FRANZ CELL TEST OF NANO GLUTATHIONE WITH HPLC ANALYSIS

Transcription

1 FRANZ CELL TEST OF NANO GLUTATHIONE WITH HPLC ANALYSIS Analysis Conducted BY CC RESEARCH (P) Ltd., For Nanoceutical Solutions Inc. USA 2950 Thousand Oaks drive, Suite 3, San Antonio, Texas

2 EXECUTIVE SUMMARY OF DRUG DIFFUSION STUDIES (% DRUG DIFFUSED) The following table shows the percentage of drug diffused from time 10 seconds to 90 seconds. Each time point tested were at 10 seconds increments up to 90 seconds. These results show that % drug diffusion started increasing steady state and reached to peak and then declining thereafter. These results indicate time kinetics and fast diffusion rate of the nano particles. TABLE 1: DIFFUSION STUDIES OF NANO GLUTATHIONE (D50) TIME (SECONDS) % DRUG DIFFUSED % % % % % % 70 8% % % 2 Nano Glutathione Diffusion Study

3 DIFFUSION RESULTS OF NANO GLUTATHIONE Diffusion: Movement of a fluid from an area of higher concentration to an area of lower concentration. Diffusion is a result of the kinetic properties of particles of matter. The particles will mix until they are evenly distributed. Materials used: PBS buffer solution Franz diffusion apparatus Sheep mucosal membrane Syringe HPLC Instrument Preparation of PBS buffer solution: Measure a volume of 800 ml of ddh2o with a graduated cylinder and transfer to an Erhlenmyer flask. Add a magnetic stir bar to the Erhlenmyer flask and place the flask on a magnetic stir plate Adjust the speed of the magnetic stir bar so that oxygen is not introduced into the solution while it is rapidly mixed. Transfer to the flask: 8g of NaCl, 0.2 g KCl 1.44 g of Na2HPO g of KH2PO4 Ensure that the ph meter has been properly calibrated and rinse the ph probe with double distilled H2O. Remove the excess water from the probe tip (without touching the probe tip) with a clean paper towel. Place the ph probe into the solution. Slowly add 1 M HCl dropwise with a transfer pipette and allow the HCl to fully dissolve into the solution. Stop stirring the solution. Measure the ph with the ph meter. Repeat until the ph of the solution is 7.4. Pour the solution into a fresh graduated cylinder and adjust the final volume to 1 liter with double distilled H2O. Store the PBS solution at room temperature. The PBS Solution is sterile; when using the PBS Solution, ensure that sterile techniques are employed. Nano Glutathione Diffusion Study 3

4 Drug Diffusion Study: The Nano Glutathione diffusion studies through membrane experiments were conducted by using vertical type diffusion cell (Franz type) having receptor compartment 60ml volume with 10.18cm2 area. The receptor compartment was filled 60ml of phosphate buffer ph 7.4; the activated mucosal membrane was mounted on the flange of the diffusion cell receptor compartment. The whole assembly was kept on a magnetic stirrer and solution in the receptor compartment was constantly and continuously stirred using a magnetic bead and at 37 1oC maintained. permeation study of gel through the sheep mucosal membrane was performed using Franz diffusion cell and membrane assembly, at 37 C ± 1 C and 50 rpm. This temperature and rpm was maintained by magnetic stirrer. The tissue was stored in Krebs buffer at 4 C upon collection. After the buccal membrane was equilibrated for 30 min with the buffer solution between both the chambers, the receiver chamber was filled with fresh buffer solution (ph 7.4), and the donor chamber was charged with 5mL (1mg/ ml) of drug solution. Aliquots (5mL) were collected at predetermined time inter wells up to 45min and the amount of drug permeated through the mucosa was then determined by measuring the values at 215 nm using HPLC method. The medium of the same volume (5mL), which was pre-warmed at 37 C, was then replaced into the receiver chamber. The experiments were performed and values were used to calculate flux (J) and permeability coefficient (P). J = (dq/dt) A t P = (dq/dt) ΔCA Where, J is Flux (mg.hrs-1cm-2) P is permeability coefficient (cm/h) dq/dt is the slope obtained from the steady state portion of the curve ΔC is the concentration difference across the mucosa and A the area of diffusion (cm2) 4 Nano Glutathione Diffusion Study

5 METHOD DEVELOPMENT FOR NANO GLUTATHIONE BY USIN HPLC CHROMOTOGRAPHIC CONDITIONS: Column Mobile Phase Flow rate : INERTSIL ODS C *4.6mm. : Acetonitrile: water (50:50 v/v). : 0.8 ml/min Injection Volume : 10 µl Wavelength : 215 nm Temperature : 5o C ± 2 Runtime Rt : mins : w2.215 mins. Preparation of Linearity Solutions: Weigh Accurately 10 mg of L-glutathione and take it into a 10 ml of volumetric flask to this add 3 ml of diluent and make up the solution up to the mark with same solution. (1000 µg/ ml) From the above stock solution take 0.1 ml into a 10 ml of volumetric flask make up the solution with diluent up to the mark.(10 µg/ml). From the Above solution take a series of solutions 0.3ml,0.6ml,0.9ml,1.2ml,1.5ml,1.8 ml into different 10 ml volumetric flasks and make up the solution with diluent to get a concentration range of 0.3 µg/ml to 1.8 µg/ml. (*mobile phase is used as a Diluent) The diffused samples are injected into HPLC by maintaining above chromatographic conditions, from the data obtained area of the peak were calculated and the % drug release. Samples were tested as per the Client requirement. Franz tests were retested up to 45 min to determine % drug diffuse and Flux rate. Nano Glutathione Diffusion Study 5

6 DIFFUSION STUDIES OF NANO GLUTATHIONE LINEARITY METHOD: Preparation of stock solution 10 mg of Nano Glutathione working standard was accurately weighed and was transferred into a 10ml clean dry volumetric flask, add about 2ml of diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. Preparation of Level I (20ppm of Nano Glutathione) 0.2 ml of stock solution was taken in to 10ml of volumetric flask and diluted up to the mark with diluent. Preparation of Level II (40ppm of Nano Glutathione) 0.4 ml of stock solution was taken in to 10ml of volumetric flask and diluted up to the mark with diluent. Preparation of Level III (60ppm of Nano Glutathione) 0.6 ml of stock solution was taken in to 10ml of volumetric flask and diluted up to the mark with diluent. Preparation of Level IV (80 ppm of Nano Glutathione) 0.8 ml of stock solution was taken in to 10ml of volumetric flask and diluted up to the mark with diluent. Preparation of Level V (100 ppm of Nano Glutathione) 1.0 ml of stock solution was taken in to 10ml of volumetric flask and diluted up to the mark with diluent. Procedure Each level was injected into the chromatographic system and peak area was measured. Plot a graph of peak area versus concentration (on X-axis concentration and on Y-axis Peak area) and the correlation coefficient was calculated. Acceptance criteria Correlation coefficient should be not less than Nano Glutathione Diffusion Study

7 Chromatograms showing linearity level-1 to level 5 (20ppm-100 ppm of Nano Glutathione) injections Nano Glutathione Diffusion Study 7

8 TABLE 2: DIFFUSION STUDIES OF NANO GLUTATHIONE (D50) SI.No Peak name Rt Area 1 Glutathione Glutathione Glutathione Glutathione Glutathione Average SD %RSD % TABLE 3: Linearity Results Nano Glutathione: S.No Linearity Level Concentration Area 1 I 20 ppm II 40 ppm III 60 ppm IV 80 ppm V 100 ppm Correlation Coefficient Nano Glutathione Diffusion Study

9 y=1079.3x R 2 = The linearity study was performed for concentration range of 20µg-100µg Nano Glutathione and the correlation coefficient was found to be (NLT 0.999). Chromatogram Optimized Chromatographic conditions: Column : Symmetry C18 (4.6 X 150 mm ;5µm Waters). Column temperature : 250C Flow rate : 1 ml/min. Injection volume : 20 µl. Wavelength : 238 nm. Run time : 10 min. Diluent : mobile phase. Mobile phase composition : methanol: water (60:40%v/v). Injector : Rheodyne. Stationary phase : C18 (4.6 X 150 mm; 5µm Waters) Operating temperature : Room temperature. Nano Glutathione Diffusion Study 9

10 Peak Name RT Area Height Injection SampleName 1 glutathione D50 10 sec 10 Nano Glutathione Diffusion Study

11 Peak Name RT Area Height Injection SampleName 1 glutathione D50 20 sec Peak Name RT Area Height Injection SampleName 1 glutathione D50 30 sec Nano Glutathione Diffusion Study 11

12 Peak Name RT Area Height Injection SampleName 1 glutathione D50 40 sec Peak Name RT Area Height Injection SampleName 1 glutathione D50 50 sec 12 Nano Glutathione Diffusion Study

13 Peak Name RT Area Height Injection SampleName 1 glutathione D50 60 sec Channel: 2487 Channel1; Injection:5; Result Id: Peak Name RT Area Height Injection SampleName 1 glutathione D50 70 sec Nano Glutathione Diffusion Study 13

14 Channel: 2487 Channel1; Injection:5; Result Id: Peak Name RT Area Height Injection SampleName 1 glutathione D50 80 sec Channel: 2487 Channel1; Injection:2; Result Id: Nano Glutathione Diffusion Study

15 FLUX VALUES Time(sec) Dose(mg/ml) Area(f1) CONCNE TRATION % Drug release Flux The flux value for 3 minutes was for 45 minutes it was , the decrease in the values was observed due to super saturation Nano Glutathione Diffusion Study 15

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