A New Spectrophotometric Method for the Determination of Eflornithine Hydrochloride in Parenteral Formulation

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The proposed method reports a new method for the determination of DFMO in parenteral pharmaceutical. The aim of this investigation was to develop a new, sensitive, cost effective and validated method for the determination of DFMO in pure form and in parenterals by spectrophotometry.

2. Materials and Methods

2.1. Materials

Eflornithine hydrochloride and 200mg vials (Ornidyl^®) were gifts from Wintac Limited, Bangalore (India). All reagents applied as solvents were analytical grade and obtained from Sigma-Aldrich (USA). Solvent utilized throughout the project was 95% ethanol. Water was always distilled.

2.2. Instrumetation

The equipments used were the following: A model Shimadzu UV-1601 with a fixed slit width of 2nm using a pair of 1cm matched quartz cells recording double beam UV- visible spectrophotometer connected to a computer loaded with Shimadzu UVPC; Mettler H51 analytical balance (Zürich, Switzerland).

2.3. Spectrophotometric Measurements
2.3.1. Preparation of Solutions
2.3.1.1. Stock and Working Standard Solutions

A stock solution of 1.0mg mL^-1 was prepared by dissolving 100mg of DFMO in a 100mL of ethanol. Working standard solutions of DFMO of 100µg mL^-1 were prepared by appropriate dilution of the stock solution with ethanol. This solution was prepared daily.

2.3.1.2. Sample Solutions

Twenty vials of (Ornidyl 200mg mL^-1) were used. The sample vial (SVP) containing 200mg mL^-1. Pipetted out 1ml and diluted to 100ml with ethanol. Now further dilutions were done with ethanol to get the final concentration of 100µg mL^-1. Appropriate dilutions were made using the same diluent.

2.3.1.3. Determination of Absorption Maxima (lambda max) of Eflornithine (DFMO)

2.0mL aliquot of standard DFMO solution of 10μg mL^-1was pipetted into a 10mL volumetric flask. The volume was made up to 10mL with ethanol and solution was then scanned in the range of 200 to 400nm against the reagent blank. The absorbance values are recorded and graphically represented in (Figure 2). On scanning the absorption maxima of (DFMO) 20μg mL^-1 was found out to be 283 nm.

2.3.1.4 Procedure for Standard Curve

Aliquots of 0.4mL, 0.8mL, 1.2mL, 1.6mL, 2.0mL, 2.4mL, 2.8mL and 3.2mL of 100µg mL^-1 solution of DFMO was pipetted into each of 10mL volumetric flasks. The volume was made up to 10mL with ethanol. The absorbance of solution was measured at 283nm against ethanol as blank. The Zero-order absorption spectra and Calibration curve for DFMO was shown respectively in (Figure 3 and Figure 4)

Figure 2. Absorption spectra of DFMO (20 μg mL^-1)

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Figure 3. Zero-order absorption spectra for DFMO, showing λ max at 283nm

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Figure 4. Calibration curve of DFMO at 283nm

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2.3.2. Method Validation

The analytical method was validated according the International Conference for Harmonization (ICH) guidelines ^[18] under the optimized experimental conditions: linearity, accuracy, precision, specificity.

2.3.2.1. Linearity

The linearity was evaluated by linear regression analysis, which was calculated by the least squares regression method.

2.3.2.2. The Limit of Detection (LOD) and Quantification (LOQ)

The limit of detection was calculated by LOD = 3.3σ/S, where σ is the standard deviation of the response of the blank or the standard deviation of intercepts of regression lines and S is the slope of the calibration curve. The limit of quantification was calculated by LOQ = 10σ/S under the ICH guidelines ^[18]

2.3.2.3. Precision and Accuracy

In order to assess the intra and inter-day precision and accuracy of the assay, were prepared as described above. The inter-day precision and accuracy were determined by analyzing the three level samples on 3 different days. Precision was expressed as the relative standard deviation (RSD %). Accuracy was expressed as the mean relative error (RME %).

2.3.2.4. Recovery

The recovery method was performed by adding known amounts of the studied compounds to a known concentration of the commercial pharmaceutical parenteral (standard addition method).

3. Result and Discussion

3.1. Linearity and Range

The proposed method showed good linearity in the concentration range of 4 - 32µg mL^-1. For DFMO method, the regression equation was A= 0.0489C + 0.0006 with r=1 (Figure 4). Analytical parameter for the determination of eflornithine hydrochloride was shown (Table 1).

Table 1. Analytical Parameters for the Determination of DFMO Using the Proposed Method^a⁾

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3.2. Accuracy and Precision

Accuracy and precision was determined from DFMO sample at three different concentrations in the calibration range in six replicates. The relative standard deviation (RSD) on the absorbance from three replicates solutions was found to be between 0.11 and 0.65%. The data is summarized in (Table 2).

Table 2. Intra-day and Inter-day Precision Accuracy of DFMO Proposed Method^a⁾

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3.3. Recovery Study of Proposed Method

The recoveries of the standard addition method (Table 3) suggested that high accuracy of the proposed methods. The study was performed by increasing standard addition of known amounts of studied drugs to an unknown concentration (constant volume) of the standard drug. Known concentration DFMO was taken which is dissolved in ethanol and the volume make with ethanol. A constant volume of the unknown solutions was added to each of eight 10ml volumetric flasks. Then a series of increasing volumes of working standard solutions were added. Finally, each flask was made up to the mark with ethanol for DFMO and mixed well. The concentration of the working standard solutions added should be chosen to increase the concentration of the unknown by minimum 50% in each succeeding flask. The resulting mixtures were analyzed and recoveries were found to be between % 99.08 and 100.23.

Table 3. Result of Recovery Studies by Standard Addition Method^a)

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3.4. Ruggedness (Reproducibility)

The ruggedness of the method was determined by carrying out the experiment by three different analysts. The average value of % RSD of the findings for determination of DFMO was found to be 1.014. This showed that the method was reproducible by three different analysts (Table 4).

Table 4. Reproducibility of the method by three different analysts

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3.5. The Limit of Detection (LOD) and Quantification (LOQ)

Calibration curve was repeated for 6 times and the standard deviation (SD) of the intercepts was calculated.

Then LOD and LOQ were measured as follows

LOD = 3.3 × SD/slope of calibration curve

LOQ = 10 × SD/slope of calibration curve

SD = Standard deviation of intercepts

The values of LOD and LOQ are given in Table 1.

3.6. Assay of Commercial Formulation

Applicability of the method was tested by analyzing the commercially available formulation as Ornidyl. The data is summarized in (Table 5) was found to be very close to the label value of commercial pharmaceutical formulation, which showed that the method was applicable for determination of DFMO from their parenteral formulations.

Table 5. Analysis of Commercial Formulation

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