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2011 © The Japan Society for Analytical Chemistry
Development and Validation of a RP-HPLC Method for the Determination of Zidovudine and Its Related Substances in Sustained-release Tablets Jucimary V. dos SANTOS,* Luís A. E. Batista de CARVALHO,** and M. Eugénia PINA*† *Centro de Estudos Farmacêuticos (CEF), Pharmaceutical Technology Laboratory, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal **Unidade de I&D “Química-Física Molecular”, Chemistry Department, Faculty of Sciences and Technology, University of Coimbra, 3004-535 Coimbra, Portugal A reversed-phase high-performance liquid chromatography (RP-HPLC) method for the rapid and accurate quantification of zidovudine (AZT) in sustained release tablets during stability testing was developed. A Waters RP-18 XTerraTM® column, using a water:methanol (80:20, v/v%) mobile phase at a flow rate of 1.0 ml min–1, and UV detection at 266 nm, was employed. The method of validation parameters indicate a linear range of between 40.0 to 220.0 μg ml–1 with an LOQ of 1.985 μg ml–1 and an LOD of 0.655 μg ml–1 for the analyte. The degradation products of AZT were isolated and characterized for the first time. There was a very little decline of antiviral by heat, and AZT did not completely degrade either by acid or alkaline hydrolysis. On the other hand, oxidation caused a higher degradation stress in the drug. Finally, the degradation products resulting from stress studies were not found to interfere with the detection of antiviral, which is an advantage of the presently proposed method. (Received May 20, 2010; Accepted November 27, 2010; Published March 10, 2011)
Zidovudine (AZT), 3′-azido-3′-deoxythymidine, is a thymidine analogue belonging to the class of drugs called nucleosideanalogue reverse transcriptase inhibitors, used in the treatment of acquired immunodeficiency syndrome (AIDS). A literature review revealed some analytical methods for the determination of AZT and its main metabolite in biological fluids.1–4 However, there are few methods for the determination of AZT in pharmaceutical dosage forms.5–8 Over the years, analytical validation methods have been redefined, and it can be argued that the concepts continue to evolve and are constantly under the responsibility of international organizations that establish procedures to enable the production of quality products, ensuring both efficacy and safety during handling and storage.9–12 The main goals of analytical validation methods are to assure reproducibility, reliability and suitability for the purpose for which it was planned.13,14 From this perspective, the development and characterization of sustained-release AZT-containing tablets requires the validation of an analytical method, inasmuch as the presently available official guidelines do not establish any methodology for the identification and quantification of this particular active substance in dosage forms.9–12 The present work intends to fill this gap: a modification of the reversed-phase high-performance liquid chromatography (RP-HPLC) method, as described in the United States Pharmacopeia, was developed and validated (according to Hubert et al.)15,16 for the determination of AZT (either in the pure form or in sustainable release tablets) and of its degradation products. The key point of this study is that the peaks corresponding to AZT degradation products, as well as those due to excipients, To whom correspondence should be addressed. E-mail:
[email protected];
[email protected]
†
do not interfere with the analysis. Additional advantages of the current method over the reported procedures are the elimination of a phosphate buffer in the mobile phase, and the replacement of a decrease in the methanol content in the aqueous eluent (from 50:50 to 20:80 (v/v)) as a means for reducing any organic contamination, which is an extremely important parameter for the lifetime of the resin, and therefore for the benefit of HPLC analysis. The purpose of stability testing is to provide evidence on how the quality of a drug substance or drug product varies with time, under the influence of a variety of environmental factors, such as temperature, humidity and light, as well as to establish a re-test period for the drug substance or expiration date for the drug product, and recommend storage conditions. Notwithstanding the diversity of active principles, drug degradation follows some common pathways. By examining the structural features of a drug molecule, possible degradation routes may be found to occur to a certain extent, which can assist in the design and execution of degradation studies.17–20 The photochemical degradation of pharmaceutical compounds may seriously affect the quality of products, and is usually associated with a reduction of the pharmacological activity and/or the occurrence of side effects. The light sensitivity of drugs in solid preparations has been investigated,21–23 having been verified that both the photo- and chemical-stability depends on the composition’s formulation. However, increasing interest in this matter is evident, since the number of light-sensitive drugs is growing and the International Conference Harmonization (ICH) guidelines require that photostability testing should be carried out.18,19 Considering that analyses of the degradation products under stress conditions are useful for establishing degradation pathways, developing and validating suitable procedures,
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the present work describes analytical parameters aimed to achieve an alternative for the quantification of AZT and its degradation products, including dosage forms, in accordance with ICH recommendations.
Experimental Reagents Drug: AZT lot No. 061218 was supplied by CIPLA (India). Polymers: Eudragit® RS PO and Eudragit® RL PO, Röhm Pharma (Germany); ethylcellulose, Sigma-Aldrich (USA); K15M and hydroxypropylmethylcellulose, Methocel® Diluent: lactose Methocel® K100M, Colorcon (UK). monohydrate, Granulac® 200, Meggle, Wasserburg (Germany). Glidant: Talc. Lubricants: magnesium stearate, Magnesia GmbH (Germany). Methanol, sodium hydroxide and hydrochloric acid were products of Merck (Germany). Hydrogen peroxide was obtained from Maialab (Portugal). HPLC-grade water was provided by a Milli-Q® water purification system, Millipore Corporation ProgrardTM (USA), and all solutions were prepared daily. Apparatus A HPLC system consisted of an Agilent Technology Model HP 1100 (Agilent, USA) with the following parts: a quaternary pump (G1311A); an HP 1100 vacuum degasser (G1322A); an HP 1100 autosampler with injector fitted with a 20-μl loop (G1313A); an HP 1100 UV/Vis detector (G1314A); and an Agilent integrator equipped with a column oven (G1328A). A reversed-phase column (RP-18 XTerraTM®, 250 mm × 4.6 mm i.d.; Waters, Ireland) 5 μm, with a security guard cartridge (Purospher® STAR RP-18, 4.0 mm × 3.0 mm i.d.; Merck, Germany) 5 μm, was used at 30 ± 2° C. Chromatographic data were processed using the ChemStation software (Agilent, USA). For disintegration of the AZT tablets, an ultrasonic bath was used (Bandelin Sonorex Super RK 255 H, England). Chromatographic conditions Firstly, elution with a mobile phase containing methanol:water (20:80, v/v) was carried out for 3 min. A linear gradient was then applied for 5 more min, up to a methanol:water ratio of 50:50 (v/v), after which elution with a methanol:water (20:80, v/v) gradient was used. The mobile phase was filtered under a vacuum, using a 0.45-μm membrane filter PVDF (Tracer®, Teknokroma, Spain), and degassed prior to use. The column was equilibrated for at least 1 h before starting the assay. The C. The samples were flow rate was 1.0 ml min–1 at 30 ± 2° monitored at 266 nm. A 20-μl volume of the sample was injected into the HPLC system. The retention time can be decreased by adding a less-polar solvent (methanol) into the mobile phase to reduce the surface tension of water. Gradient elution uses this effect by automatically reducing the polarity and the surface tension of the aqueous mobile phase during the course of analysis. Then, it decreases the retention of the later-eluting components so that they elute faster, giving narrower (and taller) peaks for most components. This also improves the peak shape for tailed peaks, as the increasing concentration of the organic eluent pushes the tailing part of a peak forward. This also increases the peak height (the peak looks “sharper”), which is important in trace analysis. Analytical procedure Preparation of the stock solution.
For the preparation of a
Table 1 AZT formulations containing cellulose derivatives and polymethacrylates Component Zidovudine HPMC K 100M HPMC K 15M Eudragit® RS PO Eudragit® RL PO Lactose Talc Magnesium stearate
Formulation/mg F1
F2
F3
300.0 19.0 19.0 19.0 19.0 — 2.0 2.0
300.0 23.0 23.0 — — 30.0 2.0 2.0
300.0 19.0 19.0 — — 38.0 2.0 2.0
standard stock solution (1000 μg ml–1), the necessary AZT was weighed (analytical balance KERN 770, Germany) and dissolved in methanol. This solution was protected from light and stored at 4° C for no longer than 1 week. Preparation of the standard solutions. Ten standard solutions were prepared, in the concentration range 40.0 to 220.0 μg ml–1, by the dilution of appropriate volumes of the stock solution. The samples were filtered through a 0.22-μm nylon membrane and kept under dark conditions. Sample preparation. AZT stability studies were performed under either hydrolytic (neutral, alkaline, acid), oxidative, or thermal stress conditions. Drug photostability was also accessed. All AZT solutions were prepared to yield 120 μg ml–1 starting concentrations. The freshly controlled samples were prepared in the mobile phase, kept under dark conditions, and analyzed in triplicate. AZT solutions for neutral (ultrapure water), acid (2 M hydrochloric acid) and alkaline (2 M sodium hydroxide) degradation studies were both refluxed for 24 h and bubbled with nitrogen gas. Solutions for oxidation studies were prepared in H2O2, either 3 or 9%, and refluxed for 24 h in order to assist AZT oxidation. From this moment on they were subjected to thermal stress at 20, 25, 30, 40, 50 and 60° C for one month. Distinctive formulations of the matrix tablets are given in Table 1. For their analysis, three tablets were accurately weighed, ground in a mortar; an average mass of 380 mg of the powder was transferred to a 100-ml volumetric flask; 20 ml of water was added and shaken by mechanical agitation to disperse the powder. Around 30 ml of methanol was added and sonicated for 10 min. This mixture was diluted with water to 100 ml and homogenized; 4 ml of the resulting solution was placed into a 100-ml volumetric flask, and diluted with water to complete the volume. After mixing, a portion of the solution was filtered through a 0.22-μm nylon filter, discarding the first 2 ml of the filtrate. Photostability studies were carried out under the following conditions: the AZT powder was carefully placed in a Petri dish to give a thin layer as homogenous as possible. On the other hand, solutions of both pure drug and of the formulations containing excipients (F1, F2 and F3) were also placed in a light chamber (Suntest CPS/CPS+ from Atlas Material Testing Technology, Germany) and exposed to a light source for 22 h, resulting in an overall illumination of 1.2 million lux per hour C. and an integrated near-ultraviolet energy of 250 Wh m2 at 30° Control samples, which were protected with aluminium foil, were also placed in the light cabinet and exposed concurrently.
ANALYTICAL SCIENCES MARCH 2011, VOL. 27 Table 2 Linear regression data for AZT Parameter Wavelength/nm Concentration range/μg ml–1 Injection levels Regression equationa Determination coefficient, R2 Limit of detection/μg ml–1b Limit of quantification/μg ml–1b Standard error of slope Standard error of intercept Significance level
Result 266 40.0 to 220.0 10 y = 43.68x + 20.38 0.9998 0.655 1.985 0.064 8.671 p < 0.005
a. y, Analyte peak area; x, analyte concentration. b. See text.
Method validation The optimized chromatographic conditions were validated by evaluating specificity, linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), robustness and system suitability, in accordance with ICH guidelines Q2 (R1).11 Specificity In order to demonstrate the specificity of the method, the AZT standard solution and a placebo solution, prepared by dissolving the excipients (Eudragit® RS PO and Eudragit® RL PO, ethylcellulose, Methocel® K15M, Methocel® K100M, lactose monohydrate, talc and magnesium stearate), were mixed in a 1:1 proportion. After filtration, both the standard solution and the mixture were injected in HPLC and chromatograms were recorded. Linearity A standard stock solution of the drug was diluted in order to prepare ten standard AZT solutions, in the concentration range 40.0 to 220.0 μg ml–1. A 20-μl volume was then injected automatically in the chromatograph. A linear regression of the AZT peak area values (y) versus the concentration in μg ml–1 (x) was performed. Three sets of such solutions were prepared, and each set was analyzed to plot the calibration curves. The linear coefficients, slope standard error, determination coefficient, standard error of the fit, residual sum and standard error in the residuals were calculated, in order to assess any statistical significance among the data (Table 2). Regression linear and one-way analysis of the variance (ANOVA) was used to test the variation of the data in the samples. Any differences between the parameters were considered to be significant if p < 0.05, followed by a Bonferroni comparison t-test. A statistical analysis was performed with SPSS Statistics for Windows.24 Precision, limit of detection, and limit of quantification The precision of the HPLC method was assessed by carrying out repeatability (instrument and method precision) and intermediate precision tests. The repeatability was evaluated by analyzing ten solutions containing a known quantity of analyte. The inter-day precision was assessed by testing three concentrations (40, 120 and 220 μg ml–1) over 3 consecutive days. The LOD was determined from the calibration curve, using the following equation: LOD = 3.3 Sab
where Sb is the y-intercept standard deviation and a is the line
285 slope (Table 2). The limit of quantification (LOQ) was calculated using LOQ = 10 Sab
The test solutions at LOD and LOQ concentrations were injected six times in the chromatograph, and the %RSD of the peak area of replicate injections was calculated. Accuracy The accuracy of the method was checked for three known concentration levels (40, 120 and 220 μg ml–1) added to a mobile phase containing all components of the tablets (1:1, v/v), and peak area was recorded. All analyses were repeated six times, and the recoveries and respective standard deviations were calculated. Robustness To demonstrate the robustness of the method, deliberate small changes were made around the optimal values, such as: temperature, flow rate and methanol content. The temperature was varied between 20° C (RT) and 40° C, while the flow rate was changed from 1.0 to 1.2 ml min–1 and the methanol content was varied from 20 to 30%. The effect on the retention time and peak parameters was studied. The AZT was analyzed at 40 μg ml–1 (low level), 120 μg ml–1 (center point) and 220 μg ml–1 (high level), in accordance with the calibration curve. The following data were collected for each testing: (a) concentration of the sample, (b) retention time of the sample peaks, (c) sample peak height, (d) sample peak width and (e) sample peak asymmetry. The values presented are the average of three measurements. System suitability The system suitability parameters were defined with respect to replicate injections of the standard, repeatability and resolution of the AZT peak, using reference standard and tests solutions.
Results and Discussion Method validation The validation results of the proposed method were acceptable. As demonstrated in Fig. 1, no substances interfered with any of the typical chromatograms (prepared in the presence and absence of excipients), from the standard solution and from the placebo solution. Thereby, it is possible to conclude that the method is selective for the separation and determination of AZT in the tablets. The response of the analyte was linear over the concentration range 40.0 to 220.0 μg ml–1. The calculated statistical parameters indicate that the calibration line fits well into the model, and that it is significantly linear. The determined LOD and LOQ values indicate a satisfactory sensitivity of the AZT quantification (Table 2). The results for chromatographic precision, such as the repeatability and intermediate precision of the method, were very satisfactory. The repeatability was evaluated by performing ten replicate injections of a sample at the nominal standard concentration (Table 3), where the peak-area RSD maximum (%) was 0.73, which was considered to be acceptable. The inter-day precision was assessed by injecting the same three concentrations (40, 120 and 220 μg ml–1) over 3 consecutive days. The RSD of the sample response factor
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inter-day was calculated for separate preparations at the nominal standard concentration of the samples, resulting in %RSD values of 0.21, 0.64 and 0.14%, respectively (Table 3). The mean recoveries for lower, intermediate and high concentrations were found to be 40.11 ± 0.72, 121.29 ± 0.32 and 219.39 ± 0.23 μg ml–1, respectively. The excellent % recovery results, near to 100%, and their low standard deviation values (SD < 1.0), reflect a high accuracy of the analytical method. Deliberate variations of some parameters (temperature, flow rate and composition of the mobile phase) were found not to result in significant changes in the overall resolution, as can be seen from the response surface (Fig. 2), indicating the robustness of the method. This fact was evidenced for the three concentrations studied. The factor that affects peak symmetry is the methanol content; however, the method continues to be robust because the asymmetry parameters are consistently lower
than 1.2, as required by ICH11 and FDA10 guidelines (Tables 4, 5, 6). Changes in the organic phase can have an impact on the retention time, as shown in Table 6; the retention time (tR) decreases with increasing % methanol. The randomized robustness design in tabular form is illustrated, along with the results of the study. The t-test (tvalue was 0.1662, 0.1690 and 0.1737 for the temperature, flow rate and mobile phase, respectively) and analysis of variance (ANOVA)24 were used in order to determine the impact of the three variables: (i) temperature, Fobs = 1.89 and p = 0.1318; (ii) flow rate, Fobs = 1.12 and p = 0.3998; and (iii) mobile phase, Fobs = 1.93 and p = 0.1243. An F critical value of 2.59 was obtained, which means that these variables are not significantly different from one another, and produced the same results. Formulations Table 7 presents the one-way ANOVA and calculations of the t-values (0.099) obtained, which were found to be less than the tabulated t-values (1.645), indicating that statistically there was no significant difference between the results of quantification of AZT in the formulations (F1, F2 and F3). Determination of the forced degradation of products The ICH Q1A(R2) guideline18 requires stress testing to be carried out, in order to elucidate the inherent stability characteristics of the active substance. Susceptibility to
Fig. 1 HPLC chromatograms of AZT (120.0 μg ml–1): standard solution (A) and a mixture with placebo solution (B, see text). Table 3 Data precision (n = 10) AZT concentration/ μg ml–1
Repeatability, %RSD Day 1
Day 2
Day 3
Intermediate precision, %RSD
40.0 120.0 220.0
0.17 0.73 0.40
0.23 0.50 0.10
0.38 0.50 0.39
0.21 0.64 0.14
Fig. 2 Response surface calculated for the robustness of the RP-HPLC method.
Table 4 Robustness study (temperature)a Temperature vs. [AZT]/ μg ml–1 RT 30° C 40° C
40 120 220 40 120 220 40 120 220
Recovery, %
Retention time/ min
Peak height, h
Peak width, w
Symmetry, S
100.05 ± 0.13 100.74 ± 0.84 100.41 ± 0.21 99.53 ± 0.55 99.06 ± 0.37 100.40 ± 0.51 99.64 ± 0.64 99.23 ± 0.76 100.42 ± 0.53
8.58 ± 0.68 8.58 ± 0.67 8.58 ± 0.67 8.38 ± 0.69 8.38 ± 0.69 8.38 ± 0.69 8.38 ± 0.00 8.38 ± 0.00 8.38 ± 0.00
140.13 ± 0.70 410.28 ± 0.93 755.70 ± 0.21 140.03 ± 0.82 405.90 ± 0.34 749.28 ± 0.39 142.03 ± 0.25 409.83 ± 0.22 758.18 ± 0.43
0.1937 ± 0.5817 0.1923 ± 0.1588 0.1929 ± 0.3111 0.1920 ± 0.3646 0.1919 ± 0.7824 0.1930 ± 0.2553 0.1884 ± 0.5155 0.1898 ± 0.1995 0.1905 ± 0.3785
1.10 ± 1.25 1.06 ± 1.15 1.07 ± 0.83 1.10 ± 1.16 1.08 ± 1.16 1.09 ± 1.01 1.09 ± 0.28 1.07 ± 0.27 1.08 ± 0.30
a. Mean ± RSD (three measurements). RT: Room temperature.
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Table 5 Robustness study (flow rate)a Flow rate vs. [AZT]/ μg ml–1 1.0 ml min–1 1.1 ml min–1 1.2 ml min–1
40 120 220 40 120 220 40 120 220
Recovery, %
Retention time/min
Peak height, h
Peak width, w
Symmetry, S
99.53 ± 0.55 99.06 ± 0.37 100.40 ± 0.51 99.49 ± 0.38 101.32 ± 0.78 100.70 ± 0.88 99.43 ± 0.52 100.04 ± 0.86 100.82 ± 0.95
8.38 ± 0.69 8.38 ± 0.69 8.38 ± 0.69 8.35 ± 0.65 8.35 ± 0.65 8.35 ± 0.65 8.35 ± 0.69 8.35 ± 0.69 8.35 ± 0.69
140.03 ± 0.82 405.90 ± 0.34 749.28 ± 0.39 137.38 ± 0.19 402.33 ± 0.29 740.00 ± 0.22 137.80 ± 0.18 399.95 ± 0.95 736.53 ± 0.29
0.1920 ± 0.3646 0.1919 ± 0.7824 0.1930 ± 0.2553 0.1944 ± 0.6433 0.1949 ± 0.3851 0.1941 ± 0.3133 0.1956 ± 0.886 0.1966 ± 0.1834 0.1973 ± 0.2210
1.10 ± 1.16 1.08 ± 1.16 1.09 ± 1.01 1.09 ± 0.64 1.12 ± 1.63 1.11 ± 1.54 1.12 ± 0.14 1.11 ± 0.45 1.12 ± 0.55
Recovery, %
Retention time/min
Peak height, h
Peak width, w
Symmetry, S
99.53 ± 0.55 99.06 ± 0.37 100.40 ± 0.51 100.18 ± 1.25 102.09 ± 0.56 101.14 ± 0.62 102.17 ± 0.34 101.32 ± 0.32 100.60 ± 0.17
8.38 ± 0.69 8.38 ± 0.69 8.38 ± 0.69 6.70 ± 0.00 6.70 ± 0.00 6.70 ± 0.00 5.80 ± 0.00 5.80 ± 0.00 5.80 ± 0.00
140.03 ± 0.82 405.90 ± 0.34 749.28 ± 0.39 132.90 ± 0.31 381.80 ± 0.68 704.68 ± 0.47 150.90 ± 0.30 436.63 ± 0.32 803.13 ± 0.76
0.1920 ± 0.3646 0.1919 ± 0.7824 0.1930 ± 0.2553 0.2025 ± 0.2436 0.2034 ± 0.6674 0.2068 ± 0.7390 0.1770 ± 0.3390 0.1780 ± 0.2270 0.1795 ± 0.6950
1.10 ± 1.16 1.08 ± 1.16 1.09 ± 1.01 1.00 ± 0.30 1.00 ± 0.69 0.99 ± 0.71 0.95 ± 0.27 0.94 ± 0.62 0.93 ± 0.33
a. Mean ± RSD (three measurements).
Table 6 Robustness study (HPLC mobile phase)a % Methanol vs. [AZT]/ μg ml–1 20% 30% 40%
40 120 220 40 120 220 40 120 220
a. Mean ± RSD (three measurements).
oxidation is one of the required assays. Also, evaluations of both hydrolytic and photolytic stabilities are required.19 An ideal stability-indicating method is one that quantifies the standard drug alone, and also resolves its degradation products. The guideline depicts the basic acceptance criteria for evaluating validation experiments based on any practical experience for chromatographic procedures, which may be used at different levels either in qualitative identity testing, assays, semi-quantitative limit tests or quantitative determination of impurities. The parameters for robustness testing of the given procedures and quality assurance of HPLC testing have been described in the ICH Q2(R1) guideline.11 According to the USP 31/NF26,6 the profile of the impurities has been defined in relation to the sources of the identified drug. The impurities detected by HPLC are limited to 0.1%. The limits for these impurities have been fixed at the minimum level permitted by the analytical method, in accordance with the requirements laid down in system conformity. Studies of AZT stability were performed, under hydrolytic (alkaline, acid, neutral), oxidative or thermal stress conditions. The photostability was also studied (AZT powder, AZT solution and the formulations containing excipients solutions). Figure 3 represents chromatograms of the forced degradation products of AZT. This drug was found to be more unstable under basic and acidic stress conditions, evidencing degradation products at tR = 2.6, 2.9 and 3.9 min (acid hydrolysis), and tR = 2.5 and 9.7 min (alkaline hydrolysis), corresponding to a decrease of the drug concentration of around 23% upon reflux for 24 h. In contrast, AZT presented good stability under neutral conditions, without signals of degradation products (Table 8). Dunge et al.25 studied the kinetics of decomposition of AZT, and found that this drug was stable in water upon reflux for up to 5 days.
Table 7 Tablets AZT content statistical analysis AZT tablet
Amount founda/mg
Assay, %
F1 Mean t F F2 Mean t F F3 Mean t F
298.14 ± 1.53 1.211 (2.064) 1.257 (3.259) 300.81 ± 1.89 0.356 (2.064) 1.152 (3.259) 300.60 ± 1.74 0.881 (2.064) 1.091 (3.259)
99.38 ± 0.61 100.27 ± 0.53 100.20 ± 0.60
a. The values in parenthesis are the tabulated values of t and F at P = 0.05.
Studies using either 3 or 9% H2O2 solutions demonstrated antiviral lower stability in oxidative media (18.42 ± 0.96% and 21.92 ± 1.23%, respectively). Furthermore, the AZT solutions showed good stability when incubated at different temperatures (Table 9). For photolytic studies (Table 10), in turn, the AZT content was further reduced on solution, as compared to the powder. In fact, the compound is more affected by light when solubilized than in the solid network. Regarding the control samples, no degradation was observed in the absence of light.25 The extended-release formulations (F1, F2 and F3) were separately put into sealed transparent flasks. After 22 h of day-light irradiation, the AZT concentration was 97.78 ± 1.75% in F1, 97.70 ± 1.14% in F2, and 97.59 ± 1.19% in F3, while pure AZT showed 82.97 ± 0.96%. Interestingly enough, the extended-release formulations (F1, F2 and F3) evidenced a lower degradation than the drug alone.
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ANALYTICAL SCIENCES MARCH 2011, VOL. 27 Table 9 AZT stability at different temperaturesa Temp./° C
Rec., %
Temp./° C
Rec., %
4 20 25 30
100.03 ± 0.93 100.07 ± 0.61 100.03 ± 1.19 100.03 ± 1.11
40 50 60
99.29 ± 1.41 98.74 ± 1.23 97.46 ± 1.16
a. Mean ± RSD (three measurements).
Table 10 AZT photostability testsa Sample
Degradation, %
AZT powder AZT solution F1 F2 F3
17.03 ± 1.24 28.61 ± 0.96 2.22 ± 1.75 2.30 ± 1.14 2.41 ± 1.19
a. Mean ± RSD (three measurements). Fig. 3 HPLC chromatograms of AZT degradation products of: neutral hydrolysis (A); acid hydrolysis (B); base hydrolysis (C); 3% H2O2 oxidative medium (D); 9% H2O2 oxidative medium (E); photolytic conditions (F); AZT reference chromatogram (G).
Table 8 AZT degradation studies for a 24-h perioda Stress condition
Degradation, %
Acid hydrolytic Neutral hydrolytic Alkaline hydrolytic Oxidative 3% H2O2 Oxidative 9% H2O2
23.04 ± 0.84 0.00 ± 0.65 18.27 ± 1.43 18.42 ± 0.96 21.92 ± 1.23
a. Mean ± RSD (three measurements).
Conclusions In the present work, a RP-HPLC method for the determination of AZT was developed and validated, in accordance with the ICH parameters (specificity, linearity, precision, accuracy, limit of detection, limit of quantification, robustness and system suitability). The proposed method separates the drug from its degradation products, formed during stability testing, in a reasonable analysis time and with acceptable chromatographic parameters. Indirectly, using the recommended ICH stress tests, it is possible to gain some insight about the AZT degradation pathways. Hopefully, this report on a stability indicating method and the degradation of AZT may be helpful for multiple generic manufactures of this drug worldwide, by sparing them from unnecessary repetition of the same studies.
Acknowledgements J. V. S. acknowledges financial support by the Programme Alβan, the EU Programme of High Level Scholarships for Latin America (scholarship No. E06D100103BR).
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