Senior research scientist at Analytical Research & Development Department, Neuland Pharma Research, R&D Centre Bonthapally, Hyderabad.

S.D. Mankumare

Pemetrexed is an anticancer drug indicated in the treatment of pleural mesothelioma as well as non small cell lung cancer (1). Its chemical structure is shown in 

. It is a folate analog metabolic inhibitor that exerts its action by disrupting folate dependent metabolic processes essential for cell replication. 

studies have shown that pemetrexed inhibits thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase, which are folate-dependent enzymes involved in the 

 biosynthesis of thymidine and purine nucleotides (2-4).

The literature reports only a few high-performance liquid chromatographic (HPLC) methods for determination of pemetrexed disodium and its metabolites in biological fluids (5-9). Genotoxicity refers to any deleterious change in the genetic material regardless of the mechanism by which the change is induced. Genotoxic impurities are defined as an impurity that has been demonstrated to be genotoxic. Detection, quantitation, and control of potentially genotoxic impurities to very low levels below the identification threshold presents considerable challenges for synthetic and analytical chemists in the development, manufacture, and control of API, impurities in the API, and the drug product. The aim of this study was to develop and validate a rapid, economical, and sensitive HPLC method for the determination of related substances in pemetrexed disodium (form-IV) in bulk drug samples. A robust, rapid, sensitive, and validated analytical method is needed for day-to-day analysis, in routine analysis in quality control laboratories of the drug in bulk drug samples, and which can be applicable for pharmaceutical dosage forms.

Materials and method
  Chemicals and reagents.

 Pemetrexed disodium (form-IV) bulk drug (99.79% purity) and genotoxic impurities end product by product EPBP-2 (85.84% purity), EPBP-3 (98.49% purity), EPBP-5 (87.98% purity), and intermediate IM-7 (99.69% purity) were provided by R&D Lab, Neuland Pharma Research Private Limited. Acetonitrile (HPLC grade) and trifluoroacetic acid (TFA) (HPLC grade) were obtained from Merck, India.

The HPLC system consisted of a Waters 2695 HPLC system separation module with Waters 2487 dual-wavelength absorbance detector. Data acquisition was performed by Empower Alliance software. The method is a gradient reverse-phase HPLC, which employs a reversed-phase column (250 x 4.6 mm, 5.0 μm, Zorbax SB-Phenyl, Agilent) with mobile phases A and B at ambient temperature. Mobile phase A consisted of 0.03% TFA in degassed and filtered Milli-Q water. Mobile phase B consisted of 0.025% TFA in degassed and filtered acetonitrile. The analysis was performed at a wavelength of 228 nm using a dual wavelength absorbance detector. The sample concentration was 1.0 mg/mL and injection volume was 5 µL with a run time of 40 min. 

Pemetrexed disodium (form-IV) is soluble in water. The drug can be separated on a reverse-phase column (Zorbax SB-Phenyl, Agilent). The optimization of the method development was done by gradient elution. The following gradient program was developed during method development (see 

Table I: Gradient program for separation of pemetrexed disodium (form-IV).

To prepare 0.1M sodium acetate solution, approximately 3.402 g of sodium acetate was weighed and transferred into a 250-mL volumetric flask containing water, and the volume was made up with water. Pemetrexed disodium (form-IV) standard solution was prepared by dissolving 25 mg of pemetrexed disodium (form-IV) standard in a 25-mL volumetric flask, and the volume was made up with the diluent. The diluent was acetonitrile and water in a 1:1 ratio. Pemetrexed disodium (form-IV) sample solution was prepared by dissolving 25 mg of the pemetrexed disodium (form-IV) sample in a 25-mL volumetric flask, and the volume was made up with the diluent.

EPBP-2 impurity solution was prepared by dissolving 10 mg of EPBP-2 impurity in a 100-mL volumetric flask and the volume made up with the diluent. EPBP-3 and EPBP-5 impurity solutions were prepared by dissolving 10 mg of EPBP-3 and EPBP-5 impurities respectively in a 100-mL volumetric flask and the volume made up with the diluent.

IM-7 impurity solution was prepared by dissolving 10 mg of IM-7 impurity in a 100-mL volumetric flask containing 95.0 mL of 0.1 M sodium acetate solution and 5.0 mL of acetonitrile.

The peak shapes and symmetry of pemetrexed disodium (form-IV) and impurities were acceptable with the gradient elution method. Pemetrexed disodium (form-IV) and impurities peaks were resolved with a resolution greater than 1.5 at a gradient flow rate. The method developed could determine related impurities at lower levels than the specification limit of not more than 0.10% with respect to the test concentration of 1.0 mg/mL. The developed method was successfully applied to estimate the amount of pemetrexed disodium (form-IV) related impurities.

The system suitability was assessed by duplicate analysis of the drug, which was spiked with all impurities at a concentration of 0.1% with respect to the test concentration of 1.0 mg/mL. The acceptance criteria were that the resolution between IM-7 and EPBP-3 should not be less than 1.5 and the tailing factor for pemetrexed disodium (form-IV) peak should not be more than 1.5. The theoretical plate count of pemetrexed disodium (form-IV) should not be less than 2000.

The reference solution for impurities quantification was assessed by the six replicate injections of all four impurities and a standard at a concentration of 0.1% with respect to the test concentration of 1.0 mg/mL. The acceptance criteria were that the % relative standard deviation (RSD) for the peak area of each component should not be more than 5.0.

The limit of detection (LOD) and limit of quantification (LOQ) were determined by calculating the signal-to-noise (S/N) ratio followed by comparison of test results from samples with known concentrations of analyte with those of blank samples. By the approach described, the minimum level at which the analyte can be reliably detected was established. The LOD and LOQ of the impurities and sample were determined by preparing 0.1% of reference solution. Approximately 5 µL of solutions were injected into the chromatographic system and the S/N ratio was calculated using the following equation:

The S/N ratio should be established to approximately 3:1 for LOD and approximately 10:1 for LOQ.

Linearity is the ability of the method to elicit test results that are directly proportional to analyte concentration within a given range. A series of seven solutions at concentrations of the LOQ, 25%, 50%, 75%, 100%, 125%, and 150% were prepared using the impurities standard stock solutions. Linearity of the method was demonstrated. The target concentration was 0.1% for these solutions with respect to the test concentration 1.0 mg/mL. Approximately 5.0 µL of each of the prepared solutions were injected into the chromatographic system and measured for six times at levels 1, 5, and 7 and for three times at levels 2, 3, 4, and 6. The average peak area response was calculated. A plot of data was established for analyte response versus concentration. The degree of linearity was estimated by calculating the regression coefficient (R2), % of Y-intercept, residual sum of squares, and Y-intercept/response at 100% of working concentration. 

The acceptance criteria were that the system suitability criteria should be met and % RSD for area of impurities should not be more than 5.0. The % RSD for the peak area of all components in the reference solution should not be more than 5.0. The regression coefficient for each impurity should not be less than 0.99 and % of Y- Intercept should be ± 5.0.

The accuracy of an analytical method expresses the closeness of agreement between the value that is accepted either as a conventional true value or an accepted value and the value found. Accuracy was calculated with respect to the prepared solution at the levels of the LOQ, 25%, 50%, 75%, 100%, 125%, and 150% of the limit concentration of 0.1% with respect to the test concentration 1.0 mg/mL. The accuracy of the method was demonstrated through a recovery experiment on samples at concentration level of the LOQ to 150%, and the recovery was calculated in each of the case using 

The acceptance criteria were that the % of recovery for the LOQ % solution should be 100 ± 20 and % of recovery for the 25-150% solutions should be 100 ± 15.

Precision of the method was demonstrated under three categories. 

 A single individual preparation of pemetrexed disodium (form-IV) drug substance was prepared with target concentration of approximately 1.0 mg/mL spiked with 0.1% of all known impurities for system precision. Six replicate injections of the preparation, each approximately 5.0 µL, were injected into the chromatographic system and the chromatograms were recorded. The RSD for the peak area and content of all four impurities from replicate injections were calculated.

 Six individual preparations of pemetrexed disodium (form-IV) drug substance were prepared with target concentration of 1.0 mg/mL spiked with 0.1% of all known impurities. Approximately 5.0 µL of the preparations were injected  into the chromatographic system and the chromatograms were recorded. The RSD for the peak area and content of all four impurities of the six different preparations were calculated.

 The ruggedness of an analytical procedure is a measure of its capacity to remain unaffected by intra-laboratory variations. It provides an indication of the method’s reliability during normal usage. Demonstration of ruggedness was conducted by different analysts, on different systems and columns, and on different days under similar conditions at different times. Six individual preparations of pemetrexed disodium (form-IV) drug substance were prepared with target concentration of about 1.0 mg/mL spiked with 0.1% of all known impurities and each were analyzed (see 

The acceptance criteria for system, method, and intermediate precision were that the system suitability criteria should be met and the % RSD for the peak area of each component in the reference solution from the six replicate injections should be not more than 5.0%. The % RSD for the content of each impurity should not be more than 5.0 and the cumulative % RSD for the content of each impurity obtained from the ruggedness study and method precision should not be more than 10%. 

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. Two parameters of the method (i.e., flow rate and wavelength of detection) were challenged at both lower and higher levels of the normal operational standard conditions (method precision conditions) and the analytical results of all the analytes of interest were evaluated. Six individual preparations of pemetrexed disodium (form-IV) drug substance were prepared with target concentration of about 1.0 mg/mL spiked with 0.1% of all known impurities and each were analyzed. The parameters selected with their higher and lower tested values are shown in 

Table II: Tabulated representation of robustness

Method    precision (standard conditions)

Articles

The authors describe the development of a stability-indicating reverse-phase high-performance liquid chromatographic (RP?HPLC) method for the quantitative determination of potential genotoxic impurities present in pemetrexed disodium (form-IV). The chromatographic separation was achieved on a RP?HPLC column (Zorbax SB-Phenyl, Agilent) at ambient temperature with gradient elution using mobile phase A (trifluoroacetic acid [TFA] in water) and mobile phase B (TFA in acetonitrile).