Development of a Rapid Chemical Identification System (RCIS) for the Detection of Fraudulently Labelled 5-nitroimidazole Products

Purpose: A simple, reliable and rapid chemical identification system (RCIS) consisting of three colour reactions based on the functional groups in the molecule and two TLC methods was developed for preliminary detection of the 5–nitroimidazole drugs.Methods: Three members of this group of drugs (tablet form) available in the Nigerian market and labelled MA and MB for metronidazole, T A and TB for tinidazole and S for secnidazole, were used. The extraction of the active ingredient from the solid dosage form was performed using acetone. The reaction of the extracted drug with zinc and 1M hydrochloric acid at 100 oC converted the nitroimidazole group to a characteristic primary aromatic amine. TLC methods A and B were carried out on GF254 plates (5cm x 10cm) to further identify the individual members of the group. TLC method A with mobile phase consisting of acetone, ethyl acetate and ammonia (100:5:1) and method B with mobile phase consisting of acetone, chloroform and ammonia (100:15:1) were developed for the identification.Results: The aromatic character of 5-nitroimidazole was highlighted in nitric acid when combined with sulphuric acid resulting in an orange colour. 40% sodium hydroxide gave the alkali-induced characteristic orange colour of aromatic amino compounds. All the samples of the various brands gave characteristic colours that distinguished the compounds of the 5-nitroimidazole group as primary or secondary nitroimidazole compounds.Conclusion: Using the developed method, fraudulently labelled product 5-nitroimidazole antiprotozoal and antibacterial agents can now be detected in approx. 40 min with limited reagents and a simple TLCtechnique. The method is rugged, simple, and should be particularly handy for use in detecting substandard products of the drug in the drug distribution chain where sophisticated equipment are often not available.Keywords: 5–nitroimidazole; Fraudulently labeled; Chemical identification system; Antiprotozoal; Antibacterial

Fluorescence polarisation for high-throughput screening of adulterated food products via phosphodiesterase 5 inhibition assay.

The surge in the consumption of food products containing herbal aphrodisiacs has driven their widespread adulteration. A rapid screening strategy is, therefore, warranted to curb this problem. This study established an enzyme inhibition assay to screen phosphodiesterase 5 (PDE5) inhibitors as adulterants in selected food products. Fluorescein-labelled cyclic-3',5'-guanosine monophosphate was utilised as substrates for the PDE5A1 enzyme, aided by the presence of nanoparticle phosphate-binding beads on their fluorescence polarisation. The sample preparation was optimised to improve the enzyme inhibition efficiency and applied to calculate the threshold values of six blank food matrices. The assay was validated using sildenafil, producing an IC50 of 4.2 nM. The applicability of the assay procedure was demonstrated by screening 55 distinct food samples. The results were subsequently verified using confirmatory liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis. Altogether, 49 samples inhibited the PDE5 enzyme above the threshold values (75.7%-105.5%) and were registered as potentially adulterated samples. The remaining six samples were marked as nonadulterated with percentage inhibition below the threshold values (-3.3%-18.2%). The LC-HRMS analysis agreed with the assay results for all food products except for the instant coffee premix (ICP) samples. False-positive results were obtained for the ICP samples at 32% (8/25), due to possible PDE5 inhibition by caffeine. Contrarily, all other food samples were found to produce 0% (0/30) false-positive or false-negative results. The broad-based assay, established via a simple mix-incubate-read format, exhibited promising potential for high-throughput screening of PDE5 inhibitors in various food products, except those with naturally occurring phosphodiesterase inhibitors such as caffeine