Eco-Friendly Facile Spectrophotometric Determination of 5-Fluorouracil in A Spiked Human Blood Plasma, Pharmaceutical Preparations, and Spiked Environmental Waste-Water Samples

Ahmad NR and Rahman GN

Published on: 2025-09-24

Abstract

A simple, accurate, precise, rapid, economical, and sensitive ultra violet spectrophotometric method has been developed for the determination of 5-Fluorouracil in pharmaceutical preparations and environmental wastewater samples, which shows maximum absorbance at 268 nm in distilled water. Beer's law was obeyed in the range of 1-20 μg/ml, with a molar absorptivity of 1.35x10⁴ L.mol^-1.cm^-1, the relative standard deviation of the method was less than 1.5%, and the accuracy (average recovery %) was 100 ± 1.2. The Sandal’s sensitivity of the colored complex is 18.346 ng/cm², and LOD and LOQ are 2.725 and 8.992 ng/cm², respectively. No interference was observed from common excipients and additives that often accompany 5-Fluorouracil in pharmaceutical preparations. The method was successfully applied to the determination of 5-Fluorouracil in pharmaceutical formulations (creams), biological blood samples, and industrial wastewater samples. The proposed method was validated by sensitivity and precision, which proves suitability for the routine analysis of 5-Fluorouracil in true samples.

Keywords

5-Fluorouracil; Spectrophotometry; Pharmaceutical preparations; Blood plasma; Environmental water samples

Introduction

5-Fluorouracil (Figure 1) is [5-Fluoropyrimidine-2,4(1H,3H)-dione], also known as Efudix. Description. A white, almost odorless powder. Solubility: Soluble in water; very slightly soluble in alcohol; practically insoluble in chloroform and in ether. 5-Fluorouracil (5-FU) is a low-molecular-weight anticancer drug in clinical use for several solid tumors in humans. Caution: 5-Fluorouracil is an irritant, and contact with skin and mucous membranes should be avoided. Actions and uses. 5-Fluorouracil is a cytotoxic agent. It is an analogue of uracil, a component of ribonucleic acid, and is believed to exert its effect in malignant disease by interfering with nucleic acid biosynthesis. It is used alone or in conjunction with radiotherapy in the palliative treatment of cancers of the gastrointestinal tract, breast, and respiratory tract [1-5].

Figure 1: Chemical Structure of 5-Fluorouracil.

C₄H₃FN₂O₂ = 130.08

A literature survey reveals that numerous methods have been published for quantitative analysis of 5-Fluorouracil alone and in combination with other drugs such as several gas-chromatographic methods have been reported for the analysis of 5-Fluorouracil (5FU) in biological fluids [2] and the titrimetric method [3]. Spectrophotometric methods [6-8], the LC-MS-MS method [9], and high-performance liquid chromatography (HPLC) methods [10-14] can be used for the determination of drugs and for purposes of control throughout the entire manufacturing process of drugs, as well as quality control of the finished product. It has the advantages of being sensitive, selective, rapid, accurate, and reproducible. The present paper reports the development of a new UV method for determination of 5-Fluorouracil in creams and environmental water samples.

Experimental

Apparatus

A Shimadzu UV-1700 pharm spec (double beam) spectrophotometer with 1.0 cm quartz cells was used for absorption measurement.

Reagents

All chemicals used were of analytical or pharmaceutical grade, and 5-Fluorouracil standard material was provided from AL-hokamaa Company for Pharmaceutical Industries (HPI), Mosul, Iraq.

5-Fluorouracil Standard Solution 50 ppm

This solution was prepared by dissolving 50 mg of 5-Fluorouracil in 1000 ml of distilled water in a calibrated flask.

Determination of Absorption Maxima

The standard solution of 5-Fluorouracil (10 µg/ml) was scanned in the range of 220-300 nm, which shows maxima located at 268 nm (Figure 2). Therefore, a 268 nm wavelength was selected for the construction of the calibration curve.

Figure 2: Absorption Spectra of 10µg/ml 5-Fluorouracil against Distilled Water.

Recommended Procedure

From the absorption maxima, a calibration curve was prepared in the concentration range of 1-20 µg/ml. The absorbance was measured at 268 nm against water as a blank. The concentration of the sample solution can be determined by using the calibration curve.

Procedures for Pharmaceutical Preparations (Creams)

Disperse a quantity of the creams containing 1.0 mg of 5-Fluorouracil in 20 ml of methanol (80%) in a 125-ml separating funnel containing 50 ml of petroleum ether. Heat the contents over a water bath (40-60°C) and shake gently for 20 minutes. Allow the layers to separate and transfer the lower layer to a 50 ml flask. Repeat the extraction with a further 2×10 ml of 80% methanol. Dilute the combined extracts to 100 ml with the distilled water to get a 10 µg/ml solution. Measure the absorbance at 268 nm using distilled water as a blank, and the concentration was calculated by using the calibration curve of this method.

Procedure for Real Water Samples

To demonstrate the practical applicability of the proposed method, real water samples were analyzed by this method. Industrial wastewater from Al-Hokamaa Company for Pharmaceutical Industries (HPI) in Mosul, Iraq, was fortified with concentrations in the range of 5, 10, and 20 µg/ml of 5-Fluorouracil. The fortified water samples were analyzed as described above for the recommended procedure, and the concentration was calculated by using the calibration curve of this method.

Analysis of Plasma Samples

Human blood samples were collected in dry and evacuated tubes (which contained saline and sodium citrate solution) from different healthy volunteers. The samples were handled at room temperature and were centrifuged for 10 min at 1500 rpm for the separation of plasma within 60 min of collection. The samples were then transferred to polypropylene tubes and stored at -20°C until analysis.

The plasma samples obtained at room temperature as described in plasma sample preparation were allowed to liquefy at room temperature before processing. The plasma samples were spiked with 5-FU and a fixed amount (5, 10, 20 μg/ml), and the tube was briefly shaken. Then the mixture was vortex mixed with ether. The ethereal layer was evaporated to dryness on a water bath under a gentle stream of nitrogen gas at 40°C. The residue was dissolved in a suitable amount of water and measured as recommended procedure.

Result and Discussion

UV-visible spectrophotometry is still considered to be a convenient and low-cost method for the determination of pharmaceuticals [16-26].

The method used for the determination of 5-Fluorouracil in pharmaceutical preparations and environmental wastewater samples was found to be sensitive, simple, accurate, and reproducible. Beer's law was obeyed in the concentration range of 1-20 µg/ml (Figure 3) with a correlation coefficient of 0.994, an intercept of 0.002, and a slope of 0.0545. The conditional molar absorptivity was found to be 1.35x10⁴ l/mol.cm, and Sandell’s sensitivity was 18.346 ng.cm^-2. Limit of detection (LOD) and limit of quantification (LOQ) values were used to describe the method sensitivity. The limit of detection and quantification was evaluated as [27-29], so the proposed method was sensitive.

LOD = 3.3 × Standard deviation of intercept/slope.

LOQ = 10 × Standard deviation of intercept/Slope

The limit of detection was 2.725 ng/ml, and the limit of quantification was 8.992 ng/ml, as the lowest standard concentration that could be determined with acceptable accuracy.

Figure 3: Calibration Curve of 5-Fluorouracil.

The accuracy and precision of the method, a pure drug solution, were analyzed at three different concentrations, each determination being repeated six times. The relative error (%) and relative standard deviation values are summarized in table 1. From table 1, the values of standard deviation were satisfactory, and the recovery studies were close to 100%.The RSD% value is less than 1.4, indicative of the accuracy of the method.

Table 1: Accuracy and Precision of the Proposed Method.

5-Fluorouracil taken µg/ml))	Er (%)^a	RSD (%)
5	1.1	1.3
15	0.9	1.1
20	1	1.3

a: Mean of six determinations

Analytical Application

The proposed method was satisfactorily applied to the determination of 5-Fluorouracil in its pharmaceutical preparations, cream samples, blood plasma samples, and wastewater samples. The results of the assay of the pharmaceutical preparations reveal that there is close agreement between the results obtained by the proposed method and the label claim. Table 2, the results of water samples Table 3 shows that the recovery values obtained were close to 100%.

Table 2: Determination of 5-Fluorouracil Formulations.

Pharmaceutical formulations	Proposed method found*	Label amount
5-Fluorouracilnin Cream (HPI)	4.97%	5%

*Mean of ten determinations.

Table 3: Determination of 5-Fluorouracil in Industrial Wastewater Samples.

Wastewater samples	Added µg/ml	*Found µg/ml**	Recovery %(n=10)
Industrial wastewater	5	5.05	101
	10	9.97	99.7
	20	20.15	100.75

* mean value of ten determinations.

Analysis of Blood Plasma Samples

The proposed method was applied to the determination of 5-FU in spiked plasma samples. The results obtained for precision and accuracy at the three different concentrations in plasma are recorded in Table 4. Low values of relative standard deviation indicate high precision of the proposed method and the results of blood samples show that the recovery values obtained were close to 100%.

Table 4: Determination of 5-Fluorouracil in Blood Plasma Samples.

Blood Plasma Samples	Added µg/ml	*Found µg/ml**	Recovery %(n=10)
Human blood samples	5	5.01	100.2
	10	9.99	99.9
	20	20.03	100.15