(M1130-09-53) Comparing Chromatic and Fluorometric Methods for Gentamicin Sulfate Detection by Ninhydrin Complexing and o-phthaldialdehyde Reagent

Purpose: Gentamicin sulfate (GS) is a well-known aminoglycoside used for treating many common pathogens such as E. coli, P. aeruginosa, and multi-drug administrations for staphylococcal and enterococcal infections.¹ Studying GS can be challenging in laboratory settings due to the lack of fluorophores or chromophores for spectroscopic detection. While high-performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LCMS) assays have been developed, these methods require lengthy sample preparation and high levels of expertise to execute.² To ease experimental burden, an alternate fluorometric assay using o-phthaldialdehyde (OPA) has been developed.³ While relatively faster and easier to use than HPLC and LCMS, OPA uses organic solvents and toxic chemicals with reduced sensitivity ranges. The aim of this study was to optimize a fast, sensitive, and easily reproducible chromatic method for GS detection using ninhydrin and compare its performance to that of OPA.

Methods: GS solutions were diluted to 0.5-500µg/mL for calibration in 750µL aliquots of 1xPBS. The ninhydrin method was adapted from Frutos et al.⁴ It involved preparing a 1-10 mg/mL stock solution of ninhydrin in 1xPBS. 750µL of the ninhydrin stock was then added to GS aliquots to achieve a total volume of 1.5mL. The reaction was facilitated and optimized by placing samples in a heat block set between 75-95ºC. The samples were heated for 14-16min, during which the solution changed color from yellow to violet. Immediately, samples were transferred to an ice bath to cool for 10min. Analysis was conducted using UV-Vis spectrometry at λmax as determined by spectral scanning.

The OPA method was modeled after Gubernator et al.³ To prepare the OPA reagent, 0.2g of o-phthaldialdehyde was dissolved in 1mL methanol. The solution then was mixed with 19mL of 0.4M boric acid buffer. A sample of the OPA reagent was then diluted by adding 1mL reagent to 8mL methanol. To facilitate the reaction, 80µL of GS was combined with 48µL of methanol and 72µL of diluted OPA reagent in a 96-well plate and incubated for 10min with a plate cover. Immediately after, the sample was analyzed via fluorescence microplate reader at 340nm excitation and 455nm emission.

Four reaction parameters were examined to optimize the ninhydrin method: temperature, time, ninhydrin concentration, and drug stability. Temperatures studied included 75, 85, and 95ºC. Time of reaction was varied +/- 1min. Ninhydrin stock concentrations included 1,2, 4, and 10mg/mL. Drug stability was quantified by heating stock solutions of GS sulfate at 95ºC for 15min prior to reacting them with the ninhydrin complex.

Results: λmax for the ninhydrin method was determined to be 403nm (Figure 1). Using the Beer Lambert law, sensitivity was established from 2 to 200µg/mL with a determination coefficient r² > 0.998 (Figure 2.A). These results were achieved 3 times with less than 3% difference at all concentrations to confirm reproducibility. The OPA method was sensitive from 0.5 to 40µg/mL and occasionally up to 80µg/mL. Its determination coefficient was r² > 0.997 (Figure 2.B).

The optimization experiments suggested that the best conditions for ninhydrin complexing were to heat solutions for 15min at 95ºC using a 2mg/mL ninhydrin stock (Figure 3). Temperature should be strictly adhered, as decreased heating significantly reduced sensitivity. GS was shown to be extremely stable at high temperatures, incurring no significant change in concentration when heated prior to ninhydrin complexing. Additionally, the reaction could be carried out +/- 1min, incurring differences less than 5%. On the other hand, the 10min incubation period with OPA had to be strictly adhered in order to achieve reliable results. This could be due to OPA’s use of organic solvents. Incubation time, slight changes in reagent concentration, and risk of methanol evaporation may contribute to lack of accuracy.

Conclusion: Both OPA and ninhydrin are valid methods for the detection of GS sulfate. While OPA is more sensitive at concentrations less than 2µg/mL, ninhydrin complexing offers a broadened detection up to 200µg/mL. Ninhydrin’s calibration curves are reproducible, thus eliminating the need to generate one for each trial. Its reaction conditions are safer and more flexible, easing the burden of strict organic solvents, toxic chemicals, and strict incubation required by OPA. Its major limitation involves storage and reliance on heat. Ninhydrin is light-sensitive, and thus its solutions can only be stored up to 24 hours. OPA reagent can be stored at 4ºC for 2 weeks. GS’s thermal stability makes it well-suited for ninhydrin complexing at 95ºC. Ultimately, the results of this study support the potential use of ninhydrin complexing for GS detection, particularly in laboratory settings requiring quick, reproducible, and sensitive methodology accessible to a broad range of experimental expertise.

References: [1] Bruno et al. StatPearls (2021); [2] Hari et al. J Appl Pharm (2019); [3] Gubernator et al. Int J Pharm (2006); [4] Frutos et al. J Pharm Biomed (2000).

Acknowledgements: Disclosures: Cecilia M. Nepple (N), Meghna Krishnagiri Tupal (N), Dinesh Dhamecha (N), Dmitry Gil (N), Keith Wannomae (N), Shannon Hugard (N), Rachel Connolly (N), Ebru Oral (1-Corin, Zimmer Biomet, Mako, Iconacy, Renovis, Arthrex, ConforMIS, Meril Healthcare, Exactech), Orhun K. Muratoglu (1-Corin, Zimmer Biomet, Mako, Iconacy, Renovis, Arthrex, ConforMIS, Meril Healthcare, Exactech, Cambridge Polymer Group; 4-Cambridge Polymer Group, Orthopedic Technology Group, Alchimist)


Figure 1. Reaction diagram of the GS-ninhydrin complex. The primary amine groups in GS, highlighted purple, react with ninhydrin’s hydroxy groups to form the chromophore known as “Ruhemann's purple.” Abbreviations: GS = gentamicin sulfate


Figure 2.A. Optimized calibration curve of GS concentrations using ninhydrin complexing. The ideal range is 2 to 200μg/mL. Figure 2.B. Optimized calibration curve for gentamicin concentrations using o-phthaldialdehyde. Sensitivity spans from 0.5 to 40μg/mL. Abbreviations: GS = gentamicin sulfate


Figure 3. Table summarizing the advantages and disadvantages of the ninhydrin and OPA methodologies for gentamicin sulfate detection. Note the primary distinctions are bolded for emphasis. Abbreviations: OPA = o-phthaldialdehyde; UV-Vis = ultraviolet and visible light