Ramesh Panchagnula, PhD, is a professor in the department of pharmaceutics, National Institute of Pharmaceutical Education and Research, Sector-67, Ph-X, SAS Nagar-160 062, Punjab, India, tel. -91 172 214 682 or 214 687, fax -91 172 214 692.

Ramesh Panchagnula, PhD

Mefenamic acid (MA) [N–(2, 3–xylyl) anthranilic acid] is a high-dose, non-steroidal anti-inflammatory agent available in immediate-release capsule and tablet formulation. It is poorly soluble in aqueous media, sticking to any surface, creating problems in granulation and tabletting processes.

				 Mefenamic acid shows bioavailability ranging from 28–86% in capsule form. 

The difference in bioavailability was found to be a result of the variation in dissolution rate involving multiple factors, such as the particle size and packing of the drug, and the de-aggregation rate of MA.

  Our studies revealed that MA from different manufacturers has different crystal properties. The complete solid-state characterization of MA has also been performed.

  We found that crystallization of MA from ethyl acetate (EA) at a fast cooling rate generated a new crystal form (or a crystal defect). To explore the feasibility of preparing this new polymorph, this study was conducted by altering the polarity of EA with different organic solvents under various crystallization conditions.  

Ethanol (E), EA and tetrahydrofuran (THF) were of absolute, analytical and HPLC grade, respectively.  

 Potassium bromide (KBr) used for recording Fourier Transform infrared (FTIR) spectra was FTIR grade. 

. Recrystallized samples of MA were viewed/photographed under light and polarized light microscopes at various magnifications (×10, ×25 and ×45).			

MA samples recrystallized from various solvents at varying degrees of supersaturation, cooling rate and agitation speed were screened for the required polymorph under a hot stage microscope (Leica DMLP; 

) at a constant heating rate from 32–250 °C to observe any transition.  

 The surface morphology of MA-recrystallized samples was studied using an SEM (D–6000; 

). Samples were fixed on a cylindrical aluminium stub with conductive double-sided adhesive tape and sputter-coated with gold.  

 Thermal analysis of MA samples was recorded in a differential scanning calorimeter (DSC 821; 

) using Mettler STARe software with the Solaris operating system (version 5.1).  

Samples of 1–5 mg were weighed directly into aluminium crucibles sealed with pin-holed lids and scanned at 25–300 °C with a heating rate of 10 °C/min under nitrogen purging (80 mL/min). The temperature axis, heat flow and cell constant were previously calibrated with a pure sample of indium. An empty aluminium crucible sealed with a pin-holed lid was taken as the reference cell. 

 This was performed on the weighed quantities of samples by heating from 25–300 °C at the constant rate using a thermogravimetric analyser (

). Any weight loss occurring before the melting point of the drug can be a result of solvent residues occluded in drug crystals during recrystallization.  

A pellet was prepared by mixing 2–5% of the sample with KBr using a mortar and pestle, and compressed at a pressure of 15000 psi. Spectra were recorded in the transmission mode in the region of 4000–4500 cm

 To minimize preferred orientation, all samples were prepared by passing through a sieve (number 60) for pXRD analysis. The pXRD pattern of recrystallized samples was recorded at room temperature using an X-ray diffractometer (

) with Cu kα1 radiation operated at 350 kv. Diffractograms were recorded over an angular range from 5–50° 2θ in a continuous scan mode using step size of 0.04° 2θ and a step time of 0.5 s. 

Intrinsic dissolution rate (IDR) studies. 

IDR studies of recrystallized samples were performed using rotating disc apparatus with optimized parameters such as sample weight, compression force and stirring speed. Sample powder weighing 100 mg was compressed using a hydraulic press at a force of 3000 psi for 1 min to yield pellets of surface area 50.27 mm

  that would not disintegrate during the test.  

Study conditions include a 1000 mL jar, containing 900 mL of borate buffer maintained at 37 °C and 100 rpm. A sample volume of 5 mL was withdrawn at specific time intervals and analysed for MA.  

 Equilibrium solubility of MA acid was determined in two solvent mixtures EA:E (10:90% v/v) and EA:THF (50:50% v/v) at 32 °C and 60 °C. The drug was added to 5 mL solvent mixtures (kept in vials) until no more drug was dissolved at the specified temperatures, and closed with aluminium foil and rubber caps to prevent vaporization. All solutions were kept under constant stirring at 100 rpm at constant temperature in a water bath (

). At set time intervals, a fixed amount of solution was withdrawn with a microlitre syringe (

) and analysed by UV-vis spectrophotometry at 280 nm (DU640i; 

Recrystallization of MA was performed from various solvents with differing degrees of supersaturation, cooling rates and with or without stirring. For recrystallization, the crystallizer was designed by using a 500 mL beaker jacketed with a 1000 mL beaker, whose inlet and outlet were connected to a circulator water bath (

) capable of controlling the temperature to 0.1 °C/min. Both the inner beaker containing sealed ampoules and outer beakers were closed separately.  

Recrystallization was performed in sealed ampoules containing magnetic beads and 15 mL drug solutions of varying concentrations in different solvents. The entire crystallizer containing ampoules was placed on a magnetic stirring plate and the temperature increased to 70 °C, at which point all the drug in ampoules was solubilized. Cooling was then performed at slow and fast rates with the aid of a circulator bath, with and without stirring. The crystals were filtered and dried in a dessicator for 12 h. 

To study the effect of harvesting time on polymorph generation, crystals from drug solutions of EA were filtered at two different times, and a visible mass of crystals was seen in solution after fast cooling. The crystals were kept in a dessicator for 12 h and analysed. 

Solvents showing less solubility and high solute–solvent interactions are preferred for metastable polymorph generation.

  Furthermore, an appropriate balance between solubility, and hydrogen bond donating and accepting propensity of solvents is necessary.  

MA solubility increases with decreasing polarity of solvent and has very high solubility in THF; therefore to achieve a balance between solubility and solute–solvent interactions, mixtures of solvents were prepared (THF:EA [50:50% v/v]; E:EA [90:10% v/v]). 

The generation of a metastable polymorph was demonstrated in a previous study of EA at different recrystallization conditions (i.e., the degrees of supersaturation were 50.25 mg/mL, 53.00 mg/mL and 55.75 mg/mL, and the cooling rate was 20 °C/h with stirring). 100% EA was also selected for recrystallization.

MA equilibrium solubility was determined at 32 °C and 60 °C in two solvent mixtures using a shaker water bath. The equilibrium solubility of MA increased with decreasing solvent polarity, as expected, at both temperatures. In addition, equilibrium solubility at 60 °C was determined to decide the degree of supersaturation for recrystallization experiments, while 32 °C was used to calculate the theoretical yield of recrystallization.

Characterization of recrystallized samples.

Recrystallization of MA was performed in solvent–solvent mixtures (E:EA, EA and THF:EA) of three different polarities at three degrees of supersaturation and two cooling rates, with and without stirring. HSM and DSC screened all the crystals for new crystal properties and polymorphic forms. However, a significant difference in recrystallized product with respect to solid state was observed only in crystals obtained from E:EA mixture at various recrystallization conditions. 

These crystals were further characterized by additional techniques to confirm the existence of the new polymorph. 

All crystals were observed under light and polarized microscopes to identify any change in morphology as well as optical properties. Since all the samples showed birefringence under a polarized microscope, the possibility of the amorphous form being produced at a fast cooling rate was ruled out.  

There was a marked difference in the size of crystals obtained under different conditions where crystal size increased with decreasing cooling rate, unstirred conditions and increased degree of supersaturation. Some surface grooves were also seen for THF:EA and EA-recrystallized samples.  

Crystals from EA and THF:EA showed a wide transition range of about 10 °C between 150–215 °C, without recrystallization. Other than this, samples recrystallized from EA with the shortest harvesting time did not show any transition before melting. There was a sharp melting of crystals obtained from E:EA under stirred conditions at 222 °C while the melting point of MA is 231 °C (Figure 1).  

 Figure 1 HSM of crystals from E:EA under stirred conditions. All the crystals showed a sharp melting between 220â223 Â°C.			

Furthermore, the melted product recrystallized at 223 °C and could not be seen clearly since melting and recrystallization were almost simultaneous.  

Melting without any transition appears to be caused by too high a heating rate. The sharper melting and recrystallization events of crystals from E:EA can be attributed to melting at 222 °C, followed by recrystallization at 223 °C leading to melting at 231 °C. A similar trend of solid–liquid transition was also observed for carbamazepine, which melted at 175.92 °C, recrystallized at 181.66 °C and melted at 192.1 °C.

The wide transition of crystals from EA and THF:EA is representative of solid–solid transition. This was observed at 150 °C for solid–solid transition of monoclinic retinoic acid to its triclinic form followed by its complete melting at 183 °C.

However, a different trend in transition and melting of recrystallized product from the E:EA mixture and other solvents indicated the possibility of a different crystal form. 

In light microscopy, some grooves were seen on the crystal surface of samples from THF:EA and EA compared with those from the E:EA and the United States Pharmacopeia (USP) reference sample, hence SEM photomicrographs of representative recrystallized and USP reference samples were recorded. The reference sample was observed to be an aggregated microcrystalline mass with columnar plate-like properties.			

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