A Stability-Indicating LC Method for Assay of Topotecan Hydrochloride
G. Saravanan1,&, M. V. Suryanarayana1, N. Balaji1, N. Someswararao2, N. M. Sekhar3 1
2 3
Analytical Research, High Potent Active Pharmaceutical Ingredients Operations, Dr Reddy’s Laboratories, Hyderabad 500072, India; E-Mail:
[email protected] Department of Inorganic and Analytical Chemistry, Andhra University, Visakhapattanam 530003, India Process Research, High Potent Active Pharmaceutical Ingredients Operations, Dr Reddy’s Laboratories, Hyderabad 500072, India
Received: 10 August 2007 / Revised: 10 October 2007 / Accepted: 23 October 2007
Abstract This paper describes the development of a stability-indicating high-performance liquid chromatographic (HPLC) method for quantitative determination of topotecan hydrochloride, a semi-synthetic derivative of camptothecin and anti-tumor drug with topoisomerase I-inhibitory activity. Chromatographic separation was achieved on a C18 column with a mixture of phosphate buffer and acetonitrile as mobile phase. The method was validated for linearity, accuracy, precision, and robustness. Forced degradation studies were performed by treating bulk samples of topotecan hydrochloride with acid (0.5 M hydrochloric acid), base (0.5 M sodium hydroxide), oxidizing agent (10% v/v hydrogen peroxide), heat (60 C), and UV light (254 nm).
Keywords Column liquid chromatography Solution and mobile phase stability Forced degradation Validation Topotecan hydrochloride
Introduction Topotecan hydrochloride, (S)-10-[(dime thylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[30 ,40 :6,7]indolizino[1,2-b]-
quinoline-3,14-(4H,12H)dione monohydrochloride, is a drug used for chemotherapeutic treatment of some types of cancer, mainly ovarian cancer or smallcell lung cancer (SCLC). It is also
sometimes used to treat acute myeloid leukemia. Topotecan hydrochloride was originally developed from the wood of a Chinese tree. Topotecan is a topoisomerase 1 inhibitor, which means it works by blocking the enzyme topoisomerase I, an enzyme which stops cells from repairing DNA when they divide. Cancer cells need to make and repair DNA to grow and multiply. Blocking the action of this enzyme damages the DNA and so the cells die. As far as we are aware no stabilityindicating analytical method for topotecan hydrochloride bulk drug is available in the literature, although analytical methods are available for the camptothecin analogs and for study of the stability and compatibility of topotecan hydrochloride with common infusion solutions and containers [1–9]. It was therefore felt necessary to develop a stability-indicating LC method for quantitative determination of topotecan hydrochloride in the bulk drug. This paper also describes the validation of the stability-indicating method and discusses forced degradation of the drug under stress conditions—acid hydrolysis, basic hydrolysis, oxidation, heat, and UV light.
Limited Short Communication DOI: 10.1365/s10337-007-0468-3 2007 Friedr. Vieweg & Sohn Verlag/GWV Fachverlage GmbH
a
CH3 N
pared by use of a Millipore Milli Q plus water-purification system. For assay determination a working solution (50 lg mL1) of topotecan hydrochloride was prepared in 0.01 M aqueous potassium dihydrogen phosphate (adjusted to pH 3.0 with phosphoric acid)–acetonitrile, 85:15. Impurity stock solution (a mixture of Imp-A and Imp-B at 1.0 mg mL1) was prepared in the same solvent.
. HCl
CH3
HO
O
N N
O H3C O
HO
b O
N
Chromatography
N O HO
O
c HO O
N N
O HO
O
Fig. 1. The chemical structures of topotecan hydrochloride and its impurities: a topotecan hydrochloride, (S)-10-[(dimethylamino) methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[30 , 40 : 6,7] indolizino(1,2-b)quinoline-3,14-(4H,12H)dione monohydrochloride; b Imp-A, (S)-4-ethyl-4hydroxy-1H-pyrano[30 ,40 :6,7]indolizino(1,2-b) quinoline-3,14(4H,12H)dione; c Imp-B, (S)-4ethyl-4,9-dihydroxy-1H-pyrano[30 ,40 :6,7]indolizino(1,2-b)quinoline-3,14(4H,12H)dione
The LC system used for method development, forced degradation studies, and method validation was a Waters model 2489 chromatograph equipped with a photo diode-array detector. The output signal was monitored and processed by use of Empower software (Waters) on a Pentium computer (Digital Equipment). Compounds were separated on a 150 mm · 4.6 mm i.d., 5-lm particle, Inertsil ODS 3V (r) column. The mobile phase was a gradient prepared from 0.01 M potassium dihydrogen phosphate in water, adjusted to pH 3.0 with phosphoric acid (component A) and acetonitrile (component B). The gradient was (time/%B): 0/15, 8/15, 15/80, 16/15, and 25/15. The injection volume was 20 lL, the mobile phase flow rate 1.5 mL min1, the column temperature 25 C, and the detection wavelength 267 nm.
Specificity
Experimental Chemicals and Reagents Samples of topotecan hydrochloride and its two impurities, Imp-A and Imp-B (Fig. 1) were obtained from the Process Research Department of the High Potent Active Pharmaceutical Ingredients Operations of Dr Reddy’s Laboratories, Hyderabad, India. HPLC-grade acetonitrile, potassium dihydrogen phosphate, and phosphoric acid were purchased from Merck Schuchardt, Germany. High-purity water was pre-
The specificity of a method is its ability to measure analyte response in the presence of potential impurities. The specificity of the HPLC method for topotecan hydrochloride was assessed in the presence of known impurities Imp-A and Imp-B. Forced degradation of the bulk drug was also performed to provide an indication of the stability-indicating property and specificity of the method. Degradation was attempted under the action of UV light (254 nm), heat (60 C), acid (0.5 M hydrochloric acid), base (0.5 M sodium hydroxide), and oxidizing agent (10% v/v hydrogen peroxide) to evaluate the ability of the
method to separate topotecan hydrochloride from its degradation products. For the heat and light experiments the study period was 10 days, for treatment with acid and oxidizing agent it was 48 h, and for treatment with base it was 3 h. A peak-purity test was conducted on stressed samples of topotecan hydrochloride by use of the PDA detector. Assay studies were performed by comparing stressed samples with qualified reference standard and the mass balance (percentage assay + percentage degradation) was calculated. Assay was also calculated for bulk sample by spiking it with the two impurities (Imp-A and Imp-B) at the specification level (0.15%).
Method Validation Precision
The precision of the method was evaluated by performing six independent assays of a test sample of topotecan hydrochloride, comparing the results with those from analysis of a qualified reference standard, and calculating the % RSD of the assay. The intermediate precision of the method was also evaluated—by a different analyst using a different instrument in the same laboratory [10, 11].
Linearity
Linearity was assessed by triplicate analysis of six test solutions, prepared from the stock solution, at concentrations from 25 to 150% of the assay analyte concentration (12.5, 25, 37.5, 50, 62.5, and 75 lg mL1). The calibration plot was drawn by plotting topotecan hydrochloride average area against concentration, expressed as a percentage, and the relationship between peak area and concentration was determined by least-squares linearregression analysis. Accuracy
The accuracy of the method was evaluated by triplicate analysis of three concentrations, 25, 50, and 75 lg mL1, in Limited Short Communication
Topotec an - 4. 991
0 .1 4 0 .1 2
AU
0 .1 0 0 .0 8 0 .0 6 0 .0 4
Control sample
0 .0 2 0 .0 0 2 .0 0
4 .0 0
6 .0 0
0 .1 0 0 .0 8 AU
1 0 .0 0
1 2 .0 0
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1 6 .0 0
1 8 .0 0
Topotec an - 5.608
0 .1 2
8 .0 0
0 .0 6 0 .0 4 0 .0 2
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2 2 .0 0
2 4 .0 0
Acid hydrolysis
0 .0 0 2 .0 0
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0.10 0.08 AU
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Topot ec an - 5. 430
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2 0 .0 0
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Basic hydrolysis
0.00 2. 00
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Oxidative degradation Topot ec an - 5.023
AU
0 .1 5 0 .1 0 0 .0 5 0 .0 0 2 .0 0
4 .0 0
6 .0 0
8 .0 0
1 0 .0 0
1 2 .0 0
1 4 .0 0
1 6 .0 0
1 8 .0 0
2 0 .0 0
2 2 .0 0
2 4 .0 0
Time (min)
Fig. 2. Typical HPLC chromatograms obtained after degradation studies
bulk drug sample, and calculation of percentage recovery. Robustness
To determine the robustness of the method the experimental conditions were deliberately altered [6, 7]. The mobile phase flow rate was usually 1.5 mL min1. To study the effect of slight changes of flow rate on resolution it was changed by ±0.2 units to Limited Short Communication
1.3 mL min1 and to 1.7 mL min1 while the other conditions were held constant. The effect of column temperature on resolution was studied at 20 and 30 C instead of 25 C, with the other conditions being held constant. Stability in Solution and in the Mobile Phase
The stability in solution was assessed by leaving test solutions of the sample and reference standard in tightly capped
volumetric flasks at room temperature for 2 days and analyzing the solutions every 6 h. Stability in the mobile phase was also assessed by analysis of freshly prepared sample solutions in the mobile phase and freshly prepared reference standard solutions at 6 h intervals for 2 days. The mobile phase was prepared was kept constant during the study period. For both set of experiments RSD (%) of the assay of topotecan hydrochloride was calculated for the study period.
Results and Discussion Optimization of Chromatographic Conditions The main objective of the chromatographic method was mutual separation of Imp-A, Imp-B, and topotecan hydrochloride. Different stationary phases (C18, C8, and cyano) and different mobile phases (phosphate, sulfate, and acetate buffers of different pH (7–10) and acetonitrile and methanol as organic mobile phase modifiers) were investigated. Under the conditions selected topotecan hydrochloride, Imp-A, and Imp-B were well separated, i.e. the method was specific for topotecan hydrochloride and the two impurities.
Results from Forced Degradation Studies No significant degradation of topotecan hydrochloride was observed when the drug was subjected to acidic and basic hydrolysis, heat, UV light, or oxidizing conditions (Fig. 2). Before the experiments the assay of the drug was 99.7%. After the experiments the assay of the drug was 99.3, 99.4, 99.7, 99.5, and 99.3%, respectively, and the respective mass balance (% assay + % degradation products) was 99.6, 99.8, 99.8, 99.6, and 99.7%. Peak-purity tests confirmed the topotecan hydrochloride peak obtained from all the stressed samples was homogeneous and pure. Assay of topotecan hydrochloride was unaffected by the presence of Imp-A and Imp-B, confirming the stability-indicating nature of the method.
precision the assay was 99.6, 99.4, 99.0, 98.9, 99.7, and 98.9%. The RSD of the assay values were well within 1.0%, confirming the high precision of the method. Linearity
The calibration plot for the assay method was linear over the range tested, i.e. 12.5–75 lg mL1 and the correlation coefficient was >0.999. When linearity was checked over the same concentration range for three consecutive days the results showed there was excellent correlation between peak area and analyte concentration. Accuracy
Recovery of topotecan hydrochloride from bulk drug samples ranged from 99.0 to 101.0% (w/w).
The assay of the topotecan hydrochloride during assessment of method precision was 99.2, 99.3, 99.4, 98.9, 99.7, and 98.9%. During study of intermediate
Acknowledgments The authors wish to thank the management of Dr Reddy’s group for supporting this work and wish to acknowledge the Process Research Group for providing the samples for our research. We would also like to thank colleagues in the separation science division of Analytical Research of High Potent Active Pharmaceutical Ingredients for their cooperation in carrying out this work.
References Robustness
When flow rate and column temperature were deliberately varied the resolution between Imp-A and Imp-B remained >3.0 at that between topotecan and Imp-A remained >10. Changing the conditions had no significant effect on the result from the assay, confirming the robustness of the method. Stability in Solution and in the Mobile Phase
The RSD for assay of topotecan hydrochloride during experiments to study its stability in solution and in the mobile phase was within 1.0%. Assay values ranged from 99.1 to 99.8%. No significant change in assay was observed during the experiments, confirming the drug is stable in these solutions for up to 48 h.
Validation Precision
pletely validated and satisfactory results were obtained for all the method validation data tested. The method is stability-indicating and so can be used for assessing the stability of bulk drug samples of topotecan hydrochloride.
Conclusions This gradient RP-LC method developed for quantitative determination of topotecan hydrochloride is precise, accurate, and selective. The method was com-
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