Development and Validation of a Liquid Chromatographic Method for Determination of Efavirenz in Human Plasma 2007, 65, 359–361
A. Lakshmi Sailaja, K. Kishore Kumar, D. V. R. Ravi Kumar, C. Mohan Kumar, N. M. Yugandhar, G. Srinubabu & Centre for Biotechnology, Andhra University College of Engineering, Visakhapatnam 530003, India; E-Mail:
[email protected]
Received: 15 September 2006 / Revised: 16 November 2006 / Accepted: 21 November 2006 Online publication: 16 January 2007
Abstract A simple, rapid, and sensitive high-performance liquid chromatographic method for estimation of efavirenz in human plasma has been developed and validated. Chromatography was performed with C18 analytical column and 50:50 acetonitrile–phosphate buffer (pH 3.5) as mobile phase. Compounds were monitored by UV detection at 247 nm. The retention time for efavirenz was 6.45 min and that for the internal standard, nelfinavir, was 2.042 min. Response was a linear over the concentration range of 0.1 lg–10 lg mL)1 in human plasma. The method was simple, specific, precise and accurate and was useful for bioequivalence and pharmacokinetic studies of efavirenz.
Experimental
Keywords
Chemicals and Reagents
Column liquid chromatography Validation Human plasma Efavirenz
Introduction Acquired immunodeficiency syndrome (AIDS) was first recognized in 1981 and the human immunodeficiency virus (HIV), which causes AIDS, was identified in 1983 [1]. HIV/AIDS is currently the fourth greatest cause of death worldwide. It is estimated that 40 million people are infected with HIV and 22 million have died from the disease [2]. Efavirenz, (S)-6-chloro-4(cyclopropylethynyl)-1,4dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one, empirical formula C14H9
Full Short Communication DOI: 10.1365/s10337-006-0170-x 0009-5893/07/03
pretreatment, consisting of solid-phase extraction with subsequent elution. Complicated instrumentation comprising a combination of two HPLC systems or gradient elution is also necessary [3–6]. A novel approach for estimation of efavirenz in human plasma is reported in this paper. The method was validated in accordance with recently published FDA guidelines for validation of bioanalytical assays [8].
ClF3NO2, is a novel anti HIV agent. The structure of efavirenz is shown in Fig. 1. Methods for analysis of antiviral agents have recently been published [3–7]. Reversed-phase high-performance liquid chromatography (RP-HPLC) with ultraviolet (UV) or mass spectrometric (MS) detection is commonly used. The methods of Rezk et al. [4] and Simon et al. [6] require 500-lL samples for determination of plasma levels; those of Aymard et al. [5] and Titier et al. [7] require 1-mL samples. All the methods use complicated time-consuming sample
Acetonitrile and methanol were HPLC grade from Qualigens (Mumbai, India). Other reagents were of analytical grade. Deionized water was produced by use of Millipore (USA) equipment. Buffer was prepared by dissolving 7.8 g sodium dihydrogen orthophosphate in 1 L water and adjusting the pH to 3.5 with orthophosphoric acid. Efavirenz and nelfinavir were obtained from Hetero Drugs (Hyderabad, India). Stock solutions of both compounds (100 lg mL)1) were prepared in 50:50 methanol–water. Serial dilutions were prepared with the same solvent mixture. Quality control (QC) samples containing low, medium, and high concentrations of the drug (denoted LQC, MQC, and HQC, respectively) were prepared in blank plasma.
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Fig. 1. The structure of efavirenz
Chromatography HPLC was performed with Shimadzu equipment comprising two LC-10AT VP pumps, an SPD-10A variable-wavelength programmable UV–visible detector, and a VP CTO-10AS VP column oven. Compounds were separated on a 75 cm · 4.6 mm i.d., 3.5-lm particle size, zorbax C18 column (waters). The mobile phase was 50:50 ACN–phosphate buffer at pH 3.5 and the flow rate was 0.8 mL min)1. Data were acquired and processed with Class VP software.
Construction of Calibration Plot The calibration plot was prepared by spiking blank plasma with 0.1–10.0 lg mL)1 efavirenz then extracting the drug. Efavirenz solutions of appropriate concentration in drug-free plasma containing 100 lL internal standard (nelfinavir) solution (50 lg mL)1) were added to 900 lL drug-free plasma in clean borosilicate glass tubes and the mixtures were vortexed for 25 s. The solutions obtained (500 lL) were transferred to clean glass test tubes and 3.0 mL tertiary butyl methyl ether (TBME) was added. The mixtures were shaken with a vibramaxer at 2,000 rpm for 20 min then centrifuged for 5 min with 4,000 rpm at 4 C. The organic layers (2.5 mL) were transferred to a sample vial, evaporated with a stream of nitrogen for 15 min at 40 C, reconstituted with 0.5 mL mobile phase, vortexed and then analysed by HPLC. Analysis was performed in duplicate for each concentration.
Sample Analysis Human blood was centrifuged, after addition of CPDA anticoagulant to separate plasma. Efavirenz was isolated
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Fig. 2. Representative chromatograms obtained from efavirenz at the HQC (top) and LQC concentrations
from the plasma by liquid–liquid extraction followed by evaporation as described above.
and the internal standard. This shows that the assay procedure is specific to efavirenz. All human plasma samples used to prepare calibration standards and QC samples were free from interfering peaks.
Results and Discussion Specificity
Linearity of the Calibration Plot
Representative chromatograms obtained from processing of LQC and HQC samples are presented in Fig. 2. The chromatograms shown are unmodified. Although the peaks seem slightly broad they are quite reproducible and the width did not affect the results. No interfering peaks were observed in the blank at retention times corresponding to drug
A standard calibration plot of eight points, obtained from solutions containing 0.1, 0.2, 0.5, 1, 3.0, 5, 8, and 10 lg mL)1, drawn with a weighting factor of 1/x, was derived from the observed response. Calibration plot data for efavirenz and IS in human plasma revealed the calibration plot was linear in this concentration range; the correlation coefficient was 0.9983.
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Precision and Accuracy The limit of detection of the assay was 0.08 lg mL)1. Inter day and intraday precision and accuracy were assessed by replicate analysis of the three QC samples. The mean RSD for intraday and interday assay reproducibility (n = 6), listed in Table 1, were within the range of acceptability [8]. Coefficients of variation for each sample processed were also included in Table 1.
Recovery Absolute recovery was determined by comparing the efavirenz to internal standard peak-area ratio obtained by extraction of the quality-control standards with the ratio obtained by injection of standard solutions of the same concentration. Recoveries were greater than 78% (Table 2).
Stability Short-Term Stability
Six aliquots of each of the low and highconcentration quality-control samples were thawed and left at room temperature for 6 h, this time being based on the expected duration of analysis. Comparison of the results from these six samples with results from six freshly spiked samples prepared from calibration standards revealed that there was no significant difference between the response of freshly spiked standards and samples of efavirenz after 6 h, indicating the analyte was stable at room temperature (Table 3). Autosampler Stability
The stability of processed samples during the time they were in the autosampler was determined, again using six aliquots of each of the low and high-concentration quality-control samples. The stability of the drug and the IS were assessed over an 18-h period, the expected run time for batches of validation samples. Comparison of the results from these six samples with six freshly spiked samples prepared from calibration standards revealed that efavirenz and IS can remain in the autosampler for at least 18 h with no significant reduction in the nominal values (Table 3).
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Table 1. Results from determination of the accuracy and precision of analysis of efavirenz in the quality-control samples Intra-day -1
Theoretical concentration (lg mL ) Measured concentration (lg mL-1) RSD (%)
0.2 0.19 8.6
Table 2. Absolute recovery of efavirenz from human plasma Sample concentration (lg mL-1)
Absolute recovery (%) mean ± SD (n = 6)
0.2 5.15 7.85
78.28 ± 5.15 81.28 ± 3.51 80.93 ± 2.18
5.15 5.12 5.2
7.85 7.93 5.8
0.2 0.185 8.3
5.15 5.18 5.8
7.85 7.78 5.6
Table 3. The stability of efavirenz Sample concentration (lg mL)1)
Concentration found (lg mL)1; n = 6)
Short-term stability (6 h) 0.198 0.2 7.49 7.85 Freeze–thaw cycles First 0.195 0.2 7.45 7.85 Second 0.189 0.2 7.37 7.85 Third 0.184 0.2 7.35 7.85 Stability in autosampler (18 h) 0.187 0.2 7.49 7.85 Long-term stability for 30 days at )20 C 0.176 0.2 7.29 7.85
Freeze–Thaw Stability
Analyte stability was determined after three freeze–thaw cycles for six aliquots of each of the low and high-concentration quality-control standards. The samples stored at the intended storage temperature ()20C) for 24 h then thawed unassisted at room temperature. When completely thawed the samples were frozen for 12–24 h under same conditions. This freeze–thaw cycle was repeated twice and the solutions were then analyzed. Use of a newly constructed calibration plot and six replicate analysis of low and highconcentration quality-control samples (comparison samples) analyzed with the stability samples in the same run showed there was no significant difference between the freeze–thaw samples and the freshly spiked samples (Table 3).
Inter-day
and rapidity are the main advantages of this method. The method can be used in routine analysis for its intended purpose.
References
Long-Term Stability
Testing of the long-term (30-day) stability of efavirenz in human plasma showed the drug was highly stable—the mean results from the tested samples were within the acceptance criteria [8] compared with the initial values. These results indicate that storage of efavirenz in plasma at temperatures below )20C is adequate and there is no stability problem (Table 3).
Conclusion The HPLC assay described here is a simple, selective, precise, and accurate means of quantification of efavirenz in human plasma. Sensitivity, simplicity,
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