Fluconazole is a drug (triazole) antifungal, as depicted in Scheme 1 that is used to treat superficial and systemic candidiasis, as well as cryptococci infections in individuals with acquired immunodeficiency syndromes (AIDS) . It works by inhibiting the ergosterol production, a major part of the fungal cell membrane like antibiotics . Fluconazole is less lipophilic than other azoles (e.g., ketoconazole, itraconazole and miconazole). It was first synthesized in 1982. The inclusion of a halogen–phenyl group boosts antifungal action and water solubility, resulting in increased bioavailability . Used to protect people from getting yeast infections before a bone marrow transplant who are at risk for getting them because they are receiving chemotherapy or radiation treatment. Even today, fluconazole is frequently used to treat fungi, including C.  infection with albicans. Studies on fluconazole resistance mechanisms are widespread. Nevertheless, despite comprehensive awareness of these pathways, not all therapy failures may be attributed to the resistance development. Indeed, it has been discovered that several isolates that were fluconazole sensitive in vitro cannot be effectively treated in the patient. Some clinical isolates of C. are well-known to have C. fluconazole is more tolerated by some albicans than others. When medication concentrations are greater than their minimal inhibitory concentration, these are characterized by enhanced growth, known as residual growth . There are numerous methods to determine fluconazole such as mass spectroscopy , Uv-spectrophotometry , NIR spectroscopy, and chemo metrics .
Scheme 1. The structure of fluconazole
Materials and methods
The results from incident light attenuation from 0 to 180 degrees were collected, this experiment involved building a flow cell (the cell that the material flow in it) with a home-built NAG-4SX3-3D analyzer. The data from the output signals was recorded by using a potentiometric recorder. A peristaltic pump (Ismatec) with a sample loop and a six-way injection valve is used (Teflon, variable length). A Shimadzu UV spectrophotometric and turbidimetric equipment was used to conduct the routine operations.
The solutions were prepared with distilled water and only analytical reagent-grade ingredients were used. By dissolving 1.5314 g of fluconazole (C13H12F2N6O, molecular weight 306.271 g.mol-1) in 250 mL of distilled water, fluconazole, with a standard concentration of 0.02 M and a molecular weight of 306.271 g.mol-1, was produced. A 0.012 M standard solution was made by dissolving 5.475 g of phosphomolybidic acid (H3Mo12PO40.12H2O), which has a molecular weight of 1,825.25 g.mol-1 in 250 mL of distilled water.
As can be seen in Figure 1, there are two distinct pathways making up the manifold strategy for identifying fluconazole in medicines. In the acidic medium, fluconazole and phosphomolybidic acid react to produce a pale yellow precipitate. A drug sample segment with a sample loop capacity of 175 µL is carried to the injection point through the first line from the manifold, which is a carrier stream of distilled water moving at a flow rate of 2.5 mL.min-1 and connected to an injection valve. The phosphomolybidic acid is supplied at a rate of 2.5 mL.min-1 (3 mmol.L-1) via the second line. The mV vs. time response data is collected by a locally designed turbidimeter (NAG-4SX3-3D Analyzer)  when the pale yellow precipitate forms at the Y-junction of the two lines. Scheme 2 depicts a similar ion pair mechanism for Flu-PMA  in acidic media. The obtained results are indicated in Figure 2.
Figure 1. The design of the used manifold to estimate fluconazole
Scheme 2. The proposed reaction for fluconazole with phosphomolybidic acid system
Figure 2. The continuous repeated on-line measurements
Results and Discussion
The optimal conditions for producing the reaction's ultimate result, a pale-yellow precipitate, depicts in Figure 1 employing a flow injection manifold system to provide the optimum accuracy and sensitivity. We had the most success when ignored all other variables and concentrated just on these spots for enhancement.
Effect of variable concentration of phosphomolybdic acid
The stock solution is diluted with distilled water. Phosphomolybdic acid solutions with concentrations ranging from (2-8) mmol.L-1 were produced and carried out three times each. Figure 3 illustrates how the NAG-4SX3-3D analyzer's energy transducer response varies with phosphomolybdic acid concentration. When utilizing different concentrations of the reagent phosphomolybdic acid from (2 to 6) mmol.L-1, the increased in S/N energy transducer response might be due to the dispersion of the precipitate particle as the phosphomolybdic acid concentration increased. As a consequence, 6 mmol.L-1 for phosphomolybdic acid was the best concentration.
Figure 3. The reagent concentrations impact the response profile
Effect of different medium (salts and acids)
Fluconazole -phosphomolybdic acid was tasted by using the characteristics of the fluconazole system. a carrier stream was used to test the impact of different solutions. Along with the aqueous medium, the other solution media with a 50 mmol.L-1 concentration include CH3COOH, tartaric acid, ascorbic acid, HCl, HNO3, H2SO4, KCl, CH3COONH4, NH4Cl, NaNO3, NaCl, and Na2CO3. Figure 4 shows a reduction in S/N response caused by the tested material. This could be the outcome of the agglomeration growth or an increase in terms of density and compactness overall. All chemicals except H2SO4 cause the S/N response to go down. This is because H2SO4 creates tiny solid particles that shorten the distances between spaces and reduce the light amount that gets through. Due to the increased number of gaps between agglomerations of particle, the incident light intensity rises as a result. H2SO4 was selected as the optimum carrier stream for fluconazole in this investigation because it was satisfactory for the sensitivity and produced a stronger response.
Effect of H2SO4 concentration
From 30 to 150 mmol/L of H2SO4 were produced as solutions. An open valve mode was employed. Each experiment was carried out three times and the results indicated that the attenuation of incoming light rose as the concentration of H2SO4 did. This was due to the nucleus-like morphology of some microscopic particles, which helped them clump together into dense blocks. More than 100 mmol.L-1 of incident light will be attenuated, and micro size solubility will rise as a result. It was found that a carrier stream of 100 mmol.L-1 H2SO4 worked best, as shown in Figure 5.
Figure 4. The effect of different medium
Figure 5. Effect of different concentrations of sulfuric acid on the response
The peristaltic pump was used to adjust the flow rates of the carrier stream and reagent for the fluconazole detection at 5 mmol.L-1. As demonstrated in Figure 6, 2.5 mL.min-1 of flow rate was selected for the carrier and reagent lines as an adequate flow rate for this particular project, despite the fact that a higher flow rate (˂2.5 mL.min-1 for the carrier stream and reagent) decreased sensitivity and resulted in a broader base of response (increase ΔtB).
Variable sample volumes were used with open valve mode. Figure 7 displays that 175 µL was the best sample volume for fluconazole, which is characterized by sharpness and a smooth response profile, as well as economy and reduced analysis time. Up to the sample volume of 175 µL for fluconazole as sample volume grew, so did the peak of the reaction, but the response profile remained unchanged. There was a continuous growth in peak maxima as the volume was increased to over 175 µL. The effects are illustrated in Figure 7.
Figure 6. The influence of flow rate in mL.min-1
Figure 7. The sample volume response profile to the energy transducer response when fluconazole was detected by using the NAG-4SX3-3D analyzer
Effect of reaction loop lengths
The system utilized was fluconazole (5.0 mmol.L-1), phosphomolybdic acid (6.0 mmol.L-1), and sulfuric acid (100 mmol.L-1). There was a look at how much of a role the reaction coil plays. How long a reaction coil is plays a role in how well and how completely chemical reactions are carried out. We used coils of varying lengths (from 0 to 30 cm) and volumes (from 0 to 942 µL), which were all connected in series through the Y-junction of the fluid system (Figure 8 and Table 1). Other factors, such as base width (ΔtB), widening at peak maxima, and the departure time for sample segment from injection valve to measure cell are also responsible. This may be because of the continuously increasing effect of dilution and dispersion on the precipitated segment and the continuously longer time duration of precipitate species in front of the detector.
Figure 8. The response of coil volume in different volumes
Study of Y-junction point
In the process, the Y-junction is required for reactant mixing. Before measuring the cell directly in the flow system, the Y-junction was attached. The effect of changing the Y-junction in different parameters on the response profile was explored. In Figure 9, we can see how the Y-junction is affected by the energy response of the S/N transducer. Particle agglomeration, regulation, and regular distribution were tested with a series of mixing chambers of varying volumes and a larger diameter at the point where they entered the flow tube. However, as interspatial distances increase and particle scattering and dispersion diminish our ability to block incoming light from increasing the energy transducer, in Figure 10, the height of response measurement as a manifold unit with two 3 mm (I.D.) entry and a 3 mm (I.D.) output was, therefore, suggested as a possible solution for removing the premix chamber or junction point. The information provided here suggests that the reactant mixing and the formation of precipitate particles in a sulfuric acid medium can occur without the use of a Y-junction.
A scatter plot is used to estimate the linear dynamic range for fluconazole's effect on S/N energy transducer response variation
With established optimal parameters, a collection of fluconazole solutions starting from 0.001to 13 mmol.L-1 were created.
Figure 9. Response profile of Yz (mV) - tmin(10mm) for the determination of fluconazole using NAG-4SX3-3D analyzer via the formation of pale yellow precipitate of fluconazole
Figure 10. A) A system used to measure fluconazole, B) Some time-based profiles and potentiometric scanning speed 1 cm.min-1
Figure 10a displays the response profile for this experiment. The linear calibration graph range for the fluctuation of the energy transducer response of the NAG-4SX3-3D Analyzer with fluconazole concentration was (0.001-6) mmol.L-1, according to a scatter plot diagram (Figure 10b). With a correlation value (r) of 0.9989, the height of the response grew as the analyte concentration rose until it reached 10 mmol.L-1. Figure 11 illustrates the variance ranges for the system (i.e. the scatter plot (0.001- 13) mmol.L-1, dynamic range (0.001-10) mmol.L-1, and working range (0.001-8) mmol.L-1. The obtained results are presented in Table 2.
Figure 11. Influence of fluconazole concentration on the response
Detection limit (LOD)
Fluconazole's detection limit was found by using three approaches: By using a scatter plot's lowest concentration as a starting point, both practically and theoretically, one can utilize the progressive dilution [11, 12]. Via phosphomolybdic acid as a precipitant, Table 3 lists the estimation of the detection limit for fluconazole.
The measurement's repeatability was compared with the relative standard deviation expressed as a percentage [14, 15]. At a constant fluconazole concentration, a set of six injections were measured. Fluconazole was administered at two different concentrations (2.5 and 13 mmol.L-1). As demonstrated in Figure 12, the relative standard deviation was less than one percent.
A newly devised methodology the NAG-4SX3-3D analyzer for the fluconazole detection by using precipitating agents, phosphomolybdic acid, was compared with the prior published approaches, including UV-spectrophotometric  and turbidimetric methods.
A spectrophotometric technique employing a quartz cell to measure absorbance in the range of 0.001- 2 mmol.L-1 at λmax = 260 nm. The scatter plot in Figure 13a displays the UV spectrum of fluconazole at 260 nm, Figure 13b shows that the optimum linear range is between 0.001 and 1 mmol.L-1, with a correlation value of 0.9986 and an R2 % of 99.73 % (n=16) (the number of measurement). 0.0003 mmol.L-1 (0.3 mol.L-1 corresponding to 0.0919 g/sample) was the detection limit.
Fluconazole interaction with PMA (2 mmol.L-1) concentration is used in the turbidimetric approach. The scatter plot in Figure 14a shows that the PMA system's appropriate linear range is (0.01-1) mmol.L-1, with a correlation coefficient of 0.9869 and an R2 % of 97.72 (n= 13). (Number of measurement). The system's scatter plot was depicted in Figure 14b and the system's results were summarized in Table 4. The detection limit was 0.007 mmol.L-1 (7 µmol.L-1 and 21.4389 µg/sample).
Figure 12. Profile of eight successive readings with repeatability for 2.5 mmol.L-1 and 13 mmol.L-1 concentrations of Fluconazole
Figure 13. A) Absorbance of UV-spectrum of fluconazole 0.0003 mmol.L-1 at λmax = 260 nm, B) The scatter plot via [Fluconazole] by utilizing the traditional technique at λmax=260 nm (0.001-2) mmol.L-1, n=20
Figure 14. A) The optimal PMA concentration utilized in the turbidimetric approach is displayed graphically, B) [Fluconazole turbidimetric]'s technique scatter plot
Determination of fluconazole in drugs by using NAG-4SX3-3D analyzer
Four pharmaceutical samples were analyzed for fluconazole by using the cutting-edge NAG-4SX3-SD analyzer. Two techniques were used in tandem with the continuous flow injection analysis via NAG-4SX3-3D analyzer: The UV measurement of absorbance at max=260 nm and turbidimetric measurement at 0-180° for the pale-yellow precipitate. We employed all three measuring techniques, and then utilized the standard addition procedure to analyze the data (Figure 15). The NAG-4S3-3D analyzer, an absorbance spectrophotometer, and the turbidimetric method were utilized. The (t-test) (Figure 16), which compares two main paths, and Table 5 show the practical content of an active component at the 95 percent confidence level and determination efficiency. Table 5 compares the created technique NAG-4SX3-3D analyzer with the officially declared amount (150 mg) by the initial computing t values  for each individual company and comparing them to tabulated t-values. The observed result demonstrated that the mean value and the cited value were significantly different. Following the establishment of this groundwork, the developed method can be used in the same way as the conventional addition techniques. When the calculated t-value is less than the tabulated t-value, it means that the created approach is statistically indistinguishable from the mentioned method by the company. These results show a major discrepancy between the mentioned and observed concentrations of the active component. Once this groundwork is laid, the generated method can be used in the same way as the conventional addition techniques. There are no substantial inconsistencies between the manufactured approach and the declared way by the corporation, as the computed t-value is smaller than the tabulated t-value. By using a paired t-test with a significance threshold of 0.05 (2-tailed), the NAG-4SX3-3D analyzer's results were compared with those from the UV-spectrophotometric method by using Shimadzu (double beam UV-1800) spectrophotometry and turbidity with a turbidity meter (HANNA, Hungary). Attenuation of incident light is measured from 0 to 180 degrees and is presented in Table 6. We assume that drugs from different companies are all part of the same population and disregard any distinctions between them. The data demonstrate that at the 5% significance level, there was no statistically significant difference between the developed methodology and the UV-spectrophotometric method, or the turbidity method, both of which are regarded as more traditional methods. The results are displayed in Table 6.
A one-way ANOVA (F-test) treatment was used by the researchers, which just has one variable but compares three or more means. To measure fluconazole in various pharmaceuticals, the NAG4SX3-3D analyzer was evaluated and contrasted with three conventional techniques (the official technique, turbidity, and the UV-spectrophometric method) (Figure 17 and Table 7) .
Figure 15. Using four distinct companies, the profile-time for the common addition approach for the fluconazole-phosphomolybdic acid system
Figure 16. Individual t-test results and comparisons among newly created methodology, the turbidity method, and the UV reference method were used to compare the results between practically content and the claimed valve
Figure 17. The consolidated data from three independent, in addition to the referenced value, and four independent samples used in the ANOVA
No potential conflict of interest was reported by the authors.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
All authors contributed to data analysis, drafting, and revising of the paper and agreed to be responsible for all the aspects of this work.
Sarah Faris Hameed 0000-0002-7607-3498
How to cite this manuscript: Nagham Shakir Turkie, Sarah Faris Hameed*. Analysis and detection of incident light attenuation with a continuous flow injection by using the precipitating reaction of fluconazole and phosphomolybidic acid by NAG4SX3-3D instrument. Asian Journal of Green Chemistry, 6(3) 2022, 255-272. DOI: 10.22034/ajgc.2022.3.6