Different. RP HPLC Method for Simultaneous Estimation of Lamivudine,. Tenofovir Disoproxil Fumarate and Efavirenz in Tablet Formulation1. K. Anandakumara ...
ISSN 10619348, Journal of Analytical Chemistry, 2013, Vol. 68, No. 9, pp. 815–821. © Pleiades Publishing, Ltd., 2013.
ARTICLES
RPHPLC Method for Simultaneous Estimation of Lamivudine, Tenofovir Disoproxil Fumarate and Efavirenz in Tablet Formulation1 K. Anandakumara, G. Abiramia, S. Murugana, and B. Ashokb a
Department of Pharmaceutical Analysis, Adhiparasakthi College of Pharmacy Melmaruvathur,Tamil Nadu, 603319 India bDepartment of Statistics, Adhiparasakthi College of Nursing Melmaruvathur, Tamil Nadu, 603319 India Received September 19, 2011; in final form September 20, 2012
Abstract—A simple, rapid, and precise reversedphase highperformance liquid chromatographic method for the simultaneous determination of lamivudine, tenofovir disoproxil fumarate and efavirenz in bulk and tablet dosage form has been developed and validated. Chromatography was performed on a 150 mm × 4.6 mm i.d., 5µm particle, Phenomenex Luna C18 column with 30 : 45 : 25 (v/v/v) acetonitrile : methanol : water as mobile phase at a flow rate of 0.5 mL/min. UV detection was done at 258 nm; lamivudine, tenofovir diso proxil fumarate and efavirenz were eluted with retention times of 3.27, 4.58 and 10.90 min, respectively. The method was validated in accordance with ICH guidelines. Validation revealed the method is specific, rapid, accurate, precise, reliable and reproducible. Calibration plots were linear over the concentration ranges 1–6 µg/mL for lamivudine and tenofovir disoproxil fumarate and 2–12 µg/mL for efavirenz. Limits of detec tion were 0.05, 0.09 and 0.11 µg/mL and limits of quantification were 0.15, 0.28 and 0.34 µg/mL for lamivu dine, tenofovir disoproxil fumarate and efavirenz, respectively. The high recovery and low coefficients of vari ation confirm the suitability of the method for the simultaneous determination of these three drugs in bulk and tablets. Keywords: high prefomance liquid chromatography, lamivudine, tenofovir disoproxil fumarate, efavirenz, method validation DOI: 10.1134/S1061934813090025 1
Lamivudine (LAM), chemically (2R,5S)4amino 1[2(hydroxylmethyl)1,3oxathiolan5yl]2(1H) pyrimidinone [1] is converted intercellularly in stages to the triphosphate. This triphosphate halts the DNA synthesis of retroviruses, including HIV, through com petitive inhibition of reverse transcriptase and incor poration into viral DNA [2]. Tenofovir disoproxil fu marate (TDF) is converted intercellularly to the diphosphate that halts the DNA synthesis of HIV through the same mechanism [2]. Chemically it is a bis(isopropyloxy)carbonyloxymethyl ester of (R)9 (2phosphonomethoxypropyl) adenine with fumaric acid [1]. Efavirenz (EFV) is chemically designated as (4S)6chloro4(cyclopropylethylnyl)1,4dihydro 4 (trifluoromethyl)2H3,1benzoxazin2one [1]. HIV Protease inhibitors are antiretrovirals that act by binding reversibly to HIVProtease thereby preventing the cleavage of the viral precursor polyproteins. This re sults in the formation of immature viral particles inca pable of infection other cells [2]. The combination of LAM, TDF and EFV is used in pharmaceutical prep arations for the treatment of AIDS. The chemical structures of LAM, TEN and EFV are shown in Fig. 1.
1 The article is published in the original.
Several methods were reported for the estimation of LAM, TDF and EFV [3–8] individually and in combination with other antireteroviral drugs [9–15]. Simultaneous estimation of LAM, TDF and EFV by UV methods also reported [16]. Because of no chromatographic method for the simultaneous determination of LAM, TDF and EFV in a combined dosage form has yet been reported, it was essential to develop such a method for bulk and tablet formulations. The method described is rapid, economical, precise, and accurate and can be effec tively used for routine quality control analysis of tab lets. The developed method was validated as per ICH norms [17–19]. EXPERIMENTAL The instrument and chromatographic conditions. Shimadzu HPLC system (Shimadzu corporation Ky oto, Japan) consisted of a pump (LC10 ATvp solvent deliver module, SPD10 Avp UVVisible detector) run under Winchrome software, with manual injecting fa cility programmed at 20 μL capacity per injection was used. The column used was Phenomenex Luna C18 (150 mm × 4.6 mm, 5.0 μm particle size). Different
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O
NH2
N
O HO
N
S (a)
O NH2
O
N
N N
N
O
O P O O
O
O •
O O
OH
HO O
O
(b)
F F F Cl
O N H
O
(c) Fig. 1. Chemical structure of (a) LAM, (b) TDF and (c) EFV.
mobile phases were tested in order to find the best con ditions for separation of LAM, TDF and EFV. The mobile phase contained acetonitrile : methanol : water (30 : 45 : 25, v/v/v) and the flow rate was maintained at 0.5 mL/min. UV detection was carried out at 258 nm. The mobile phase and samples were filtered through a 0.45 μm membrane filter. Mobile phase was degassed by Sonica ultrasonic cleaner (model 2200 MH) prior to use. All determinations were performed at ambient temperature. Materials and reagents. Pharmaceutical grade working standards LAM, TDF and EFV were obtained from Strides arco labs, Bangalore, India. All chemicals and reagents were of HPLC grade and were purchased from Qualigens India Pvt. Ltd., Mumbai, India. Solution preparation. LAM, TDF (20 μg/mL) and EFV (40 μg/mL) were prepared by transferring an ac curately weighed 20 mg of LAM, TDF and 40 mg of EFV reference standards to 100 mL volumetric flasks separately. The compounds were dissolved with 50 mL of HPLC grade methanol and sonicated for 2 min. The solution was diluted up to the volume with meth anol. Further dilutions were made by diluting 2.5 mL into 25 mL with mobile phase. LAM, TDF and EFV test stock solution was pre pared for the analysis of a tablet dosage form. Twenty tablets (Trioday, Cipla Ltd., Goa, India, containing
300 mg of LAM, 300 mg of TDF and 600 mg of EFV) were weighed individually and their average mass was determined. The tablets were crushed to a fine powder and amount of tablet powder equivalent to 40 mg of EFV was transferred to a 100 mL volumetric flask and dissolved in 100 mL of HPLC grade methanol. The so lution was sonicated for 15 min with swirling. After son ication, the solution was filtered through 0.2 μm mem brane filter. From the clear solution, further dilutions were made by diluting 7.5 mL into 50 mL with mobile phase to obtain the solution containing 3 μg/mL of LAM, 3 μg/mL of TDF and 6 μg/mL of EFV. Method development. The HPLC procedure was optimized with a view to develop a simultaneous assay method for LAM, TDF and EFV. The mixed standard stock solution (10 μg/mL of each of the compound) was injected in HPLC. Different ratios of acetonitrile, methanol and water were tried. Method validation. The method was validated ac cording to the TCH guidelines/ The folloing valida tion parameters were used such has linearity, accuracy, precision specificity, limit of detection, limit of quan titation and robustness. Linearity of the method was studied by injecting the mixed standard solutions in the concentration range of 1–6 μg/mL for LAM and TDF and 2–12 μg/mL for EFV six times into the LC system keeping the injec
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Table 1. Statistical analysis for calibration graphs of pure standard and analyte in sample matrix Item
Values for LAM, TDF and EFV, respectively*
Ftest for homoscedasticity evaluation
0.89, 0.934 and 2.36 (9.01)b
Ftest for linerity evaluation
0.001, 0.013 and 0.147 (3.8)d
tTeste to compare intercepts
0.335, 0.442 and 0.211 (2.45)
f
tTest to compare slopes
0.98, 0.51 and 1.066 (2.45)
g
Interceptt
0.465, 0.232 and 0.466 (122)
Confidence interval for the intercept
0.465, 0.232 and 0.466 (122)
* ⎯The values in parentheses correspond to critical values at α = 0.05 and the following degrees of freedom: a(ν1 = 2, ν2 = 2), b(ν1 = 4, ν2 = 5), c(ν1 = 3, ν2 = 6), d(ν1 = 4, ν2 = 8), e(ν = 6), f(ν = 6). g⎯The values in parentheses correspond to the standard deviation.
tion volume constant. The peak areas were plotted against the corresponding concentrations to obtain the calibration graphs. Robustness was studied by evaluating the effect of small but deliberate variations in the chromatographic conditions. The conditions studied were flow rate (al tered by ±0.1 mL/min), and mobile phase composi tion ratio of 30 : 50 : 20 and 30 : 40 : 25, v/v/v (aceto nitrile, methanol, water). These chromatographic variations were evaluated for resolution between LAM and TDF, TDF and EFV and % assay of drugs. The fil ter compatibility was studied by comparing % assay of test solution filtered through 0.2 and 0.4 μm mem brane filters. System suitability parameters with respect to theo retical plates, tailing factor, repeatability and resolu tion between LAM, TDF and EFV peaks were defined. Accuracy of the method was studied by recovery experiments. To the preanalysed drug sample (LAM, TDF and EFV combination tablets) known amounts of LAM, TDF and EFV raw material corresponding to 80, 100 and 120% of label claim were added (standard addition method), mixed and the drugs were extracted and determined by running chromatograms in opti mized chromatographic conditions. The experiment was performed in triplicate and % recovery and % RSD were calculated. RESULTS AND DISCUSSION Method development and optimization. The HPLC procedure was optimized with a view to develop a suit able LC method for the determination of LAM, TDF and EFV in fixed dose combined dosage form. Initial ly, methanol and water in different ratios were tried. But LAM was eluted in less than 2 min and EFV gave broad peak, so acetonitrile was added and mixtures of acetonitrile, methanol and water in different ratios were tried. It was found that acetonitrile : methanol : water in ratio of 30 : 45 : 25 (v/v/v) gave acceptable retention times (3.27 min for LAM, 4.58 min for TDF and 10.90 min for EFV), Number of theoretical plates and JOURNAL OF ANALYTICAL CHEMISTRY
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good resolution were reached at the flow rate of 0.5 mL/min. Validation. For linearity, before performing regres sion, the homoscedasticity of the calibration standards was verified using a Cochran’s test. The ccalc values were 0.00115, 0.00010 and 2.08918 for LAM, TDF and EFV, respectively. These test values were smaller than the critical value, ctab(α = 0.05; k = 3; n = 6) = 5.99. Thus, the variances of the calibration standards were considered to be homoscedastic and ordinary least squares could be used to estimate the regression lines. Regression analysis was performed using Microsoft Office Excel XP. Equations of the calibrations lines for LAM, TDF and EFV were AreaLAM = 9.18 × 105x + 1.77 × 104, AreaTDF = 7.05 × 105x + 4.68 × 103 and AreaEFV = 8.04 × 105x + 2.16 × 103, respectively. The corresponding values of the slopes and intercepts with their 95% confidence limits were 9.18 × 105 ± 9.5 × 103 and 1.77 × 104 ± 1.8 × 104 for LAM, 7.05 × 105 ± 7.5 × 103 and 4.7 × 103 ± 2.6 × 104 for TDF and 8.04 × 105 ± 9.6 × 103 and 2 × 103 ± 4 × 104 for EFV. The correlation co efficients were 0.9995, 0.9995 and 0.9997 for LAM, TDF and EFV, respectively. Visual observation of the calibration curves gave the impression that they were linear. The lackoffit test results for the calibration data of LAM, TDF and EFV were Fcalc = 1.036, 1.100 and 1.234, respectively. These values were smaller than the critical value, Ftab(α = 0.05; df1 = 2, df2 = 17) = 3.59. Thus, straight lines were considered adequate to describe the relationships between the peak areas and the concentrations for each compound. From the above it is observed that the straight line model is cor rect for the considered calibration ranges and the in tercept of the calibration lines is not significantly dif ferent from zero (Table 1). Precision was evaluated at the repeatability and in termediate precision levels. For repeatability analysis, six independent portions of a tablet dosage form were processed through the full analytical method and re sults were evaluated obtaining a % RSD value of 0.69. Intermediate precision was evaluated with a new series of six portions of the same sample used in the repeat No. 9
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ANANDAKUMAR et al. (а)
0.12
AU
–0.02 0
min
15.00
(b)
4.56
AU
10.90
3.27
0.12
1
3
2
–0.02 0
min
15.00
Fig. 2. RPHPLC chromatogram of (a) placebo containing tablet matrix without drugs and (b) tablet formulation showing LAM (peak 1), TDF (peak 2) and EFV (peak 3) from the solution of tablet dosage form. Mobile phase: acetonitrile, methanol, water (30 : 45 : 25, v/v/v). Detection at 258 nm.
ability assay, processed on a different day, one week lat er and by a different analyst. The corresponding % RSD was 0.62. A statistical Ftest was applied to compare the variance with the one computed in the re peatability analysis. The computed F value was equal to 1.29, whereas the critical Fcrit(5,5, α = 0.05) is 5.05. Therefore, it can be concluded that no differences ex ist between the variances obtained. On the other hand, both variances are lower than 2%, indicating the ex tremely good repeatability of the proposed methodol ogy. In addition, an % RSD value was computed using the mean values of the analysed series; the resulting RSD was 0.90. Owing to the fact that the latter value is lower than 2.8%, the maximum value accepted for
AOAC in a recommendation for active ingredients, we conclude that the intermediate precision can be con sidered as excellent. Specificity was performed by injecting of the pla cebo to demonstrate the absence of interferences with the signals of LAM, TDF and EFV, as shown in Fig. 2a. On the other hand, the chromatogram of the solution of tablet dosage form with the three com pounds showed clear, compact and wellseparated peaks of LAM, TDF and EFV, as shown in Fig. 2b. Moreover, in Fig. 2b, it is seen that no other peaks are eluted besides the three active compounds. Therefore, the method was considered specific.
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Table 2. Summary of validation parameters Validated parameter Precision Repeatability, n = 6
LAM
Mean, % assay
TDF
EFV
100.02
99.82
100.18
1.16
0.93
0.64
0.9995 (1–6 µg/mL)
0.9995 (1–6 µg/mL)
0.9997 (2–12 µg/mL)
LOD, µg/mL
0.05
0.09
0.11
LOQ, µg/mL
0.15
0.28
0.34
RSD, %
Coefficient of determination Linearity, n = 6
Table 3. Robustness study Mobile phase, acetonitrile: methanol: water
Percent assay of the drug
30 : 50 : 20 (v/v/v) 30 : 45 : 25 (v/v/v) LAM TDF EFV
100.16 100.42 100.28
99.89 100.74 99.86
Membrane filter pore diameter, µm
Flow rate, mL/min 0.6
0.5
0.2
0.4
100.20 100.31 100.16
99.90 100.80 100.26
100.48 100.92 100.80
100.12 99.98 100.08
Limit of quantification (LOQ) which represents the concentration of analyte at S/N of 10 and limit of detection (LOD) at which S/N is three were deter mined for the proposed method and results are given in Table 2. LOD and LOQ were computed based on the standard deviation of the response and the slope.
System suitability was evaluated by six standard solution injections, following ICH guidelines. Evalua tion of analyte peak parameters provided high quality results (Table 4). The results agree with those specified in USP and ICH guidelines, demonstrating that the chromatographic system is adequate and reliable.
Robustness study was conducted by deliberate changes in mobile phase composition and flow rate, revealed that there was no significant variation in % assay (Table 3), retention time, tailing factor and reso lution. The studied membrane filters were found suit able for assay of drug product as there was no signifi cant change in % assay values. Stability for LAM, TDF and EFV was found to be 4, 3.30 and 2.45 h.
The accuracy of the method was done by recovery studies. The different concentrations of the solutions were injected and the recovery of the known amount of the added analyte was computed for each sample. Val ues of % recovery, % RSD and standard error of mean (SEM), indicating the method was found to be accu rate, are listed in Table 5. A statistical student’s ttest was applied, which allows us to conclude that no sig nificant difference exists between the recovery ob
Table 4. Data for the evaluation of the system suitability Parameters
LAM
TDF
EFV
Acceptance criteria
Retention time, min, RT
3.27
4.58
10.90
–
Asymmetrical factor
1.69
1.61
1.35
£2
Tailing factor
1.44
1.38
1.24
£2
3046.71
4481.15
8658.96
>2000
3.21
4.91
13.07
10 > k > 1
Theoretical plates per unit Capacity factor Resolution
Between LAM and TDF 2.91
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Between TDF and EFV 9.02 2013
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ANANDAKUMAR et al.
IP and the developed method was done by performing repeatability study on the market sample. Statistical evaluation of the obtained results showed no signifi cant difference in terms of % assay, which was con firmed using student’s ttest and Ftest (Tables 6 and 7).
Table 5. Recovery studies Component
Recovery, %
% RSD
SEM
LAM
TDF
EFV
80
99.99
101.06
98.31
100
100.83
100.06
98.76
120
100.79
98.90
99.21
80
1.12
1.02
0.62
100
0.96
1.06
0.98
120
0.86
0.62
0.92
80
0.11
0.26
0.05
100
0.21
0.60
0.24
120
0.30
0.39
0.05
An UV method has been published for simultaneous determination of LAM, TDF and EFV in bulk and in tablet formulation. In this method, Beer’s law was obeyed in the ranges of 5–30, 5–30 and 10–60 μg/mL for LAM, TDF and EFV, respectively [16]. But the de veloped HPLC method was obeyed in the concentra tion ranges of 1–6, 1–6 and 2–12 μg/mL for LAM, TDF and EFV, respectively. By comparing LOD and LOQ, this indicates that the developed HPLC method is more sensitive compared to the reported UV method.
tained and the ideal value of 100 at a confidence level of 95%.
The developed new HPLC method provides a sim ple, precise, accurate, selective and sensitive for the simultaneous estimation of LAM, TDF and EFV in bulk and in tablet dosage form. It can be effectively applied for routine quality control analysis of these three drugs.
***
ACKNOWLEDGMENTS
In recent years, LC methods have been described for the analysis of LAM, TDF and EFV in tablet dos age form individually in IP [1]. IP method in terms of assay can be considered time consuming, expensive, cumbersome and tedious because it involves gradient elution, elaborates sample preparation, use of buffer and a flow rate of 1 mL/min. Comparative study of the
The authors are thankful to Strides acro Labs, Ban galore, India for supplying souvenir samples of LAM, TDF and EFV raw materials. The authors are also thankful to Dr. T. Vetrichelvan Principal, Adhiparasakthi College of Pharmacy, Melmaruvathur for providing necessary facilities to carry out the research work.
Table 6. Statistical evaluation of results obtained from analysis of marketed formulation by proposed and IP Pharmacopeial methods Assay value of LAM, % Parameter
Mean, n = 6 SD
Assay value of TDF, %
Assay value of EFV, %
developed method
IP method
developed method
IP method
developed method
IP method
100.02
100.05
99.82
99.90
100.18
100.22
1.16
0.63
0.93
0.44
0.65
0.56
t test ttab = 2.015 (P = 95%, n = 6)
tcal = 0.056; tcal < ttab*
tcal = 0.190; tcal < ttab*
tcal = 0.114; tcal < ttab*
F test Ftab = 12.8 (at n – 1)
Fcal = 3.390; Fcal < Ftab**
Fcal = 4.467; Fcal < Ftab**
Fcal = 1.347; Fcal < Ftab
Notes: * Shows the mean values differ insignificantly. ** Shows that the difference between the dispersions is not significant. JOURNAL OF ANALYTICAL CHEMISTRY
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Table 7. Comparative parameters and results of assay of market sample by IP and developed methods Parameter Assay, %
Specifications as per IP Developed method NLT 98–102% of labeled LAM = 100.02 amounts of LAM, TDF and EFV TDF = 99.82 EFV = 100.18 % RSD For replicate injections is not LAM = 1.16 more than 2.0% TDF = 0.93 EFV = 0.65 Phenomenex Luna ODS Column Stainless steel column25 cm × 4.6 mm, packed with octadecylsi 150 mm × 4.6 mm, particle lyl silica gel, 5 µm size 5 µm 0.5 mL/min Mobile phase Flow rate Isocratic Elution Volume required to analyze six samples About 500 mL Reagentt Easily available HPLC– grade reagent Sample preparation – About 20 min Total time consumed for – Approx 1.5 h per sample, six analysis injections each
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IP pharmacopeial method LAM = 100.68 TDF = 100.21 EFV = 100.18 LAM = 1.21 TDF = 0.98 EFV = 0.74 Stainless steel column 25 cm × 4.6 mm, packed with octadecylsilyl silica gel, 5 µm 1 mL/min Gradient About 1000 mL Buffer solution should be prepared About 1 h 15 min Approx 4.5 h per sample, eighteen injections each
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