A simple, accurate, precise, rapid, economical and sensitive Ultra violet spectrophotometric v spectrophotometric method has been developed for the determination of 5-Fluorouracill 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 molar absorptivity of 1.35x104 L.mol-1.cm-1, relative standard deviation of the method was less than 1.5%, and accuracy (average recovery %) was 100 ± 1.2. The Sandals sensitivity of the colored complex is 18.346 ng/cm2 and LOD, LOQ are,2.725,8.992 ng/cm2 respectively. No interference was observed from common excipients and additives often accompany with 5-Fluorouracill in pharmaceutical preparations. The method was successfully applied to the determination of 5-Fluorouracill 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-Fluorouracill in true sample   

5-Fluorouracill (Figure. 1) is [5-Fluoropyrimidine-2,4(1H,3H)-dione], Other name (Efudix). Description. A white, almost odorless powder. Solubility. Soluble in water; very slightly soluble in alcohol; practically insoluble in chloroform and in ether. 5-Fluorouracill (5-FU) is a low-molecular-weight anticancer drug in clinical use for several solid tumors in humans, Caution. 5-Fluorouracill is irritant and contact with skin and mucous membranes should be avoided. Actions and uses. 5-Fluorouracill 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 gastro-intestinal tract, breast, and respiratory tract. [1-5].

C₄H₃FN₂O₂ = 130.08

Figure 1: Chemical structure of 5-Fluorouracill.

Literature survey reveals that numerous methods have been published for quantitative analysis of 5-Fluorouracill alone and in combination with other drugs such as Several gas-chromatographic methods have been reported for the analysis of 5-Fluorouracill (5FU) in biological fluids [2], Titrimetric method [3]. Spectrophotometric methods [6-8], LC–MS-MS Method [9], high performance liquid chromatography HPLC methods [10-14], can be used for 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-Fluorouracill in creams and environmental water samples.

Experimental

Apparatus

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

Reagents

All chemical used were of analytical or pharmaceutical grade and 5-Fluorouracill standard material was provided from AL-hokamaa company for pharmaceutical industries (HPI) Mosul-Iraq. 

5-Fluorouracill standard solution 50ppm

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

Determination of absorption maxima

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

Figure 2: Absorption spectra of 10µg/ml 5-Fluorouracill against distilled water.

Recommended procedure

From the absorption maxima, 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-Fluorouracill  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 C҆) 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 2X10 ml of methanol 80%. Dilute the combined extracts to 100ml with the distilled water to get 10µg/ml solution. Measure the absorbance at 268  nm using distilled water as 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 waste water from AL-hokamaa company for pharmaceutical industries (HPI) Mosul-Iraq, were fortified with the concentrations in the range of 5,10,20 µg/ml of 5-Fluorouracill. The fortified water samples were analyzed as described above for 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 – 20Cْ  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 of (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 gentle stream of nitrogen gas at 40Cْ. The residue was dissolved in suitable amount of water and measured as recommended procedure.

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-Fluorouracill 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 correlation coefficient of 0.994, intercept of 0.002 and 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 for describe the method sensitivity The limit of detection and quantification were 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(2.725ng/ml) and the limit of quantification (8.992ng/ml) as the lowest standard concentration which could be determine with acceptable accuracy

Figure 3: Calibration curve of 5-Fluorouracill

The accuracy and precision of the method, a pure drug solution was 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 than1.4 indicative of accuracy of the method.                   

a: Mean of six determinations

Table I: Accuracy and precision of the proposed method.

The proposed method was satisfactorily applied to the determination of 5-Fluorouracill in its pharmaceutical preparations creams samples, blood plasma samples and wastewater samples, the results of the assay of the pharmaceutical preparations revels 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 show that the recovery values obtained were closed to 100%. 

Table 2: Determination of 5-Fluorouracill formulations                                                                                         

* mean value of ten determinations. 

Table 3: Determination of 5-Fluorouracill in industrial wastewater samples

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 closed to 100%   

               * Mean value of ten determinations

Table 4: Determination of 5-Fluorouracill in Blood Plasma samples           

The developed method is found to be highly sensitive, accurate, simple, precise and economical, and can be used for routine quality control analysis of 5-Fluorouracill in pure form, pharmaceutical formulations, Blood Plasma Samples and environmental waste water samples

The author wishes to express gratitude to AL-hokamaa company for pharmaceutical industries (HPI) Mosul-Iraq, for providing gift samples of 5-Fluorouracill standard materials and pharmaceutical preparations.