© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 35 https://doi.org/10.1093/chromsci/bmab029 Advance Access Publication Date: 15 March 2021 Article
Article
Design of Experiment (DoE)-Approach Based RP-HPLC Analytical Method Development and Validation for Estimation of Efavirenz in Bulk and Formulations
Paramita Saha, and Murali Monohar Pandey *
Industrial Research Laboratory, Department of Pharmacy, Birla Institute of Technology & Science, Pilani, (BITS Pilani), Pilani Campus, Jhunjhunu, Rajasthan 333031, India
*Author to whom correspondence should be addressed. Email: [email protected] Received 10 August 2020; Editorial Decision 15 February 2021
Abstract
Present study reports design of experiment (DoE) based development and validation of a simple, rapid and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method for estimation of efavirenz (EFZ), a non-nucleotide reverse transcriptase inhibitor (NNRTs), used in the treatment of acquired immunodeficiency syndrome (AIDS). Plackett–Burman design was explored to screen the critical method variables (CMVs) for the RP-HPLC method. A response surface Box–Behnken design was employed to optimize the screened CMVs which affect the analytical responses (ARs) of RP-HPLC method. Using the optimized CMVs the HPLC method was developed and validated according to International Conference on Harmonization (ICH) guidelines.
EFZ in marketed formulation was estimated using the validated method. Acetonitrile proportion, pH of the phosphate buffer and mobile phase flow rate were the CMVs and retention time and number of theoretical plates were the ARs for the study. The optimized chromatographic parameters were acetonitrile proportion in mobile phase: 51.17%v/v, pH of phosphate buffer:
4.04 and flow rate: 1.25 mL/min. Use of these optimized parameters resulted in retention time of 11.031 min and 9,498.787 number of theoretical plates as ARs of the HPLC method. The method was further validated in harmony with current ICH guidelines Q2 (R1). The method was capable of the successful estimation of EFZ in marketed formulation. The study depicts successful development and validation of a simple RP-HPLC method of EFZ using DoE approach.
Introduction
Efavirenz (EFZ) is chemically (S)-6-chloro-4-(Cyclopropylethynyl)- 1,4-dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one, with a molecular weight of 315.675 g/mol and empirical formula C14H9ClF3NO2 (Figure 1). EFZ is a noncompetitive type non- nucleotide reverse transcriptase inhibitor (NNRTs), mainly selective to human immunodeficiency virus (HIV) type 1 (1). It is used in combination with tenofovir disoproxil fumarate and lamivudine in the treatment of acquired immunodeficiency syndrome (AIDS) (2). The drug acts by inhibiting activity of reverse-transcriptase
enzyme. EFZ essentially inhibit the activity of viral RNA-directed DNA polymerase.
Various analytical methods have been reported for quantification of EFZ using reversed-phase high-performance liquid chromatog- raphy (RP-HPLC) method. Most of these research works are for simultaneous estimation of EFZ along with tenofovir disoproxil fumerate, lamivudine, lopinavir, ritonavir and many other anti-HIV drugs (3–5). Usami and group used a solvent system comprised of acetonitrile-methanol-0.02 M tetramethylammonium perchlorate in 0.2% trifluoroacetic acid for simultaneous separation of EFZ,
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Figure 1.Structure of EFZ.
lopinavir and ritonavir (3). Appala and Shabana reported simulta- neous separation of EFZ with emtricitabine and tenofovir disoproxil fumarate using combination of methanol–water as mobile phase and retention time was found to be 5.875, 8.800 and 12.020 min for emtricitabine, tenofovir disoproxil fumarate and EFZ, respectively (5). There is dearth of literature on RP-HPLC methods for estimation of EFZ alone in bulk and pharmaceutical dosage forms. Furthermore, these methods carry limitations of the presence of high amount of organic solvents in mobile phase (1), low sensitivity with a linearity range in microgram level (6). Vianaet al. (1) used 70%v/v acetonitrile (ACN) and pure water in the mobile phase. Usage of water, instead of buffer, is associated with some limitations. Presence of various excipients in the formulations may change pH of the mobile phase (as water does not have buffering capacity) which might result in the changes in ionization behavior of drug. This may eventually affect the important chromatographic responses such as peak shape, peak area, retention time and number of theoretical plates (7). In the present study, we have employed design of experiment (DoE) approach to obtain best experimental conditions for the RP-HPLC method. We have reduced the amount of organic solvent (51%v/v ACN) in mobile phase and also optimized the pH of buffer and flow rate which helped in achieving a sensitive, ecofriendly and economical analytical method for the routine analysis of EFZ in bulk and formulation.
Furthermore, use of buffers in our method helped to attain better chromatographic responses.
A number of commercial oral formulations like Sustiva®, Stocrin®and many more generic formulations containing only EFZ are available in the market. So, there is a pressing need of evaluating EFZ alone using a simple solvent system in HPLC method (6). HPLC method development is conventionally a time-consuming and tedious process. HPLC method is typically developed by varying one process or system factor in consideration at a time and the effect of those parameters on anticipated outcome of the method like peak area, retention time, tailing factor etc. are observed. This process of method development predominantly necessitates a considerable number of experimental trials. Considering these factors and an indent to the current need as per food and drug administration (FDA) (8), we planned to employ DoE approach to optimize various parameters in the development of RP-HPLC method for EFZ. DoE is a systematic development approach that starts with screening of different process parameters and underlining the progress of an optimized process or product development. Use of DoE approach leads to identification of critical method variables (CMVs) and significantly affecting
analytical responses (ARs) of RP-HPLC method (9). Optimization approach of DoE allows to obtain the best experimental designs which provide highest performance for the method (10). Plackett–
Burman design (PBD) is the most commonly applied screening design in chromatographic method development, whereas Box–Behnken design (BBD) is mostly employed for optimization of the method.
PBD is commonly preferred for screening over other screening designs such as factorial design etc. because it has been proven that PBD analyze same number of factors more efficiently with less number of runs in comparison to the factorial design (11). Waghmare and Kashid (12) reported development and validation of alone EFZ by applying DoE but that method has limitations of sensitivity (5–
25 μg/mL) and use of higher amount (72% v/v) of organic solvent.
Objective of this current study is to report a simple, rapid, sensitive and economical RP-HPLC determination method using DoE approach for the estimation of EFZ. This study incorporates screening of critical analytical variables affecting the ARs and opti- mization of the analytical method of EFZ employing PBD and BBD, respectively.
Experimental
Materials and reagents
HPLC grade acetonitrile (ACN) and Emplura®grade o-phosphoric acid (OPA) were purchased from Merck Chemical Company (Mum- bai, India). Sodium dihydrogen phosphate and sodium hydroxide were used of analytical grade and purchased from SISCO Research Laboratories (SRL) Pvt. Ltd (Delhi, India). EFZ was kindly gifted by Hetero Healthcare (Hyderabad, India) and EFZ IP 600 mg Tablet manufactured by Aurobindo Pharma (Hyderabad, India) was pur- chased from market. Water used for preparing buffer was prepared using Milli-Q®Reference water purification system. Three different molar concentration of buffers were prepared by taking accurately weighed amount of sodium dihydrogen phosphate in Mili-Q®water and pH of the buffers were adjusted using 0.1 (N) OPA or 0.1 (M) sodium hydroxide solution.
Instrumentation and chromatographic conditions The HPLC system (LC-2010HT, Shimadzu Corporation, Japan) comprised of pulse-free solvent system delivering two pumps, 5-line degasser, block heating type column oven, sample cooler (LC-2010CHT), intelligent autosampler with UV-visible detector of dual wavelength. The software used was LC solution, version 1.6.
Chromatographic separation of EFZ was done using BDS HYPERSIL C18 column, (250 mm×4.6 mm) and particle size of silica was 5 μ (Thermo Scientific, Mumbai, India). The mobile phase used was ACN and 10 mM phosphate buffer pH 4.0 (51:49 v/v) in isocratic mode at a flow rate of 1.25 mL/min. Detection wavelength used was 254 nm and injection volume was 20 μL, column temperature was mentioned at 30◦C. All the samples were filtered through 0.45 μ membrane filter.
Methods
Preparation of stock solution, calibration curve standards and quality control (QC) samples
EFZ stock solution for both screening and optimization experiments was prepared by dissolving 10 mg of EFZ into 10 mL of ACN to get a final concentration of 1 mg/mL. Working solution was prepared
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Table I.List of the factors and their levels employed for Plackett–
Burman design
Levels of factors studies Levels
High Low
(+1) (−1)
ACN proportion (%) 60 50
Column temperature (◦C) 30 25
Molarity of buffer (mM) 12.5 7.5
Injection volume (μL) 20 10
pH 6 4
Flow rate (mL/min) 1.25 0.75
Detection (nm) 256 252
by stepwise diluting the stock solution to get a concentration of 10 μg/mL using mobile phase. From the 10 μg/mL working solution 500 ng/mL solution was prepared by stepwise dilution with mobile phase and used in both screening and optimization experiments.
For preparing CS of EFZ 10 μg/mL solution was further stepwise diluted to get 100, 200, 500, 1000, 1500 and 2000 ng/mL solutions using the mobile phase. The QC samples at three concentration levels—low QC (LQC, 150 ng/mL), medium QC (MQC, 900 ng/mL) and high QC (HQC, 1800 ng/mL)—were prepared by suitably dilut- ing 10 μg/mL working standard solution with mobile phase.
Preparation of sample solution
For preparation of EFZ sample solution, 10 marketed tablets of EFZ IP 600 mg were finely powdered and a mass equivalent to 10 mg EFZ was taken. It was dissolved in 10 mL of ACN to get a 1 mg/mL sample stock solution. The solution was centrifuged at 15,000 rpm for 10 min and supernatant was taken and appropriately diluted with the mobile phase to get sample solution of 2000 ng/mL. The resultant solution was used for the assay of EFZ in its marketed tablet dosage form.
Experimental design Factor screening study
A screening study was conducted using PBD to identify the CMVs which significantly affect the ARs i.e., retention time, number of theoretical plates, tailing factor (10%) and height equivalent to theo- retical plate (HETP) of HPLC method. Percentage of ACN in mobile phase, column temperature, molarity of buffer, injection volume, pH of the buffer, flow rate and detection wavelength are the dependent variables which actually affect the ARs of an HPLC method. All the dependent variables were varied in two levels represented as+1 and − 1 (Table I) which denote high and low levels, respectively.
A total of 12 experiments were suggested by the Design-Expert® Software version 8.1 as shown inSupplementary Table SI. All the 12 experiments were executed to screen the CMVs which significantly affect the ARs of HPLC method.
Optimization study
As per the screening study, factors critically affecting ARs of the HPLC method were selected and optimization study was conducted employing BBD. Percentage of ACN in mobile phase, pH of buffer and flow rate were found to be significant CMVs for the ARs i.e., retention time and number of theoretical plates of HPLC
method. All the three critically affecting dependent variables were varied in three levels and represented as high (+1), intermediate (0) and low (−1), respectively. For percentage of ACN in mobile phase (+1) = 60%v/v, (0) = 55%v/v and (−1) = 50%v/v, for pH of buffer (+1)= 6, (0)=5 and (−1)=4 and for flow rate (+1)=1.25 mL/min, (0)=1.00 mL/min and (−1)=0.75 mL/min were taken as actual value. Design matrix with total 17 experimental runs as suggested by Design-Expert®when 33BBD was employed as shown inSupplementary Table SII. The goal of optimization study was to attain a minimum retention time (response 1=R1), with a maximum number of theoretical plates (response 2=R2), which demonstrate a good, sharp, well-resolved peak of the analyte (EFZ).
Method validation Specificity
Specificity of the analytical method was checked by determining EFZ content in a marketed formulation (EFZ IP 600 mg Tablet, Aurobindo Pharma Ltd., Hyderabad). For this, EFZ sample solution was prepared using EFZ IP 600 mg Tablet as described in the earlier section entitled “Preparation of sample solution”. A total of 400 μL of 10 μg/mL of EFZ stock solution was diluted to 2 mL to acquire 2000 ng/mL EFZ reference standard solution. The drug content of EFZ sample solution was determined and evaluated against the reference standard solution. The analysis was conducted in triplicates.
Calibration curve (CC), linearity and range
Calibration curves of EFZ were obtained using six calibration stan- dards in a range of 100–2000 ng/mL. Calibration curves (n=6) were plotted by taking analyte concentration on X-axis and the peak area on Y-axis and linearity of the method was determined. Linearity can be defined as the ability of a specific method to generate test results which were directly proportional to the drug concentration within a range.
Accuracy
Accuracy of the method was calculated using all three QC lev- els. Accuracy was represented as percentage bias i.e., deviation of observed concentration from the nominal concentration. Observed concentration was calculated from area utilizing the average regres- sion equation obtained from the six calibration curves.
Precision
The intraday (n=3) and interday (n=18) precision was evaluated utilizing all the three QC levels. For intraday precision study, QC samples were analyzed on two different time of same day in tripli- cate, while interday precision study was done on three subsequent days. Precision study data were represented as % relative standard deviation (% RSD).
Limit of detection and limit of quantification
Limit of detection (LOD) is defined as the minimum concentration of standard solution that can be determined using the method. Limit of quantification (LOQ) is the lowest concentration which can be accurately quantified by the developed method. LOD and LOQ were calculated using standard deviation of response and the slope obtained from calibration curve. LOD and LOQ were calculated using the following formulas:
LOD=3.3σ
S (1)
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LOQ=10σ
S (2)
where
σ =standard deviation of the y-intercepts of the six calibration curves,
S=the mean of the slopes of the six calibration curves.
System suitability
System suitability test is used to make sure that HPLC system is adequately specific and reproducible for the analytical runs. Test was conducted by injecting consecutive six injections of any sam- ple (500 ng/mL). System suitability parameters include peak area, retention time, tailing factor (10%), HETP and theoretical plate and expressed as %RSD.
Stability studies
The solution state stability of EFZ was performed at atmospheric temperature (Bench top stability) for 24 h by comparing QC samples with freshly prepared solutions. Autosampler stability (25◦C) was examined by injecting QC samples instantly after preparation from stock solution and reinjecting QC samples after storing in autosam- pler for 24 h. Short-term and long-term stability were carried out at 4◦C and−20◦C for a period of 7 days and 1 month, respectively.
Results
Experimental study Factor screening study
Factor screening studies using PBD helped to find out the method variables which were critically affecting ARs of the HPLC method.
Furthermore, the statistical analysis of data was performed using Design-Expert® with help of Pareto charts. As shown inFigure 2, Pareto charts recommend a highly remarkable influence of the factors i.e., ACN proportion (A), pH (E) and flow rate (F), on both ARs i.e., retention time (Figure 2A) and number of theoretical plates (Figure 2B) of the HPLC method as these were found to be greater than t-value limit and Bonferroni limit in the Pareto charts with high statistical significance. As shown in Figure 2A, all the three factors i.e., ACN proportion (A), pH (E) and flow rate (F) negatively affected retention time and the number of theoretical plates as well (Figure 2B). These three factors as significantly influencing ARs were then selected as CMVs for method optimization studies using BBD.
Optimization study
Method optimization study implementing BBD was performed to obtain an optimized and validated chromatographic method. Experi- mental results acquired after executing all the 17 runs were suggested by Design-Expert®software using 33BBD is shown inTable II. The statistical analysis of data was done using Design-Expert® soft- ware. Obtained data were fitted into different models and the final model was selected depending on highest F-value,P-value and r2for future experiments. Model was validated using two-way analysis of variance.
Statistical analysis of data
Statistically evaluated data and fit of the model for response R1and response R2are shown inTable III. For both of the response i.e.,
Table II. Experimental results obtained for the Box–Behnken design
Run R1 R2
Retention time Number of theoretical plates (min)
1 5.825 9,865.141
2 8.895 11,545.525
3 13.596 14,629.521
4 14.541 13,615.384
5 4.911 93,81.876
6 13.068 12,063.297
7 15.273 10,960.671
8 7.283 12333.838
9 8.906 11,430.318
10 8.911 11,526.413
11 6.063 10,316.647
12 7.420 11,983.152
13 10.349 10,799.469
14 9.495 12,397.363
15 19.434 12,701.615
16 8.922 11,411.206
17 8.904 11,500.752
retention time (R1) and number of theoretical plates (R2), quadratic model was observed to be significant with a model F-value of 355.183 and F-value of 1038.311, respectively. Adjusted R2 (R2 adj) value evaluates the goodness of fit of the model. Greater the value, higher is the correlation between model-predicted values and actual values.
The model equation for response R1and response R2in terms of coded factors are as follows:
R1= +8.9076−3.98625∗A−0.32075∗B−3.386∗ (3) C−0.47675∗A∗B+1.12525∗A∗C+1.470575∗
A2+0.400575∗B2+0.669075∗C2
R2= +11482.84−455.927∗A−1169.52∗B−1220.27∗ (4) C+159.3805∗A∗B−278.335∗A∗C+112.0533∗B∗
C−245.702∗A2+569.4946∗B2+82.94035∗C2 where A, B and C are the critical factors in the design.
Response surface plots and desirability function
Figure 3demonstrates the 3D-response surface plots for both the ARs, i.e., retention time (R1) and number of theoretical plates (R2).
Response surface curves illustrate the relationship between CMVs and ARs of the chromatographic method. On retention time both the factor A and factor B has a negative effect when kept flow rate (factor C) at the intermediate level. The same is derived from coefficient of factors in polynomial equation.Figure 3Ashows the 3D-response surface plot for effect of factor A and factor C on the same AR (R1). The plot shows same negative effect of both factors on R1, keeping factor B at the intermediate level. Figure 3Bplot demonstrates the effect of factor B and factor C on retention time (R1) when factor A is kept constant at intermediate level. This plot depicts factor B has no significant effect on retention time (R1) but
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Figure 2.Pareto charts representing the influence of various factors critically on the AR,Aretention time andBnumber of theoretical plates.
factor C has a negative effect on that AR (retention time). The same is confirmed from coefficient of factors in polynomial equation. Effect of CMVs, i.e., ACN proportion (factor A), pH (factor B) and flow rate (factor C) on AR and number of theoretical plates (R2), are shown inFigures 3C and D.Figure 3Cportrays the effect of factor A and factor B on R2,when factor C was kept constant at intermediate level. Both the CMVs have negative impact on R2 as shown in Figure 3C.Figure 3D shows the 3D-response surface plot for the effect of factor A and factor C on number of theoretical plates (R2).
The plot shows same negative effect of both factors on R2keeping
factor B at the intermediate level. Further, there is a negative effect of factor B and factor C on R2when factor A is kept constant at intermediate level. The same is confirmed from coefficient of factors in the polynomial equation for R2.
Determination of optimized method and its validity
The goal of optimized method was to obtain a minimum retention time with a maximum number of theoretical plates. A desirability function (0.913) was obtained for an optimized method with min- imum retention time and maximum number of theoretical plates.
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Table III. Regression coefficients and statistical analysis for Box–Behnken design
Response model Factor Factor coefficient P-value R2 Adjusted R2 Model
F-value R1: Retention
time
Intercept 8.9076 0.0001 0.9978 0.9950
A-ACN Proportion
-3.98625 0.0001
B-pH -0.32075 0.0127
(Quadratic model) C-Flow Rate -3.386 0.0001
AB -0.47675 0.0101 355.183
AC 1.12525 0.0001
BC -0.26675 0.0918
A2 1.470575 0.0001
B2 0.400575 0.0197
C2 0.669075 0.0015
R2: Number of theoretical plates
Intercept 11482.8428 0.0001 0.9992 0.9982
A-ACN Proportion
-455.926875 0.0001
B-pH -1169.517375 0.0001 1038.311
(Quadratic model) C-Flow Rate -1220.26975 0.0001
AB 159.3805 0.0006
AC -278.33525 0.0001
BC 112.05325 0.0040
A2 -245.7024 0.0001
B2 569.4946 0.0001
C2 82.94035 0.0153
The optimal conditions for CMVs were following: ACN proportion (factor A): 51.17% v/v, pH (factor B): 4.04, flow rate (factor C):
1.25 mL/min and responses were retention time (R1): 11.031 min and number of theoretical plates (R2): 9,498.787. Using the above- mentioned optimized chromatographic conditions, six trials were conducted and responses were compared with predicted values given by the model using unpaired t-test. Statistical test result showed that mean values and standard error values for retention time (R1) and number of theoretical plates (R2) were not significantly different, at aP-value<0.05 from the predicted values given by the model.
Method validation Specificity
Specificity of the developed HPLC method was evaluated by using marketed formulation of EFZ of 2000 ng/mL as sample solution and compared with reference solution of EFZ. Peak area for the sample solution of marketed EFZ and reference solution of EFZ were 72,304 mV∗min and 72,435 mV∗min. Statistical results showed that difference in both the cases of peak area and retention time of sample solution of marketed EFZ as compared with reference solution of EFZ were not significant, at aP-value of<0.05.
Calibration curve (CC), linearity and range
All the six calibration curves depicted linearity in the range 100–2000 ng/mL for EFZ. The CC points were 100, 200, 500, 1000, 1500 and 2000 ng/mL. Regression equation for the analyte was determined to be: y=36.288x−145.03. Regression coefficient (R2) for EFZ over the above specified range was observed to be 0.9996. Statistical evaluation of the analytical curves yielded the adjusted R2 (R2adj) value of 0.9992 which proves that fit of the
model is good. F-calculated (1037.71) value was observed to be significantly higher than that of F-critical (19.16), at aP-value of 0.00096 which indicates that the regression is significant between peak area and concentration. The “lack of fit” for the model was found to be insignificant (F-value=3081.78,P-value=0.79399), which further indicates that the regression model between peak area and concentration of analyte is significant and valid.
Accuracy
Accuracy data are shown inTable IV. Percentage bias varies between
−0.389 and 0.410 for all the QC samples indicating accuracy of the developed HPLC method. % Recovery for LQC, MQC and HQC was obtained to be 100.410±1.230, 99.366± 0.361 and 99.661±0.524, respectively, for the developed method. The overlaid chromatogram of all the QC samples is shown inFigure 4.
Precision
Intermediate precision data are shown inTable V. The % RSD of intraday precision and interday precision was observed to be not more than 1.237 and 1.028, respectively. The % RSD values are within the acceptance limit of ICH guideline Q2 (R1), which indicates that quantification method of the analyte is precise (13).
LOD and LOQ
LOD and LOQ were calculated to find out the sensitivity of the method. As per formulae (1) and (2), LOD and LOQ of the method were found to be 5.01 and 15.19 ng/mL. To further validate the theoretically obtained LOD and LOQ of the proposed method, an independent analysis of samples was performed using the following concentrations: 15, 20, 30, 40, 50, 60, 70, 80, 90 and 100 ng/mL.
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Figure 3.3D-response surface plots representing
Aretention time (min) as a function of acetonitrile proportion (%v/v) (A) and flow rate (mL/min) (C).
Bretention time (min) as a function of pH (B) and flow rate (mL/min) (C).
Cnumber of theoretical plates as a function of acetonitrile proportion (%v/v) (A) and pH (B).
Dnumber of theoretical plates as a function acetonitrile proportion (%v/v) (A) and flow rate (mL/min) (C).
Table IV.Accuracy and absolute recovery data
QC levela Predicted conc.b Mean accuracyc % Recoveryd
Range Mean±SD %RSD Mean±SD %RSD
LQC 147.433–152.062 150.615±1.846 1.226 0.410 100.410±1.230 1.226
MQC 888.835–897.929 894.291±3.244 0.363 -0.634 99.366±0.361 0.363
HQC 1784.144–1808.285 1792.999±9.940 0.526 -0.389 99.661±0.524 0.526
Standard deviation (SD), % relative standard deviation (% RSD), n=6 samples in all cases.aLQC, MQC, HQC are 150, 900, 1800 ng/mL of EFZbPredicted concentrations of EFZ were calculated using linear average regression equation, given in ng/mL.cAccuracy is given as % bias=[100×(predicted concentration−nominal concentration)/nominal concentration].
d% Recovery=[(Peak area of standard/peak area of analytical standard of same concentration)×100].
At 15 and 20 ng/mL concentrations, no peak was found at the retention time of EFZ. Peak of the drug was observed from 30 ng/mL concentrations onwards but % RSD values for 30 and 40 ng/mL concentrations were found to be greater than the acceptable limit.
From concentration 50 ng/mL onwards, the % RSD values were found to be within acceptable range (<2%). This result depicts that although the theoretical LOD and LOQ of the proposed analytical method were calculated to be 5.01 and 15.19 ng/mL, respectively,
but practically LOD and LOQ were observed to be 30 and 50 ng/mL, respectively.
System suitability
System suitability data are shown in Table VI. System suitability was expressed as %RSD for peak area, retention time, tailing factor (10%), HETP and number of theoretical plates. %RSD values for
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Figure 4.Overlaid chromatogram of blank, LQC (150 ng/mL), MQC (900 ng/mL) and HQC (1800 ng/mL).
Table V.Results of intermediate precision
QC levela Intraday
repeatability (% RSD) (n=3)
Interday repeatability (%
RSD) (n=18)
Day 1 Day 2 Day 3
LQC 1.237 0.711 0.106 1.028
0.515 0.433 0.151
MQC 0.498 0.399 0.218 0.389
0.422 0.545 0.540
HQC 0.312 0.175 0.275 0.442
0.126 0.451 0.686
% relative standard deviation (% RSD); Intraday repeatability was assessed by replicate analysis (n=3) twice a day at each QC level.aLQC, MQC, HQC are 150, 900, 1800 ng/mL
peak area, retention time, tailing factor (10%), HETP and number of theoretical plates were found to be<2%, which indicates that the chromatographic system is reproducible and suitable for analysis.
Stability studies
Auto sampler, short-term and long-term stability studies were per- formed for all the three QC samples for EFZ and results are shown in Table VII. No significant difference was found between concentration of all the three QC samples on zero day, bench top stability (24 h), auto sampler stability (25◦C), short-term stability (4◦C, 7 days) and long-term stability (−20◦C, 1 month). % RSD observed for the estimated concentration of stability studies samples and the nominal concentration was not more than 1.5% for all the three QC samples, which is within the acceptable limits.
Discussion
This current study emphasizes on optimization, development and validation of RP-HPLC method for estimation of EFZ in bulk and formulation. We actuated to optimize the method using the concept of DoE as pharmaceutical industries and various regulatory guidelines
currently suggesting for developing the analytical method using the principle of quality by design. A systemic and easy screening of method variables and their optimization was possible due to the application of DoE approach and that also provided a better under- standing about the CMVs with respect to the ARs. PBD facilitated the selection of three CMVs for the analytical method from various method variables which depicted significant effect on both the ARs (retention time and number of theoretical plates) using a single experiment. Response surface methodology (RSM)-based BBD was further used and helped in finding the optimum ACN proportion in mobile phase, pH of buffer and flow rate for the analytical method and to establish a mathematical relationship between CMVs and ARs. Under these three conditions an optimized retention time (11.031 min) with a high number of theoretical plates (9,498.787) was obtained, which is an indication that a time-saving and specific RP-HPLC method was developed. A consistent and acceptable % RSD (<2%) values of different validation parameters indicate that the developed RP-HPLC method is accurate and precise in a good agreement with the regulatory guidelines. Furthermore, the validated method obtained low LOD and LOQ values, which indicate the high sensitivity of this method as compared to the previously reported
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Table VI. System suitability using 500 ng/mL of EFZ
Parameters Mean SD %RSD
Peak area 17,944.333 99.835 0.556
Retention time 11.106 0.061 0.549
Tailing factor (10%) 0.961 0.011 1.099
HETP 15.212 0.176 1.159
Number of theoretical plates 9441.132 106.802 1.131
Table VII. Stability studies for EFZ in various conditions QC samples
Nominal Conc.
Mean measured Conc.a Nominal Conc.b % RSD % Recoveryc
ng/mL ng/mL ng/mL
Bench top stability at room temperature (24 h)
150 151.144 150.003 1.059 99.245
900 892.922 894.348 0.551 100.160
1800 1798.566 1805.384 0.573 100.379
Auto sampler stability
150 151.144 152.259 0.519 100.738
900 892.922 903.357 0.498 101.169
1800 1798.566 1807.225 0.312 100.481
Short-term stability (4◦C, 7 days)
150 151.144 148.487 0.925 98.243
900 892.922 876.886 0.315 98.204
1800 1798.566 1768.696 0.571 98.339
Long-term stability (−20◦C, 1 month)
150 151.144 152.788 0.666 101.088
900 892.922 881.214 0.156 98.689
1800 1798.566 1761.290 0.193 98.097
amean measured concentration of EFZ on day zero for LQC=150 ng/mL, MQC=900 ng/mL and HQC=1800 ng/mLbnominal concentration of EFZ during stability studies; % relative standard deviation (% RSD);c% Recovery=[(Nominal conc./Mean measured conc.)×100].
RP-HPLC methods for the drug. Stability study of EFZ in different stability conditions depicted that the drug is stable in solution form up to a month. The developed method was found to be specific for the quantification of EFZ in presence of formulation excipients and finally applied for the estimation of drug content in marketed formulation of EFZ.
Conclusion
This article presents development and validation of a simple, sensi- tive, rapid and accurate RP-HPLC analytical method for estimation of the anti HIV drug, EFZ. Employment of DoE approach for screening of parameters helps to find out the critical parameters affecting ARs of HPLC method for EFZ, whereas optimization design of DoE software helps to optimize the precise conditions required to develop final analytical method for EFZ. Quantification of EFZ was accurately performed using ACN and 10 mM phosphate buffer pH 4.0 (51:49 v/v) as mobile phase at a flow rate of 1.25 mL/min with a retention time of 11.031 min. Method can detect a minimum of 30 ng/mL and quantify a minimum of 50 ng/mL of EFZ present in a sample. The developed method was found to be suitable for accurate quantification of EFZ in bulk, in-house formulation and marketed formulation.
Supplementary data
Supplementary data are available at Journal of Chromatographic Scienceonline.
Acknowledgment
The authors are thankful to Birla Institute of Technology and Science, Pilani (BITS Pilani) for providing the funds to carry out this research work.
Conflict of interest
The authors declare no conflict of interest.
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