IDENTIFICATION AND QUANTITATIVE ANALYSIS OF WARFARIN FORMS IN ORAL SUSPENSION USING RAMAN SPECTROSCOPY

In the present study micro-Raman Spectroscopy was employed in order to establish a novel, fast and reliable method for quantitative analysis of the API (Active Pharmaceutical Ingredient) Warfarin Sodium which is present in commercial oral suspension (syrup). It was found that the amorphous Warfarin sodium precipitates as a crystalline form after its dissolution in either water or in the mixture of the liquids that constitute the placebo of the oral suspension. The existence of the new crystalline form was verified by recording the respective Raman spectrum and XRPD pattern. A quantitative method, using Raman spectroscopy, for the precipitated new crystalline form in the commercial syrup was also developed. The ratios of warfarin’s crystalline form vibration peaks’ areas at 1573 and 1612 cm -1 to the area of a strong placebo peak at 923 cm -1 were calculated. A calibration line was constructed, using as standards, dispersions of syrup and placebo. Limits of Detection (L.O.D) and Quantification (L.O.D) were calculated and found to be 0.0414 and 0.1243 mg/mL respectively. INTRODUCTION Active Pharmaceutical Ingredient (API) Warfarin Sodium can be found in three formulations: as a tablet, oral suspension (syrup) with strength of 1 mg/mL and dilution for injections. Warfarin Sodium is the sodium salt of 3-(αacetonylbenzyl)-4-hydroxycoumarin. The pharmacologic function of the compound is an anticoagulant that inhibits the synthesis of warfarin K-dependent coagulation factors. The treatment aims at preventing further extension of the formed clots and secondary thromboelic complications that may result in serious and possible fatal sequelae [1]. Two solid forms of Warfarin sodium, amorphous and crystalline clathrate [2], are known to exist. The crystalline clathrate form is an Warfarin Sodium-isopropyl alcohol complex. It is has been also reported that Warfarin Sodium is a true 2-propanol solvate, not a clathrate, which it is transformed to the amorphous state through an intermediate crystalline step [3]. Both forms are used as APIs in pharmaceutical formulations and the most common method for quantitative analysis, adopted also by Pharmacopoeia [4], is High Pressure Liquid Chromatography (HPLC), which is not capable to identify if the measured quantity refers to crystalline or to amorphous warfarin sodium. In this work it is suggested that Raman Spectroscopy, a vibration technique, can be successfully used for quantitative determination of the API being much less cumbersome and time consuming than the suggested HPLC method. Also as oppose to XRPD, it can differentiate between the amorphous and the crystalline warfarin. The present work focus in: (a) developing a methodology for quantitative analysis of the oral suspension of Warfarin Sodium using Raman Spectroscopy, (b) investigating the presence of a new crystal form of the compound which is not yet characterized. 10ο ΠΑΝΕΛΛΗΝΙΟ ΕΠΙΣΤΗΜΟΝΙΚΟ ΣΥΝΕΔΡΙΟ ΧΗΜΙΚΗΣ ΜΗΧΑΝΙΚΗΣ, ΠΑΤΡΑ, 4-6 ΙΟΥΝΙΟΥ, 2015 EXPERIMENTAL Chemicals and reagents Warfarin sodium amorphous, the final oral dispersion having strength of 1 mg/mL, the placebo mixture as well as placebo’s individual excipients were provided by a Greek Pharmaceutical company. All aquatic solutions were prepared in Milli-Q distilled water that had resistance of greater than 17 MΩ-cm. Preparing the mixtures For the construction of the calibration line five dispersions were prepared. The first four were made by adding placebo to commercially available syrup (25% syrup-75% placebo, 50% syrup50% placebo, 75% syrup25% placebo and 100% syrup) and the last one by enriching the placebo with solid API in order to achieve a concentration 120% compared to syrup’s concentration. For the experiments of API’s dissolution and subsequent re-crystallization, a pseudo-placebo was used which was prepared by mixing, in the appropriate ratios, the three major liquid excipients of the oral suspension (water 66%-liquid maltitol 27%-propylene glycol 4%). Instrumentation Raman Spectroscopy: A Renishaw, InVia, Raman microscope equipped with a laser with a 785 nm excitation line was used. The laser line was focused by Olympus oblective lens (20x) onto the samples surface. The system was equipped with a CCD detector. The power of the incident laser was 250mW. The typical spectral resolution was 2 cm -1 . A Windows-based software was used (WiRE © 2.0) to obtain the spectra. Instrument response (laser power and the wavenumber) was checked by recording the peak of Si. Recording the spectra: Raman spectra were acquired by focusing a laser line at through the objective lens (20x) of an optic microscope onto the sample’s surface. Prior to each Raman recording one drop of each dispersion was placed with a Pasteur pipette on a slide with gold substrate which served as reflectance surface (mirror). Each drop placed on the golden substrate was approximately 0.30 mL. The material deposited on the slide had a diameter of approximately 1.5 cm (Fig 1a). The drops were dried in the oven for 30 minutes at 100 °C. After solvents’ evaporation (water and part of propylene glycol and water in liquid maltitol) a gel formation was obtained in which the strength of warfarin was approximately triple. The slide was placed on a home-made apparatus (Fig. 1b) and the sample was rotated. In this way it was possible to collect the Raman signal from the circumference of a circle formed during sample’s rotation, minimizing the under-sampling problems. The recorded spectra were the sum of 5 scans which were acquired in the region 250-2000 cm -1 . Fig 1a. On the left the apparatus with the gold slide on, under the objective lens of micro Raman; on the right the gold slide with the drop of syrup on. Fig 1b. Home-made rotating apparatus for minimizing the undersampling problems 10ο ΠΑΝΕΛΛΗΝΙΟ ΕΠΙΣΤΗΜΟΝΙΚΟ ΣΥΝΕΔΡΙΟ ΧΗΜΙΚΗΣ ΜΗΧΑΝΙΚΗΣ, ΠΑΤΡΑ, 4-6 ΙΟΥΝΙΟΥ, 2015 XRPD PXRD patterns were obtained at room temperature using an X-ray diffractometer (Bruker AXS D8 Advance with Bragg-Bretano geometry), LynxEye detector and Cu Ka spectral line (l=1,540562 A) with the generator voltage and current set at 40 kV and 40 mA, respectively. The sample holder, had a central circular cavity of diameter 2 cm in which the filtered crystals being on the filter, were placed. Samples were scanned from 5 to 30° 2θ at a step size of 0.02(θ) and scan speed 1s/step. Instrument performance was checked against the recording of corundum reference sample A13-B73 provided by Bruker. Optic Microscope A Leica (DM 2500 M) optic microscope was used to obtain pictures of Warfarin Sodium, in reflection mode, using the appropriate Leica oblective lens. A color video camera (Leica,model DFC 420C) was used to relay the microscope image to a computer image processor, which in turn employed a Windows based software (LAS © V3.8) for image acquisition and analysis. RESULTS AND DISCUSSION API identification and stability The spectrum of solid form of the amorphous Warfarin Sodium added in the suspension exhibits a single strong peak at 1606 cm-1 (Fig. 2; red line). After its dispersion in placebo, being mostly a mixture of water, propylene glycol and liquid maltitol along with other solid excipients, two API peaks at 1573 and 1612 cm -1 appear. The presence of the two peaks indicates a possible transformation of the amorphous Warfarin Sodium to another state. 50