A rapid, ideal, and eco-friendlier protocol for quantifying proline.

Area of Science:

• Biochemistry
• Analytical Chemistry

Background:

• Proline is a crucial stress marker routinely quantified using a hazardous toluene-based protocol.
• Toluene poses negative health impacts, necessitating the development of a safer alternative.

Purpose of the Study:

• To develop and validate a reliable, eco-friendlier protocol for proline quantification.
• To compare the sensitivity and efficiency of the new protocol against existing methods.

Main Methods:

• Proline quantification via ninhydrin reaction at 100°C.
• Absorbance measurement of the proline-ninhydrin condensation product directly in the reaction mixture.
• Optimization of reagent concentrations (ninhydrin in glacial acetic acid) and reaction times.

Main Results:

• The new protocol, measuring absorbance in the reaction mixture (λmax 508 nm), showed higher sensitivity than toluene-based methods (λmax 513 nm).
• Ninhydrin in glacial acetic acid, without H3PO4, yielded higher proline-ninhydrin product quantity and faster reaction rates.
• The optimized protocol uses 1 ml proline sample, 2 ml 1.25% ninhydrin in glacial acetic acid, heated at 100°C for 30 min.

Conclusions:

• The developed protocol is simple, rapid, reliable, cost-effective, and eco-friendlier than existing toluene-based methods.
• Eliminating toluene and optimizing reaction conditions enhances proline quantification sensitivity and efficiency.