Quantitative determination of imatinib stability under various stress conditions.

OBJECTIVE: A simple, rapid, reverse phase and stability-indicating high-performance liquid chromatography (HPLC) method for the estimation of imatinib in solution and in plasma under forced degradation conditions was developed.
MATERIALS AND METHODS: The method employed isocratic elution using a Waters Atlantis C18 (5 μ, 4.6 mm × 150 mm) HPLC column. The mobile phase consisted of acetonitrile and 10 mM KH2PO4 buffer in the ratio of 35:65 (v/v, pH = 4.6), which was delivered using isocratic flow at a rate of 1 mL/min. The injection volume of 50 μL and imatinib was monitored using UV detection 270 nm after a clean-up step with diethyl ether and with a total run time of 6 min.
RESULTS: The method was validated in solution as well as in plasma, and the response was found to be linear in the concentration range of 0.5-20 μg/mL. The coefficient of correlation was found to be >0.99. Forced degradation studies revealed that imatinib undergoes degradation under different stress conditions. DISCUSSION: The developed HPLC method could effectively resolve degradation product peaks from imatinib except at neutral pH. Further, no interference was found at the retention time of imatinib from any plasma components, indicating selectivity of the developed method. The limits of detection and quantitation of the method were 0.025 and 0.5 μg/mL, respectively.

Imatinib is a competitive inhibitor of the Bcr–Abl and c-kit tyrosine kinases, and it is used for the treatment of Philadelphia chromosome–positive chronic myeloid leukemia (CML) and for other malignant pathologies such as lymphoblastic leukemias and gastrointestinal stromal tumors.[12] Chemically, imatinib is 4-[(4-methyl-1-piperazinyl) methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl] amino]-phenyl] benzamide methane sulfonate) [Figure 1].[3]

Figure 1

Chemical structure of imatinib

Stability testing of drugs is a crucial requirement and is needed to establish the safety and efficacy of the active substance. As part of the good manufacture practice (GMP) regulations, the Food and Drug Administration (FDA) requires that drug products bear an expiration date determined by appropriate stability testing. The International Conference on Harmonization (ICH) guidelines, which have been incorporated as law in the U.S, EU and Japan, require that the drug substances should be tested for those features that are susceptible to change during storage and are likely to influence the quality, safety and/or efficacy by validated methods. The stability of drug products needs to be evaluated over time in the same container closure system in which the drug product is marketed. In some cases, accelerated stability studies can be used to support tentative expiration dates in the event that full shelf-life studies are not available. When a firm changes the packaging of a drug product (e.g., from a bottle to unit-dose), stability testing must be performed on the product in its new packaging, and expiration dating must reflect the results of the new stability testing. An ideal stability-indicating method is one that quantifies the drug per se and also resolves its degradation products.[4-6]

The stability assessment of any promising drug candidate plays a vital role in the process of new drug development. Many environmental conditions, such as heat, light and moisture, as well as the inherent chemical susceptibility of a substance to hydrolysis or oxidation can play an important role in pharmaceutical stability. These studies also provide essential information supporting pharmaceutical formulation development. Furthermore, they help to define storage and handling conditions. The exposition of the drug substance to extreme external conditions helps to reveal and identify the likely degradation products. The stability of drugs should be determined in the aqueous media of different pH, at different temperatures, and other extreme external conditions (UV light, dry and wet heat) in order to describe the degradation behavior of the said compound. Hydrolytic cleavage of certain functional groups in a drug molecule constitutes an important route for clearance of compounds from the body. Incubation with plasma in vitro can quickly determine whether a compound is susceptible to plasma degradation and to what extent. It also supports structure modification studies to reduce plasma degradation.[7]

Several analytical procedures have been developed to measure imatinib in biological fluids and pharmaceutical dosage forms using different detection techniques, including high-performance liquid chromatography (HPLC)-UV and liquid chromatography and mass spectracopy (LC-MS/MS) methods.[8-12] The aim of the current work is to quantitively test the stability of imatinib under various physical and chemical stress conditions as well as its enzymatic hydrolysis in plasma samples using a simple HPLC method developed and validated in our laborator.

Materials and Methods

Chemicals and reagents

Gift sample of Imatinib mesylate powder (99% pure) was generously supplied by Novartis Pharmaceuticals (Switzerland). Omeprazole was purchased from Sigma (St. Louis, MO, USA). Methanol, acetonitrile and sodium dihydrogen phosphate of HPLC grade were obtained from BDH Laboratory (BDH Chemicals Ltd., Poole, UK). All other reagents and chemicals used in the assay were of the highest purity available for analytical research; water was of Milli-Q quality.

Imatinib assay and quantitation

Stock solutions of imatinib and omeprazole (internal standard) were prepared in methanol to yield a final concentration of 1 mg/mL. Working standards of imatinib were prepared fresh every day by diluting the aliquots of the stock solutions with sodium dihydrogen phosphate (10 mM) buffer to make final concentrations of 100 and 1 μg/mL. For omeprazole, the stock was further diluted in methanol to 100 μg/mL. All the solutions were stored at -80°C for the duration of the study. Calibration standards were prepared by diluting different concentrations of imatinib working standards with the phosphate buffer/plasma to give final concentrations of 0.5, 1, 5, 10, 15 and 20 μg/mL. Two concentrations of quality control samples (QCs) containing 1.5 μg/mL and 15 μg/mL were also similarly prepared.

In another set of experiments, human plasma samples (0.5 mL) were spiked with different concentrations of imatinib (i.e., 0.5, 1, 5, 10, 15 and 20 μg/mL), 25 μL of internal standard (omeprazole, 100 μg/mL; internal standard) was added and the samples were vortex mixed for 10 s in 10-mL glass tubes. Diethyl ether (4 mL) was added to each tube and the drugs were extracted by vortexing the tubes for 5 min. The samples were then centrifuged at 3000 g for 10 min, the ether layer was transferred to another tube and evaporated to dryness under a gentle stream of nitrogen with temperature set at 40°C. The residue was reconstituted in 100 μL of acetonitrile and an aliquot of 50 μL was injected onto the column.[911]

The HPLC system consisted of a Waters model 717 autosampler, M-600 dual piston solvent delivery pump and M-2487 dual UV absorbance detector (Waters Corp., Milford, MA, USA). The mobile phase consisted of potassium dihydrogen phosphate (10 mM) and acetonitrile in the ratio of 60:40, respectively. The mobile phase (pH 5.6) was delivered at a flow rate of 1 mL/min after degassing. A Waters Atlantis dC18 column (5 micron, 4.6 mm × 150 mm) was utilized to elute the compound of interest. A UV scan of imatinib gave a λmax = 270 nm, and this wavelength was used to analyze the drug. Signal output was captured using Millennium32 software, version 3.05 (Waters Corp.).

Method Validation

Linearity was established by triplicate injections of aqueous solutions or plasma samples containing drug in the concentration range of 0.5-20 μg/mL. The precision and accuracy of the assay was determined using quality control (QC) samples of two known imatinib concentrations (1.5 and 15 μg/mL), which were processed fresh each day along with the calibration curve standards everyday for 3 days. Six replicates of each QC were analyzed on 3 days, and the intra- and inter-assay means, standard deviations and coefficients of variation (CV) were calculated. For the purpose of recovery studies in plasma, water (0.5 mL) was used to spike the samples instead of plasma. The lower limit of detection (LLOD) was set three times above the baseline noise. The lower limit of quantification (LLOQ) was calculated as LLOQ = 10*(SD/Y), where SD is the standard deviation and Y is the slope of the calibration curve.[45]

Stability Studies

For stability studies, two solutions of imatinib were prepared to give final concentrations of 4 and 16 μg/mL. Aliquots of 10 mL of each concentration were separately stored under different conditions to study the effect of temperature, humidity, pH and UV radiation. For temperature effect, these samples were kept at room temperature (24°C) in dark, 4°C and 40°C. Another set of samples was stored in a humid atmosphere of >90% humidity produced with the help of saturated solution of potassium chloride in a closed container. The effect of acidic, neutral and alkaline milieus was studied by adjusting the pH of the two imatinib concentrations to 4, 7 and 10 with the help of hydrochloric acid (0.1 M) and sodium hydroxide (5 M) solutions measured using a pH meter.[91112]

For studying the effect of UV exposure, the two imatinib solutions were kept under direct short UV light in a closed clear glass container. Aliquots of samples kept under different conditions were collected and analyzed at different time points. For enzymatic hydrolysis studies, concentrations of 4 and 16 μg/mL of imatinib were prepared in plasma as described above and placed in a water bath with temperature adjusted to 37°C. Aliquots of 2 mL were obtained at different time points, and plasma samples were processed using the developed method.[1112]

Results

Imatinib assay and separation

Imatinib and omeprazole were separated within 5 min of the chromatographic run. The peaks were well resolved and the retention times for imatinib and omeprazole were approximately 2.4 and 3.6 min, respectively [Figure 2]. Standard curves, in triplicate, for imatinib in phosphate buffer and plasma were linear over the range of 0.5-20 μg/mL. This method is capable of detecting up to 0.25 μg/mL of imatinib. The LLOQ was 0.5 μg/mL and the CV was found to be 1.96%. The mean coefficient of correlation (r2) for the standard curves was >0.99, and this shows the linearity of the calibration curve in the investigated range. The calculated intra- and inter-day CVs for imatinib QC samples were less than 15% in all experiments.

Figure 2

A typical chromatogram of standard imatinib and omeprazole (I.S.)

Stability of imatinib

The effect of various physical and chemical stressors on imatinib stability is illustrated in Tables 1 and 2. Imatinib demonstrated a good thermal stability for at least 1 week, with only < 7% degradation at 40°C. Similarly, exposing the drug to high humid environments (>90% humidity for two consecutive days) revealed its stability under the harsh conditions as no significant change in the peak area was observed. The pH effect showed that the drug is quite stable in both acid (i.e., pH = 4) and alkaline milieu (i.e., pH = 10); however, it degrades to a great extent (~35-40% loss) at a neutral pH [Table 2]. Imatinib was also found to be stable under constant irradiation of short UV light for 4 h, with only ~15% decomposition.

Table 1

Stability of imatinib under various stress conditions

Table 2

Enzymatic stability of imatinibin human plasma

DISCUSSION

A validated HPLC method was developed and evaluated for the analysis of imatinib, which is evident from the results. The response of the drug was linear over the calibration range (r2 = 0.996). The precision of the method was indicated by the relative standard deviation (RSD) values for the inter- and intra-day runs, which were <0.6 and 1.86, respectively [Table 3]. Accuracy was determined from the percentage recovery between the peak areas obtained from drug-spiked plasma sample and samples prepared in water. The recovery percentage for the two different levels of concentrations was in the range of 83.5-86.0%.

Table 3

Intra-and inter-day precision and accuracy of imatinib in phosphate buffer and in plasma

Stress testing of drugs can help in identifying the likely degradation products and hence the degradation mechanism. Indeed, the stability studies have received considerable attention because of their importance in the development and quality control of pharmaceutical products. In the present work, stability of imatinib mesylate under forced thermal, humid, alkaline, neutral and acidic conditions as well as under direct short wave UV light was studied [Figure 3]. It is evident that the drug is quite stable under these stress conditions, with the exception when it was exposed to a neutral pH. This seems to be the case of hydrolysis of amide group through a nucleophilic attack. There are a few reports in the literature about the stability of imatinib under stressed conditions. Szczepek and colleagues studied imatinib under hydrolytic, oxidative and photolytic conditions using HPLC, MS and nuclear magnetic resonance (NMR) to identify and analyze the degradation products. Our results are in line with those of Szczepek's work, i.e. imatinib is quite stable under acidic and basic conditions, but in a neutral environment. This degradation at pH 7.0 may be due to different experimental conditions like the use of a different solvent.

Figure 3

(a) High-performance liquid chromatography chromatograms of temperature (b) humidity and (c) pH induced forced degradation of imatinib