Enantiomeric separation and validation of D-isomer in Pemetrexed disodium–An anti-cancer agent using Chiral HPLC
S. Hemchand*1, R. Ravi Chandra Babu1 , Mukthinuthalapati Mathrusri Annapurna2
1GITAM Institute of Science, GITAM (Deemed to be University), Visakhapatnam, India
2Department of Pharmaceutical Analysis & Quality Assurance, GITAM Institute of Pharmacy,
GITAM (Deemed to be University), Visakhapatnam, India
*Corresponding Author E-mail: hemchand.suryadevara@gmail.com
ABSTRACT:
Pemetrexed disodium is an anti-cancer agent. It is an anti-folate drug used for the treatment of malignant pleural mesothelioma and non-small cell lung cancer. In the present study the authors have proposed a chiral HPLC method for the separation and determination of Pemetrexed disodium and its D-isomer using Chiralpak AD-H (250 x 4.6 mm, 5 µm) column within a run time of 30 mins. A mixture of n-Hexane: Ethanol: Isopropyl alcohol: TFA (250:650:100:1) was the final optimized mobile phase composition after many trials for the separation of D-isomer of Pemetrexed disodium (at 35°C) using Waters Alliance 2695 series HPLC system with 2998 photodiode array detector (UV detection at 240 nm). The method is very much useful for the pharmacokinetic studies as well as the metabolite study in vitro and in vivo for the determination of therapeutic activities of optical isomers.
KEYWORDS: Pemetrexed disodium, HPLC, D-isomer, Validation.
INTRODUCTION:
Pemetrexed disodium is a disodium salt of (2S)-2-[[4-[2-(2-amino-4-oxo-3,7-dihydropyrrolo[2,3-d] pyrimidin-5-yl)ethyl]benzoyl]amino]pentanedioate (Figure 1A) and is available as hepta hydrate or hemi penta hydrate whereas the (D-Isomer impurity) is chemically N-[4-[2-(2-Amino-4, 7-dihydro-4-oxo-1H-pyrrolo [2, 3-d] pyrimidin-5-yl) ethyl] benzoyl]-D-glutamic acid disodium hemi penta hydrate (Figure 1B). Pemetrexed disodium was approved for the treatment of cancer. It can be used alone or also in combination with other drugs for the treatment of malignant pleural mesothelioma for patients who cannot be treated using surgery and also certain types of non-small cell lung cancer for patients whose disease is advanced and metastasized1-5. Pemetrexed disodium was determined by using
Waters Alliance 2695 series HPLC system with 2998 photodiode array detector (UV detection at 240 nm) along with Chiralpak AD-H (250 x 4.6 mm, 5 µm) column was used for the determination of D-isomer in Pemetrexed disodium in the present chiral-LC study and the method was validated (ICH guidelines) 14-16.
Figure 1A: Chemical structure of Pemetrexed disodium
Figure 1B: Chemical structure of D-Isomer of Pemetrexed disodium
MATERIALS AND METHODS:
Pemetrexed disodium and its related substances were procured and HPLC grade acetonitrile, n-Hexane, Ethanol, Isopropyl alcohol, methanol, TFA, DEA, Propanoic acid, sodium hydroxide, hydrochloric acid and hydrogen peroxide were purchased from Merck (India). Milli-Q water was used for the entire work. Stock solution containing Pemetrexed disodium and D- isomer was prepared in mobile phase and diluted using the diluent and stored. During the robustness study the above solution was spiked with four related substances available, stored and diluted as per the necessity.
Chromatographic conditions
The D-isomer was separated from Pemetrexed disodium and quantified using Chiralpak AD-H (250 x 4.6 mm, 5 µm) column within a run time of 30 mins. A mixture of n-Hexane: Ethanol: Isopropyl alcohol: TFA (250:650:100:1) was the optimized mobile phase composition after many trials for the separation of D-isomer of Pemetrexed disodium (at 35°C) using Waters Alliance 2695 series HPLC system with 2998 photodiode array detector (UV detection at 240 nm). The system was operated on isocratic mode with flow rate 0.5 ml/min with an injection volume 5µl.
Method validation17
A series of linearity solutions containing D-isomer (1.3523-11.3067 µg/mL) and Pemetrexed disodium standard (0.9437-9.9702 µg/mL) at different concentrations at LOQ level, 0.075%, 0.12%, 0.15%, 0.18% and 0.225% were prepared with respect to the working concentration. The peak area was noted and the linearity graph was drawn with concentration of the solutions on X-axis and the mean area on Y-axis. The slope, y-intercept and correlation coefficient of the calibration curve were calculated. Precision studies (n=6) were performed for the mixture of Pemetrexed disodium and its D-isomer and the % RSD of the mean peak area was determined. Accuracy study was performed for Pemetrexed disodium and its D-isomer (i.e. 50%, 100% and 150%). Robustness was also performed by varying flow rate (0.1 ml/min), mobile composition and column temperature (5°C).
Assay of Pemetrexed disodium injection
Pemetrexed disodium is available with brand names Alimta (Eli Lilly and Company, India) (Label claim: 100 mg and 500 mg); Pexate (Miracalus Pharma Pvt Ltd) (Label claim: 100 mg), Giopem (GLS Pharma Ltd) (Label claim: 100 mg and 500 mg) as solution for injection. Two different brands were chosen and extracted and diluted with mobile phase as per the requirement and the percentage purity of Pemetrexed disodium was determined.
RESULTS AND DISCUSSION:
A specific and simple chiral RP-HPLC method was developed and validation for the separation and determination of Pemetrexed disodium and its D-isomer using Chiralpak AD-H (250 x 4.6 mm, 5 µm) column within a run time of 30 mins.
Method optimization
A Chiral RP-HPLC method has
been developed for the separation and quantification of Pemetrexed disodium and its
D-isomer. During optimization a mixture of solvents containing n-Hexane, Ethanol, TFA, DEA, Propionic acid, Isopropyl
alcohol were used in different composition with different flow rates,
concentration etc. and finally n-Hexane: Ethanol: Isopropyl alcohol: TFA
(250:650:100:1) / 35 / 240 was chosen for the present work. The observations recorded during the trials were
shown in Table 1 and that of the optimized chromatographic conditions Table 2. Pemetrexed disodium and its
D-isomer were eluted at 9.632 ± 0.001 min and 12.272 min respectively (Figure 2).
Table 1: Method optimization
|
Trial |
Mobile phase (v/v) / Column temp (°C) / λ (nm) / Flow rate (mL/min) |
Observations and conclusions |
|
1 |
n-Hexane : Ethanol : TFA :DEA: Propionic acid (350:654:1:1:10) / 27 / 240 / 1 |
Concentration (2 mg/mL) and injection volume (10 µL). D-isomer impurity and one of the process related unknown impurities are closely eluting. Mobile phase was used as diluent. |
|
2 |
n-Hexane: Ethanol: TFA (400: 600: 1) / 27 / 220 and 240 |
Peak broadening and two wavelength maxima for Pemetrexed peak at 220 nm and 240 nm were observed and 240 nm was selected to monitor D-isomer. |
|
3 |
n-Hexane: Ethanol: TFA (400 : 600 : 1) / 40 / 240 |
D-isomer and unknown impurity were merged together. |
|
4 |
n-Hexane: Ethanol: TFA: Propanoic acid (350 : 650 : 1:10) / 40 / 240 |
D-isomer is resolved but D-isomer was eluted at the tailing of Pemetrexed peak. |
|
5 |
n-Hexane: Ethanol :TFA: DEA: Propanoic acid (350: 650 : 1:1:10) / 40 / 240 |
All the peaks were resolved but the solution is un-stable in the diluent. |
|
6 |
n-Hexane: Ethanol: TFA: DEA: Propanoic acid (350: 650: 1:1:10) / 40 / 240 |
Solution are stable for 24 hrs even at room temperature. New impurity, Alanine derivative was identified. Methanol was used as diluent. |
|
7 |
n-Hexane: Ethanol: Isopropyl alcohol: TFA (250:650:100:1) / 35 / 240 / 0.5 |
Alanine derivative impurity and D-isomer resolved with USP resolution of 1.4 and Pemetrexed disodium and D-isomer also well resolved with resolution 3.8. |
|
8 |
n-Hexane: Ethanol: Isopropyl alcohol: TFA (250:650:100:1) / 35 / 240 |
Concentration (5 mg/mL) and injection volume (5 µL) were changed. S/N ratio of D-isomer is 250. |
|
9 |
n-Hexane: Ethanol: Isopropyl alcohol: TFA (250:650:100:1) / 35 / 240 |
S/N ratio of D-isomer peak is 220 and resolution between Pemetrexed and D-isomer peak is 2.9. Method optimized. |
|
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Trial 1: Overlay Chromatograms of spike solution with samples |
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Trial 2: Chromatogram of sample solution |
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Trial 3: Chromatogram of sample solution |
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Trial 4: Chromatogram of sample solution |
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Trial 5: Chromatogram of sample solution |
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Trial 5: Chromatogram of Pemetrexed API spiked with D-isomer at different time intervals |
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Trial 6: Chromatogram of Pemetrexed spiked solution (Initial) |
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Trial 6: Chromatogram of Pemetrexed spiked solution (24hrs) |
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Trial 6: Chromatogram of Blend solution of D-isomer and Alanine derivative of Pemetrexed impurity at lower level |
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Trial 7: Chromatogram of Pemetrexed spiked with D-isomer and Alanine derivative of Pemetrexed impurity |
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Trial 8: Chromatogram of D-isomer impurity and Pemetrexed |
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Trial 9: Chromatogram of methanol as blank |
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Trial 9: Chromatogram of system suitability of Pemetrexed and D-isomer |
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Trial 9: Chromatogram of Pemetrexed disodium |
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Figure 2: Chromatograms observed during method optimization (Trials) |
Table. 2. Optimized chromatographic conditions
|
Column |
Chiral pak AD-H (250 x 4.6 mm, 5 µm) |
|
Detector |
PDA detector |
|
Mobile phase (v/v) |
n-Hexane : Ethanol : Isopropyl alcohol :TFA (250:650:100:1) |
|
Mobile phase program |
Isocratic |
|
Flow rate |
0.5 mL/min |
|
Detection Wavelength |
240 nm |
|
Sample temperature |
Ambient |
|
Sample Temperature |
15°C |
|
Injection volume |
5 µL |
|
Diluent |
Methanol |
Method validation
Linearity
Pemetrexed disodium and its D-isomer have shown linearity over 0.9437-9.9702 and 1.3523-11.3067 µg/mL (Table 2) with linear regression equations y = 19600x - 765.3 (R2 = 0.9969) (Figure 3A) and y = 17033x - 2285.9 (R2 = 0.9986) (Figure 3B) respectively. The relative retention time (RRF) for Pemetrexed disodium and D-isomer was calculated basing on the slope ratio (API to D-isomer) method. The relative response factor was found to be 1.00 and 1.15 for Pemetrexed disodium and its D-isomer respectively. As the relative response factor for Pemetrexed disodium D-isomer is within the range 0.8-1.2 and therefore the relative response factor will be considered as 1.00 for D-isomer.
Table 3: Linearity
of Pemetrexed disodium and D-isomer
|
Pemetrexed disodium |
D-isomer |
||
|
Conc. (µg/mL) |
Mean peak area |
Conc.
(µg/mL) |
Mean peak area |
|
0.9437 |
14210 |
1.3523 |
18592 |
|
3.3234 |
67291 |
3.7689 |
63868 |
|
5.3174 |
102605 |
6.0302 |
99124 |
|
6.6468 |
132496 |
7.5378 |
129360 |
|
7.9762 |
158809 |
9.0454 |
152370 |
|
9.9702 |
189891 |
11.3067 |
187967 |
|
Slope |
19600 |
Slope |
17033 |
|
y-intercept |
-765.3 |
y-intercept |
-2285.9 |
|
Correlation coefficient |
0.9969 |
Correlation coefficient |
0.9986 |
Figure 3A: Linearity of Pemetrexed disodium
Figure 3B: Linearity of D-isomer
Pemetrexed disodium sample (5.0 mg/mL) was injected to estimate the content of D-isomer. D-isomer at three concentration levels i.e. 50%, 100% and 150% w.r.t. specification limit of test concentration was spiked with Pemetrexed disodium sample solution (5.0 mg/mL). Each level was prepared in triplicate and each of nine solutions was injected once and amount of D-isomer obtained in each solution was calculated (Table 4). The % recovery was 82.1-97.9 which is within the acceptance criteria (80.0%-120.0%) the % RSD is 6.3 for D-isomer with indicating that the acceptance criteria (<10) was satisfied.
Table 4: Accuracy
|
Spiking level |
Amount Added (µg/mL) |
Amount obtained (µg/mL) |
% Recovery |
|
50% |
3.2725 |
2.7172 |
83.0 |
|
2.8349 |
86.6 |
||
|
2.6867 |
82.1 |
||
|
100% |
6.5450 |
6.4087 |
97.9 |
|
6.2583 |
95.6 |
||
|
5.8023 |
88.7 |
||
|
150% |
9.8175 |
8.2429 |
84.0 |
|
8.3537 |
85.1 |
||
|
8.5936 |
87.5 |
||
|
RSD |
6.3% |
||
Precision
Six replicate sample solutions of Pemetrexed disodium (5.0 mg/mL) in diluent containing 0.15 % of D-isomer w.r.t. sample concentration were prepared and each spiked sample solution was injected and RSD was calculated D-isomer content. Intermediate precision of the method was determined by analyzing six samples spiked with D-isomer on different day, by different analyst, different columns and using different equipment. The % D-isomer was found to be 0.14 and the % RSD was zero in both method precision and intermediate precision studies (Acceptance criteria: RSD should not be more than 10.0%).
Solution of Pemetrexed disodium and its D-isomer at LOQ level was injected in six replicates and RSD for the peak areas of Pemetrexed disodium and D-isomer was calculated. The RSD of peak areas was found to be 8.2% for D-isomer and 11.1% (Table 5) for Pemetrexed disodium indicating that the method is precise (Acceptance criteria: RSD of peak areas should not be more than 15.0% for each analyte).
Table 5: Precision of Pemetrexed disodium and D-isomer
|
S. No. |
D-isomer |
Pemetrexed disodium |
|
1 |
16459 |
13735 |
|
2 |
13928 |
11791 |
|
3 |
13274 |
10554 |
|
4 |
14084 |
11547 |
|
5 |
14113 |
14148 |
|
6 |
13322 |
12879 |
|
Mean |
14197 |
12442 |
|
RSD |
8.2% |
11.1% |
Robustness
The effect of column temperature, flow rate, mobile phase composition on system suitability were summarised in Table 6. As per the system suitability criteria, for the robustness study is the resolution between Pemetrexed disodium and D-isomer peak should not be less than 2.0 and that of S/N ratio for D-isomer peak should be more than 50. As small variations of the chromatographic conditions did not affect the system suitability and relative retention time (RRT) of D-isomer it indicates that the method is robust. Pemetrexed disodium solution was spiked with D-isomer and the related substances such as acid intermediate, alanine derivative of Pemetrexed, DMF derivative of Pemetrexed and Pemetrexed diethyl ester to prove the specificity of the method during robustness study (Figure 4). It was observed that the D-isomer was well separated from that of Pemetrexed disodium even in presence of the related substances.
Table 6: Robustness of Pemetrexed disodium and its D-isomer
|
Condition |
System suitability solution |
Specificity solution |
||
|
S/N value D-isomer (NLT 50) |
Resolution (NLT 2.0) |
Pemetrexed disodium Retention Time (min) |
RRT of D-isomer |
|
|
As per method |
80 |
3.2 |
9.18 |
1.28 |
|
Lower column (Temp. 30°C) |
82 |
3.2 |
9.29 |
1.30 |
|
Higher column (Temp. 40°C) |
88 |
3.3 |
9.01 |
1.26 |
|
Lower Flow rate (0.4 mL/min.) |
86 |
3.5 |
11.51 |
1.29 |
|
Higher Flow rate (0.6 mL/min.) |
73 |
3.1 |
7.63 |
1.28 |
|
Mobile phase organic ratio variation (275 : 650 : 100 :1.0) %v/v |
98 |
3.3 |
9.38 |
1.30 |
|
Mobile phase organic ratio variation (225 : 650 : 100 :1.0) %v/v |
59 |
3.0 |
8.84 |
1.27 |
|
Mobile phase organic ratio variation (250 : 715 : 100 :1.0) %v/v |
64 |
2.8 |
8.36 |
1.26 |
|
Mobile phase organic ratio variation (250 : 585 : 100 :1.0) %v/v |
78 |
2.9 |
9.45 |
1.30 |
|
Mobile phase organic ratio variation (250 : 650 : 110 :1.0) %v/v |
121 |
3.2 |
9.17 |
1.29 |
|
Mobile phase organic ratio variation (250 : 650 : 90 :1.0) %v/v |
73 |
3.2 |
9.28 |
1.29 |
|
Mobile phase organic ratio variation (250 : 650 : 100 :1.1) %v/v |
70 |
3.2 |
8.63 |
1.28 |
|
Mobile phase organic ratio variation (250 : 650 : 100 :0.9) %v/v |
61 |
3.1 |
9.40 |
1.29 |
|
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Pemetrexed disodium spiked with its D-isomer and related substances |
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Lower flow rate – 0.4 mL/min |
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Higher flow rate – 0.6 mL/min |
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Lower column temperature - 30°C |
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Lower column temperature - 40°C |
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Higher n-Hexane content |
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Lower n-Hexane content |
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Higher Ethanol content |
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Lower Ethanol content |
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Higher Isopropyl alcohol content |
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Lower Isopropyl alcohol content |
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Higher TFA content |
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Lower TFA content |
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Figure 4 : Representative chromatograms observed during robustness study |
Assay of Pemetrexed disodium
Two different brands of Pemetrexed disodium formulations were analyzed using the above optimized conditions. Pemetrexed disodium has shown 99.89-100.13 purity range and there is no interference of the excipients with the drug peak.
Specificity
Specificity of the method was determined by injecting the analyte spiked with all the known related substances expected to be present in Pemetrexed disodium. Separate solutions of diluent, Pemetrexed disodium standard and known related substances (D-Isomer, Alanine derivative of Pemetrexed, DMF derivative of Pemetrexed, Acid intermediate and Pemetrexed diethyl ester) and a spiked solution containing all the mentioned components were injected at 0.15% level for D-Isomer, Alanine derivative of Pemetrexed, DMF derivative of Pemetrexed, Acid intermediate and Pemetrexed diethyl ester into HPLC. Table 7 shows retention time, RRT and peak purities for D-isomer in the spiked solution. No peaks were observed in the diluent (Figure 5A) at the retention time of D-isomer. D-isomer peak was well resolved (Figure 5C) from nearby peak in spiked solution (Figure 5B) as shown in the chromatogram. The purity angle is less than purity threshold indicating the peak is spectrally pure. Peak purity plots of D-isomer in both standard and spiked solution were shown in Figure 5D and Figure 5E. The LOD and LOQ of Pemetrexed disodium and D-Isomer were shown in Figure F and Figure G.
Table 7: Specificity
|
Component |
Retention time (min) |
RRT |
Purity angle |
Purity threshold |
|
D-isomer standard |
11.52 |
----- |
3.451 |
4.138 |
|
D-isomer in spiked solution |
11.56 |
1.25 |
2.111 |
2.705 |
|
Pemetrexed disodium in Spiked solution |
9.22 |
1.00 |
|
|
|
Alanine derivative in Spiked solution |
12.70 |
1.38 |
|
|
|
DMF derivative in Spiked solution |
8.36 |
0.91 |
|
|
|
Acid intermediate in Spiked solution |
12.55 |
1.36 |
|
|
|
Pemetrexed diethyl ester in Spiked solution |
24.12 |
2.62 |
|
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A. Blank |
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B. Chromatogram of Pemetrexed disodium and D-isomer in combined solution |
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C. Chromatogram of D-isomer standard |
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D. Purity plot of D-isomer standard |
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E. Purity plot of D-isomer (combined solution) |
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F. LOD for Pemetrexed disodium and D-isomer |
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G. LOQ for Pemetrexed disodium and D-isomer |
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Figure 5: Specificity of Pemetrexed disodium and D-isomer |
CONCLUSIONS:
A new Chiral RP-HPLC method was developed for the separation and quantification of Pemetrexed disodium and its D-isomer in presence of related substances. The method was validated (ICH guidelines) by linearity, precision, accuracy and robustness and it is mostly helpful in the determination of therapeutic activity of the metabolites during the drug absorption as well as in the pharmacokinetic studies. The proposed method is specific and the system suitability parameters are within the acceptable criteria.
ACKNOWLEDGEMENT:
The authors are grateful to (Eli Lilly and Company (India) for providing the gift samples of Pemetrexed disodium and its D-isomer in presence of related substances. There is no conflict of interest.
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Received on 12.01.2019 Modified on 29.01.2019
Accepted on 04.02.2019 © RJPT All right reserved
Research J. Pharm. and Tech 2019; 12(2):773-786.
DOI: 10.5958/0974-360X.2019.00136.7