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

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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 the duration of PMQ exposure^5,6. To lower the risk, the WHO has recommended a single low dose (0.25 mg base/kg) of PMQ for malaria treatment. However, this is still challenged by the variations in patient drug sensitivity^5,7. Dose monitoring is necessary to assess the pharmacokinetics after PMQ administration, which can effect dosage adjustment for a successful treatment with limited toxicity.

High-performance liquid chromatography (HPLC) is the most widely used technique for PMQ clinical determination. Endoh et al. reported a HPLC system with a UV detector for serum PMQ quantification using a C-18 polymer gel column⁸. In their system, serum proteins were first precipitated with acetonitrile, and then the PMQ in the supernatant was separated for HPLC. The calibration curve was linear over the concentration range from 0.01-1.0 μg/mL⁸. Another method based on a reverse-phase HPLC with UV detection at 254 nm has been reported for the quantification of PMQ and its major metabolites⁹. The calibration curve for PMQ was linear in the range between 0.025-100 μg/mL. An additional liquid-liquid extraction with mixed hexane and ethyl acetate as organic phase was used for PMQ separation with percentage recovery reached to 89%⁹. More recently, Miranda et al. developed an UPLC method with UV detection at 260 nm for PMQ analysis in tablet formulations with a detection limit at 3 μg/mL¹⁰.

Though HPLC methods exhibit promising sensitivity in drug determination and the sensitivity can be further improved if the HPLC is equipped with a mass spectrometer, there are still some disadvantages. Direct drug measurements in biological fluids are usually inaccessible by HPLC, since many biomolecules can greatly influence the analysis. Additional extractions are required to remove endogenous molecules before HPLC analysis^11,12. Moreover, PMQ detection by a HPLC-UV detector is typically performed at its maximum absorption wavelength (260 nm).; however, there are many endogenous molecules in biological fluids with a strong absorbance at 260 nm (e.g., amino acids, vitamins, nucleic acids and urochrome pigments), thus interfering with PMQ UV detection. There is a need to develop simple and cost-effective methods for PMQ determination with reasonable sensitivity and selectivity.

The Griess reaction was first presented in 1879 as a colorimetric test for nitrite detection^13,14,15,16. Recently, this reaction has been extensively explored to detect not only nitrite but also other biologically relevant molecules^17,18,19,20. We have previously reported the first systematic study of an unexpected Griess reaction with PMQ (Figure 1). In this system, PMQ is able to form colored azos when coupled with substituted anilines in the presence of nitrite ions under acidic conditions. We have further found that the color of azos varied from yellow to blue when increasing the electron donating effect of the substituent on anilines²¹. A UV-vis absorption based colorimetric method for PMQ quantification has been developed through the optimized reaction between 4-methoxyaniline and PMQ. This method has shown great potential for sensitive and selective detection of PMQ in bio-relevant fluids. Here, we aim to describe the detailed procedures for PMQ determination based on this colorimetric strategy. 

Protokół

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 at room temperature.
3. Monitor the reaction with a silica gel coated thin-layer chromatography (TLC) plate. Use a dichloromethane (DCM)/methanol (MeOH) mixture (vol/vol = 5:1) as the eluent for TLC. The azo product exhibits colored spots on the TLC plate, which is easy to distinguish by naked eyes. Stop the reaction when the PMQ spots disappear on TLC.
4. Adjust the reaction mixture to pH >10 by NaOH (2 M) on an ice bath. Use a 50 mL separation funnel to extract the mixture 3 times with 20 mL of ethyl acetate for each, combine and concentrate the organic phase under vacuum using a rotary evaporator.
   NOTE: Before extraction, adjust the pH value of reaction solutions over 10. This can maintain the primary amine as its non-ionized form, thus facilitating extraction.
5. Purify the residues by flash chromatography with reverse-phase silica gel under normal pressure, using MeOH/H₂O as the eluent. Dry the product solution through lyophilization to give desired azo products.
   NOTE: The same reaction can also be performed in diluted HCl solutions (0.2 M).

2. UV-Vis Measurements and Theoretical Calculation

1. Dissolve pure azo (50 μM) in distilled water or in 5% H₃PO₄ solution (pH 1.1), respectively. Record UV-vis absorption spectra (250-700 nm) on a spectrophotometer at room temperature (25 °C). Export the data as .xls/.xlsx files for further analysis.
2. Perform all theoretical calculations for PMQ itself and azo products using the Gaussian 16 program. Use time dependent density functional theory (TD-DFT) with a 6-31G basis set. Include solvent effects by polarizable continuum model (PCM) formalism using water.
   1. Use software (e.g., Chemdraw Office) to draw the structures and then save the structure as a Gaussian input file (.gif).
   2. Open the gif file with Gauss View and click the button Calculate. Select Gaussian Calculation Setup, Opt+Freq, and ground state-DFT-B3LYP-6-31G; then click Submit. The geometry optimization will generate a .log file.
   3. Following the procedure above, use Gauss View to open this log file. Click Calculate-Gaussian Calculation Setup and select energy and TD-SCF-DFT-B3LYP-6-31G-Singlet only. Then Submit. The energy calculation will generate another log file and a cube file.
   4. Use Gauss View to open the log file from the energy calculation. Click Results-UV/Vis to see the predicted absorption.
   5. Use Gauss View to open the cube file. Click Results and select surface and contours-surface actions and new surface to see the orbits.
3. Compare the results from both experimental measurement and Gaussian calculation. Calculate the percent error between the calculated and measured values, according to the following equation.
                 Error = | (W_max cal.-W_maxexper.)/ W_maxexper. | × 100%
   where W_max cal. represents the maximum absorbance wavelength from theoretical calculation and W_max exper. represents the wavelength from experimental result.

3. PMQ Determination

1. PMQ measurement using a 96-well plate (Figure 5)
   1. Dissolve 4-methoxyaniline in 0.2 M HCl for a 200 mM aniline solution, R1. Dissolve sodium nitrite in distilled water to obtain a 5 mM solution, R2. Keep all the solutions in the fridge at 4 °C before use.
   2. Add 100 µL of R1 into a 96-well plate, and add 50 µL of PMQ containing sample into the plate to mix with R1. Then, add 50 µL of R2 into the plate. Mix the solutions by repeated pipetting.
   3. Keep the plate at room temperature for 15 min, and then record the UV-vis absorbance at 504 nm. Repeat 3x for each test.  The azo product is stable with room light exposure; it not necessary to keep the plate under dark.
   4. Export the data as .xls/.xlsx files for further analysis.
2. Calibration curve for direct PMQ measurement in a urine sample
   1. Prepare PMQ solutions using synthetic urine with PMQ concentrations at 0, 1, 2, 5, 10, 20, 50, 100, 200 μM, respectively.
   2. Add 100 µL of R1 into a 96-well plate, and add 50 µL of PMQ urine solution to mix with R1. Then, add 50 µL of R2 to the above mixture. Mix the solutions by repeated pipetting. Keep the plate at room temperature for 15 min, and then record the UV-vis absorbance at 504 nm.
   3. Generate a calibration curve based on the absorbance I₅₀₄ and PMQ concentrations. Use the values from the wells without PMQ as a blank, and subtract the blank values from all tests before data processing.
   4. Perform a linear fit to generate the linear equations as Y = aX+b, where Y is the absorbance intensity at 504 nm, X is the concentration of PMQ, a is the slope, and b is the y-intercept of the linear line.
3. Calibration curve for direct PMQ measurement in a human serum sample
   1. Prepare PMQ solutions using human serum with PMQ concentrations at 0, 1, 2, 5, 10, 20, 50, 100, 200, μM respectively.
   2. Add 100 µL of R1 into a 96-well plate and add 50 µL of PMQ serum solution to mix with R1. Add 50 µL of R2 to the above mixture and mix the solutions by repeated pipetting. Keep the plate at room temperature for 15 min and then record the UV-vis absorbance at 504 nm. Export the data as .xls/.xlsx file for further analysis.
   3. Generate a calibration curve based on the absorbance I₅₀₄ and PMQ concentrations. Use the values from the wells without PMQ as a blank, and subtract the blank values from all tests before data processing.
   4. Perform a linear fit to generate the linear equations as Y = aX+b, where Y is the absorbance intensity at 504 nm, X is the concentration of PMQ, a is the slope, and b is the y-intercept of the linear line.
4. PMQ extraction from serum
   1. Add a certain amount of PMQ into human serum to simulate PMQ-containing serum. For PMQ extraction, add 6 mL of mixture of ethyl acetate/hexane (7:1 v/v) into 2 mL of PMQ-containing serum in a 15 mL centrifuge tube.
   2. Add 100 µL of sodium hydroxide (2 M) solution to the extraction system. Violently shake the tube using a vortex mixer for 30 s. Collect the organic layer and concentrate it using a rotary evaporator under vacuum.
   3. Redissolve the residue with 200 µL of distilled water and remove insoluble lipid components by filtration through a disk-shaped membrane with 220 nm pore size. Use the final solution for test.
5. Determine PMQ from the serum with extraction
   1. Follow steps 3.2 or 3.3 to generate the calibration curve for PMQ in distilled water. Extract PMQ from PMQ-containing serums according to step 3.4.
   2. Add 100 µL of R1 and 50 µL of PMQ solution into a 96-well plate. Add 50 µL of R2 to above mixture, and mix the solutions by repeated pipetting.
   3. Keep the plate at room temperature for 15 min and record the UV-vis absorbance at 504 nm. Use the wells with R1 and R2 but without PMQ as controls. Export the data as .xls/.xlsx files for further analysis.
   4. Subtract the control values from the absorbance values I₅₀₄ for each test, and then use the result for concentration calculations according to the liner equation from the calibration curve.
      NOTE: The limit of detection (LOD) for PMQ in all cases can be calculated according to a standard method²². Calculation was based on the calibration function: LOD = 3.3 × SD/b, where SD is the standard deviation of the blank and b is the slope of the regression line

Wyniki

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...

Dyskusje

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...

Materiały

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