HPLC with Diode-Array Detection for Determination of Leflunomide in Tablets 2006, 63, 283–287
D. S. Miron1,&, C. Soldattelli1,2, E. E. S. Schapoval1,2 1
Programa de Po´s-graduac¸a ˜o em Cieˆncias Farmaceˆuticas, Faculdade de Farma´cia, Universidade Federal do Rio Grande do Sul, Porto Alegre RS, Brazil; E-Mail:
[email protected] 2 Departamento de Produc¸a ˜o e Controle de Medicamentos, Faculdade de Farma´cia, Universidade do Rio Grande do Sul, Porto Alegre RS, Brazil
Received: 15 December 2005 / Revised: 12 January 2006 / Accepted: 30 January 2006 Online publication: 28 February 2006
Abstract We have developed and validated a simple HPLC method for analysis of leflunomide in tablets. Method conditions were determined by assay of a photodegraded sample of leflunomide. Optimum chromatographic performance was obtained with a C18 column and acetonitrilewater as mobile phase. Comparison of spectra recorded with a diode-array detector during elution of the leflunomide peak enabled determination of method specificity. The method is highly sensitive (detection limit 10 ng mL)1) and robust to deliberate variation of the conditions (RSD of peak area < 2.0%). Precision and accuracy were adequate over the concentration range 10 to 100 lg mL)1. These results show the proposed method is suitable for its intended use.
Leflunomide (Fig. 1) is a prodrug used for treatment of active rheumatoid arthritis. It is an isoxazole derivative marketed as 10, 20, and 100-mg coated tablets [8, 9]. This paper reports the development and validation of an HPLC method for determination of leflunomide in tablets which can be used for quality control. In addition to the typical performance characteristics used for validation of an analytical method, this paper also reports results from prevalidation and assessment of the peak-purity profile by diode-array detection.
Keywords Experimental
Column liquid chromatography Diode-array detection Leflunomide
Introduction The quality of pharmaceutical products is of foremost concern in health care. In this context, validation of analytical methods is highly important in development and routine analysis. Assays used for drug quantification must generate accurate and reliable analytical results, to ensure the quality of pharmaceutical products. The tests performed during validation can provide analytical data enabling estimation of the variability and sensitivity of the proposed assays and are commercially essential, because many
Short Communication DOI: 10.1365/s10337-006-0739-4 0009-5893/06/03
Chemicals
decisions, for example batch release, involve analytical measurements [1–3]. HPLC is widely used for analysis of pharmaceutical products, mostly because of its capacity to analyze mixtures. Diode-array detection increases the power of HPLC and is an elegant option for assessing method specificity by comparison of spectra recorded during peak detection [4, 5]. In addition, forced degradation of samples must be considered during development and optimization of chromatographic conditions, particularly when degraded products are unknown or not available [6, 7].
Leflunomide reference substance was kindly supplied by Aventis Pharma (Sa˜o Paulo, Brazil); it was stated to contain 100.2% leflunomide. Tablets labeled to contain 20 mg leflunomide were purchased commercially. Acetonitrile for chromatography was purchased from Merck (Darmstadt, Germany). Deionized water was obtained from a Millipore Milli-Q filtration/purification system.
Instrumentation and Analytical Conditions HPLC analysis was performed with a Shimadzu HPLC system, equipped with
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Robustness
H3C O O F
F N NH
F
Fig. 1. Chemical structure of leflunomide
an SCL-10Avp system controller, an LC-10ADvp pump, an SPD-M10Avp photodiode-array detector, a CTO10ACvp column oven (set at 25 C), and a SIL-10ADvp auto injector. Class VP 6.12 SP2 manager system software was used to control the equipment and to calculate data and responses from the HPLC system. Reversed-phase chromatography was performed on a Metachem Metasil ODS column (250 mm · 4.6 mm i.d., 5 lm particle size). The injection volume was 20 lL and isocratic elution at a flow-rate of 1.0 mL min)1 was achieved by use of acetonitrile–water, 1:1 (v/v), as mobile phase; the run time was 12 min. Leflunomide was determined by UV detection at 254 nm.
Standard Solutions and Linearity Standard solutions (1 mg mL)1) of leflunomide were prepared by dissolving 20 mg standard in 20 mL acetonitrile. This solution was diluted with acetonitrile–water, 6:4 (v/v) to furnish five concentrations (range 10 to 100 lg mL)1) for assessment of linearity.
Specificity Specificity was evaluated by testing the effect of placebo on the determination of leflunomide. The mixture of tablet excipients was treated similarly to homogenized tablet powder to prepare placebo solution. A sample of leflunomide exposed to light for three days in an intensified-light cabinet equipped with a 30-W Ecolume ZW lamp (254 nm) was assayed to evaluate method specificity. To test peak purity, spectra were recorded at a frequency of 1.28 s)1. Leflunomide chromatographic peaks were also corrected for background, and the wavelength range 210 to 280 nm was used to calculate a peak-purity index.
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Robustness was assessed by testing the susceptibility of measurements to deliberate variation of the analytical conditions. Thus, different columns were used and pH, mobile phase composition, and flow rate were modified. In this test three injections of photodegraded leflunomide were performed for each variation of method conditions.
Detection Limit Detection limit was determined by reducing the concentration of a standard solution until the leflunomide peak response was approximately three times greater than the noise.
Accuracy Accuracy was tested by assaying five different solutions, two replicates each, containing 10.0, 25.0, 50.0, 75.0, and 100.0 lg mL)1 standard spiked with placebo solution.
Preparation of Sample Solutions For precision testing, accurately weighed amounts of homogenized powdered tablets equivalent to 10 mg leflunomide were placed in six 20-mL volumetric flasks. Acetonitrile–water, 6:4 (v/v), was added and the mixtures was stirred rapidly for at least 20 min to ensure drug extraction was complete. Samples were then filtered and diluted to obtain a final leflunomide solution of approximately 50 lg mL)1.
Results and Discussion Prevalidation Study To determine the HPLC conditions for validation, studies were performed with photodegraded leflunomide solution and properties suitable for measurement of system suitability were considered [10, 11]. This study began with acetonitrile–water, 1:1 (v/v), as mobile phase, and a flow rate of 1.0 mL min)1. Different columns were tested and capacity factor, resolution, and number of theoretical plates for the
leflunomide peak were found to be greater for a C18 column than for a C8 column (Fig. 2). Peak symmetry was satisfactory for both columns. The greater resolution and excellent number of theoretical plates resulted in choice of the C18 column. The mobile phase acetonitrile-to-water ratio had a substantial effect on, mostly, capacity factor and resolution. Despite longer retention times, acetonitrile–water, 1:1 (v/v), was preferred because capacity factor and resolution decreased if the amount of acetonitrile in the mobile phase was increased. The flow rate had no significant effect on capacity factor, resolution, and theoretical plate number. At a flow rate of 1.0 mL min)1 the retention time was approximately 9.0 min for the leflunomide peak; this was regarded as satisfactory for routine analysis with good potential for stability studies.
Method Validation Specificity was assessed by analyzing chromatograms obtained from the placebo and determining leflunomide peak purity for samples obtained after forced degradation. The placebo solution afforded two peaks with low retention times with no potential to interfere with the leflunomide peak. Analysis of the photodegraded sample of leflunomide showed the peak-purity index was close to 100% (peak-purity index of 1.00), confirming the absence of other substances coeluting with leflunomide. The similarity curve and threshold curve functions improved the sensitivity and reliability of peakpurity evaluation by using all the spectra acquired during elution of a peak [4]. The similarity curve did not intersect the threshold curve, indicating that no impurity was coeluting with leflunomide (Fig. 3). Robustness was assessed by analysis of the results obtained after deliberate variation of the method conditions. There was little variation of responses that define system suitability test limits (resolution, tailing factor, capacity factor, and theoretical plates) and no significant interference with leflunomide measurement. Peak-area variations were calculated and low RSD values were indicative of the robustness of the method (Table 1). These results are indicative of the reliability of the method and its potential for transfer to other laboratories and instruments.
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Fig. 2. HPLC chromatograms obtained from photodegraded leflunomide: (a) C18 column; (b) C8 column
Fig. 3. (a) Diode-array peak purity profile. (b) Chromatogram of leflunomide exposed to light stress for 3 days
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Table 1. Results from peak-area determination in robustness test Condition
Value )1
Flow rate (mL min )
0.95 1.05
Mobile phase composition (% acetonitrile)
48 52
pH of water used in mobile phase
Peak area
Mean (n = 3)
RSD (%)
1218237 1172440
1194213
1.9
1171254
1194146
2.0
1161059 1200414
1184479
1.7
1173402 1174325
1179897
0.9
1219221
5.0 7.3 Hypersila LiChrospherb
C18 column
y ¼ 76203x þ 32026ðr ¼ 0:9999Þ
Method conditionsc
1191963
a
Keystone – BDS Hypersil C18 (250 mm · 4.6 mm, 5 lm particles) Merck – LiChrospher 100 RP-18 (250 mm · 4.0 mm, 5 lm particles) c Flow rate = 1.0 mL min)1; 50% acetonitrile; pH 6.2; column Metachem C18 b
60000
Residuals
20000 0
0
25
50
75
100
–20000 –40000 –60000
Concentration (µg mL–1) Fig. 4. Residual plot. Dotted lines represent ±3 · RSE
100
% found
99,5 99 98,5 98,3
98
98,4
97,9
97,5 Mean and 95% confidence Interval
97 0
1
2
3
4
5
Time (Day) Fig. 5. Results from determination of precision
Linearity was established by preparing solutions of leflunomide at five different concentrations ranging from 10 to 100 lg mL)1, each with three replicates. The
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where y is the peak area of leflunomide detected at 254 nm and x is the concentration of leflunomide (lg mL)1). The standard error of the slope and intercept were 154 and 8887, respectively. There was no obvious pattern in the plot of residuals and no outlying points were observed (Fig. 4) [12]. The regression standard error (RSE) is a good indicator of performance and the relative standard error of the slope is comparable with the relative standard deviation obtained in precision studies [3]. The calculated regression precision (Eq. 2) was satisfactory (Vx0 = 0.48%). Vx0 ¼
40000
concentrations corresponded to 20–200% of method concentration (50 lg mL)1). The equation obtained for leflunomide from regression analysis was:
ð1Þ
RSE 100% b x
ð2Þ
where RSE is the regression standard error, b is the slope, and x is the mean leflunomide concentration. The intercept was expressed as a percentage of the analytical signal at 100% method concentration. The value of 0.84% obtained was regarded as adequate and revealed the low relevance of the intercept. The experimental detection limit of 10 ng mL)1 shows the method is quite sensitive for leflunomide detection. This sensitivity can be associated with the high molar absorption of leflunomide at 254 nm and the large theoretical plate number for the leflunomide peak (N > 13 000). Method accuracy was determined by investigating the recovery of leflunomide at five levels, each with two replicates, ranging from 20 to 200% of method concentration from solution-spiked placebo. Results indicated recoveries were excellent, ranging from 99.38 to 102.41% (mean 100.52%). Precision was adequate (RSD = 0.89%). The repeatability of injection was evaluated on every analysis day by performing six replicate injections of standard solution at the method concentration. The RSD was always below 0.62%. Precision was determined for tablet sample solutions by performing six replicate analyses at the method concentration on three different days. On the third day precision was assessed using another HPLC system (with UV detection). Figure 5 shows the low variability of results obtained on the same day; RSD = 0.87% for the third day. The amounts of
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leflunomide found on the three different days were equivalent (ANOVA probability value = 0.448) with satisfactory intermediate precision (RSD = 0.72%).
Conclusion This HPLC method has good sensitivity and accuracy for quantification of leflunomide and separation from its photodegraded products. Sample degradation under stress conditions was essential for assessing method specificity and led to process optimization. Diode-array detection provides much more information about the samples than conventional UV detection and enabled evaluation of leflunomide peak purity. Validation showed the precision and robustness of
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the method were satisfactory. RSD < 1.0% was obtained from precision tests; this variability is in accordance with that expected for HPLC quantification of tablet dosage forms. Finally, the method can be regarded as validated and adequate for quantification of leflunomide in tablets.
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