Optimized Griess Reaction for UV-Vis and Naked-eye Determination of Anti-malarial Primaquine

Summary

This protocol describes a novel colorimetric method for antimalarial primaquine (PMQ) detection in synthetic urines and human serums.

Abstract

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening infections caused by P. vivax and ovale. However, PMQ has unwanted adverse effects that lead to acute hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. There is a need to develop simple and reliable methods for PMQ determination with the purpose of dosage monitoring. In early 2019, we have reported an UV-Vis and naked-eye based approach for PMQ colorimetric quantification. The detection was based on a Griess-like reaction between PMQ and anilines, which can generate colored azo products. The detection limit for direct measurement of PMQ in synthetic urine is in the nanomolar range. Moreover, this method has shown great potential for PMQ quantification from human serum samples at clinically relevant concentrations. In this protocol, we will describe the technical details regarding the syntheses and characterization of colored azo products, the reagent preparation, and the procedures for PMQ determination.

Introduction

PMQ is one of the most important antimalarial drugs, it works not only as a tissue schizontocide to prevent relapse but also as a gametocytocide to interrupt disease transmission^1,2,3,4. Intravascular hemolysis is one of the concerning side effects of PMQ, which becomes extremely serious in those deficient in G6PD. It is known that the G6PD genetic disorder is distributed worldwide with a gene frequency between 3-30% in malaria endemic areas. The severity of PMQ weakness depends on the degree of G6PD deficiency as well as the dose and t....

Protocol

1. Synthesis of Colored Azos

1. In a 25 mL round bottom flask (RBF), dissolve aniline (0.1 mmol) and primaquine bisphosphate (45.5 mg, 0.1 mmol) into 10 mL of H₃PO₄ solution (5% v/v). Put the RBF on an ice bath, add a stir bar with the proper size into the solution, and put the RBF on a stir plate.
   NOTE: For the synthesis of azo 3g (Figure 2), use 0.2 mmol of primaquine bisphosphate.
2. Dissolve NaNO₂ (6.9 mg, 0.1 mmol) in 1 mL of cooled water and then add into the reaction mixture dropwise. Remove the ice bath, and keep the reaction mixture stirred ....

Results

To optimize the reaction conditions (Figure 2), various anilines were used to couple with PMQ through the Griess reaction. We have achieved a series of azos with different colors. It has been found that anilines with an electron donating substituent can cause a red-shift in the UV-vis absorption spectrum. Theoretical calculations were carried out through time dependent density functional theory (TD-DFT). As presented in Figure 2A, the calculation result was in g.......

Discussion

We described a colorimetric method for convenient PMQ quantification. It is potentially the most simple and cost-effective current method. More importantly, this method offers enables naked-eye based PMQ measurement without using any equipment.

The optimized Griess reaction for PMQ detection can generate a red color azo with a maximum absorption at 504 nm. The potential influence from UV-vis absorption of endogenous biomolecules is limited, thus making the method promising for direct measureme.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4-Methoxyaniline	Aladdin	K1709027
2,4-Dimethoxyaniline	Heowns	10154207
3,4-Dimethoxyaniline	Bidepharm	BD21914
4-Methylaniline	Adamas-beta	P1414526
4-Nitroaniline	Macklin	C10191447
96-wells,Flat Botton	Labserv	310109008
Gaussian@16 software	Gaussian, Inc		Version:x86-64 SSE4_2-enabled/Linux
Hydrochloric acid	GCRF	20180902
Marvin sketch (software)	CHEMAXON		free edition: 15.6.29
Phosphoric acid	Macklin	C10112815
Primaquine bisiphosphate	3A Chemicals	CEBK200054
Sodium nitrite	Alfa Aesar	5006K18R
Sulfonamides	TCI(shanghai)	GCPLO-BP
Varioskan LUX Plate reader	Thermo Fisher		Supplied with SkanIt Software 4.1