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

Primaquine is a well-known anti-malarial drug. The determination of primaquine in biological fluids is often required to assess the pharmacokinetics. Currently, the major techniques for Primaquine determination are based on HPLC.

The procedures of HPLC can be quite complicated and time-consuming. So this method offers a simple way for rapid detection of Primaquine. This Griess method is potentially the most simple and cost-effective way for Primaquine quantification.

Moreover, this method can offer possibilities for naked-eye Primaquine detection without the requirement of any equipment. Besides serum and urine samples, this method could also be used to quantitatively determine Primaquine in pharmaceutical formulations, for example, the determination of Primaquine in tablets for manufacturing quality control. Demonstrating the procedure will be Miss Wu Yalan and Wu Shengjun, two graduate student from my lab.

Start by dissolving 0.1 millimoles of aniline and primaquine biphosphate into 10 milliliters of 5%phosphoric acid solution in a 25 milliliter brown bottom flask. Put the flask on an ice bath, and add a stir bar. Then put the ice bath on a stir plate.

Dissolve 6.9 milligrams of sodium nitrate and one milliliter of cooled water and add it to the flask drop-wise. Remove the ice bath and keep the reaction mixture stirring at room temperature. Monitor the reaction with a silica gel-coated, thin-layer chromatography or TLC plate, using a dichloromethane methanol mixture as the eluent.

The azo product exhibits colored spots on the TLC plate. Stop the reaction when the PMQ spots disappear. Adjust the pH of the reaction mixture to greater than 10 by adding sodium hydroxide solution to an ice bath.

Then, use a 50 milliliter separation funnel to extract the mixture three times with 20 milliliters of ethyl acetate per extraction. When finished, combine and concentrate the organic phase under vacuum using a rotary evaporator. Purify the residues using flash chromatography with reverse phase silica gel under normal pressure.

Then, dry the solution by lyophilization to obtain the desired azo product. To measure the UV vis absorption spectra, dissolve the pure azo in distilled water or 5%phosphoric acid, and use the spectrophotometer to record the absorption spectra at room temperature. To measure PMQ absorption, start by dissolving 4-methoxyaniline in 0.2 molar hydrogen chloride for a 200 millimolar aniline solution.

Then dissolve sodium nitrite in distilled water to obtain five millimolar solution. Keep the solutions at four degrees Celsius. Add 100 microliters of the aniline solution to a 96-well plate.

Then add 50 microliters of the PMQ-containing sample, and mix it with the aniline. Add 50 microliters of the sodium nitrite solution to the plate, and mix the contents by pipetting. Keep the plate at room temperature for 15 minutes, then measure the absorbance of the solution at 504 nanometers.

Export the data as a spreadsheet file for further analysis. To construct a calibration curve for PMQ measurements in urine samples, prepare PMQ solutions in synthetic urine at concentrations of zero, one, two, five, 10, 20, 50, 100, and 200 micromolar. Prepare the PMQ detection reaction, and measure absorbance as previously described.

Then generate a calibration curve based on the 504 nanometer absorbance and the PMQ concentrations. Subtract the absorbance values of the samples without PMQ from the test measurements, and perform a linear fit to generate a linear equation y equals x plus b, where y is the absorbance and tensity, and x is the concentration of PMQ. To construct a calibration curve for PMQ measurement in human serum samples, prepare PMQ solutions in human serum with PMQ concentrations of zero, one, two, five, 10, 20, 50, 100, and 200 micromolar.

Prepare the PMQ reaction. Measure absorbance, and construct the calibration curve as previously described. Add six milliliters of seven to one ethyl acetate hexane into two milliliters of PMQ-containing serum in a 15 milliliter centrifuge tube.

Add 100 microliters of two molar sodium hydroxide solution to the extraction system, and vortex the tube for 30 seconds. Then, collect the organic layer and concentrate it with a rotary evaporator under vacuum. Dissolve the residue in 200 microliters of distilled water, and filter through a disc-shaped, 220 nanometer pore-sized membrane to remove insoluble lipid components.

Construct the calibration curve for PMQ in distilled water, then prepare a detection reaction with the extracted solution in a 96-well plate as previously described, and measure absorbance. Then, determine the concentration of PMQ according to the linear equation from the calibration curve. Reaction conditions were optimized by using various anilines to couple with PMQ through the Griess reaction.

Theoretical calculations were carried out, and the results were in good agreement with optical measurements. It was determined that 4-methoxyaniline was optimal for the PMQ detection reaction, due to its good performance and reaction rate, product solubility, and stability. Moreover, the azo product for 4-methoxyaniline is red in color, which is easy to distinguish with naked eyes.

The effects of pH on the UV vis absorption of the azo product and the PMQ solutions were tested. While increasing pH from 1.0 to 7.0 does not change absorbance, basic pH has a significant effect. Calibration curves were constructed for the detection of PMQ in urine and serum samples.

A linear relationship was found when PMQ ranges from zero to 200 micromolar in urine, and from 10 to 200 micromolar in serum. To simulate a real PMQ-containing serum, PMQ was added into human serum at 0, 0.2, 0.5, 1.0, 2.0 micromolar concentrations. Using this protocol, the concentrations recovered were found to be 0.02, 0.14, 0.44, 0.90, and 1.78 micromolar, resulting in 90%recovery for concentrations over 0.5 micromolar.

It should be noted that the time for the reaction to reach its saturated intensity is temperature-dependent. Normally, 10 to 20 minutes is enough for temperature between 20 to 30 degrees Centigrade. However, to conclude our example of Griess chemistry for primaquine detection.

Similar design is also suitable for other relevant molecules that have similar chemical reactivity as primaquine.