Article Type: Research Article Article Citation: Romana Mahivish, Manjunath SY, and Hemant Kumar T. (2021). RP-HPLC
METHOD FOR THE ESTIMATION OF ZILEUTON IN TABLET FORMULATION. International
Journal of Research -GRANTHAALAYAH, 9(1), 141-149. https://doi.org/10.29121/granthaalayah.v9.i1.2021.2276 Received Date: 01 November 2020 Accepted Date: 31 December 2021 Keywords: Zileuton RP-HPLC Tablet Dosage
Form A simple, rapid, accurate and precise RP-HPLC method was developed and validated for the determination of zileuton in table dosage form. Chromatographic analysis of the drug was achieved on Cyberlab HPLC comprising of LC- 100P pump, a variable wavelength programmable LC-UV100 UV detector and SCL system controller. Flowrosil C18 column (250 mm x 4.6 mm, 5 μ) as stationary phase with mobile phase consisting of Methanol: Acetonitrile: 1% GAA in the ratio of 70:10:20 v/v. The method showed a good linear response in the concentration range of 5-30 μg/ml with correlation coefficient of 0.9993. The flow rate was maintained at 1.0 ml/min and detection was carried out at 230 nm. The retention time was 3.12 min. The method was statistically validated for accuracy, precision, linearity, ruggedness, robustness, solution stability, selectivity and sensitivity. The results obtained in the study were within the limits of ICH guidelines and hence this method can be used for the determination of zileuton in tablet formulation.
1. INTRODUCTIONZileuton
[R, S (±) N-(1-(benzo [b]-thien-2-yl) ethyl)-N-hydroxyurea] (Fig. 1) is a
racemic mixture having approximately equal therapeutic activities which
selectively and reversibly inhibits 5-lypoxygenase potentiating leukotrienes
(LT’s – LTA4, LTB4, LTC4, LTD4 and
LTE4); mostly indicated in inflammatory diseases akin to psoriasis,
rheumatoid arthritis, asthma, multi-ple sclerosis,
uveitis and inflammatory bowel syndrome [1]. Zileuton is a very slightly
soluble compound without any ionizable functional group. Zileuton is used for
the prophylaxis and chronic treatment of asthma in adults and children 12 years
of age and older. Contraindications are active liver disease or resistant
elevation in transaminase at least 3 times the history of allergic reactions to
zileuton or any of its inactive ingredients. Zileuton is a minor substrate of
CYP1A2, 2C8/9, 3A4, and a weak inhibitor of CYP 1A2[2]. The drug has been shown to increase the
serum concentration or effects of theophylline, propranolol and warfarin,
although significant increase in prothrombin time is not obvious. It is advised
that the doses of each medication be monitored and/or reduced accordingly.
Literature survey reveals analytical methods reported for estimation of
zileuton in API, pharmaceutical dosage form and biological fluid includes
spectrophotometry [3],[4],[5],[6],[7],[8],[9],[10], RP-HPLC [11],[12],[13] and LC-MS [14],[15]. Obviously, HPLC methods [16],[17] are superior in compared to
spectrophotometric methods in terms of accuracy and sensitivity. The reported
HPLC methods available for quantification of zileuton have a drawbacks of long
retention times and not completely validated. As long retention time need a
more consumption of mobile phase. So, there is a need of a HPLC method, where
the retention time is less. The
purpose of this study was to develop a simple, rapid, precise, and accurate
RP-HPLC method for the estimation of zileuton in tablet dosage form. Figure 1: Structure
of Zileuton 2.
MATERIALS
AND METHODS:
Instrumention Chromatographic
separation was performed on a Cyberlab HPLC system
equipped with a Flowrosil C18 column (250 × 4.6 mm,
with 5 μm particle), single pumps, variable wave
length detector and Rheodyne injector with 20 μl loop volume. ‘LC solution’ software was used to
collect and process the data. Ultra sonicator
(Citizen ultra sonicator) was used for sonicating the drug and
sample solution.
Digital weighing balance (SHIMADZU AUX 220) used for weighing. ELICO SL 244
double beam UV-VIS spectrophotometer. Materials Zileuton
raw material was obtained from Yarrow Chem. products, Mumbai, India. Tablet
formulation GRILUTO-CR (Cadila Healthcare Limited,
Goa, India) containing Zileuton 600 mg was purchased from local pharmacy. All
reagents and solvents used were analytical grade. Double distilled water was
obtained from a millipore purification unit. HPLC
grade Methanol was obtained from Merck life science Pvt Ltd, HPLC grade
Acetonitrile was obtained from Merck life science Pvt Ltd. Preparation of Mobile phase Mobile phase
was prepared by mixing 700 mL of HPLC grade methanol, 100 ml of HPLC grade
acetonitrile and 200 mL of 1% Glacial acetic acid (prepared by mixing 2 ml of
glacial acetic acid in 200 ml of of HPLC grade water).
The mobile phase was sonicated for 10 min and filtered through the 0.45 μm membrane filter. Preparation of standard stock solutions The
standard stock solutions of 100 μg/mL of the
drug were prepared by dissolving 50 mg of pure drug in the mobile phase in a 50
mL volumetric flask and the volume was made up to the mark with mobile phase.
Resulting solutions were further diluted with mobile phase to obtain a final
concentration of 100 μg/mL and stored under
refrigeration. Aliquots of standard stock solutions were put in a 10 mL
volumetric flask and diluted up to the mark with mobile phase. In such a way,
the final concentrations of the drug were in the range of 5-30 μg/mL. Preparation of sample solution To
determine the content of zileuton in tablet dosage form (Label claim: 600
mg/tablet) ten tablets were accurately weighed and triturated to fine powder.
The powder weight equivalent to 100 mg of zileuton was taken and dissolved in
100 ml of mobile phase. The solution was sonicated for few minutes and filtered
through 0.22 μ membrane filter. Form the resulting solution (1 ml) was
transferred to a 10 ml volumetric flask and diluted up to the mark with mobile
phase. From this 2 ml was transferred to a 10 ml volumetric flask and diluted
up to the mark with mobile phase. A 20 µL of the filtrate was injected into
chromatographic system. The peak area of the zileuton was determined and
concentration was found using linear regression equation obtained from calibration
curve. Chromatographic conditions The chromatographic system used for method
development and validation includes the LC-P100 pump, variable wavelength
programmable LC-UV100 UV detector and SCL20A system controller at CYBERLAB
HPLC. A Rheodyne injector 7725i equipped with a 20 μL loop was used and the data was recorded and
evaluated using LC solution software version 5.0. Separation was performed at Flowrosil C18 (250 × 4.6 mm i.d., 5µm) at the ambient
temperature. A mixture of Methanol: Acetonitrile: 1% GAA in the ratio of 70:10:20 v/v
was found to be the ideal mobile phase for the ideal chromatographic
analysis of zileuton. The solvent mixture was filtered through a 0.22 μ membrane filter and sonicated before use. It is pumped through the column
at a flow rate of 1.0 mL / min. The injection volume is maintained in the
column at 20 µL and room temperature. The column was balanced by pumping the
mobile phase through the column for at least 20 min before injecting the drug
solution. The detection was monitored at 230 nm. Run time is set to 10 minutes.
Optimized chromatographic conditions are shown in Table 1. Figure 2: Chromatogram
of standard solution of zileuton Table 1: Optimized chromatographic
conditions
Method Validation The
developed method was validated as per ICH guidelines26 by evaluating
linearity, accuracy, precision, robustness, ruggedness, detection limit,
quantification limit and stability. Coefficients of variation and relative
errors of less than 2 % were considered acceptable. System Suitability Test Before
performing validation experiments, system suitability test (SST) has to be
applied to indicate that HPLC system and method are capable of providing data
with admissible quality. SST was performed by investigating capacity factor,
tailing factor, theoretical plates number, and also relative standard deviation
(RSD) of the peak areas. Stability Stability was assessed by analyzing QC standard solutions after keeping
them at room temperature for 48 h. Obtained results were investigated as
recovery values and compared to the freshly prepared solutions. Linearity A stock
solution of zileuton of 1000 μg/mL was prepared
with mobile phase. From it, various working standard solutions were prepared in
the range of 5 to 50 μg/ml and injected into
HPLC. It was shown that the selected drug had linearity in the range of 5–30 μg/mL. The calibration plot (peak area of zileuton versus
zileuton concentration) was generated by replicate analysis (n=6) at all
concentration levels and the linear relationship was evaluated using the least
square method within Microsoft Excel® program. Accuracy The
accuracy of the method was carried out using one set of different standard
addition methods at different concentration levels, 50 %, 100 % and 150 %, and
then comparing the difference between the spiked value (theoretical value) and
actual found value. Precision The
precision of the method was ascertained from the peak area obtained by actual
determination of six replicates of a fixed amount of the drug (20 μg/mL). The precision of the assay was also determined
in terms of intra- and inter-day variation in the peak areas of a set of drug
solutions on three different days. The intra- and inter-day variation in the
peak area of the drug solution was calculated in terms of relative standard
deviation (RSD). Robustness Robustness
of the proposed method for zileutone was carried out
by the slight variation in flow rate, analytical wavelength and mobile phase
ratio. The percentage recovery and RSD were noted for zileutone. Ruggedness The test
solutions were prepared as per test method and injected under variable conditions.
Ruggedness of the method was studied by different analysts. Detection limit and quantification limit The limit
of detection (LOD) and limit of quantification (LOQ) were established based on
the calibration curve parameters, according to the following formulas: LOD=3.3SD/slope LOQ=10SD/slope or
detection limit=3.3σ/s, quantification limit=10σ/s, where σ is
the standard deviation of y-intercept of regression line, and so is the slope
of the calibration curve. Specificity The
specificity of the proposed method was determined against blank and placebo
applications. Here mobile phase was used as blank and excipients like starch,
lactose, magnesium stearate were used as placebo. 3.
RESULTS
AND DISCUSSION
Method validation System Suitability
Test After
setting the optimum conditions, system suitability parameters for the developed
method were determined and compared with recommended limits. To determine the
parameters, the study was performed with standard solution of 50 µg/ml
concentration and the results were acquired from six injections. System suitability parameters of the method were
demonstrated in Table 2. According to the
results, all of the system suitability parameters were within the recommended
limits and the method was found to be suitable for the analysis. Table 2: Results of system suitability
test (n = 6)
Stability The sample solution stability was
analyzed by injecting the same solution at 0, 12, 24, and 48 h. Identical
change was not observed in the developed method. Also, results were found
within acceptable limits (% RSD < 2), which are summarized in Table 3. Table 3: Stability data of zileuton (standard
solutions)
Linearity and sensitivity Linearity study was performed with calibration standards with 5,
10, 15, 20, 25 and 30 µg/ml concentrations. The standards were injected in triplicate.
Calibration curves were obtained by plotting the peak areas against the given
concentrations. The calibration curve was evaluated by the determination
coefficient. The determination coefficient (R2) of the
calibration curves was 0.9993. Therefore, the calibration curve for zileutone was found to be linear within the range of 5–30
µg/ml concentrations as shown in Fig.3. The regression equations were
calculated from the calibration graphs. The
sensitivity of the analytical method was evaluated by determining the limits of
detection (LOD) and quantitation (LOQ). The values of LOD and LOQ are given in
Table 4. The low values of LOD and LOQ indicates the sensitivity of method. Table 4: Spectral and statistical data for
determination of Zileuton by proposed RP-HPLC method.
aY = mx + c, where x is the
concentration (µg/ml). Figure
3: Calibration curve of zileuton Accuracy To study the reliability, the suitability, and the accuracy of the
method, recovery experiments were carried out. Known quantities of the pure
drug were added to the preanalyzed sample to make
samples at the levels of 50 %, 100 %, and 150 %, and were assayed by the
proposed method. Accuracy was calculated as the percentage of recovery. The recovery and relative
standard deviation for each of the analytes are given Table 5. From the
recovery studies it is evidence that the
method is highly accurate and can give excellent results. Table 5: Accuracy results
Precision The
precision was demonstrated at three levels: repeatability, intermediate
precision, and reproducibility (between laboratories’ precision). Each level of
precision was investigated by 3 sequential replicates of injections of three
concentrations of 10, 20 and 30 µg/mL. The precision
was expressed as relative standard deviation (RSD) or
coefficient of variation (CV). The results of three levels of precision are
shown in Table 6. The developed method
was found to be precise as the RSD values for repeatability, intermediate precision
and reproducibility studies were < 2 %, respectively as recommended by ICH
guidelines (ICH Q2 (R1), 2005). Table 6: Precision results
Robustness and ruggedness Robustness
of the method was studied by deliberate variations of the analytical parameters
such as flow rate (1.0±0.1 mL/min), mobile phase composition (± 5 % organic
phase) and analytical wavelength (±2 nm). The results are given in Tables 7.
The result shown that have the negligible effect on retention time, recoveries
and peak area of zileutone indicating the developed
method is robust. Ruggedness of the method was carried out by different
analysts. The results are displayed in Table 8. There is no variation in peak
areas and retention time of zileutone from studies
carried out by two analysts as indicated by % RSD < 2 gives the method
rugged. Table 7: Robustness studies
Table 8: Ruggedness studies
Mobile phase
stability The stability of the mobile phase was evaluated, so the mobile phase
was stored at 4–8 °C for 1 week. The aged mobile phase was compared using a
freshly prepared one. The mobile phase was stable up to 1 week at 4–8 °C. Specificity Specificity
is the ability to unequivocally assess the analyte in the presence of
components that may be expected to be present. Typically, these might include
impurities, degradants or matrix. Specificity of an analytical method is its
ability to accurately and specifically measure the analyte of interest without
interference from blank or placebo. The peak purity of Zileuton was assessed by
comparing the retention times of standard zileutone
and the sample, and good correlation was obtained between the retention time of
the standard and sample. Placebo and blank were injected and there were no
peaks. There is no interference of blank and placebo on drug peaks hence, the
method is specific. Sample
Analysis The
developed and validated method was applied for analysis of tablet formulation
contain zileutone. The
sample was analyzed in triplicate. Analysis results were evaluated using a
calibration curve. The amount of zileutone in the
samples was calculated from calibration curve equation and recovery and RSD
values were determined. The results of analysis are given in Table 9. The recoveries were in good agreement with the label
claims. The chromatogram obtained were clear as shown in Fig. 6. It was concluded that the method can be applied
successfully for the analysis of zileutone in tablet dosage form Figure 4: Chromatogram of zileuton sample solution Table 9: Assay results from commercial
formulation
* Average
of five determinations 4.
CONCLUSION
The
proposed method for the estimation of zileuton was validated as per the ICH
guidelines and it is simple, specific and reliable. Furthermore, this simple
and rapid RP-HPLC method can also be used successfully for the determination of
zileutone in pharmaceutical formulations without any
interference from the excipient. SOURCES OF FUNDING
This
research received no specific grant from any funding agency in the public,
commercial, or not-for-profit sectors. CONFLICT OF INTEREST
The
author have declared that no competing interests exist. ACKNOWLEDGMENT
Authors are thankful to the
Management of Srikrupa Institute of Pharmaceutical
Sciences for providing necessary laboratory facilities for carrying out this
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