Validated Analytical Method for the Estimation of Gemcitabine from its Pharmaceutical Formulation by RP-HPLC
Survi Mishra, S. T. Narenderan, B. Babu*, Kuntal Mukherjee, S. N. Meyyanathan
Department of Pharmaceutical Analysis, JSS College of Pharmacy, Udhagamandalam, Tamil Nadu,
JSS Academy of Higher Education & Research, India.
*Corresponding Author E-mail: babu@jssuni.edu.in
ABSTRACT:
A simple, sensitive and rapid high performance liquid chromatographic method has been developed for the determination of Gemcitabine. Chromatographic separation was achieved on anInertsil ODS-3V column (250mm x 4.6mm; 5΅m) with a mobile phase consisting of water: acetonitrile (90:10; pH 3.5 adjusted with orthophosphoric acid) with a flow rate of 1.0ml/min (UV detection at 270nm). Linearity was observed over the concentration range of 1-120΅g/ml with a regression equation y=19773x+ 86063 and having a regression value (R2)= 0.998. The limit of detection (LOD) and limit of quantification (LOQ) values found to be 10ng/ml and 20ng/ml, respectively. The percentage recovery of the marketed formulation was found to be 100.08%. Validation revealed that the preferred method was specific, accurate, precise, reliable, robust, reproducible and suitable for the quantitative analysis.
KEYWORDS: Gemcitabine, RP-HPLC, Validation, LOD, LOQ, Robustness.
1. INTRODUCTION:
Gemcitabine (2-deoxy-2, 2-difluoro cytidine) HCl (Figure 1) has a molecular formula C9H11F2N3O4HCl and a molecular weight of 263.201g/mol1. Gemcitabine is a white to off-white solid. Gemcitabine is a prodrug that enters the cell by means of nucleoside transporters and becomes active through an intra-cellular transformation catalyzed by deoxycytidine kinase to its diphosphate and triphosphate derivatives. The triphosphate derivative is incorporated into the DNA strand, inhibiting thymidylate synthetase which inhibits DNA synthesis and chain elongation, contributing to the antineoplastic activity of the drug1. It is an antitumor agent used for the treatment of a wide spectrum of solid tumours including pancreatic, non-small-cell lung cancer, breast and bladder cancer2. It is soluble in water, slightly soluble in methanol and practically insoluble in acetone. Gemcitabine Hydrochloride is not able to maintain an optimum concentration in the body, because of its very short half-life (short infusions 32 to 94 minutes, long infusions 245 to 638 minutes) and highly hydrophilic nature.
Very few HPLC methods deal with the determination of Gemcitabine in the presence of degradation products from forced degradation studies, and those that do are time-consuming, with complicated mobile phases and gradient elution leading to baseline shifting3. The developed isocratic HPLC method was validated according to the International Conference on Harmonization (ICH) guidelines4.
Figure 1: Chemical structure of Gemcitabine.
An extensive literature survey has revealed that several methods are reported for the combination with other drugs. Overall the presented method was to establish specific, precise, accurate, optimized and validated RP-HPLC method which can be used for the quantification of Gemcitabine. A literature survey reveals that only a few methods based on ultraviolet spectroscopy5, HPTLC6, and HPLC7-14 are available for determination of drug in the formulation. Although several HPLC15-23 and LC-MS/MS24-36 methods have been reported for estimation of drug and its metabolites in biological fluids, these methods are complicated, costly, and time-consuming in comparison to a simple HPLC-UV method.
2. MATERIALS AND METHOD:
a. Chemicals and Reagents:
Gemcitabine Hydrochloride was acquired as a gift sample from Ranbaxy, Hyderabad. The marketed formulation of the drug was procured from the local market. All reagents were of analytical grade unless stated otherwise. Acetonitrile of HPLC grade was purchased from Merck Laboratories Pvt. Ltd. HPLC grade water was gained from Milli-Q Reverse Osmosis (RO) water purification system. Orthophosphoric acid and triethylamine AR grade were procured from Sisco Research Laboratories Pvt. Ltd.
b. HPLC Instrumentation and Conditions:
High-Pressure Liquid Chromatography instrument administered with anInerstsil ODS-3V column (250mm x 4.6mm; 5΅m); an LC-2010A HT solvent delivery system, a universal loop injector of injection capacity of 10΅l, and an UV-Visible Spectrophotometer (𝜆max 270nm) was enlisted in the study. Data acquisition was performed using LC-Solution software. Chromatographic analyses were achieved by applying the mobile phase of water: acetonitrile (90:10; pH adjusted to 3.5 using orthophosphoric acid). The mobile phase was constructed daily and sieved through 0a .45΅m membrane filter. The temperature of the column was maintained at 25±2ᴼC.
c. Preparation of Standard and Stock Solutions:
1. Gemcitabine Standard Solutions:
An exactly weighed quantity (100mg) of Gemcitabine was transferred into a 100ml calibrated volumetric flask and the volume was made up to with a suitable volume of water.
2. Calibration Standards:
Calibration standards were composed a new using either stock or intermediate working solutions of Gemcitabine and internal standard. Standard solutions of concentration of 20.0, 40.0, 60.0, 80.0, 100.0 and 120.0΅g/ml were prepared. All the above-introduced solutions were carried out using 100΅g/ml as standards. These solutions were further put on sonication for degassing. These solutions were further inspected to avoid degradation.
3. Preparation of the mobile phase:
The mobile phase was prepared by dissolving water: acetonitrile (90:10; pH modified to 3.5 applying orthophosphoric acid) and degassed.
4. Wavelength Selection:
100mg of the standard solution was concocted in 100ml of water. From that stock solution, 1΅g/ml of the solution was prepared in water by diluting 1ml of the stock solution to 10ml of water. After preparing, the solution was exposed to UV (Shimadzu-1700) for the scrutinization of wavelength between 200-400nm. The λmax of the drug Gemcitabine was found to be at 270nm.
Figure 2: UV Spectrum of Gemcitabine
3. METHOD VALIDATION:
The RP-HPLC method which has been developed for the assessment of Gemcitabine was validated as stated in ICH Q2 (R1A) guidelines. The parameters of the validation are specificity, linearity, precision and accuracy, detection limit and quantification limits.
a. Specificity:
For method validation is specificity, the capability of an analytical method is to differentiate the analyte from other chemicals in the sample. The specificity of the technique may be estimated by knowingly attaching impurities into a sample by carrying out the analyte and testing how well the method can recognize the analyte.
b. Linearity:
The linearity of an analytical method specifies if the acquired response is linearly proportional to the concentration of the analyte within a definite range. The linearity of the suggested method was analyzed over a range of 20-120΅g/ml. The working standards were composed in the mobile phase from 1mg/ml standard stock solution and were injected in triplicate under optimized chromatographic conditions and the chromatograms were noted. The linearity was established based on the correlation co-efficient acquired by framing a graph with a concentration in ΅g/ml at the x-axis and peak area of Gemcitabine at the y-axis.
c. Precision and Accuracy Studies:
The precision of the evolved method can be estimated by intra-day and inter-day studies.6 independent injections of 3 non-identical concentrations i.e. 20, 60, 120΅g/ml (LQC, MQC & HQC) level were used to study the precision of the presented method. Intra-day precision, as well as repeatability, was scrutinized by administering the samples on the same day and the inter-day precision study was accomplished by introducing the same samples on 2 different days. The mean values and the values of %RSD were calculated.
d. Limit of Detection and Limit of Quantification:
The sensitivity of the method intent to how liable is the procedure of observing the lowest possible concentration of an analyte in the absence of any noise. This is evaluated by the framework of LOD and LOQ. Limit of Detection is the smallest concentration of the analyte that can be detected by the developed method which involvesan estimable response (signal to noise ratio 3) whereas Limit of Quantification is the smallest concentration of the analyte which produces a response that can be promptly quantified (Signal to noise ratio 10).
LOD and LOQ can be calculated by the formula:
LOD = 3.3 σ/S
LOQ = 10 σ/S;
Where σ = Standard deviation of the response; S = Slope of the deviation curve.
e. Robustness and Ruggedness:
The ruggedness and robustness of the evolved method were studied by leading about small changes in the exploratory surroundings (analyte, reagent source and columns of varying brands) and chromatographic conditions (pH, mobile phase composition, mobile phase ratio and flow rate).
f. System suitability:
The system suitability examination is used to confirm that an analytical method was acceptable for its intended purpose the day analysis was done. It is a necessary parameter to safeguard the quality of the method for accurate measurements. Chromatographic parameters viz; Number of Theoretical Plates (N), Retention time (Rt), Resolution (Rs) and Peak Asymmetric Factor (A) were scrutinized on administering 6 replicates of the standard Gemcitabine at a concentration of 120΅g/ml.
4. RESULTS AND DISCUSSION:
The presented method was organized by optimizing the chromatographic conditions by including diverse trial runs modifying the mobile phase composition, ratio of the mobile phase, pH, column, column length to achieve symmetrical analyte peak at an adequate short run time. Originally, various ratios of acetonitrile and water were engaged as the mobile phase for separations, which revealed peak asymmetry. Throughout the literature survey of the GCB, there were no satisfactory stability-indicating assay methods available with wide range of linearity for the determination of GCB in existence of its degradation products employing RP-HPLC. The present work was focused to develop a stability-indicating RP-HPLC method for the determination of GCB in presence of its degradants. The better resolution was attained with the utilization of a combination of water and acetonitrile (90:10 v/v) and C18 column was acquired for the analysis as it has assumed a preferable separation of the analytes.
a. METHOD DEVELOPMENT:
The proposed method was outlined by optimizing the chromatographic conditions by relating to various trial runs changing the mobile phase composition, ratio of the mobile phase, pH, column, column length to attain symmetrical analyte peak at an adequately short run time.
Figure 3: Typical HPLC Chromatogram of Gemcitabine Standard
Eventually, a symmetrical analyte peak with a justifiable short run time was fulfilled by engaging water and acetonitrile in a ratio of 90:10 v/v at a flow rate of 1ml/min, with an Inertsil ODS-3V Nitrofurantoin column (250mm x 4.6mm, 5΅) being made use of as the stationary phase and checked at a wavelength of 270nm.Gemcitabine was eluted at 4.8 minutes. The mobile phase was processed by straining through a 0.45΅ PTFE (Poly Tetra flour ethylene) membrane filter before including into the HPLC system. The chromatograms were noted and handled on a Class VP Data Station.
b. Accuracy and Precision:
The Accuracy method was demonstrated as % mean recovery for three different concentration levels (20, 60, 120΅g/ml) by standard addition method. Triplicate analyses were achieved at an individual level. Percent mean recovery was studied. The % RSD for precision studies ranged from 0.98 to 2.50 %. The developed method was also used for the assessment of the marketed Gemcitabine injectable formulation Table 1.
Table 1: Precision Studies
|
S. No |
Concentration Added(΅g/ml) |
Intra - day (΅g/ml) |
Inter-day(μg/ml) |
|
Amount found ±RSD (n=6) |
Amount found ±RSD (n=6) |
||
|
1. |
20 |
19.5 ±2.25 |
19.0 ±2.50 |
|
2. |
60 |
59.1 ±1.30 |
58.8 ±1.10 |
|
3. |
120 |
119.9 ±1.00 |
119.1±0.98 |
c. Linearity:
A calibration curve was planned for 6 different concentrations of drug versus corresponding peak area. The graph exhibited outstanding correlation linking the concentrations and peak area when noticing within the range of 20 ΅g/ml to 120 ΅g/ml for the drug. The correlation co-efficient for Gemcitabine was 0.998.
Figure 4: Linearity of Gemcitabine.
d. Detection Limit and Quantification Limit:
The Detection and Quantitation limit constitutes the sensitivity of the proposed method. The LOD and LOQ were found to be 10ng/ml and 20ng/ml, respectively indicating the sensitivity of the method.
e. Specificity:
The specificity test displayed that the used excipients did not interfere with the peak of the main compound. No peaks were eluted along with the retention time of Gemcitabine (Figure 3). Hence, the results showed that the developed method was selective for determination of Gemcitabine in the formulation.
f. System Suitability:
6 replicates of the standard at working concentration were injected to observe changes in separation, retention time and asymmetry of the peaks and validate the system suitability parameters. The system suitability was found to be within limits and is summarized in Table 2.
Table 2: System Suitability Studies:
|
Sl. No |
Parameters |
Gemcitabine |
|
1. |
Retention Time (min) |
4.8 |
|
2. |
Theoretical Plates (N) |
10495 |
|
3. |
Tailing Factor |
0.5 |
|
4. |
Asymmetry Factor |
1 |
|
5. |
Linearity Range (μg/ml) |
20-120 |
|
6. |
Slope |
19773 |
|
7. |
Correlation Coefficient |
0.998 |
|
8. |
LOD (ng/ml) |
10 |
|
9. |
LOQ (ng/ml) |
20 |
Assay of the marketed formulation:
The assay of the formulation was performed as per the Indian Pharmacopeia. According to Pharmacopeia, the test solution was prepared by determining in Liquid Chromatography as described in the Related Substances with the following modifications.
The reference solution was injected i.e. 10mg of the substance was weighed for examination in a small vial and 4.0ml of 16.8 percent w/v solution of potassium hydroxide in methanol was sonicated for 5minutes was added. The mixture was found to be cloudy. Heated at 55ᴼC for a minimum 6 hours to produce Gemcitabine impurity B. Allowed to cool, then the entire contents of the vial was transferred to 100.0ml volumetric flask by washing with a 1.0 percent v/v solution of orthophosphoric acid. Diluted to 100.0ml with a 1.0 percent v/v solution of orthophosphoric acid and mixed. The test was not valid unless the resolution between the peaks due to Gemcitabine impurity and Gemcitabine is not less than 8.0.
Reference solution (b) and Test solution (b) was injected. Test solution (b) i.e. by dissolving 50mg of the substance under examination in water and diluted to 25.0ml with water and from the above mixture, taken about 1.0ml of test solution was diluted to 20.0 ml with water. Reference solution (b) i.e. a 0.01 percent v/v solution of Gemcitabine Hydrochloride RS in water.
Calculate the content of C9H12ClF2N3O4.
Gemcitabine Hydrochloride intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins complies with the following additional requirements.
The recovery percentage of the marketed formulation was found to be 100.08%.
Table 3: Recovery studies in Formulation:
|
Drug |
Label claim |
Amount taken for assay(μg/ml) |
Amount obtained(μg/ml) ±RSD (n=6) |
Percentage Recovery (%) |
|
Gemcitabine |
200 mg |
20 |
18.9 ±3.21 |
94.50 |
|
200 mg |
60 |
59.2 ±1.20 |
98.66 |
|
|
200 mg |
120 |
120.1±0.09 |
100.08 |
Figure 5: Typical Chromatogram of Marketed Formulation
5. CONCLUSION:
A rapid, simple, sensitive, precise, accurate RP-HPLC method was progressed for Gemcitabine and the evolved method was validated as per ICH Q2 (R1A) guidelines. The % RSD of < 2.50 % indicated the method to be precise. The drug showed good linearity over concentrations ranging from 20 to 120 ΅g/ml. Recovery studies that were accomplished exhibited the accuracy of the method. The mean recovery of the validation ranged between 94.50 to 100.08 %. Thus it may be concluded that an accurate, precise and rapid RP-HPLC method have been developed and validated for routine quantification of Gemcitabine in bulk and pharmaceutical formulations.
6. CONFLICTS OF INTEREST:
The authors declare that there are no conflicts of interest.
7. ACKNOWLEDGEMENT:
The authors are grateful to the Ranbaxy, Hyderabad for providing the standard drug Gemcitabine as a gift sample.
8. REFERENCES:
1. URL: http://www.ebi.ac.uk/chebi/searchId.do?chebiId=CHEBI: 175901
2. Barton-Burke M. Gemcitabine: a pharmacologic and clinical overview. Cancer Nurs. 1999, 22, 176183.
3. Mastanamma S et al. A stability indicating RP-HPLC method for the estimation of Gemcitabine HCl in injectable dosage forms. J Chem,2010; 7: 239244.
4. Lanz C et al. Rapid determination of Gemcitabine in plasma and serum using Reversed-Phase HPLC.J Sep Sci 2007; 30: 18111820.
5. D. G. Sankar et al. UV Spectrophotometric determination of Temozolamide and Gemcitabine, Asian Journal of Chemistry, 2007; vol. 19, no. 2, pp. 16051607.
6. S. L. Borisagar et al. A Validated Stability-Indicating HPTLC method for the estimation of Gemcitabine HCl in its dosage form, Journal of Planar Chromatography, 2012; vol. 25, no. 1, pp. 7780.
7. J. V. L. N. S. Rao et al. RP-HPLC analysis of Gemcitabine in pure form and in pharmaceutical dosage forms, Asian Journal of Chemistry, 2007; vol.19, no. 5, pp. 33993402.
8. H. Xu, J. Paxton et al. Development of a gradient High Performance Liquid Chromatography assay for simultaneous analysis of hydrophilic Gemcitabine and lipophilic curcumin using a central composite design and its application in liposome development, Journal of Pharmaceutical and Biomedical Analysis, 2014, vol. 98, pp. 371378.
9. S. S. Bansal et al. Validated RP-HPLC method for the simultaneous analysis of Gemcitabine and LY-364947 in liposomal formulations, Current Drug Targets,2013, vol.14, no. 9, pp. 1061-1069.
10. Q. Zhou et al. Analysis of Gemcitabine liposome injection by HPLC with evaporative light scattering detection, Journal of Liposome Research, 2012; vol. 22, no. 4, pp. 263269.
11. V. Rajesh et al. Simultaneous estimation of Gemcitabine Hydrochloride and Capecitabine Hydrochloride in combined tablet dosage form by RP-HPLC method. E-Journal of Chemistry, 2011; vol. 8, no. 3, pp. 12121217,
12. S. Mastanamma et al. A Stability-Indicating RP-HPLC Method for the estimation of Gemcitabine HCl in injectable dosage forms, E-Journal of Chemistry, 2010; vol. 7, no. S1, pp. S239S244.
13. S. Kudikalaet al. RP-HPLC Method for the estimation Gemcitabine in API and parenteral dosage form, Journal of Scientific Research in Pharmacy, 2014; vol. 3, pp. 1618.
14. N. Devanaboyina et al. A novel RP-HPLC method development and validation for analysis of Gemcitabine in bulk and pharmaceutical dosage form, International Journal of Pharma Sciences, 2014, vol. 4, pp. 522525.
15. C. Lanz, M et al. Rapid determination of Gemcitabine in plasma and serum using reversed-phase HPLC, Journal of Separation Science, 2007; vol.30, no. 12, pp. 1811- 1820.
16. M. N. Kirstein et al., High-performance Liquid Chromatographic Method for the determination of Gemcitabine and 2΄,2΄-difluorodeoxyuridine in plasma and tissue culture media, Journal of Chromatography B, 2006; vol. 835, no. 1-2, pp. 136142.
17. R. Losa et al. Simultaneous determination of Gemcitabine di- and triphosphate in human blood mononuclear and cancer cells by RP-HPLC and UV detection. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 2006; vol. 840, no. 1, pp. 4449.
18. R. Losa, M. I. Sierra et al. Development and validation of an ion pair HPLC method for Gemcitabine and 2΄,2΄-difluoro-2΄-deoxyuridine determination, Analytica Chimica Acta, 2005; vol. 528, no. 2, pp. 255260.
19. B. Yılmazet al. Comparison of zero- and secondorder derivative spectrophotometric and HPLC methods for the determination of Gemcitabine in human plasma, Il-Farmaco, 2004; vol. 59, no. 5, pp. 425429.
20. B. Keithet al. Measurement of the anti-cancer agent Gemcitabine in human plasma by high-performance liquid chromatography, Journal of Chromatography B, 2003; vol. 785, no. 1, pp. 6572.
21. B. Yilmaz et al. Simultaneous determination of Gemcitabine and its metabolite in human plasma by High-Performance Liquid Chromatography, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 2003; vol. 791, no. 1-2, pp. 103109.
22. K. B. Freeman et al., Validated assays for the determination of Gemcitabine in human plasma and urine using High-Performance Liquid Chromatography with Ultraviolet Detection, Journal of Chromatography B: BiomedicalApplications,1995; vol. 665, no. 1, pp. 171181.
23. N.-M. Lin et al. Determination of Gemcitabine and its metabolite in human plasma using High-Pressure Liquid Chromatography coupled with a Diode Array Detector, Acta Pharmacologica Sinica, 2004; vol. 25, no. 12, pp. 15841589.
24. Y. Xuet al. Measurement of the anticancer agent Gemcitabine and its de-aminated metabolite at low concentrationsin human plasma by Liquid Chromatography-Mass Spectrometry, Journal of Chromatography B, 2004; vol. 802, no. 2, pp.263270.
25. E. Marangon et al. Simultaneous determination of Gemcitabineand its main metabolite, dFdU, in plasma of patients with advanced non small-cell lung cancer by High-Performance Liquid Chromatography-Tandem Mass Spectrometry, Journal of Mass Spectrometry, 2008; vol. 43, no. 2, pp. 216223.
26. H. Khour et al. Simultaneous determination of Gemcitabine and Gemcitabine-squalene by Liquid Chromatography-Tandem Mass Spectrometry in human plasma, 2007; Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, vol.858, no. 1-2, pp. 7178.
27. L. D. Vainchtein et al. Validated assay for the simultaneous determination of the anti-cancer agent Gemcitabine and its metabolite 2΄,2΄-difluorodeoxyuridine in human plasma by High-Performance Liquid Chromatography with Tandem Mass Spectrometry, Rapid Communications in Mass Spectrometry, 2007; vol. 21, no. 14, pp. 23122322,
28. E. Marangon et al. Simultaneous determination of Gemcitabine and its main metabolite, dFdU, in plasma of patients with advanced non-small-cell lung cancer by high-performance liquid chromatography-tandem mass spectrometry, Journal of Mass Spectrometry,2008; vol. 43, no. 2, pp. 216223.
29. H. Khoury et al. Simultaneous determination of Gemcitabine and Gemcitabine-squalene by Liquid Chromatography-Tandem Mass Spectrometry in human plasma, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 2007; vol. 858, no. 1-2, pp. 7178.
30. L. D. Vainchtein et al. Validated assay for the simultaneous determination of the anti-cancer agent Gemcitabine and its metabolite 2΄,2΄-difluorodeoxyuridine in human plasma by High-Performance Liquid Chromatography with Tandem Mass Spectrometry, Rapid Communications in Mass Spectrometry, 2007; vol. 21, no. 14, pp. 23122322,
31. R. Honeywell et al., The determination of Gemcitabine and 2΄-deoxycytidine in human plasma and tissue by APCI Tandem Mass Spectrometry, Journal of Chromatography B, 2007; vol. 847, no. 2, pp. 142152.
32. S. Nussbaume et al. Simultaneous quantification of ten cytotoxic drugs by a validated LCESI- MS/MS method, Analytical and Bioanalytical Chemistry, 2010; vol. 398, no. 7-8, pp. 30333042.
33. IFPMA, ICH validation of analytical procedures: text and methodology Q2 (R1), in Proceedings of the International Conferenceon Harmonization, 2005; pp. 113, IFPMA, Geneva, Switzerland.
34. R. Honeywell et al. The determination of Gemcitabine and 2΄-deoxycytidine in human plasma and tissue by APCI Tandem Mass Spectrometry, Journal of Chromatography B, 2007; vol. 847, no. 2, pp. 142152.
35. S. Nussbaumer et al. Simultaneous quantification of ten cytotoxic drugs by a validated LCESI-MS/MS method, Analytical and Bioanalytical Chemistry, 2010; vol. 398, no. 7-8, pp. 30333042.
36. IFPMA, ICH Validation of Analytical Procedures: Text and Methodology Q2 (R1), in Proceedings of the International Conferenceon Harmonization, 2015; pp. 113, IFPMA, Geneva, Switzerland.
Received on 27.03.2019 Modified on 25.04.2019
Accepted on 21.05.2019 © RJPT All right reserved
Research J. Pharm. and Tech. 2019; 12(11): 5407-5412.
DOI: 10.5958/0974-360X.2019.00938.7