HPTLC detection of falsification in drugs

Mrs. Carole Vrignaud and her colleagues at the Anti-counterfeiting Laboratory (LCAC) at Sanofi Tours, France, employ chromatographic separation techniques, especially HPTLC, to develop new qualitative analytical methods for detection of falsified drugs, in a variety of sample matrices. The world drug market is about 1000 billion € (2019). The World Health Organization (WHO) considers that half of the drugs sold through the internet are fakes. This is why Sanofi group in 2008 created this unique laboratory to fight against drug falsification using various analytical methods, including HPTLC. The main objective is to confirm the presence of active ingredients (API) or excipients (preservatives, flavoring…) in each Sanofi formulation. HPTLC is also often used to detect degradation of products stored under inappropriate conditions, as well as unexpected compounds, allowing characterization of the falsified products. To detect falsification of drugs, a generic HPTLC method was developed.

HPTLC is well suited for a rapid parallel screening of many samples. Up to 10 samples can be analyzed in less than one hour. The developed method is simple and can be perform ed with little consumption of solvents and reagents.

Standard solutions

20 mg of API no. 1, 55 mg of API no. 2, 53 mg of API no. 3, and 51 mg of impurity of API no. 2 are individually dissolved in 10 mL of water (each).

Sample preparation

One tablet of each suspected case of falsification is milled, and then extracted with 7.5 mL of water by vortexing. After centrifugation, the filtered supernatant is transferred into a 5 mL volumetric flask and filled up to the mark.

Chromatogram layer

HPTLC plates silica gel 60 F₂₅₄ (Merck), 20 x 10 cm, pre-washed with methanol, are used.

Sample application

Samples and standard solutions are applied as bands with the Automatic TLC Sampler (ATS 4), 14 tracks, band length 8.0 mm, distance from left edge 20.0 mm, distance from lower edge 8.0 mm. 1.0 μL of sample solutions and 3.0 μL of standard solutions are applied.

Chromatography

In the ADC 2 with chamber saturation (20 min) and after conditioning at 33% relative humidity for 10 min using a saturated solution of magnesium chloride, development with dichloromethane – ethanol – water – formic acid 9:9:1.5:0.25 (V/V) to the migration distance of 50 mm (from the lower edge), followed by drying for 5 min.

Post-chromatographic derivatization

First, the plates are manually sprayed with Dragendorff’s reagent (commercial solution) and dried for 60 s at room temperature. Second, the plates are manually sprayed with sodium nitrite solution (5%) and dried for 30 s at room temperature.

Documentation

Images of the plates are captured with the TLC Visualizer in UV 254 nm before derivatization and in white light after each derivatization step.

Densitometry

Absorbance measurement is performed with the TLC Scanner 3 and visionCATS at 272 nm, slit dimension 5.0 mm x 0.2 mm, measurement speed 20 mm/s, spectra recording from 200 to 450 nm.

Results and discussion

A representative plate with real samples is shown. During method development simulated and spiked samples gave the same R_F values as the standards and were well separated from matrix components. Any positive identification of API no. 2 and no. 3 can be confirmed by spectral comparison of samples and standards as well as the expected presence of impurity of API no. 2. Detection of API no. 1 (cases no. 1–7) is achieved after derivatization in white light. Results are shown for selected drug products as well as the identification of three active ingredients expected in different oral formulations. HPTLC is also used to estimate each quantity. API no. 3 is not present in the composition of the original tablets (cases no. 8–9). However, some previous cases were identified as counterfeit tablets containing API no. 3 instead of API no. 2.

In the shown real cases no falsification was detected. Nevertheless, the approach has proved suitable by several positive cases. HPTLC is therefore used at Sanofi as high throughput screening technique.

Further information is available on request from the authors.

Contact: Carole Vrignaud, 30-36 avenue Gustave Eiffel, 37100 Tours, France, carole.vrignaud@sanofi.com