Development of a Validated RP-HPLC Method for Estimation of Ethionamide in Spiked Human Plasma with UV Detection
Priya Rahade, Sandeep Sonawane*, Atharva Bhalerao, Sanjay Kshirsagar
Department of Pharmaceutical Analysis, MET’s Institute of Pharmacy, Bhujbal Knowledge City, Adgaon, Nashik – 422 003
*Corresponding Author E-mail: sandeeps.iop@gmail.com
A simple, rapid and economic RP-HPLC method was developed and validated for estimation of Ethionamide in spiked human plasma. The drug along with Guaifenesin (used as internal standard) was extracted from plasma by liquid-liquid extraction (LLE) procedure using ethyl acetate as organic solvent. The drug was well resolved from the plasma interference and internal standard in a reversed phase mode on C 18 (250 × 4.6 mm, 5 μ) column with methanol: water (40: 60 %, v/v) as mobile phase, at a flow rate of 1 mL/min. The detection was performed at 275 nm. The developed method was validated as per the US-FDA guidelines, where the weighted linear regression was used in calibration experiments to obtain the homoscedasticity. In accuracy and precision studies, intra-day and inter-day, % relative error was found between ± 15 and % RSD was less than 15 %. Stability experiments indicated that the drug remained stable after three freeze-thaw cycles.
KEYWORDS: Ethionamide, Bioanalytical, Liquid-liquid extraction, Weighted regression, RP-HPLC.
INTRODUCTION:
Ethionamide, chemically, 2-Ethyl-4-pyridine carbothioamide (Figure 1) [1], used in treatment of tuberculosis. . It is used as a part of treatment regimens to treat MDR and XDR TB [2].
Figure 1: Structure of Ethionamide
Literature survey revealed several methods for estimation of Ethionamide in Pharmaceutical formulations and in biological fluids using spectrophotometric and chromatographic techniques [3-10]. The objective of the present work was to develop a simple, rapid and economic RP-HPLC-UV based method with the use of liquid-liquid extraction for estimation of ethionamide in human plasma.
MATERIALS AND METHODS:
Chemicals and Reagents
Pharmaceutical grade Ethionamide was supplied as a gift sample from Macleods Pharmaceuticals Limited, Gujarat, India. Blank human plasma was provided as a gift sample from Dr. Vasantrao Pawar Medical College, Hospital and Research Centre, Nashik, India. Blank plasma was pooled by thoroughly mixing the plasma obtained from six different sources. methanol and acetonitrile used in analysis were of HPLC grade and all other chemicals and reagents were of analytical grade purchased from SD Fine Chemicals, Mumbai, India. Double distilled water used was freshly prepared by Double Distillation Assembly (Borosil, Mumbai, India) and further used in analysis after filtering through 0.45 µ membrane filter papers purchased from Milipore (India) Pvt. Ltd., Bengaluru, India.
Instrumentation
HPLC instrumentation consisting of pump PU-2080 plus (JASCO, Tokyo, Japan), with Rheodyne manual loop injector 7725i (injection loop capacity 100 µl) was used. Detection was carried out using UV-2075 detector (JASCO, Tokyo, Japan). Data acquisition was done by Borwin chromatography software version 1.5 (JASCO, Tokyo, Japan). All calculations were performed using Microsoft Excel 2010.
Chromatographic conditions
All chromatographic separations were carried out on Phenomenex Hyperclone C18 column (250 ´ 4.6 mm, 5 µ), using mobile phase comprising of methanol: water, (40:60 %,v/v). The flow rate was kept constant at 1 mL/min and eluent was detected at 275 nm.
Preparation of Standard Stock Solution
Quantity equivalent to 10 mg of Ethionamide was weighed and dissolved in methanol to make 10 mL. This gave 1000 µg/mL standard stock solution for Ethionamide. Quantity equivalent to 10 mg of the Guaifenesin (IS)was weighed and dissolved in methanol to make 10 mL. This gave 1000 µg/mL standard stock solution for Guaifenesin.
Preparation of Working Standard Solutions
The standard stock solution of Ethionamide was appropriately diluted with methanol to get six working standard solutions with concentrations of 1, 2, 4, 8, 16 and 32 µg/mL, respectively. The standard stock solution of Guaifenesin was diluted with methanol working standard solution of 15 µg/mL.
Liquid-liquid Extraction Experiments
Aliquot of 1 mL of blank human plasma was taken in a 20 mL stoppered glass tube and spiked with 125 µL of 100 µg/mL methanolic solution of Ethionamide and 125 µL of 15 µg/mL methanolic solution Guaifenesin (IS). This mixture was vortex-mixed for 5 min. Further, a 5 mL aliquot of LLE solvent was added to each tube and the tubes were vortex mixed for 5 min, the tubes were then centrifuged at 3000 rpm for 10 min at 4 0C in cooling centrifuge to effect phase separation. From this, 2 mL of organic layer was transferred to another tube and the solvent was evaporated to dryness. The dry residue of blank and spiked human plasma extracts was reconstituted with 500 µL of mobile phase and 100 µL were subjected to chromatographic analyses.
Preparation of calibration curve (CC) standards and Quality Control (QC) samples
Aliquots of 1 mL pooled plasma were taken in six different glass tubes of capacity 20 mL. To these tubes, 125 µL of working standard solution of ethinoamide was added and vortex mixed for 5 min to get CC standards with concentrations of 100, 200, 400, 800, 1600 and 3200 ng/mL. The QC samples were prepared similarly to get 500 ng/mL of LQC (low Quality Control), 1500 ng/mL MQC (Middle Quality Control) and 3000 ng/mL HQC (High Quality Control) samples.
Calibration runs
All CC standards were analyzed in six replicates using optimized LLE procedure and chromatographic conditions. Prior to analysis, each CC standard was mixed with 125 µL of working standard solution of 15 µg/mL of Guaifenesin. At the end of the calibration runs, the chromatograms of CC standards were processed to get the peak areas for Ethionamide and Guaifenesin. For each CC standard the area ratio of Ethionamide and Guaifenesin was calculated.
Selection of calibration model and range
The data obtained from the calibration run experiments was subjected to unweighted and weighted least square regression analysis to generate the respective calibration equations [11]. In weighted regression, weighing factors (w) of 1/x and 1/x2 were used, where x is the concentration of the CC standards of Ethionamide. In order to select the best calibration model, each calibration model and equation was evaluated with respect to % Relative error (% RE), Residual plot and homogeneity of variance (homoscedasticity) in the linear range. The calibration model with, minimum % RE, random scatter of points in the plot of residuals and no significant difference in one- way ANOVA was selected.
Validation Studies
The developed method was validated as per US-FDA Guidance for Industry: Bioanalytical Method Validation (September 2013) [12]. Selectivity was studied at the lower limit of quantification (LLOQ) at 100 ng/mL by comparing blank responses of plasma from six different sources with peak areas afforded by the LLOQ samples. The Calibration curve standards were evaluated by preparing and analyzing CC standard solutions spiked with internal standard for five days. The concentrations of each CC standard were back calculated using suggested calibration model and the deviation of the back-calculated concentrations from the nominal values was studied and expressed as % nominal.
Precision and accuracy were studied by analyzing five bioanalytical batches over five days. Each batch consisted of one blank, all CC standards and five replicates of LQC, MQC and HQC samples. The calibration equation was determined for each batch from the analysis of CC standards and was used to calculate the concentration of Ethionamide in LQC, MQC and HQC samples. The within batch and between batch accuracy and precision was determined in terms of % RE and % RSD, respectively.
Stability of Ethionamide in plasma was evaluated under various conditions viz. freeze-thaw cycles, stability at – 20 °C for 30 days and stability at room temperature for 6 h. The amount of Ethionamide in the stability samples was found out and the % nominal and % RSD of the determinations were calculated.
RESULTS AND DISCUSSION:
When Ethionamide and Guaifenesin (IS) were subjected to chromatographic analysis in mobile phases of different strengths and compositions, it was found that mobile phase comprising of methanol: water (40:60 %, v/v) gave adequate retention and resolution at flow rate of 1 mL/min. The detection was carried out at 275 nm. The retention for Ethionamide and Guaifenesin obtained at 5.467 min and 7.642 min, respectively. When liquid-liquid extraction experiments were performed using different solvents like ethyl acetate, n-hexane, dichloromethane and diethyl ether, it was found that both Ethionamide and Guaifenesin were comparably extracted with ethyl acetate. Also, when aliquots of blank plasma were extracted with ethyl acetate and chromatographed, it was found that there were no significant interfering peaks at the retention times of Ethionamide and Guaifenesin. Thus, concluded that ethyl acetate can be used as organic solvent in liquid-liquid extraction of Ethionamide and Guaifenesin. The chromatogram of blank plasma extracted in ethyl acetate and representative chromatogram of Ethionamide and Guaifenesin extracted in ethyl acetate is shown in Figure 2 and Figure 3, respectively. The extraction recovery for Ethionamide and Guaifenesin was found 52.76 % and 84.74 %, respectively.
During calibration experiments, when data obtained from Table 1 was subjected to unweighted and weighted linear regression with a weighing factor (w) of 1/x and 1/x2, unweighted regression resulted in the equation, Y = 0.0080X + 0.0004 and with 1/x and 1/x2 weights, resulted in equation, Y = 0.0125X + 0.0004 and Y = 0.0085X + 0.0004, respectively. For selection of calibration model, each of these obtained linear regression equations were evaluated for the % RE, random scatter and homoscedasticity. The analysis of calibration model is depicted in Table 2.
Figure- 2 Chromatogram of blank plasma extract
Figure- 3 Chromatogram of Ethionamide and Guaifensin (IS) extracted in ethyl acetate
Table 1: Area ratios from calibration experiments
|
CC Std No. |
Amount of Drug in ng/mL |
Mean area ration ± SD (n = 6) |
|
1 |
100 |
0.054 ± 0.005 |
|
2 |
200 |
0.110 ± 0.006 |
|
3 |
400 |
0.222 ± 0.022 |
|
4 |
800 |
0.381 ± 0.043 |
|
5 |
1600 |
0.714 ± 0.045 |
|
6 |
3200 |
1.543 ± 0.040 |
When the lower limit of quantification (LLOQ) samples were tested against the blank responses of plasma samples, it was found that peak areas were five times more than blank responses, which concluded that the method was selective for LLOQ at 100 ng/mL.
Table 2: Analysis of various calibration models
|
Unweighted regression |
Weighted regression (1/X) |
Weighted regression (1/X2) |
||||||
|
∑% RE |
Nature of residuals plot |
F5,5* value |
∑% RE |
Nature of residuals plot |
F5,5** value |
∑% RE |
Nature of residuals plot |
F5,5** value |
|
79.91791118 |
Random scatter |
64.7771073 |
3.00E-04 |
Random scatter |
0.06325981 |
3.69E-13 |
Random scatter |
6.1776E-05 |
Table 3: Standard Curve Parameters for Ethionamide
|
CC std No. |
Nominal Conc. (ng/mL) |
Mean back calculated Conc. (ng/mL) n=6 |
± SD |
% RSD |
% Accuracy |
|
1 |
100 |
110.6 |
10.6 |
9.6 |
110.6 |
|
2 |
200 |
195.6 |
6.4 |
3.3 |
97.8 |
|
3 |
400 |
388.0 |
11.5 |
3 |
97 |
|
4 |
800 |
801.5 |
11.73 |
1.5 |
100.2 |
|
5 |
1600 |
1606.0 |
6 |
0.4 |
100.4 |
|
6 |
3200 |
3197.0 |
2.7 |
0.1 |
106.6 |
From Table 3, it was concluded that the % nominal values of the back calculated concentrations of CC standards were between 95-106 %, which was in acceptable limit.
The results of assay precision and accuracy as well as extraction recovery for Ethionamide at LQC, MQC and HQC and for Guaifenesin (IS) are presented in Table 4. From this, it was observed that the intra-day %RE was between ± 15%, while the % RSD was less than 15, which was in the acceptable limit as per US-FDA guidelines. Further, the intermediate precision of the method was determined using one-way ANOVA. From Table 5, it was observed that the Fcalculated < Ftabulated at α = 0.05, indicates there is no significant difference between intra- day and inter- day precision.
Table 4: Results of accuracy and precision studies
|
|
|
Intraday n=5 |
Interday n=5 |
|
||||
|
QC Level |
Conc in ng/mL |
Mean conc found in ng/mL |
RE % |
RSD % |
Mean conc found in ng/mL |
RE% |
RSD % |
% Recovery |
|
LQC |
500 |
512.02 |
-12.02 |
1.08 |
473.72 |
26.28 |
2.81 |
54.15 |
|
MQC |
1500 |
1479.97 |
20.03 |
3.95 |
1494.84 |
5.16 |
1.85 |
51.98 |
|
HQC |
3000 |
3008.01 |
-8.01 |
1.02 |
3002.46 |
-2.46 |
1.61 |
52.95 |
|
IS |
|
84.74 |
||||||
Table 5 Result of one-way ANOVA at each QC level
|
Quality Control |
Source |
Sum of squares |
Df |
Mean squares |
Total Variance |
± SD |
F value |
|
LQC
|
Within run |
4135.31 |
20 |
206.77 |
1033.83 |
214.63 |
30.46 |
|
Between run |
25189.12 |
4 |
6297.28 |
||||
|
MQC
|
Within run |
20592.44 |
20 |
1029.62 |
5148.11 |
1370.61 |
0.85 |
|
Between run |
3497.47 |
4 |
874.37 |
||||
|
HQC
|
Within run |
53714.31 |
20 |
2685.72 |
13428.58 |
2540.49 |
0.34 |
|
Between run |
3591.79 |
4 |
897.95 |
The results of stability evaluation of Ethionamide are presented in Table 6. Analysis cycles viz. three freeze-thaw cycles, stability at -20 °C for 30 days and stability at room temperature for 6 h indicated that Ethionamide was stable in human plasma under these conditions.
Table 6: Results of stability studies
|
QC Level |
Stability at RT (6 h) |
Stability at -20°C |
Freeze thaw stability |
|||
|
|
%Nominal |
% RSD |
%Nominal |
% RSD |
%Nominal |
% RSD |
|
LQC |
102.97 |
4.60 |
103.31 |
3.74 |
103.24 |
5.52 |
|
HQC |
100.57 |
0.24 |
100.62 |
0.27 |
100.29 |
0.33 |
ACKNOWLEDGEMENTS
Authors are thankful to the management and trustees of Mumbai Educational Trust’s Bhujbal Knowledge City, Nashik, for providing necessary chemicals and analytical facilities; Macleods Pharmaceuticals, Gujarat for providing `Ethionamide as ex-gratis. Authors are also thankful to Dr. Vasantrao Pawar Medical College, Hospital and Research Centre, Nashik for providing human plasma samples.
REFERENCES
1. Indian Pharmacopoeia. Vol. I. 1996, Gaziabad Indian Pharmacopoeial Comission. 302-303.
Received on 13.07.2016 Accepted on 30.10.2016
© Asian Pharma Press All Right Reserved
Asian J. Res. Pharm. Sci. 2016; 6(4): 230-234.
DOI: 10.5958/2231-5659.2016.00032.1