Development and Validation of a Diethyl Phosphite Content in Foscarnet Sodium USP by GC MS Technique

A simple, rapid, selective, and reproducible Gas chromatographic mass spectrometry (GC-MS) method has been developed and validated for the estimation of Diethyl Phosphite content in Foscarnet Sodium USP Drug substance. The drugs were estimated using HP-5, Length-30 M, Internal diameter 0.32 mm; Film thickness 1 μ at a total flow rate of 11.9 ml/min, and column flow of 1.49 ml/min was used for the separation. Flow control mode was pressure. Column oven temperature 70°C and injector temperature 220°C. Oven program modified for proper elution of peak. The linearity range used was 0.025-0.120μg/ml and (Rt) was 6.7 min. The correlation coefficient values were found to be 0.997. Precession studies showed % RSD values less than 15.0% for all the selected concentrations. The percentage recovery of Diethyl phosphite from LOQ to 150% was found in range of 100.7 -116.7%. The content results of Phophite content were within the limits of less than 0.12 ppm. The method was validated as per the International Conference on Harmonization (ICH) guidelines. The developed method was successfully used for the quantitative analysis of commercially available dosage forms. Original Research Article Bhatale et al.; JPRI, 33(46A): 192-201, 2021; Article no.JPRI.75557 193


INTRODUCTION
Foscarnet Sodium [1] is the trisodium salt of a synthetic organic analog of inorganic pyrophosphate with antiviral [2] activity. Foscarnet selectively blocks the pyrophosphate binding site of herpesvirusspecific DNA polymerases at concentrations that do not affect cellular DNA polymerases. This agent does not require phosphorylation by thymidine kinase (TK) or other kinases and therefore is active in vitro against herpes simplex virus (HSV) TK deficient mutants and cytomegalovirus (CMV) UL97 mutants. Because foscarnet crosses the blood brain barrier, it may be used in the treatment of viral infections of the CNS.
Foscarnet is used [3] to treat cytomegalovirus (CMV) retinitis in people with AIDS. Foscarnet is also used to treat the herpes simplex virus (HSV) in people with a weak immune system. Serious side effects may include [4] Anaemia, nausea, and vomiting, disturbances in electrolyte levels and genital ulceration have also been associated with administration of the drug.

Reagents and Materials
Dichloromethane HPLC grade Di-Sodium hydrogen phosphate dihydrate (Emparta Merck) Purified water

Diluent
Used Dichloromethane as diluent.

Buffer solution
Prepared 1% of Di-Sodium hydrogen phosphate dihydrate in purified water.

Blank preparation
Pipetted 1.5 mL diluent in HS vial and added 17 mL buffer solution mixed well, allowed the layers to settle down. Pipetted out about 1 ml of the lower Dichloromethane layer to an auto sampler vial containing about 150 mg of sodium sulfite. Shook well, decant the supernatant liquid in a vial for analysis.

Standard preparation
Prepared 0.08 ppm of standard solution (Diethyl phosphite) in diluent. Pipetted 1.5 mL of this solution in HS vial and added 17 mL buffer solution mixed well, allowed the layers to settle down. Pipetted out about 1 ml of the lower Dichloromethane layer to an auto sampler vial containing about 150 mg of sodium sulfite. Shook well, decant the supernatant liquid in a vial for analysis.

Test preparation
Weighed 1000 mg of the test sample in the HS vial, added 17 mL buffer solution sonicated to dissolve. Added 1.5 mL diluent and mixed well. Allowed the layers to settle down. Pipetted out about 1 ml of the lower Dichloromethane layer to an auto sampler vial containing about 150 mg of sodium sulfite. Shook well, decant the supernatant liquid in a vial for analysis.

METHOD DEVELOPMENT
Because in foscarnet sodium Diethyl phosphite impurity(Genotoxic impurity) in liquid state and no chromophore found in structure hence Gas Chromatography Mass spectrometer method used for detection. In GC MS, non-polar stationary phases such as phenyl ie. When the HP-5, Length-30 M, Internal diameter 0.32 mm; Film thickness 1 μ as utilized, superior impurity separation, peak sharpness, and system suitability were discovered.

RESULTS AND DISCUSSION
The IP, BP, USP, and Q2 (R1) [5-8] of the ICH guideline were used in this validation and development study. For the finalization of the specified limit based on treatment duration and dose, the ICH guideline M7 (R1) 9 was used. The validation parameters for Analytical method [10][11][12][13][14][15] are explored in more detail below.

Specificity
By injecting Blank (diluent), standard (0.08 ppm Diethyl phosphite), and sample solution, the selectivity research parameter was done (666666 ppm). The chromatograms are analyzed at the same chromatograph having a mass detector as the method specifies. Table 5 contains the specificity data, as well as a chromatogram in Fig. 2. Blank (diluent) has no effect on the retention period of the Diethyl phosphite peak. All recognized and unknown peaks in the sample solution are well isolated from one another.

Limit of Detection (LOD) and Limit of Quantitation(LOQ)
The signal-to-noise ratio approach was used to calculate the LOD and LOQ conc. of Diethyl phosphite impurity in Foscarnet sodium. Injecting various concentration levels (between 10 and 100 percent) of standard solutions of Diethyl phosphite at limit level concentrations to determine the projected LOD and LOQ concentrations. 0.025 ppm was the predicted LOQ concentration value for Diethyl phosphite impurity. The LOD concentration is calculated by multiplying the predicated LOQ concentration by a factor of 0.33. Table 6 shows the predicted LOD and LOQ values.

Linearity and Range
The capacity of a method to produce test findings that are proportionate to the concentration of analyte in a given test sample is known as linearity. Standard solutions of Diethyl phosphite impurity with LOQ Level to 150 percent specified limit (including 30 50, 80, 100, 120, and 150 percent) of concentration were used in the linearity investigation.      Table 7 shows the correlation coefficient, slope, concentrations, and intercept of linearity data, and Figure 6 shows the linearity graph. Least squares linear regression analysis was used to examine the peak area versus concentration data. The Diethyl phosphite impurity has a correlation coefficient of 0.9973, which is higher than 0.99.

Precision
As stated in the technique of analysis, system precision was achieved by injecting five replicates of the standard preparation.

Accuracy
Spiking test preparation with an impurity at LOQ level, 50% level, 100 and 150 percent of specification limit concentrations was used to establish method accuracy. Table 9 shows the percent accuracy data for the Diethyl phosphite impurity. The percent accuracy observed at the LOQ level and 50% level, 100 and 150 percent is between 100.7 and 116.7 percent, which is within acceptable limits. (An accuracy of 70 to 130 percent is recommended).

Robustness
The method's robustness was tested by altering the pressure by±10% psi. The pressure is changed from3.0 psi to 2.7 psi and 3.3 psi. In the actual procedure, the column oven temperature is varied by± 5 °C from 70 °C to 65 °C and 75 °C. Table 7 displays the observed standard deviation, and percent RSD. The retention times in all of the studies above differed by ±0.2 minutes from the original retention times. For robustness studies, the percent RSD ranges from 2.05 to 3.35%. Changes in method parameters (pressure and column oven temperature) had no significant impact on system suitability criteria ie. percent RSD, according to Table 10. The values obtained are considerably within the acceptable range.

Solution Stability
The solution stability of the test preparation was tested at 25°C on a day-by-day basis for up to three days. Up to 3 days, the cumulative percent RSD values of the Diethyl phosphite impurity are substantially below acceptable limits. This implies that when stored at 25°C temperature, Analytical test preparations are stable for 3 days.

CONCLUSION
The GC-MS method for Diethyl phosphite impurity content determination of Foscarnet sodium is very precise, selective, accurate, and stable, and follows ICH criteriaQ2(R1) also has been accurately developed and validated. The selectivity of method demonstrates that the Diethyl phosphite impurity peak is fully resolved from both known and unknown impurities. With LOQ -150% level w.r.t. specification concentration, the method is linear, and the observed Correlation coefficient is 0.997 and Diethyl phosphite impurity was recovered between 100.7 and 116.7%.. System suitability, such as percent RSD, has no substantial impact on robustness. The observed outcomes were deemed to be within acceptable criteria. For all of the technical parameters that have been examined, the validated method has shown satisfactory results. As a result, the current method is specific, linear, selective, precise, robust, and stable, and can be used well in analysis.

DISCLAIMER
The company name used for this research is commonly and predominantly selected in our area of research and country. There is absolutely no conflict of interest between the authors and company because we do not intend to use this company as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.