Continued from Part 1 ...
Instruments-
Camag HPTLC system comprising of Camag IV sample applicator Linomat, Hamilton syringe 100 micro liter, Camag twin trough chamber (20x10 cm2), Camag scanner Ii V3.14 with ACTS software version 3.12, HPLC unit (Jasco PV 980) equipped with intelligent sampler (Jasco 851), Intelligent UV-Vis detector (Jasco model 875) and integrator (Jasco model 870 IT), Mass spectrometer (Schimadzu QP 1000) equipped with EI mode, FTIR spectrophotometer (Jasco model FT/IR- 45300), Differential scanning calorimeter (Schimadzu model DT040 thermal analyzer equipped with chromatopac CR 6A integrator).
Drugs, chemicals and other materials-
Silica gel 60 F 254 HPTLC plates (20x 10 cm, Merck), pseudoephedrine hydrochloride USP, pseudoephedrine sulphate USP, (from a reputed Indian company), pseudoephedrine hydrochloride reference standard (B.Knoll, Germany), phenyl ephedrine reference standard (B.Knoll, Germany), ethylene dichloride and benzene (HPLC grade, Spectra Chem., India), toluene, ethyl acetate, cyclohexane, acetone, methanol and chloroform (HPLC, E.Merck India grade), potassium hydroxide GR and ammonia (Qualigens, India) and formic acid 98/100 % (BDH grade, India ). All the chemicals were used without further treatment. All volumetric glassware used for the study was calibrated.
Stock solution of pseudoephedrine hydrochloride (reference standard) 7 μg x ul –1 (A) was prepared by dissolving 70 mg of the material in degasses distilled water in a 10 ml volumetric flask.
Identification and purity of pseudoephedrine hydrochloride test sample-
Mass spectrum, IR spectrum and DSC thermo gram of pseudoephedine (test sample) were recorded using the instruments mentioned earlier and the data obtained were compared with those of the reference sample. Due to some difficulties, the impurity profile of the pseudoephedrine HCl could not be determined.
HPTLC of pseudoephedrine hydrochloride- Preliminary experiments-
Pseudoephedine HCl was spotted on HPTLC plate using Linomat IV. The plate was developed in a twin trough chamber. A single band was obtained indicating high purity of the test sample. However, the bands either failed to migrate from the base line or they almost migrated to the solvent front. Among all the reported systems employed including ethylene chloride – methanol- benzene (70:25:5 v/v/v) showed good movement of the band except that it showed tailing. Further studied on this has been presented here.
Preparation of calibration curve, sample application, plate development and densitometric evaluation-
Stock solution A was prepared as mentioned above 1,2,5,10,15 and 20 μl of this solution corresponding to 7,14,35,70,105 and 140 μg respectively of pseudoephedrine HCl were spotted as individual bands using Camag Linomt IV sample applicator equipped with a 100 μl Hamilton syringe. The plate was developed to a distance of 80-85 mm (35-40 min) in a solvent system consisting of ethylene dichloride- methanol-benzene- formic acid (14 :4:2:1 v/v/v/v) in a Camag twin trough chamber pres-saturated with the solvent system for a period of not less than 45 minutes.
The developed plates were scanned densitometrically observing the following conditions: [I] lamp deuterium. [ii] Scanning speed 4.0 mm/s, [iii] slit dimensions 4x3 mm2, [iv] detection wave length 254 nm, [v] sensitivity 230 units and [vi] span 40 units.
The migration distance (MD) of the band was taken as the response standard during the scanning and integration of the developed bands. Peak area (AUC) was then plotted against the concentration values to obtain a calibration curve.
In the next post, I shall take up discussion on method validation and its results. To be continued in Part 3 ...
Instruments-
Camag HPTLC system comprising of Camag IV sample applicator Linomat, Hamilton syringe 100 micro liter, Camag twin trough chamber (20x10 cm2), Camag scanner Ii V3.14 with ACTS software version 3.12, HPLC unit (Jasco PV 980) equipped with intelligent sampler (Jasco 851), Intelligent UV-Vis detector (Jasco model 875) and integrator (Jasco model 870 IT), Mass spectrometer (Schimadzu QP 1000) equipped with EI mode, FTIR spectrophotometer (Jasco model FT/IR- 45300), Differential scanning calorimeter (Schimadzu model DT040 thermal analyzer equipped with chromatopac CR 6A integrator).
Drugs, chemicals and other materials-
Silica gel 60 F 254 HPTLC plates (20x 10 cm, Merck), pseudoephedrine hydrochloride USP, pseudoephedrine sulphate USP, (from a reputed Indian company), pseudoephedrine hydrochloride reference standard (B.Knoll, Germany), phenyl ephedrine reference standard (B.Knoll, Germany), ethylene dichloride and benzene (HPLC grade, Spectra Chem., India), toluene, ethyl acetate, cyclohexane, acetone, methanol and chloroform (HPLC, E.Merck India grade), potassium hydroxide GR and ammonia (Qualigens, India) and formic acid 98/100 % (BDH grade, India ). All the chemicals were used without further treatment. All volumetric glassware used for the study was calibrated.
Stock solution of pseudoephedrine hydrochloride (reference standard) 7 μg x ul –1 (A) was prepared by dissolving 70 mg of the material in degasses distilled water in a 10 ml volumetric flask.
Identification and purity of pseudoephedrine hydrochloride test sample-
Mass spectrum, IR spectrum and DSC thermo gram of pseudoephedine (test sample) were recorded using the instruments mentioned earlier and the data obtained were compared with those of the reference sample. Due to some difficulties, the impurity profile of the pseudoephedrine HCl could not be determined.
HPTLC of pseudoephedrine hydrochloride- Preliminary experiments-
Pseudoephedine HCl was spotted on HPTLC plate using Linomat IV. The plate was developed in a twin trough chamber. A single band was obtained indicating high purity of the test sample. However, the bands either failed to migrate from the base line or they almost migrated to the solvent front. Among all the reported systems employed including ethylene chloride – methanol- benzene (70:25:5 v/v/v) showed good movement of the band except that it showed tailing. Further studied on this has been presented here.
Preparation of calibration curve, sample application, plate development and densitometric evaluation-
Stock solution A was prepared as mentioned above 1,2,5,10,15 and 20 μl of this solution corresponding to 7,14,35,70,105 and 140 μg respectively of pseudoephedrine HCl were spotted as individual bands using Camag Linomt IV sample applicator equipped with a 100 μl Hamilton syringe. The plate was developed to a distance of 80-85 mm (35-40 min) in a solvent system consisting of ethylene dichloride- methanol-benzene- formic acid (14 :4:2:1 v/v/v/v) in a Camag twin trough chamber pres-saturated with the solvent system for a period of not less than 45 minutes.
The developed plates were scanned densitometrically observing the following conditions: [I] lamp deuterium. [ii] Scanning speed 4.0 mm/s, [iii] slit dimensions 4x3 mm2, [iv] detection wave length 254 nm, [v] sensitivity 230 units and [vi] span 40 units.
The migration distance (MD) of the band was taken as the response standard during the scanning and integration of the developed bands. Peak area (AUC) was then plotted against the concentration values to obtain a calibration curve.
In the next post, I shall take up discussion on method validation and its results. To be continued in Part 3 ...
#PseudoephedrineHydrochloride #HPTLC #Densitometry
