity of stock solution with ACN to follow the linearity range (100- 1600 g/ml for ART) and (1- 16 g/ml for LUME). Stock solution of 1 g/ml of pure ITZ as IS was freshly prepared in ACN. Marketed formulation was prepared as similar to pure ART and LUME, then diluted accordingly after proper separation of excipients. 5.5.1.4 Plasma extraction procedure 100 l of working stock solution of (ART and LUME) was spiked to plasma sample (100 l). This spiked plasma sample was vortex mixed for 1 min. Acetonitrile (700 ml) was added to it, shaken well and separation was done by centrifugation (8000 rpm 10 min). 1 ml of the supernatant was taken and the residues were reconstituted in a ACN (100 l). A 20 l aliquot of final preparation was injected into the HPLC column. The same procedure was followed for the plasma without spiking the stock solution and IS equal volume (20 L), of the samples that contain ART and LUME and ITZ in ACN were injected into the HPLC and the chromatograms were recorded. The responses (peak area) for the major peaks were measured and the quantity of ART , LUME and ITZ were calculated. 5.5.2 Validation Parameters 5.5.2.1 Calibration curve For linearity studies, Stock solution of 1000mg of artemether reference standard or 10mg of Lumefantrine reference standard were accurately weighed and transferred to 10ml volumetric flask and made upto mark with ACN, to obtain a solution of 10000g/ml of artemether and 1000g/ml of lumefantrine, and resultant solution was ultra sonicated of ART and LUME. 5.5.2.2 Precision and Accuracy Both repeatability (within a day precision) and reproducibility (between days precision) were determined as follows. Solutions containing lowest, intermediate, and highest
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5min. Calibration graph was prepared by plotting the mean peak area versus concentration

MATERIALS AND METHODS

quantification concentrations (LQC MQC and HQC) of the calibration curve, i.e. 100 g/ml, 400 g/ml, 1600 g/ml for ART and 1g/ml, 4g/ml, and 16g/ml for LUME were prepared. Six injections at each of the specified concentration levels were injected within the same day for repeatability, and over a period of 3 days (6 injections / day) for reproducibility. Mean and relative standard deviation were calculated and used to judge accuracy and precision of the method. Both intra-day and inter-day samples were calibrated with standard curves concurrently prepared on the day of analysis. Intra-day precision and inter-day precision for the developed methods were measured in terms of % R.S.D. which was taken for conclusion. Accuracy was calculated as the percent of ratio of ART and LUME amount found to that of the actual of LQC, MQC and HQC. 5.5.2.3 Extraction efficiency Spiked biological samples were prepared in triplicate at three concentrations LQC, MQC, HQC of ART and LUME and 1 g/mL of IS, and assayed as described above. The extraction efficiency of SC was determined by comparing the peak areas measured after analysis of spiked plasma samples with those found after direct injection of non-biological (unextracted) samples or blank sample into the chromatographic system at the same concentration levels. 5.5.2.4 Limit of detection (LOD) and limit of quantitation (LOQ) Limit of detection (LOD) and limit of quantitation (LOQ) were calculated by taking the AUC of standard and calculation is according to the 3.3s/m and 10s/m criterions, respectively, where s is the standard deviation of the AUC (n = 10) of the sample and m is the slope of the corresponding calibration curve. 5.5.2.5 Stability Blank plasma was spiked with the known amount of ART and LUME to achieve the concentration of LQC, MQC and HQC of SC (n = 3) and stored at 4 C. The stability of these samples was checked for up to 15 days by comparing the results with fresh stock prepared on the day of analysis. Further, the freezethaw (20 C/room temperature) stability of the ART and LUME spiked plasma samples were determined for three cycles. Samples were considered to be stable, if the assay values were within the acceptable limits of accuracy and precision. No internal standard was added prior to the analysis.

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5.5.2.6 System Suitability Parameters It was performed same as described for analytical method development.

5.6 GAS CHROMATOGRAPHY METHOD

5.6.1 INSTRUMENTAL: The Chromatographic system used to perform development and validation of this assay method consisted of NUCON 2014 G.C. with Flame Ionization Detector and a manual injector (New Delhi, India) connected to a multi-instrument data acquisition and data processing system, GC Solutions.

5.6.2 CHROMATOGRAPHIC CONDITIONS: Instrument Column : NUCON 5675 : Stationary phase ECTM- WAX, coated with polyimide that gives length 30m x i.d .25mm. Detector Injector H2pressure O2 pressure Syringe : FID, 220 C : Manual Injector, 220 C : 10 psi : 10 psi : 10l Hamilton syringe

notorious flexibility of the capillary column, Film thickness (0.25m),

NO2 pressure : 56 psi

5.6.3 Column Temperature programming : Rate(C/min) 20 10 40 10 Temperature(C) 40 140 160 200 240
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Hold Time(min) 10 10 5

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Table 5.1 Temperature programming for G.C.

5.6.4 Validation of the proposed method 5.6.4.1 Linearity For linearity studies, Stock solution of 100 mg of artemether reference

standard were accurately weighed and transferred to 1.5ml apendroff and made up to mark with toluene, to obtain a solution of 1000g/ml of artemether,. Calibration graph was prepared by plotting the mean peak area versus concentration of ART.

5.6.4.2 Precision and Accuracy Intra-day precision and inter-day precision for the developed method was measured in terms of % R.S.D. The experiments were repeated three times a day for intra-day precision and on three different days for inter-day precision. The concentration values for both intra-day precision and inter-day precision were calculated six times separately and percent relative standard deviation were calculated. Finally, the mean of % R.S.D. (% R.S.D. = [S/X] 100, where S is standard deviation and X is mean of the sample analysed) were taken for conclusion. For studying the accuracy of the proposed method, and for checking the interference from excipients used in the dosage form, recovery experiments were carried out by the standard addition method. This study was performed by addition of known amounts of ART to a known concentration of the commercial tablets. The amounts of standard recovered were calculated in terms of mean recovery with the upper and lower limits of percent relative standard deviation. So for the determination of accuracy of the analytical procedure three same dilution of market formulation were spiked with different concentrations of standard solution i.e. 80 %, 100 % and 120 %. Area of these solutions was observed and this observed area was plotted into calibration curve. Concentration and accuracies were calculated.

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5.6.4.3 Detection and quantitation limits (LOD & LOQ) The limits of detection and quantitation were calculated by the method based on standard deviation () and slope (S) of the calibration plot using the formula LOD = 3.3/S and LOQ = 10/S.

5.5.4.4 Percentage Purity Twenty tablets were weighed and powdered equivalent to 10 mg of ART was accurately weighed and diluted up to 1ml with toluene.

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