Dr. Gawande, Assistant Professor in the Quality Assurance Techniques at Sinhgad Institute of Pharmacy, Pune, Maharashtra, India, is involved in the development and validation of chromatographic methods for stability of pharmaceutical drugs, both standalone and fixed dose combinations.

Stability is the capacity of a drug product to remain within specification for a given time. It is a prime requirement to ensure its identity, strength, quality and purity. Forced degradation studies are an integral part of drug development programs. For detecting the number and types of degradation products that are formed under various conditions, different chromatographic techniques in conjunction with UV and MS are used.

HPTLC is especially beneficial for stability testing due to its disposable stationary phase, as well as in the case of forced degradation, when appreciable amounts of acidic, alkaline and peroxide reagents are used. Furthermore, HPTLC permits the analysis of a large number of samples in a short time. There are also multiple derivatization and detection options that can help to characterize degradation products. Apart from this, simplicity and low cost of analysis add to the benefits.

10 mg of cefixime trihydrate (CEFI) or azithromycin dihydrate (AZI) dissolved in 10 mL methanol

1. Hydrolytic degradation: 1 mL of drug standard solution was mixed with 1 mL water or 0.5 N HCl or 0.5 N NaOH, kept for 1 h at RT and neutralized with acid or base
2. Oxidative degradation: 1 mL of drug standard solution was mixed with 1 mL of H2O2 (3 and 30 %) and kept for 1 h at RT
3. Thermal degradation: The drug powder placed in a sealed glass ampoule was heated at 100 °C and 200 °C for 1 h and 2 h in a hot oven
4. Photolytic degradation (according to ICH guidance Q1B, option 2): A thin layer of solid drug powder was exposed with fluorescent cold white light (1.25 million lux h) and UV light (200 Whm-2) in a photo stability chamber

All degradation samples were dissolved (solid samples) or diluted with methanol to a final concentration of 100 μg/mL.

HPTLC plates silica gel 60 F254, aluminium backed (Merck), 20 x 10 cm, prewashed by developing first with methanol and then with the mobile phase followed by drying for 15 min with cold air

Bandwise application with Linomat 5 or Automatic TLC Sampler (ATS 4), 15 tracks, band length 8 mm, distance from left edge 20 mm, distance from lower edge 8 mm, application volumes 10.0 μL for sample solutions and 2.0 and 5.0 μL for standard solutions (CEFI resp. AZI); for preparative isolation: band length 180 mm and application volume 200.0 μL

In the Twin Trough Chamber 20 x 10 cm with chamber saturation (with filter paper) for 20 min, development with ethyl acetate – methanol – acetone – toluene – ammonia 2:10:14:1:1 to the migration distance of 80 mm (from the lower edge), drying for 15 min with cold air

For detection of AZI, the plate was immersed with the Chromatogram Immersion Device into sulfuric acid reagent (1:4 in ethanol; immersion speed 3 cm/s, immersion time 6 s), dried for 30 s with cold air, and heated at 100 °C for 5 min using the TLC Plate Heater.

TLC Visualizer under UV 254 nm, UV 366 nm, and white light

TLC Scanner 3 and winCATS, absorption measurement at 235 nm for CEFI and 530 nm for AZI, slit dimension 6.00 x 0.45 mm, scanning speed 20 mm/s, spectra recording from 190 to 550 nm

The target zones were marked with a pencil, and plates were cut carefully to separate different bands. Individual bands were cut and sonicated with methanol for extraction of degradation products. Methanol fractions were concentrated and evaporated to obtain solid residues which were analysed by Q-TOF and Ion Trap mass spectrometry in the positive ionization mode

Contact: Dr. Vandana Gawande, Department of Pharmaceutical Chemistry, STES’s Sinhgad Institute of Pharmacy, Narhe, Pune – 411041, Maharashtra, India, gawandevandana848[at]gmail.com