Chemicals and reagents
Oseltamivir phosphate API was taken from GVK Biosciences Pvt. Ltd. (Hyderabad, India).
The solvents viz. methanol, ethanol, acetonitrile, isopropyl alcohol, Dichloromethane, MTBE, Hexanes, ethyl acetate, THF, 1,4-Dioxane, DMF, and Toluene (all procured from Merck, India).
Instruments
Agilent GC 6890 N system, Agilent headspace G1888 N system, Flame ionization detector system, Waters United States of America (USA) Empower version 3 software were used for the quantification of opted Twelve organic volatile solvents in Oseltamivir phosphate API.
Chromatographic conditions
Column | : | DB-1, 60 m, 0.32 mm, and 5.0 µm |
Injector temperature | : | 180°centigrade(°C) |
Detector | : | Flame ionization detector (FID) |
Detector temperature | : | 290 °C |
Initial oven temperature | : | 40 °C |
Hold time-1 | : | 10.0 min |
Ramp rate-1 | : | 5.0 °C/min |
Oven temperature | : | 80 °C |
Hold time-2 | : | 10.0 min |
Ramp rate-2 | : | 4.0 °C/min |
Oven temperature | : | 150 °C |
Hold time-3 | : | 5.0 min |
Ramp rate-3 | : | 40.0 °C/min |
Oven temperature | : | 280 °C |
Hold time-4 | : | 6.25 min |
Carrier gas | : | Nitrogen |
Mode of injection | : | Split |
Constant Flow | : | 1.0 mL/min |
Split ratio | : | 1:5 |
Makeup | : | 30 mL/min |
Hydrogen | : | 40 mL/min |
Air | : | 400 mL/min |
Run time | : | 60.0 min |
Diluent | : | N-methyl-2-pyrrolidone (NMP) |
Headspace parameters
Oven temperature | : | 90 °C |
Loop temperature | : | 160 °C |
Transfer line temperature | : | 165 °C |
GC cycle time | : | 75 min |
Injection time | : | 5.0 min |
Loop equilibration time | : | 0.05 min |
Loop fill time | : | 2.0 min |
Pressurization | : | 2.0 min |
Vial equilibration | : | 30.0 min |
Shaking | : | High |
Vial pressure | : | ~ 15 Pounds per square inch (psi) (Nitrogen) |
Specifications of organic twelve volatile impurities
Methanol | : | Not more than 3000 ppm |
Ethanol | : | Not more than 5000 ppm |
Isopropyl alcohol | : | Not more than 5000 ppm |
Dichloromethane | : | Not more than 600 ppm |
Hexanes | : | Not more than 290 ppm |
Ethyl acetate | : | Not more than 5000 ppm |
THF | : | Not more than 720 ppm |
Toluene | : | Not more than 890 ppm |
DMF | : | Not more than 880 ppm |
1.4-Dioxane | : | Not more than 380 ppm |
Acetonitrile | : | Not more than 410 ppm |
MTBE | : | Not more than 5000 ppm |
Standard and sample solution preparations
Blank preparation Transferred 2.0 Milley Letter (mL) of diluent into a 20 ml headspace vial. Immediately crimped the vial with a septum and a cap.
Standard stock preparation
Weighed and transferred about 600 mg of Methanol, 1000 mg of Ethanol, 82 mg of Acetonitrile, 1000 mg of IPA, 120 mg of DCM, 1000 mg of MTBE, 58 mg of Hexanes, 1000 mg of Ethyl acetate, 144 mg of THF, 76 mg of 1,4-Dioxane, 176 mg of DMF, and 178 mg of Toluene into a 50 mL volumetric flask contained 20 mL of diluent dissolved and diluted to volume with diluent.
Standard preparation Transferred 1.0 mL of the stock solution into 100 mL volumetric flask containing 50 mL of diluent, dissolved, and diluted to volume with diluent.
Preparation of standard vial Transfer 2.0 mL of standard stock solution in a headspace vial and seal with an aluminum septum and crimp the cap.
Preparation Oseltamivir phosphate sample
Accurately weighed and transferred 80 Milly gram (mg) of Oseltamivir phosphate into a headspace vial. Then add 2.0 mL of diluent and immediately seal with an aluminum septum and crimp the cap.
Pharmaceutical sample preparation
Twenty tablets were weighed and powdered. Accurately weighed and transferred an amount of powder equivalent to 80 mg of Oseltamivir phosphate to a 20 mL headspace vial then added 2.0 mL of diluent and immediately seal with an aluminum septum and crimp the cap.
The Organic volatile impurity content (ppm) was calculated by the following formula:
$${\text{Calculation (ppm):}}\frac{{\text{Impurity area in test solution}}}{{\text{Impurity area in standard solution}}} \times \frac{{\text{Standard concentration (mg/mL)}}}{{\text{Sample concentration (mg/mL)}}} \times 10^{6}$$
GC-HS method development
The adopted solvents for the Oseltamivir phosphate API are associated with class-II and class-III solvents by ICH guidelines. To develop an elevated sensitive and novel method, one needs to choose the best suitable solvent and column for Oseltamivir phosphate. The method should be robust to determine the trace levels of residual solvents in the drug substance as well as the drug product. As part of method development, following the quality by design principles to have a better control method for residual solvents in Oseltamivir phosphate.
Diluent selection
This method development was started with the selection of diluent. We have sought different diluents (DMF, NMP, and Dimethyl sulfoxide). For its ability to dissolve a broad range of organic solvents and will not impede with chosen solvents, analyzed by gas chromatography, NMP was exploited as the standard as well as sample diluent.
Column selection
The Column selection for GC-HS analysis was also a very important task in the method development process. This study utilized a chromatographic basic rule “like attracts like” and focused on the polarity matching among column stationary phase and mobile phase. In this study, many columns were screened i.e., VF-1(30 m × 0.32 mm × 0.45 μm), DB-624(30 m × 0.53 mm × 3.0 μm), DB-1, 60 m, 0.32 mm × 5.0 µm and DB-624 (30 m × 0.25 mm × 0.25 μm), DB-17, DB-5, DB-Vax columns. Out of seven columns , DB-1 column gave better resolution between Ethyl acetate, MTBE, Dichloromethane, and Hexanes whereas in other columns VF-1, DB-624, DB-5, DB-Vax either MTBE peak is co-eluting with Hexanes first peak or with DCM peak. In some columns, Ethyl acetate is co-eluting with Hexane’s fraction 3. The GC-HS parameters were first optimized to achieve good retention time, acceptable resolution, and better peak shapes for the MTBE, Hexanes, Dichloromethane, and Ethyl acetate in Oseltamivir phosphate and its formulations. The DB-1 eluted sharp peaks with good peak tailing and good resolution between all peaks. It demonstrated that the DB-1 column was closely matched. Hence, the DB-1 60 m, 0.32 mm × 5.0 µm column was selected for this study.
Column oven temperature optimization:
Four different column oven temperature fluxes were tried. (1) maintained for 11 min at 40 ºC and then continued to upsurge to a temperature close of 240 ºC at a rate of 20 ºC/min and retained for 30 min; (2) maintained for 10 min at 40 ºC and then raise 80 ºC with Ramp rate 5.0 °C/min hold for 10 min and again raise 150 ºC with Ramp rate 4.0 °C/min hold for 5 min continued to upsurge to a temperature close of 280 ºC at a rate of 40 ºC/min and retained for 6.25 min. (3) maintained for 5 min at 40 ºC and then continued to upsurge to a temperature close of 240 ºC at a rate of 10 ºC/min and retained for 35 min; (4) maintained for 11 min at 40 ºC and then continued to upsurge to a temperature close of 240 ºC at a rate of 15 ºC/min and retained for 30 min;
Better separation with the good resolution was obtained with 1st oven program [maintained for 10 min at 40 ºC and then raise 80 ºC with Ramp rate 5.0 °C/min hold for 10 min and again raise 150 ºC with Ramp rate of 4.0 °C/min hold for 5 min continued to upsurge to a temperature close of 280 ºC at a rate of 40 ºC/min and retained for 6.25 min]. In 1st, 3rd, and 4th oven temperature fluxes, the peaks of all the nine organic solvents were closely eluted. Nitrogen, as a carrier gas, with a flowing stream of 1.0 mL per min and 2 mL per min were tested. A 1.0 mL per min flow stream was optimized. The remaining optimized parameters were 180 ºC temperature at injector port; 290 ºC temperature at detector port; split mode injection in 1:5 ratio; air flow and hydrogen flow were 400 mL/min and 40 mL/min, respectively. Figure 2 displays the chromatogram acquired using configured parameters.