A Novel Approach of CFIA Technique for Assaying of Furosemide (Sulfa Drug) as Antibacterial Agent in Pharmaceutical and Biological Samples Using Potassium Ferricyanide as Oxidizing Agent

Furosemide drug determination in pharmaceutical and biological urine samples using a novel continuous flow-injection analysis technique that is simple, rapid, sensitive and economical. The complex formed by the reaction of furosemide and O-phenylenediamine with oxidative agent K 3 [Fe(CN) 6 ] to produce an orange-yellow colored product at 460 nm was the basis for the proposed method. The proposed method’s linearity ranges (3-100) μg.mL -1 and (1-50) μg.mL -1 for CFIA/merging zone methods and batch .The detection limit and Limit of quantification values were 2.7502 μg.mL -1 and 9.1697 μg.mL -1 the relative standard deviation was 0.7143 %, and the average recovery is 98.80% with a verified sample throughput of 73 h -1 . The new approach was effectively employed to determination of furosemide the presence of in the pure, biological, and pharmaceutical samples.


Introduction
Furosemide has a chemical formula of (C12H11CIN2O5S) with molar mass of 330.74g/ mol.Its formula structure is shown in Figure 1.furosemide is formally a sulfonamide, an antibacterial agent [1][2].However, the intense and fast dieresis produced by this drug, has extended its application as a powerful acidic diuretic for diverse treatment in human and veterinary medicine.Furosemide is often classified as a loop diuretic due to its predominate action in the nephron [3][4] .Various analytical methods for determining FUR using many techniques have been described in the literature, electrochemical sensing method [5][6][7], HPLC technique [8][9][10], and spectroscopic methods [11][12].The method used for the analysis of furosemide in the oxidationreduction reaction of the FUR and the reagent and [K3Fe(CN)6] as an oxidizing agent using the flow injection technique to determine the drug in pharmaceutical preparations and biological samples [13][14][15][16][17] The advantages of this method are that it consumes small amounts of the drug and the reagent, has high repeatability with accurate results obtained, is determined in the visible light range, and is a very simple and stable method for drug determination.

Apparatus and FIA Manifold
All absorbance in the batch procedure was measured using a double-beam Shimadzu UV-1800 UV-VIS spectrophotometer with a 1 cm quartz cell.The suggested FI manifold was developed as a simple type with a one-channel manifold in the FIA/merging zones system technique as shown in Figure 2. The carrier stream distilled water was pumped through the injection valve seven three-way injection valve, handmade by a peristaltic pump (Master flexC/L, two-channel, USA), which travels at 90° and three Teflon loops (I.d =0.5 mm) into which the sample L1, the oxidizing agent L2, and the reagent L3 were loaded.The reaction coil is made of glass; and used to mix the ingredients (2 mm, I.D.).The modified Optima photometer 301-D+ (VIS-Spectro, single beam) (Japan) was used for all absorbance and spectral measurements during the FIA procedures.The responses (as peak height) were measured using a Kompensograph C1032 (Siemens) or an optical multimeter absorption (DT9205A, OVA, China) for the absorbance measurements.A flow cell quartz silica (1 cm) with an internal volume of 80 μL is used in the detection unit [18,19].This injection valve was utilized to pass the volumes of reagent solution and sample.The loops were constructed of flexible vinyl and 1 mm for the manifold system.The reaction coil was constructed of glass with an inner diameter of 2 mm.A single-channel manifold system in CIFA is shown in Figure 2. D.W. was used as a carrier stream that was commixed with FUR in loop1, in loop2 was K3[Fe(CN)6] and O-Ph in loop 3.In a reaction coil, all the compounds were mixed, and the carrier flow rate was (12.8) mL.min -1 with a length of 50 cm and a height absorbance of 460 nm for the yellow complex.

Chemicals and reagent
Every one of the chemicals and solvents utilized in this work was provided with the analytical grade for this project by the state organization for drug industries and medical equipment in Samara Iraq.
• Standard drug solution (M.wt 330.745 g.mol -1 ): (1000 μg.mL -1 ) Standard solution furosemide was prepared by dissolving (0.1g) of pure drug in methanol then adding 10 mL of concentration HCl, and 40 mL of distilled water.diluting to the mark in (100ml) volumetric flask with distilled water to prepare 1000 μg.mL -1 .The obtained solution was heated at 50C° until it would be clear and yield a light yellowish solution pointed to complete the acidic hydrolysis.

• Preparation of interferences
Dissolving 0.1 g from any one of the interferences including glucose, sodium citrate, cellulose, lactose, and sucrose in 100 mL of distilled water by using a 100 mL standard volumetric flask.

Lazine /Syria (40mg).
An average of one tablet weighing (40mg) of FUR was accurately weighed and finely crushed.Each weight that was taken in the previous operation was treated as pure material.[20] Additional solutions were diluted to get the concentration inside the linearity of the calibration graph.Serial dilution can be employed to make 100 μg.mL -1 of the other solution types, and the proposed method is then utilized to quantitatively quantify 10, 25 μg.mL -1

Urine samples preparation
The samples were taken from several healthy individuals, utilized immediately after 5 drops of HClO4 acid were added (to precipitate the protein), and then centrifuged at 3000 rpm.The samples were collected from a healthy volunteer and kept at 20 °C until use after gentle thawing.volume of 1 mL for urine sample preparation, then converted to a volumetric flask of 10 ml and spiked with (2.5, 5) mL of standard solution (100) μg.mL -1 and diluted with distilled water to attain (40, 50) μg.mL -1 of spiked FUR.A blank solution was prepared in the previous steps, except [21].

Batch method
Spectrophotometric determination based on oxidative 1mL (100 μg.mL-1) of the FUR drug with 1mL of O-Ph reagent in the presence of 0.5 mL of the oxidizing agent K3[Fe(CN)6] and then added to a volumetric flask of 10 mL and the volume completed with distilled water.The appearance of an orangecolored product at λmax 460 nm, against reagent blank is shown in

Mechanism of the proposed method
The suggested mechanism for the hydrolysis with coupling reaction is shown in scheme (1) [22] Scheme 1.The proposed mechanism of the complex between FUR with O-Ph

Result and discussion
This study explains the oxidation-reduction reaction between FUR and O-phenylendiamine as a reagent in the presence of the oxidizing agent K3[Fe(CN)6].To from colored product orangeyellow at λmax 460 nm opposite reagent blank, which has little absorbance at the same wavelength.

Stoichiometry study
To investigate the stoichiometric ratio of drug to reagent applied molar ratio method10 and continuous variation by using an equal concentration of FUR ( (3x10 -4 ) M and O-Ph by using increased volumes of O-Ph and added to 1mL of FUR drug, The study found that the hydrochlorothiazide to coupling reagent ratio was 1:1. as shown in Figure 6 A-B.

Study of the optimum reaction conditions:
The study of various variables and factors on the color product was done to determine the most suitable conditions for drug determination.

Effect of reagent concentration
Through the use of different volumes of the reagent O-phenylenediamine, a volume of 1mL from (8x10 -3 ) M for O-Ph was the best concentration which has the highest responses the absorbance increases with volume 2mL then decreased, as shown in Figure 4-A

Calibration curve and linearity
After ideal conditions utilizing several concentrations (1-50) μg.mL -1 of FUR were obtained by diluting the standard solution.The reaction mixture evaluated the maximum absorbance of the orange-yellow colored result at 460 nm in comparison to the reagent blank, as shown in Figure 6.
Figure 6.Linear calibration curve of FUR-OPh using spectrophotometric method

Precision and accuracy
The accuracy and precision of the suggested method have been verified by measuring the (RSD) relative standard deviation proposed and (RE)relative error values as shown in Table 1 which shows good results for accuracy and precision.

Stable Calculations
The stability constant for the proposed reaction (FUR: OPh) was computed based on the two groups of solutions that had been created; the first group had a stoichiometric amount of FUR and reagent OPh, whereas the second group had a 2-fold excess of OPh.The stoichiometry of the drug to reagent (1:1).According to the relationship, the reaction between FUR and OPh proceeds: While As, Am are the absorbance values of the aqueous solution, which include a sufficient and stoichiometric quantity of reagent [23,24], C is the molar concentration, and K is the stability constant, where (α) expresses the degree of disintegration (M) of the product, which is equivalent to the concentration of FUR.Where The (ΔG value) spontaneous of complex formation reaction was determined based on K evaluation as in Table 2 and the equation( ΔG = -RT lnK) where(ΔG), (R), (T) its mean gibbs free energy, general constant of gases (8.314J. mol -1 .K -1 ), absolute temperature (298 K) shown in the Table 2.

FIA/ MZ spectrophotometric determination
An FIA procedure was developed using the batch method for calculatingFUR.The estimation manifold used for hydrochlorothiazide was made to provide a variety of reaction conditions for magnifying the absorbance signal produced by the oxidative reaction of FUR with O-Ph in the presence of potassium ferricyanide.

Chemicals and physical variables
The trace of chemical parameters (volume of reagent, concentration of oxidative agent, and order of addition) and physical variables (the length of reaction coil sampling, flow rate, injected volume of drug, and injection time) were studied.

Effect of O-phenylendiamine
To determine the best concentration of O-phenylenediamine as a reagent was examined by injecting various concentrations (4.6х10 -4 -7.3х10 -3 ) M of OPh by utilizing a seven-three injection valve.The result is shown in Figure 7.The (2×10 -3 ) M of O-Ph was the best concentration because gives the greater value of responses and high repeatability measured as peak height in mV (n=3).

Effect of oxidized agent
injecting several concentrations of the oxidizing agent that the best concentration was (4×10 -4 ) M represented as peak height in mV (n=3), in the redox reaction between the (FUR) drug and the reagent, The highest value of absorbance is represented as peak height in mV (n=3) with excellent reproducibility, as shown in Figure 8.

Effect of mixing coil and injected volume
The effect length of the reaction coil was examined by using different lengths of it (50, 100, and 200 cm).The results showed that the better length of the reaction coil, which gave the highest absorption, was 50 cm, as shown in Figure 9-A, which was used in all subsequent experiments.After using different lengths of loops 58.88 (L1), 78.88 (L2), and 78.88 (L3) μL, the results showed that these injection volumes of the drug, oxidizing agent, and reagent are the best volumes, as they showed a high response that expresses a high peak shown in the following figure 9-B.

Effect of optimum total flow rate and sample through-put
The sampling rate was calculated based on the time needed to load the chemicals into the seventhree-way valve's loops in addition to the time needed for the highest response appearing.several flow speeds and it was found that the better speed is 9.2 mL.min -1 with a sample through-put 73 sample/h -1 .The results are indicated in Figure 11.

Purge Time
The effects of the purge time for the sample to be injected by distilled water as a carrier stream were investigated using the best chemical and physical features previously investigated.For this experiment, an open valve (injection mode) and times of 5, 10, 15, and 20 seconds were used.To get the highest response intensity with the least amount of dispersion, the purge time-the amount

Study of dispersion
The most important aspect of the flow injection technique is to control the physical dispersion phenomenon, which can be calculated through the law (D = Co/Cmax).D refers to the dispersion coefficient.While C0 is the peak height without using dilution reactions outside the FIA system, C is the peak height with the use of dilution interactions inside the FIA system.The dispersion of the FUR-OPh reaction was 1.167 and 1.170 for concentrations 25, 50.It is illustrated in Figure 13 as well as in Table 3.

Calibration curve
Utilizing the ideal experimental for FUR drug evaluation, a linear calibration curve was created in the concentration range of 3-100 μg.mL -1 over this range Beer's law was not followed, and the reaction mixture measured the maximum absorbance of the orange-colored result at 460 nm in contrast to the reagent blank as shown in Table 4 and Figure 14.

Analysis of variation (ANOVA) of the linear equation and Repeatability
To compute (yi -ŷi)2 for (n-2) degrees of freedom, calculate the assumed error, called-for regression, and the sum of squares of the difference between the response's (yi) and the appraiser's ŷi values (S2) 2. Calculate the sum of squares of the variance of values ŷi from the average value (due to regression) and then divide that result by the square root of the degree of freedom (1) to obtain the value (F), as shown in Table 4.The good repeatability of the approach is shown in Tables (5, 6).

6.1Methods validation
The analytical characteristics of the new technique (CFIA/MZ) include the detection limit LOD, LOQ [27][28],correlation coefficient (r), relative standard deviation, linear range, Standard deviation of the residuals (Sy/x), intercept (Sa) slope (Sb) with 95% confidence limits for (n-2) degrees were acquired under ideal circumstances as indicated in Table 7. Comparing the proposed FIA to the batch approach, it was found to have excellent repeatability and reproducibility on tiny subjects.Due to its speed sample throughput of 73samples/h.The developed FIA/approach which includes my thesis was a simpler and semiautomated technique than the classic method since it produced calibration curves with large linear ranges

Effect of interferences
The impact of several types of interferences; cellulose, sodium citrate, glucose, lactose, and sucrose was examined for selectivity of the suggested method, by estimating the concentration of 100μg.mL - of FUR in a presence of the interferences.The results appear in Table 7. where it was found that there was no impact from any of the excipients on the determination of FUR by using the CFIA system.

Urine samples
The FIA method used successfully to determine FUR with accuracy in samples of spiked human urine.
(100) μg.mL -1 of FUR's precision and accuracy were assessed.Every concentration was subjected to three analyses.The urine sample results in Table 8 were observed with satisfactory precision and accuracy.

Applications and assessment of suggested method
Under the proposed method, the first types of medications containing FUR that are equipped with different sources when added conventionally have been investigated.Student F-test and ttest results from the numeration comparison between the suggested approach and the spectrophotometric approach [31] revealed that the calculated t-test values were 1.3026 and 0.5337 and the F-test values were 0.5399 and 0.4678, which were less than the theoretical t-test (2.45) and F-test (9.28) via FIA, as shown in

Conclusions
FIA designs based on the combining zone approach and spectrophotometric detection wrer used successfully to find furosemide in both its pure and pharmaceutical forms.The construction of a wonderful lab-made valve, which is a crucial part of the system that was built, was easy, affordable, and efficient, with components that were readily available and cheap to clean, replace, and repair.The procedure is based on the development of an orange-yellow condensation adduct when FUR and O-Ph combine with the oxidizing agent K3[Fe(CN)6] that has been provided.The approach offers a high sample throughput and a low detection limit.The suggested techniques had the best application for the pharmaceutical preparation and adhered to Beer's law.By examining the assay of FUR, the FIA method's broad application for everyday quality monitoring is successfully demonstrated.

Figure 4 .
Figure 4. Stoichiometric study between FUR and Reagent Continuous variation (A) mole ratio (B)

Figure 7 .
Figure 7. Effect of O-Ph

Figure 9 .
Figure 9.Effect of: A\ Reaction coil, B\ Injected volume5.6 selecting the best manifold unitThe highest added sequence was D(drug)+O(oxidative agent)+R(reagent), which gave the best absorption value expressed as the highest peak.The results are indicated in Figure10.

Figure 10 .
Figure 10.Effect of the sequence of chemicals.

Figure 11 .
Figure 11.Effect of flow rate of distilled water in developed system in injection valveof time between the injection of the sample and the start of the termination of the signal-should be larger than 20 seconds.Figure12illustrates the ideal injection timing for moving the drug from the drug loop to the flow cell: when the valve is open.

Figure 12 .
Figure 12.Effect of purge time

Figure 13 .
Figure 13.Dispersion of FUR in CFIA system

Figure 14 .
Figure 14.Calibration curve of FUR using the developed CFIA system.

Table 1 .
Precision and accuracy for FUR

Table 2 .
Stability constants for FUR with O-Ph.

Table 5 .
Analysis of a nova for proposed method

Table 6 .
The repeatability of the proposed method

Table ( 7
).Analytical properties of calibration curve for FUR

Table 8 .
Determination of FUR in urine samples using proposed method