INTRODUCTION:

The PDH is an active pharmaceutical ingredient (API)in cancer treatment. It is used as a single agent or in combination with other chemotherapeutic agents for the treatment of different types of cancers such as bladder cancer, colorectal carcinoma, breast cancer and cervical cancer^1,2. It acts as a guanine derived anti-neoplastic agent.PDH also inhibits a nucleic acid synthesis via binding and inhibition of thymidylate synthase. PDH is a folate analog metabolic inhibitor that exerts its action by disrupting folate-dependent metabolic processes essential for cell replication^3,4. This new generation anti-folate is approved for the treatment of mesothelioma as well as non-small cell lung cancer.

PDH exist in a solid drug formulation either as a crystalline or amorphous form^5,6. The chemical structure of PDH, i.e.N-[[4-[2-(2-amino-4-oxo-4,7-dihydro-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]phenyl] carbonyl]-L-glutamic acid disodium hemipentahydrate, is shown in Fig. 1. The molecular formula of PDH is C₂₀H₁₉N₅Na₂O₆·(H₂O)_2½and its molecular weight is 516.42 g/mol. The three different forms of Pemetrexed Disodium viz. anhydrous, hemipentahydrate and heptahydrate are presently well established in patent publications^{7,8,9,10,11,12,13,14}. Out of that, heptahydrate form is published in the European Pharmacopoeia¹⁵.

Fig. 1: Chemical structure of PDH

MATERIAL AND METHOD:

Materials:

The sample of PDH is received from Emcure pharmaceuticals limited, analytical research center, Hinjawadi, Pune. Analytical grade glacial acetic acid and milli-Q grade purified water is used for mobile phase and diluent preparation. Analytical balance is used as Metler Toledo and high-performance liquid chromatography as Waters All the instruments are calibrated during method development and validation study.

Method:

Mobile phase:

The homogeneous mixture of 0.15% glacial acetate acid with 5.3 pH and acetonitrile in the ratio of 90:10

Diluent:

Purified water

Standard and test preparation:

Weighed 50.0mg of standard/test sample of PDH, dissolved and diluted 100mL with purified water.

The instrument method parameters for quantitative analysis of PDH are listed in Table 1.

Table 1: Instrument method parameters for quantitative analysis of PDH

Component	Specification
Instrument	HPLC equipped with injector, pump, UV/PDA detector and recorder
Detector	UV/PDA detector
Column	Zorbax SB C8, (4.6 x 150 mm) 3.5 µ
Wavelength	285 nm
Flow rate	2.0 mL/min
Injection volume	20 µL.
Column oven temperature	30°C
Auto sampler temperature	10ºC
Run time	15 min

RESULTS AND DESCUSION:

According to ICH guideline^16,17,18,19analytical method validation is performed by using different parameters. They are described as follows,

Specificity:

The specificity of method is measures the analyte response in presence of related impurities. The assay method should present discrimination of the analyte and such impurities. Specificity data is given in Table 2 and the related chromatogram is shown Fig.2. This is done by demonstrating that the assay results are unaffected due to the presence of spiked impurities with 1.0 %concentration (5ppm) compared to the pure substance (500 ppm). Blank run does not show any interfering peak with PDH peak. The PDH peak is well resolved from known impurities (Fig.2). Peak purity angle for PDH peak and the peaks of its related impurities are below threshold in individual solutions of related impurities and unspiked as well as spiked tests. This indicates that these peaks are pure (Table 2). The % relative standard deviation (%RSD) in five replicates of PDH is 0.32. Tailing factor and theoretical plates in prepared standard are 1.27 and 4047 respectively.

Fig.2: Typical chromatogram for specificity in the PDH assay over the spiked impurities

Linearity and Range:

The linearity of method is the measure the proximity of the straight line obtained from a calibration plot of response versus concentration. It is obtained by performing the experiments using different concentrations of the PDH. Thus the linearity of present HPLC method is determined by using test solution with concentrations in 80 to 120% of analyte as per ICH guidelines. The linearity data for 80, 90, 100, 110 and 120% concentrations of standard preparation is given in Table 3. The peak area verses concentration data was treated by least squares linear regression analysis. The correlation coefficient obtained was 0.99992. The %Y intercept of calibration curve is 0.16 which is much below the allowed value 2.00. This shows that there is less variation in different linearity concentration levels. All observed value and graph plotted for visual inspection (Fig.3) indicates that this method is linear for given range.

Table3: Linearity data for the PDH from 80 to 120% concentration range

Levels	Concentration (ppm)	Peak area		Average
Levels	Concentration (ppm)	Injection-1	Injection-2	Average
1	107.80	1161580	1159605	1160593
2	121.27	1311668	1312714	1312191
3	134.75	1453661	1457852	1455757
4	148.22	1602923	1599791	1601357
5	161.70	1743086	1741118	1742102
Correlation coefficient				0.99992
Intercept			10776.6960
% Y Intercept			0.16

Fig. 3: Linearity graph for the PDH in concentration range of 80 to 120 %

Precision:

Precision provides an indication of random errors and can be subdivided into repeatability and intermediate precision. The precision studies are performed when the entire analytical method procedure is finalized. System precision is studies by carrying out five replicates of PDH standard. Comparative data of % assay of method precision and intermediate precision are shown in Table 4. The % RSD of five replicate PDH standards is 1.27. The method precision of assay has been performed by injecting six individual test sample preparation. The % assay and % RSD of test sample obtained are calculated. The intermediate precision is also evaluated using different instruments and different columns by different analysts on different days in different laboratory. The % RSD for assay of repeatability and intermediate precision study is 0.09 and 0.28 respectively. The overall % RSD of twelve test preparations (six each for method precision and intermediate precision) is 0.22.

Solution Stability:

The stability of sample solution was performed at the room temperature as well as 2 to 8ºC on the day basis up to 3 days. The % assay of PDH was calculated for the study period of test preparation. Cumulative % RSD values of assay are 0.02, 0.18 and 0.24 at the room temperature and 0.05, 0.32 and 0.27 at 2 to 8ºC for days 1, 2 and 3 respectively. It is within acceptance criteria (% RSD being less than 2.00) up to 3 days. This indicates that the sample solution is stable up to 3 days, when stored at room temperature and 2 to 8ºC.

Table 4: Comparative % Assay data of repeatability and intermediate precision for the PDH

ID	% Assay
ID	Repeatability	Intermediate precision
Test Preparation-1	99.13	99.41
Test Preparation-2	99.23	99.74
Test Preparation-3	99.34	99.58
Test Preparation-4	99.22	99.01
Test Preparation-5	99.16	99.75
Test Preparation-6	99.08	99.53
Average (n=6)	99.19	99.50
SD (n=6)	0.0911	0.2741
% RSD (n=6)	0.09	0.28
Average (n=12)	99.30
SD (n=12)	0.2227
% RSD (n=12)	0.22

Robustness:

The robustness of analytical method is determined by purposely altering experimental conditions such as flow rate and column oven temperature and PH of mobile phase. The flow rate was changed by ±10%. Actual flow rate of 2.0mL/min is altered as 1.8mL/min and 2.2mL/min. The column oven temperature is changed with ±5°C from 30°C in original method. The pH of mobile phase was changed by ±0.2. Actual pH of mobile phase of 5.3 is altered as 5.1 and 5.5. The observed area, standard deviation and its % RSD are listed in Table 5. In all above cases the retention times are varied by ±0.3 mins compared to actual retention times. System suitability parameter such as tailing factor is 1.20 to 1.32. The %RSD for robustness studies are from 0.04 to 0.08. In all deliberate varied chromatographic conditions (flow rate, column oven temperature and pH of mobile phase), significant changes are not observed for the system suitability criteria like tailing factor and %RSD. The values of these criteria are well within acceptance limit.

Forced degradation Studies:

Forced degradation^20,21study gives a measure of specificity and helps in selection of stability-indicating analytical procedures. The % degradation was determined by comparing chromatogram obtained under stress condition with untreated sample. The degradation studies include thermal, photolytic, humidity, aqueous, acidic, basic and oxidative stress conditions. The degradation data under these conditions are shown in Table 6. In thermal degradation test sample was heated to 105°C for 24 h. Humidity degradation was carried out with 75% relative humidity at 40°C for 24 h, while in the photolytic degradation the test samples were exposed to near UV light of 200 W·h·m−2 intensity till the energy of 1.2 x 10⁶lux.h.The physical appearance of the test solutions remained unchanged in heat, humidity and photolytic stress condition. All above test are performed using the present analytical method for the concentration of 150 ppm of PDH. The peak area of the PDH remained constant without any degradant peak, which indicates that this molecule is stable for heat, humidity and photolytic stress. The chromatograms of the acid, alkali and oxidation degradations are depicted Fig. 4, 5 and 6. All known as well as degradants impurities are well separated from the PDH. The peak purity criteria are also satisfied for PDH, its known and degradant impurities.

The alkali degradation is performed by exposing test sample to 5mL 5N NaOH kept the solution at room temperature for 15 h. The sample is then neutralized with HCI and the analysed on HPLC. The net 17.84% degradation of PDH is observed. Oxidation degradation has been performed by adding 2.5mL 50% H₂O₂ kept the solution at room temperature for 30 min. The PDH is degraded by 11.32%. While in acid degradation study test sample exposed to 2.5mL 50% H₂O₂ kept the solution at room temperature for 30 min. The solution is then neutralized with NaOH. The 11.67% degradation of PDH is observed from the relative peak area.

Table 5: Area, standard deviation and its % RSD of robustness study for the PDH

Replicates of injections	Area of PDH
	Flow rate of mobile phase		Column oven temperature		pH of mobile phase
	1.8 mL/min	2.2 mL/min	25°C	35°C	pH-5.1	pH-5.5
Standard Inj.-1	1633671	1330093	1470500	1467044	1473342	1467927
Standard Inj.-2	1635093	1330934	1471048	1467282	1475314	1469319
Standard Inj.-3	1633281	1331728	1472069	1468218	1475527	1470139
Standard Inj.-4	1633585	1330285	1471245	1468324	1474707	1469845
Standard Inj.-5	1634237	1329559	1469998	1467981	1472697	1468977
Mean	1633973	1330520	1470972	1467770	1474317	1469241
SD	714.9663	835.4243	783.4497	573.8817	1243.5065	862.2058
%RSD	0.04	0.06	0.05	0.04	0.08	0.06
Tailing factor	1.32	1.26	1.32	1.23	1.26	1.20

Table 6: The stress conditions with % Assay and % degradation of PDH

Stress Condition	Exposure period	% Assay	% Degradation	Purity Criteria
Untreated test	-	99.68	---	Pass
Humidity degradation	40°C, 75% RH for 24 h	99.72	---	Pass
Thermal degradation	105°C for 24 h	99.96	---	Pass
Photolytic degradation	Light energy of 1.2 million lux hours and near UV 200 watt hrs./m²	99.56	---	Pass
Aqueous degradation	10 mL 24 h at room temperature.	99.60	---	Pass
Acid degradation	5 mL 5N HCI kept the solution for 2.5 h at80°C in oil bath.	88.01	11.67	Pass
Alkali degradation	5 mL 5N NaOH kept the solution at room temperature for 15 h.	81.84	17.84	Pass
Oxidation degradation	2.5 mL 50%H₂O₂ kept the solution at room temperature for 30 min.	88.36	11.32	Pass

Fig. 4: Chromatogram for the alkali degradation studies of PDH

Fig. 5: Chromatogram for the oxidation degradation studies of PDH

Fig. 6: Chromatogram for the acid degradation studies of PDH

CONCLUSION:

The RP-HPLC method was developed for the quantitative analysis of PDH in active pharmaceutical ingredients and successfully validated as per the criteria of pharmacopeia and ICH guidelines. In this method PDH peak is well resolved from known impurities. The effective chromatographic separations were achieved on Zorbax SB C8, (4.6 x 150mm) 3.5µ column with isocratic elution programme. The mobile phase is as homogeneous mixture of 0.17% glacial acetate acid with 5.3 pH and acetonitrile in the ratio of 89:11. The delivered flow rate of mobile phase is 2.0mL/min using UV/PDA detector. The column oven temperature is 30°C and the injection volume is 20.0μL. This method is linear with correlation coefficient being 0.99992 and %Y intercept of 0.16. The repeatability and intermediate precision are evaluated and % RSD for these assays is 0.09 and 0.28, respectively, with overall %RSD of 0.22. Robustness studies do not shows significant change for the system suitability criteria like tailing factor, theoretical plates and %RSD. The values of these criteria are well within acceptance limit. The heat, humidity and photolytic stress condition have not shown any change in the physical appearance of sample and the peak area of the PDH, indicating its stability for these stress conditions. In case of acid, alkali and oxidation degradations all known as well as degradants impurities are well separated from the PDH peak and the peak purity criteria are also passed.

This RP-HPLC method is specific, linear, accurate, precise, selective, robust, as well as stable and can be used for the routine analysis in other laboratories.

ACKNOWLEDGEMENT:

The author expresses gratitude to Emcure Pharmaceuticals Limited, Hinjawadi, Pune for providing the Pemetrexed disodium hemipentahydrate sample for this work.

CONFLICT OF INTEREST:

Conflict of interest declared none.

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Received on 11.10.2019 Modified on 08.12.2019

Research J. Pharm. and Tech. 2020; 13(8):3685-3689.

DOI: 10.5958/0974-360X.2020.00652.6

Eluent*	RT (min)	Area	Tailing Factor	Theoretical plate	Purity Angle	Purity Threshold	Remark (Peak Purity)
A	1.747	11287	0.99	3023	1.197	5.225	Pass
PDH	2.690	1362106	1.17	4049	0.071	0.503	Pass
F	8.295	21612	1.02	10425	1.321	1.847	Pass