Introduction

Fluconazole (FLZ, 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4triazole-1-yl)-propan-2-ol), as indicated in Scheme 1, that is commonly known as diflucanis [1], is an antifungal medication [2-4] prescribed by the United States Food and Drug Administration (FDA) to treat mucocutaneous candidiasis [5], such as esophageal candidiasis (infection of the esophagus), oropharyngeal candidiasis (infection of part of the throat), and vulvovaginal candidiasis (infection of the vulva and vagina), reduces candidiasis in people getting chemotherapy [6] and/or radiation after a bone marrow transplant. Meningitis caused by cryptococcal bacteria should be treated. Fluconazole is a fungistatic triazole drug that is used to treat systemic and superficial fungal infections. Fluconazole medication has been linked to temporary mild-to-moderate blood aminotransferase increases and clinically obvious acute drug-induced liver impairment. In 2004 and 2005, there were patent expirations. FLZ is a broad-spectrum tri azole antifungal agent that has emerged as a suitable substitute to amphotericin B in the treatment and prevention of various superficial and systemic fungal infections. It is a tri azole compound with potent antifungal activity that is currently being evaluated in clinical trials. The adverse effects [7-11] of fluconazole might range from skin lesions or skin rashes to pounding heartbeats, skin discomfort, breath shortness, dark urine, liver issues and changes in taste, stomach pain, major heart problems, abrupt disorientation, and others. A literature review reveals various techniques for determining fluconazole in biological fluids that have been documented such as UV spectrophotometric technique [12], and gas chromatography [13].

Materials and Methods

The solutions were produced using distilled water and all of the utilized chemicals were analytical reagent grade. By dissolving 1.2251 g of fluconazole in 100 mL of distilled water (C₁₃H₁₂F₂N₆O), a standard solution of 0.04 M fluconazole (molecular weight 306.271 g.mol^-1, BDH) was produced. 8.6406 g of phosphotungestic acid (H₃PW₁₂O₄₀,) having a molecular weight of 2880.2 g.mol^-1 (Hopkin and Williams LTD) was dissolved in 250 mL of distilled water to make a standard solution.

Instruments

A flow cell was used which was produced from a homemade NAG-4SX3-3D analyzer. The output from the attenuation of incident light 0–180 ^⃘ was captured (Figure 1). The signal outputs were recorded using a potentiometric recorder (Siemens, Germany), Ismatic peristaltic pump with sample loop, and six-port injection valve (Teflon, variable length). The UV spectrophotometric (Shimadzu, Japan) and turbidimetry equipment were used for the traditional techniques. The proposed mechanism was depicted in Scheme 2.

Scheme 1: Fluconazole structure

Figure 1: Diagram of system utilized to determine fluconazole via phosphotungestic acid

Scheme 2: A possible mechanism for reaction of fluconazole with phosphotungestic acid

Influence of phosphotungestic acid concentration

To determine the optimal concentration, a series of phosphotungestic acid solutions ranging from (1-10) mmol.L^-1 were produced. Fluconazole (5 mmol.L^-1) was utilized as a preliminary concentration in a 175 µL sample volume, and each measurement was performed three times. According to the results of the study, 7 mmol.L^-1 of phosphotungestic acid was required to achieve the greatest attenuation of incoming light, as indicated in Table 1 and Figure 2. It can be revealed that an increase in phosphotungestic acid can cause an increase in particle due to the accumulating action of precipitate particles, density can reach up to 7 mmol.L^-1, after which there was a decrease in incident light attenuation corresponding to the same particle growth with increased phosphotungestic acid concentration.

Table 1: Effect of phosphotungestic acid on the measurement of energy transducer response S/N for determination of fluconazole

Figure 2: Various concentrations of phosphotungestic acid and its effect on (S/N) energy transducer response in mV

Influence of acid or salt media on fluconazole - phosphotungestic acid precipitation system

Using the optimal concentration of phosphotungestic acid solution 7 mmol.L^-1, the influence of acid and salt (50) mmol.L^-1 in the reaction media on sensitivity, in general, was investigated. This investigation employed tartaric acid, sulfuric acid, acetic acid, hydrochloric acid, nitric acid, ascorbic acid, potassium chloride, ammonium acetate, ammonium chloride, sodium chloride, sodium nitrite, potassium iodide, sodium sulfate, and sodium carbonate, respectively. A preliminary physical condition was employed, with a volume of sample 175 µL with flow rate 2.5 mL.min^-1. Figure 3 depicts a distinct peak height of the measured response; leading to the conclusion that sulfuric acid can be utilized as optimum medium for the research work.

Figure 3: Effect of type of media on the profile using sample volume 175 μL, [Fluconazole]=4 mmol.L¹, [phosphotungestic acid]=7 mmol.L^-1, flow rate of carrier stream 2.5  mL.min^-1 via the attenuation measurement of incident light expressed as a peak heights

Effect of sulfuric acid concentration

Using phosphotungestic acid 7 mmol.L^-1- fluconazole 4 mmol.L^-1- sulfuric acid system, the phosphotungestic acid system was constructed with a variable concentration of sulfuric acid ranging from (10-250) mmol.L^-1  and a volume of sample 175 µL  and a flow rate of 2.5 mL.min^-1 for the carrier stream and reagent streams. Table 2 indicates a rise in peak height represented as an incident light attenuation with increasing sulfuric acid concentration, which might be connected to the creation of tiny solid particles that resulted in an increase in inter spatial distance and incoming light attenuation. Whereas up to 200 mmol.L^-1 sulfuric acid concentration, as seen in Table 2, resulted in a reduction in S/N energy transducer response, which was likely related to precipitation particle dispersion. As a result, the optimal concentration of sulfuric acid was determined to be 200 mmol.L^-1.

Table 2: Effect of variable concentration of sulfuric acid on energy transducer response (S/N) of fluconazole (4 mmol.L^-1) - phosphotungestic acid system

Physical variable

Influence of flow rate

To choose the optimum flow rate that varied from 1-4 mL.min^-1 for carrier stream and phosphotungestic acid stream with 175 µL of sample volume, the optimal concentration of the reactant: phosphotungestic acid (7 mmol.L^-1), and the initial concentration of fluconazole (4 mmol.L^-1) were used. Figure 4 summarizes the results. As demonstrated in this figure, there was a rise in peak height with an increase in Δt_b with an increase in flow rate, dispersion, and dilution resulted in a longer sample segment of the precipitate product, which was attributable to dispersion and dilution. While higher flow rates for the carrier stream and reagent resulted in a reduction in peak height, this could be due to the rapid departure of precipitate particles from the measuring cell, so the most efficient flow rate for fluconazole-phosphotungestic acid accomplishment was 2 mL.min^-1 to obtain a regular response, sharp maxima, and reduce reactant solution consumption.

Influence the volume of sample on S/N transducer energy response in mV

Using a phosphotungestic acid (7 mmol.L^-1)-fluconazole (4 mmol.L^-1) precipitation system with a 2 mL.min^-1 flow rate. An open valve mode was used to inject a variable volume (82-281) µL of sample into the injection valve, allowing for removing samples from the sample loop on a continual basis. Figure 5 displays a plot of incoming light attenuation vs time. As demonstrated in this figure, increasing the sample volume up to 82 µL resulted in a considerable drop in response height and was more detectable than a high volume. This was owing to an increase in the width Δt_B and response maxima, which may be ascribed to the continuous substantially, the section of precipitate particles in front of the detector has a greater temporal length, as well as an increase in particle size, leading precipitate particles to move slowly. Therefore, the optimal sample volume was determined to be 82 µl.

Figure 4: Profile at variable flow rate of versus t_min(d)_mm

Figure 5: Effect of the different sample volume based on the response of an energy transducer via time using flow rate 2 ml .min^-1 of reagent and carrier stream, fluconazole (4 mmol/L)- phosphotungestic acid (7 mmol/L) system

Effect of reaction coil length in µL

The influence of reaction coil length was investigated using a coil length ranging from 0 to 30 cm and an I.D of 1 mm. This length range has a volume of 0 to 942 µL, which was linked directly into the flow system following the Y-junction in Table 3. The fluconazole solution (4 mmol.L^-1) was employed as the optimal concentration for the precipitation system, with 175 µL as the injected sample volume. Table 3 clearly depcits that as coil length, Δt_B, and arrival time of injected sample from injection valve to measure flow cell increase, peak height decreases, which could be attributed to the increased effect of dilution and dispersion zones of sampling, as well as the continuous longer time duration of precipitate species in front of the detector.

Table 3: Effect of different reaction coil on the measurement of energy transducer response for determination of fluconazole

Effect of Y-junction on profile response

The Y-junction is needed in the process to mix the reactants. The Y-junction was added before the cell was measured directly the system of flow. The response pattern was examined when the Y-junction was changed in various parameters. For illustrating with a sample capacity of 175 µL and a flow rate of 2.5 mL.min^-1, fluconazole at its optimal concentration (4 mmol.L^-1) was utilized for both the carrier stream and the reagent. Figure 6 shows how the S/N transducer's energy response affects the Y-junction (meeting zone). Using varied volume mixing chambers and a larger diameter intersecting point, the effect on particle agglomeration, regulation, and regular distribution prior to the flow tube's entry was investigated. Particle scattering and dispersion, as well as increasing inter spatial distances; reduce the ability of incident light to raise the height of the energy transducer's response measurement, as indicated in Table 4. As a consequence, it was assumed that by employing a manifold unit with two 3 mm (inter diameter) entries and a 3 mm (inter diameter) output.

Figure 6: Effect of Y-junction on S/N energy transducer response versus t_min(d)_mm

Table 4: Effect of the variation of Y-junction on the attenuation of incident light by using 4 mmol.L^-1 concentration of fluconazole and 7 mmol.L^-1, concentration of phosphotungestic acid, and flow rate of the carrier stream 2 mL.min

Estimating the linear dynamic range of fluconazole on a scatter plot of the S/N energy transducer response

Both physical and chemical variables were adjusted to their best possible values in the previous section (fluconazole–phosphotungestic acid (7 mmol.L^-1)–H₂SO₄ (200 mmol.L^-1) system, 175 µL volume of sample, 82 µL reaction coil, and 2.0 mL.min^-1 as a flow rate for carrier stream and phosphotungestic acid line (Figure 7a), and a set of series 0.01–25 mmol.L^-1 solutions were prepared). The profile seen in Figure 7b is represented by a scatter-plot with a correlation coefficient of 0.9858 over the range of 0.01-18 mmol.L^-1 (i.e. picking all twenty-seven points). Table 5 shows the analytical range that explains an increase in fluconazole concentration causes an increase in precipitate particulates (i.e. deals with directly proportional between fluconazole concentration and S/N energy transducer response) up to 18 mmol.L^-1 (n = 23point) with r=0.9989 at the range of (0.01-18) mmol.L^-1. A broad maxima of the peak height was also observed above 18 mmol.L^-1, which could be attributed to an increase in precipitate particulates and their compactness, resulting in a reduction in interstitial spaces and reflecting surfaces, as well as an increase in particle size, resulting in a slower movement of particles, which resulted in a longer time duration of particles in front of the detector, resulting in a distorted response (Figure 7b).

Figure 7: a) Design of system used for the determination of Fluconazole by phosphotungestic acid, b) Some of output response versus time, potentiometric scanning speed 1cm.min^-1

The working range was 0.01-19 mmol.L^-1 (n = 24) with r = 0.9985, the scatter plot range was 0.01-25 mmol.L^-1 (n = 27) with r = 0.9858, the dynamic analytical range was 0.01-22 mmol.L^-1 (n = 26) with r = 0.9945 and the linear dynamic range was 0.01-18 mmol.L^-1 (n = 23) with r = 0.9945. As a result, to improve the mathematical construction of the evaluation, a shorter range should be employed. The best-fit linear equation for the relationship between fluconazole concentration (independent variable) and diverging light (a parameter that is dependent) has r = 0.0.9989 and a percent capital R-squared of 99.97 percent (Table 5) on the following form:

Ŷzi(mV) = 150.695 ±126.655+156.998 ±10.999[fluconazole] mmol.L^-1

Table 5: Different ranges for the Fluconazole concentration versus absorbance and scatter light using spectrophotometer and Turbidimetry method (classical methods) and NAG-4SX3-3D analyzer

The scatter plot was able to explain a large portion of the n = 27 data. The new developed methodology for determining fluconazole was compared with the existing reference methods, namely the spectrophotometric method, as demonstrated in Figure 8a and the turbidity method in Figure 8b while Figure 8c represents the optimum concentration of phosphotungestic acid reacted with  Fluconazole (4 mmol.L-1) in the presence of the best H₂SO₄ concentration of turbidimetric method, both of which are based on the absorbance measurements for a variable range of concentration, as indicated in Table 5 at λ_max 260 nm, and as depicted in Figure 9. The optimal linear range of n = 23 is 0.01 to 18mmol.L^-1. It has a correlation coefficient of 0.9989 and a percent capital R-squared of 99.79 percent (Table 5).

Detection limit 

The detection limit for fluconazole was estimated using three distinct strategies, theoretically, using the slope value; and practically, using progressive dilution for the lowest concentration from a scatter plot [14-16]. Using the precipitating agent phosphomolybdic acid, the fluconazole limit of detection was calculated, as indicated in Table 6.

Figure 8: A: Various ranges for the impact of Fluconazole concentration on incident light attenuation using the NAG-4SX3-3D analyser, B: Graphical representation shows the optimum concentration of phosphotungestic acid reacted with Fluconazole (4 mmol.L^-1) in the presence of the best H₂SO₄ concentration of Turbidimetric method, C: The scatter plot for [Fluconazole] using turbidimetric method

Figure 9: UV- spectrum for fluconazole at λ_max = 260 nm

Table 6: Detection limit of Fluconazole-phosphotungestic acid using 175 μL as an injection sample and best parameters using Fluconazole (4 mmol.L^-1)-[phosphotungestic acid] (7 mmol.L^-1)- H₂SO₄ (200 mmol.L^-1) system

Repetability

The RSD given as a percentage in Table 7 is equivalent to the measurement's repeatability. A series of six injections were tested at a constant fluconazole concentration. Two different concentrations of fluconazole were used (2 and 13 mmol.L^-1) [17-19]. The relative standard deviation, as indicated in Figure 10, was less than 0.5%.

Table 7: Repeatability of fluconazole at the best parameters with 175μl sample volume

Figure 10: Repeatability of fluconazole peak height in mV at optimum parameters with 175μl sample volume

Estimation of fluconazole in drugs utilizing NAG-4SX3-3D analyzer

Fluconazole was determined in four samples of medicines from four different firms (Fluconazole, Vassil, Bulgaria, 150 mg), (Candeur, Tabuk, Saudi Arabia, 150 mg), (Fluconazole, Bristol, UK, 150 mg), (Fluconazole, Bristol, UK, 150 mg), and (diflucan, Pfizer, France, 150 mg) using the latest advanced technique (NAG-4SX3-SD analyzer). The continuous flow injection analysis was combined with two methods: The UV-spectrophotometric measurement of absorbance at λ_max=260 nm and turbidimetric measurement of incident light attenuation at 0-180° for pale yellow fluconazole precipitate that reacted with phosphomolybdic acid using a homemade NAG-4SX3-3D analyzer. The data were mathematically addressed using the standard addition procedure, as displayed in Figure 11.

Figure 11: Peak height in mV for standard addition method for Fluconazole –phosphotungestic acid system using four different companies: Sample 1: Fluconazole, 150 mg, Vassil, Bulgaria; Sample 2: Candeur, 150mg, Tabuk, Saudi Arabia; Sample 3: Fluconazole, 150 mg, Brustol, UK; Sample 4: Dflucan, 150 mg, Pfizer, France

Table 8a shows the practical content of an active component at a 95% confidence level, as well as the (t-test) efficiency of determination (Figure 12), this depicts a comparison between two paths:

• Comparison of the created technique NAG-4SX3-3D analyzer with the official stated value (150 mg) as Table 8b by calculating t-values for each individual corporation and comparing them to the tabulated t-value, the following is a formula for estimating a hypothesis: The null hypothesis is that there is no significant difference between the means derived from four distinct sources ( ) and the stated value (µ)

i.e; : = µ (150mg )

Alternative hypothesis: the means and quoted value are significantly different.

i.e; :    µ (150mg )

Because some obtained values exceeded t table (4.3030) at 95 percent confidence level and degree of freedom = 3, the null hypothesis was rejected and the alternative hypothesis was accepted, implying that there is a significant difference between the quoted active ingredient value and the measured value.

• For the comparison of new methodology employing the NAG-4SX3-3D analyzer with two conventional methods UV-spectrophotometric method and turbidity, a paired t-test at = 0.05 (2 tailed) was used. Table 8a indicates the measurement of incident light attenuation, taking into consideration that all medications from various multiple companies belong to the same population, ignoring individual differences between manufacturers.

Assumption

Null hypothesis

: µ_{NAG-4SX3-3D analyzer} = µ_{UV- spectrophotometric} = µ_turbidity

There is no significant difference in the means of the two approaches.

Alternative hypothesis

: µ_{NAG-4SX3-3D analyzer}    µ_{UV- spectrophotometric}  µ_turbidity

The obtained data reveal that there was no significant difference between the devised approach, UV-spectrophotometric method, and turbidity method at 95 percent,  = 0.05 confidence interval, as shown in Figure 12 and Table 8b.

Statistical treatment of result by (one-way variance analysis)

The study employed a one-way ANOVA [20] (F-test) treatment, which compares three or more means, but only has one variable. The NAG4SX3-3D analyzer was evaluated for fluconazole measurement in various pharmaceuticals using a reaction of fluconazole with precipitating reagent phosphotungestic acid in the presence of H₂SO₄ and compared with three traditional methods (i.e., official method, Turbidity, and the UV-spectrophometric method), as depicted in Figure 13.

The F-test assumption for comparing three or more means. The first estimate is referred to as between – group variance (MS_B) or between – group variance (MS_B) (S_B²). The second estimate, the within-group variance (MS_W) or (S_W²), is computed using the data and is entirely unaffected by mean differences, but is dependent on variance (i.e., S_i² = n-12). If there are no variations in the means, the estimated between-group variance will be almost equal to the within-group variance, resulting in an F-value of 1. The alternative hypothesis will be accepted and the null hypothesis will be rejected if the F-test value is larger than 0.5. This process is known as variance analysis (ANOVA) [21].

The ANOVA-test was used with a significance level of  = 0.05. (95 percent confidence level). Table 9 shows the results of an ANOVA test and a comparison of four Fluconazole samples.

The hypothesizes

(Null hypothesis) (H₀)

H₀= μ _{fluconazole- Bulgaria} =μ _{Candeur- Saudia Arabia}   = μ _{fluconazole- UK}   = μ _{Diflucan – Pfizer}

In terms of the output of data, there is no significant variation between all used means of each sample from different companies. There is a considerable difference in all mean values, according to the option given.

H₁ (alternative hypothesis)

H₁: μ _{Fluconazole- Bulgaria}     μ _{Candeur- Saudia Arabia       Fluconazole – UK}    μ _{Diflucan- Pfizer}

The collected information, presented in Table 9, reveals a substantial difference between the means of samples, with F_Cal (3.559801) more than F_tab (3.490295) for the Fluconazole-phosphotungestic acid system.

As a result, the null hypothesis will be rejected, and the alternative hypothesis will be accepted. This means that there are considerable variances in the samples utilized by four separate companies (four samples).

Table 8a: Standard addition data for the estimation of fluconazole in four samples of drugs using NAG-4SX3-3D analyser for fluconazole with phosphotungestic acid system and two classical methods

Table 8b: Summary of data for practical content, efficiency (Rec %) for determination of Fluconazole in four samples of drugs and individual t- test for comparison between the means of weight with quoted value, for fluconazole-phosphotungestic acid system

Figure 12: Set of results for comparison between practically content and claimed valve (Individual t-test) and comparison between four methods (Newly developed methodology, turbidity, and the UV-spectrophotometric reference method) using Paired t-test, d: average of different between four methods (developed Newly methodology & classical methods), Wd: different between two methods, σ_n-1: standard deviation of difference (paired t-test)

Figure 13: Summed up the results for three different in addition to quoted value and four different samples for

 Table 9: ANOVA results for comparison between four different samples from four different Companies

df = degree of freedom, F_tab= F _0.95,V1,V2= F _0.95,3,12 = 3.490 at 95% confidence level, K= number of group =4, N=number of measurements or sum of the samples for the groups (i.e) =n₁+n₂+……+n_i =16, SS_B = Sum of squares between group, SS_W = Sum of squares within group, MS_B  = SS_B/ K-1 & MS_W = SSw/ N-K,  F_cal = MS_B / MS_W.

Conclusion

To investigate fluconazole in pure and pharmaceutical formulations, the recommended Turbidimetric flow-injection method is simple, quick, inexpensive, and sensitive. Fluconazole is precipitated with phosphotungestic acid in an acidic medium, yielding a white ion pair product. The precipitate is calculated by employing a linear array of 12 super white light emitting diodes as a source and three solar cells as a detector to measure the attenuation of incoming light at 0-180°. Compared with spectrophotometry and turbidimetric procedures that employ various precipitating agents, the proposed method uses less expensive apparatus and reagents. The percent R.S.D. was less than 0.5 percent, and all samples were in a good agreement, indicating that the proposed approach is accurate enough. To avoid matrix effects, the conventional additions method was utilized.

Acknowledgements

I would like to express my deepest gratitude to Prof. Dr. Issam M.A. Shakir Al-Hashimi & prof. Nagham S. Al-awadie for the invaluable guidance, insightful remarks, support, and encouragement. I am very grateful.

Funding

This research did not receive any specific grant from fundig agencies in the public, commercial, or not-for-profit sectors.

Authors' contributions

All authors contributed to data analysis, drafting, and revising of the paper and agreed to be responsible for all the aspects of this work.

ORCID:

Sarah Faris Hameed

https://www.orcid.org/0000-0002-7607-3498

HOW TO CITE THIS ARTICLE

Nagham Shakir Turkie, Sarah Faris Hameed. Determination of fluconazole using flow injection analysis and turbidity measurement by a homemade NAG-4SX3-3D Analyzer. Chem. Methodol., 2022, 6(10) 731-749

https://doi.org/10.22034/CHEMM.2022.348264.1561

URL: http://www.chemmethod.com/article_153721.html