Method Development and Validation of Cleaning Procedure For Fenbendazole Residual Determination in Manufacturing
Method Development and Validation of Cleaning Procedure For Fenbendazole Residual Determination in Manufacturing
Method Development and Validation of Cleaning Procedure For Fenbendazole Residual Determination in Manufacturing
SUNIL KUMAR YELAMANCHI V1, USENI REDDY MALLU2, KASI VISWANATH IV1*, MAHESHWARA REDDY L3
1
Department of Chemistry, K.L. University, Green Fields, Vaddeswaram, Guntur, Andhra Pradesh, India. 2Celltrionpharm, Inc. Seoul,
South Korea. 3Department of Chemistry, YSR Engineering College of Yogi, Vemana, University, Kadapa, Andhra Pradesh, India.
Email: viswanath_fed@kluniversity.in
Received: 24 February 2016, Revised and Accepted: 10 March 2016
ABSTRACT
Objectives: Cleaning validation is the methodology used to assure that a cleaning process removes residues of the active pharmaceutical ingredients
of the product manufactured in a piece of equipment. All residues are removed to predetermined levels to ensure the quality of the next product.
Today, manufactured is not compromised by waste from the previous product and the quality of future products using the equipment, to prevent cross
contamination, and as a good manufacturing practices requirement. The main aim of the study was to develop and validate a new simple, precise,
and accurate reverse phase high-performance liquid chromatographic (HPLC) method for fenbendazole (FBZF) residual determination in veterinary
active pharmaceutical ingredient manufacturing.
Methods: The method was developed using the isocratic solvent system, HPLC grade acetonitrile, and Mill-Q water in the ratio of 60:40 (volume/
volume), and 1% hydrochloric methanol is used as diluent. Successful elution of the FBZF was achieved on Waters Symmetry C-18 column with
250 mm×4.6 mm internal diameter and 5 μm particle size.
Results: The method validation was successfully applied for routine analysis for cleaning/residual samples. The developed reverse phase liquid
chromatography method was validated with respect to specificity, linearity, accuracy, precision, and high sensitivity with detection limits and
quantification limits ranging from 0.2 ppm to 15 ppm.
Conclusion: The present developed and validated method is run successfully for FBZF residual determination of cleaning samples in veterinary active
pharmaceutical ingredient manufacturing.
Keywords: Fenbendazole, Residual determination, Reversed phase high-pressure liquid chromatography and cleaning validation.
Toxicity
Despite being widely used as a dewormer in many species, toxicity
has been reported. Birds (storks, pink pelicans, vultures, pigeons, and
doves) and reptiles (vipers, turtles, and tortoises) have shown toxicity
associated with bone marrow suppression, intestinal crypt cell necrosis,
and distal villi sloughing [1]. Abortions in domestic ruminants have Fig. 1: Structure of fenbendazole
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System suitability and precision parameter From the Table 4, it is clear that the area response of FBZF versus
To verify that analytical system is working properly and can give concentration in ppm of FBZF is linear in the range of interest. The
accurate and precise results, the system suitability and precision correlation coefficient and regression coefficient calculated from the
parameters are to be set. System suitability and precision tests were regular plot are >0.999. Hence, the method is linear for the residual
carried out on freshly prepared 10 ppm standard solutions of FBZF, determination of FBZF.
and it was calculated by determining the SD of FBZF standards by
injecting standards in six replicates at 10 minutes interval. The LOD and LOQ
values of %RSD prove that the method is accurate and precise, and LOD is the lowest amount of analyte in a sample that can be detected, but
acceptance criteria are not more than 5 (5.0%) for absorbance not necessarily quantitated, under the stated experimental conditions [9].
response, not more than 1 (1.0%) for retention time. The values were
recorded in (Table 1). LOQ is the lowest amount of analyte in a sample that can be quantitated
with acceptable precision, under the stated experimental conditions.
FBZF 10 ppm standard preparation
Weigh about 50.13 mg of FBZF standard into a 50 mL volumetric flask
dissolve and diluted volume with diluent. Take 1 mL of above solution Table 2: Specificity parameters
into the 100 mL volumetric flask and make up to the mark with diluent.
Peak name RT
From the Table 1, it can be concluded that the system suitability and Blank No peaks
precision parameters meet the requirements of method validation. Blank with swab stick No peaks
10 ppm standard solution 4.753
Specificity parameter RT: Retention time
Specificity is the ability of the analytical method to assess the analyte in
the presence of components that may be expects to be present such as
impurities, degradation products, and matrix components. Table 3: Linearity different levels of concentrations
Specificity tests were carried out on above prepared 10 ppm standard Concentration Stock solution Volume
solution of FBZF, and it was determining by injecting blank, blank with in ppm to be added make up to
swab stick, and specify solution (standard solution) for FBZF material 0.2 0.02 100
at 10 ppm standard solution. 0.5 0.05 100
1.0 0.10 100
From the Table 2, Fig. 3 proves that method is specific that is there is no 3.0 0.30 100
interference of blank peaks in FBZF standard solution. 5.0 0.50 100
8.00 0.80 100
Linearity 10.0 1.00 100
The linearity of an analytical method is its ability to elicit test results 13.0 1.30 100
that are directly or by a well-defined mathematical transformation, 15.0 1.50 100
proportional to the concentration of analyte in the sample within a
given range [9].
Table 4: Linearity parameters
The developed method has been validated as per ICH guidelines; the
standard solutions of FBZF in the mass concentration range of 0.2 ppm Trial number Actual concentration (ppm) Area response
to 15 ppm were injected into the chromatographic system [9]. The 1 0.2 223073
chromatograms were developed, and the peak area was determined 2 0.5 343692
for each concentration of the drug solution. Calibration curve of 3 1.0 838731
FBZF was obtained by plotting the peak area ratio versus the applied 4 3.0 1775030
concentrations of FBZF. The linear correlation coefficient was found 5 5.0 2894229
to be 1.0 (0.999). The values and calibration curve were recorded in 6 8.0 4705246
Table 4 and Fig. 4. 7 10.0 5954186
8 13.0 7505160
Preparation of FBZF stock solution and linearity solutions 9 15.0 8575858
Weighed 100.12 mg of working standard into 100 mL volumetric flask Slope 567172223.3
dissolved and diluted up to the mark with diluent. Preparation of Correlation coefficient 0.999681792
Regression coefficient 0.999363686
different levels of concentrations.
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Fenbendazole
Theoretical LOD in mg/mL 0.6 ppm
Theoretical LOQ in mg/mL 2.0 ppm
LOD: Limit of detection, LOQ: Limit of quantification
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area glass plate, taking utmost care to avoid any spillage. Dry the Using 100 mL of accurately measured diluent recover the test sample
plate at room temperature. Using 100 mL of accurately measured from 10 cm×10 cm surface area glass plate, by gentle swirling. Filtered
diluent recover the test sample from 10 cm×10 cm surface area glass and inject into high-pressure liquid chromatography. Performed the
plate, by gentle swirling. Filtered and inject into high-pressure liquid exercise in triplicate. Finally recorded the area of the test sample
chromatography. Performed the exercise in triplicate. Finally recorded in swab recovery on stainless plate and glass plate in Table 10 than
the area of the test sample in the rinse recovery on stainless plate and calculate the % rinse recovery, % swab recovery below formula.
glass plate in Table 9.
From the Table 10 results, it can be concluded that % of rinse and %
Swab recovery of swab recovery on SS plate and the glass plate is consistently above
The swab recovery of the sampling method shall be established by 80.0 (80.0%). The values obtained above are in good agreement in
spiking a solution of known concentration on stainless steel surface. terms reliability, suitability, and accuracy of the proposed method.
Recover the spiked sample from the surface by swabbing the surface
using swab stick with the sampling agent [9]. Solution stability and mobile phase stability
To determine the stability of sample solution, the mobile phase 10 ppm
Preparation of swab spiking solution standard and cleaning sample solutions of FBZF were prepared and
Weighed about 100.67 mg of test sample taken into 100 mL volumetric analyzed immediately after preparation and after different time
flask dissolved and diluted with diluent. Further, 10 mL of this solution intervals up to 24 hrs, while maintaining the sample cooler temperature
diluted to 100 mL with diluent. Take 10 mL of the above solution into at about 30 (30°C). The results from these studies indicated the
100 mL volumetric flask. Dissolved and dilute up to the mark with standard and sample solution was stable at room temperature for at
diluent. Mixed well. least 24 hrs. The sampled chromatograms are recorded as below in
Figs. 6-11 [9].
Swab recovery study on stainless plate
Select three cleaned and dried 10 cm×10cm surface area glass plates. Record of analysis for FBZF cleaning samples
Spread 10 mL of the spiking solution on dried 10 cm×10 cm surface Triplicate FBZF cleaning samples are run successfully using this
glass plates, taking utmost care to avoid any spillage. Dry the plate at method, and the experimental results and chromatograms are recorded
room temperature. in Figs. 12-16 [9].
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RSD: Relative standard deviation, SD: Standard deviation, LOD: Limit of detection, LOQ: Limit of quantification, FBZF: Fenbendazole
From the above chromatograms or figures observes that there is the high purity of the drug, i.e., All residues are removed to predetermined
absence of FBZF content in triplicate bulk cleaning samples. Hence, levels to ensure the quality of the next product and also to prevent cross
proved this method is applicable for FBZF bulk cleaning samples. contamination and as a good manufacturing practices requirement.
Based on the above-observed results, the developed cleaning method
RESULTS validation for FBZF method is valid and run successfully the summary
and evaluation of results are in below Table 11.
As there is a growing demand of FBZF in veterinary active pharmaceutical
ingredient market, it is required to develop fast, cost effective, stable, CONCLUSION
precise, and sensitive analytical cleaning method. The primary target in
developing and validate this cleaning RP-HPLC method is to achieve the A validated reversed phase high-pressure liquid chromatography
optimum resolution between products with other products to supply cleaning method has been developed for cleaning method validation for
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