Second derivative spectrophotometric determination of cyclophosphamide in pharmaceutical formulations

A new, simple, rapid, wide applicable range and reliable second derivative spectrophotometric method has been developed for determination of cyclophosphamide (CP) in bulk and pharmaceutical dosage forms. Calibration graph is linear in the concentration range of 25 - 200 μg/ml of CP with 10 μg/ml of detection limit and correlation coefficient of 0.9976. The precision and accuracy were acceptable depending upon the values of relative standard deviation and error percentage. Developed second derivative spectrophotometric method can be directly and easily applied for analyzing pure form and commercial pharmaceutical preparations of CP. The method was compared with a standard high performance liquid chromatography (HPLC) method and can be used for the quality control of pharmaceutical preparations in Erbil City.


INTRODUCTION
Cyclophosphamide (CP) is widely used in cancer chemotherapy, mostly in combination with other antineoplastic agents, and as an immunosuppressant (Karahalil and Akkoyunlu, 2003).Cyclophosphamide (belongs to the group of alkylating agents) is a pro-drug that is activated via 4-hydroxylation by cytochromeP450s such as CYP2B6 and CYP3A4 to generate alkylating nitrogen mustards (phosphoramide mustard and the byproduct acrolein).The resultant mustards can alkylate DNA to form DNA-DNA cross-links, leading to inhibition of DNA synthesis and cell apoptosis (Malothu et al., 2009).
Several analytical methods have been reported and published for quantitative determination of CP in biological fluid and environmental samples, such as: High performance liquid chromatography (HPLC) methods (Malothu et al., 2009;Ahmad et al., 2011), gas chromatography (GC)-mass spectrometry (Sugiura et al., 2010), and after extraction of CP by solid phase extraction techniques, using solid-phase extraction and GC-MS spectrometry (Martins et al., 2004), on-line sample preparation method by micro-extraction packed sorbent (MEPS) followed by LCMS/MS (Kamel et al., 2009), liquid chromatography with diode array detector (Alcântara et al., 2010), andspectrophotometric method (Karen et al., 2009).To our knowledge, there are no derivative spectrophotometric methods concerning the determination of cyclophosphamide in pharmaceutical formulations.
Derivative spectrophotometry is an analytical technique of great utility for extracting both qualitative and quanti tative information from spectra composed of unresolved bands, and for eliminating the effect of baseline shifts and baseline tilts.It consists of calculating and plotting one of the mathematical derivatives of a spectral curve.Derivative spectrophotometry is now a reasonably prized standard feature of modern micro-computerized UV spectrophotometry (Patel et al., 2010).Derivative UV spectrophotometry has been widely used as a tool for quantitative and control analysis in agricultural, pharmaceutical and biomedical fields (Stanisz et al., 2009).The aim of this study was to develop a simple, rapid and efficient second derivative spectrophotometric method for the determination of CP in pharmaceutical formulations and compared with HPLC method that can be used for the quality control of pharmaceutical preparations in Erbil City.

Apparatus
A CECIL CE 3021 UV/Vis scanning spectrophotometer equipped with 10 mm path length quartz cell was used to record normal and second derivative spectra of CP solutions.

Chemicals
All chemicals used were of analytical reagent grade.Stock CP solution (1000 μg/ml) (Sigma-Aldrich) was prepared by dissolving 1000 mg of CP in distilled water and diluting to 1000 ml in a volumetric flask and stored in a refrigerator.Each working standard solution was freshly prepared by diluting the stock solution with distilled water.

Recommended procedure
The standard solutions were prepared by dilution of the stock solution with distilled water in a concentration range of (25 -200) μg/ml for normal and second derivative spectrophotometric methods.Distilled water was used as a reagent blank.The second derivative spectral recording were carried out using smoothing of 2 and 25 nm of interval at a scanning speed of 10 nm/sec in the wavelength range of 190 -400 nm.

Sample preparation
Amount of the CP vial content were accurately weighed and dissolved in distilled water and transferred to 25 ml volumetric flask, then diluted to the mark with distilled water.Appropriate dilutions Fakhre et al. 6533 were made to obtain a solution in the concentration range of the calibration curve, and recommended procedure was applied.

RESULTS AND DISCUSSION
The normal and second derivative spectra of the solutions of CP were recorded at the wavelength range of 190 -400 nm against reagent blank as shown in Figures 1 and 2 which show maximum absorption at 193 nm for normal spectra and 207.3 nm for second derivative spectra of the solutions, respectively.

Statistical data of the calibration curve
Under the recommended experimental conditions for second derivative spectrophotometric method the calibration graph was found to obey Beer's law in the concentration range of 25 -200 μg/ml, with the detection limit of 10.0 μg/ml.Regression analysis was made for the slope, intercept and correlation coefficient values.The regression equation of calibration curve is y = 0.0456x + 0.0982 and R 2 = 0.9976.

Precision and accuracy
The precision and accuracy of the second derivative spectrophotometric determination of CP were studied depending upon the value of the relative standard deviation percentage (RSD %) and the relative error percentage (error %) for five replicate measurements of three different concentrations, respectively.Table 1 shows the results.

Application of the method
The proposed method was applied to the determination of CP in some vials of CP samples from Baxter Company which are the only vials available in the hospitals of Erbil City.In the present method, from each vial samples (500 and 1000) mg different solutions were prepared and the recommended procedure was applied.The results are summarized in Table 2.The method was in a good agreement in comparison with the results obtained using HPLC for the standard CP (Figure 3) and qualification and determination of CP in the two vial samples as shown in Figures 4 and 5.

Conclusion
The second derivative spectrophotometric methods have been described for quantification of cyclophosphamide in pure form of CP and its ratio in the vial content, at the time there were no any UV and derivative     preparations in Erbil City.

Figure 1 .
Figure 1.Normal spectrum of CP solution.

Figure 2 .
Figure 2. Normal and second derivative spectra of CP solution.

Figure 5 .
Figure 5. Chromatogram of the second sample.

Table 1 .
Precision and accuracy of the proposed second derivative spectrophotometric method.

Table 2 .
Determination of CP in vial samples with the proposed method.Chromatogram of the first sample.