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Determination of lamivudine and zidovudine in binary mixtures using first derivative spectrophotometric, first derivative of the ratio-spectra and high-performance liquid chromatography–UV methods
Analytica Chimica Acta 466 (2002) 175–185
Determination of lamivudine and zidovudine in binary mixtures using first derivative spectrophotometric, first derivative of the ratio-spectra and high-performance liquid chromatography–UV methods Bengi Uslu, Sibel A. Özkan∗ Department of Analytical Chemistry, Faculty of Pharmacy, Ankara University, 06100 Ankara, Turkey Received 20 February 2002; received in revised form 22 May 2002; accepted 10 June 2002
Abstract Three methods are presented for the simultaneous determination of lamivudine and zidovudine. The first method depends on first derivative UV spectrophotometry, with zero-crossing and peak-to-base measurement. The first derivative amplitudes at 265.6 and 271.6 nm were selected for the assay of lamivudine and zidovudine, respectively. The second method depends on first derivative of the ratio-spectra by measurements of the amplitudes at 239.5 and 245.3 nm for lamivudine and 225.1 and 251.5 nm for zidovudine. Calibration graphs were established for 1–50 g/ml for lamivudine and 2–100 g/ml for zidovudine. In the third method (HPLC), a reversed-phase column with a mobile phase of methanol:water:acetonitrile (70:20:10 (v/v/v)) at 0.9 ml/min flow rate was used to separate both compounds with a detection of 265.0 nm. Linearity was obtained in the concentration range of 0.025–50 g/ml for lamivudine and 0.15–50 g/ml for zidovudine. All of the proposed methods have been extensively validated. These methods allow a number of cost and time saving benefits. The described methods can be readily utilized for analysis of pharmaceutical formulations. There was no significant difference between the performance of all of the proposed methods regarding the mean values and standard deviations. The described HPLC method showed to be appropriate for simultaneous determination of lamivudine and zidovudine in human serum samples. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Lamivudine; Zidovudine; First derivative spectrophotometry; Ratio derivative spectrophotometry; HPLC; Pharmaceuticals; Human serum
1. Introduction Lamivudine and zidovudine are synthetic nucleoside analogues with activity against human immunodeficiency virus (HIV). Lamivudine was initially developed for the treatment of HIV infection. The chemical name of lamivudine is (2R,cis)-4-amino-1-
∗ Corresponding author. Fax: +90-312-2238243. E-mail address: [email protected] (S.A. Özkan).
(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one. Lamivudine the (−) enantiomer of 2 deoxy-3 -thiacytidine, is a nucleoside analog in which the 3 carbon of the ribose of zalcitabine has been replaced by sulfur. The (−) enantiomer of the racemic mixture shows much less cytotoxicity than the positive enantiomer. Although, generally less potent than zidovudine in inhibiting HIV-1 and -2 replication in vitro, lamivudine has very low cellular cytotoxicity. It is rapidly absorbed with bioavailability of approximately 80% [1,2].
0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 0 5 4 5 - 7
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Fig. 1. The structural formulae of lamivudine and zidovudine.
Zidovudine is a nucleoside analogue structurally similar to thymidine. It is used in the management of AIDS and AIDS-related complex and may be given to symptomatic patients with early HIV infection or to symptomatic patients [3]. It is rapidly absorbed from the gastro-intestinal tract with a bioavailability of about 60–70%. It crosses the blood–brain barrier. The plasma half-life is about 1 h. In HIV-1 infected MT-4 cells, lamivudine in combination with zidovudine had synergistic antiretroviral activity (Fig. 1). There have been several high-performance liquid chromatographic (HPLC) method reports of the determination of zidovudine in biological samples [4–17]. Very few methods appeared in the literature for the determination of lamivudine based on HPLC in biological fluids [18–22]. Only three HPLC-tandem mass spectrometric methods have been described in the literature for the simultaneous determination of lamivudine and zidovudine in biological samples [23–25]. The reported methods require solid-phase extraction or expensive equipments, which are not economically feasible for routine use in pharmacokinetic and pharmaceutical studies, where numerous samples should be analyzed. No references were found to the simultaneous determination of lamivudine and zidovudine using spectrophotometric methods. Also, no method has been reported in literatures so far for the determination of lamivudine and zidovudine in drug dosage forms. In recent years, derivative spectrophotometry has been found to be a useful method in the determination of mixtures with two or more components having
overlapping spectra and in eliminating interference from formulation matrix by using the zero-crossing technique [26–28]. Furthermore, ratio-spectra derivative spectrophotometric method [29–31] has also been found to be useful in the estimation of drugs from their mixtures. The use of the zero-crossing method in derivative spectrophotometry for resolving a mixture of compounds with overlapped spectra produces a considerable loss of accuracy and sensitivity. This problem may be due to the fact that the measurements are taken at a very critical wavelength, the localization of which is sometimes very difficult and whose value is sometimes very small for obtaining a good analysis. Fortunately, in the present case, the above circumstances did not occur. A spectrophotometric procedure for resolving mixture, named “ratio-spectra” derivative spectrophotometry has recently been developed [32,33]. This method permits the determination of a component in their mixture at the wavelengths corresponding to a maximum or minimum and also the use of the peak-topeak between consecutive maximum and minimum. The main advantage of derivative of the ratio-spectra method may be the chance of doing easy measurements in correspondence of peaks so it permits the use of the wavelength of highest value of analytical signals (maximum or minimum). Moreover, the presence of a lot of maxima and minima is another advantage by the fact that these wavelengths give an opportunity for the determination of active compounds in the presence of other active compounds and excipients which possibly interfere the analysis. HPLC methods are useful in the determination of drugs in pharmaceutical dosage forms and biological sample. Owing to the widespread use of HPLC in routine analysis, it is important that good HPLC methods are developed and that these are thoroughly validated [34–36]. The purpose of the present study was to investigate the utility of derivative and ratio derivative spectrophotometry and HPLC in the assay of lamivudine and zidovudine in combination in the pharmaceutical preparations without the necessity of sample pre-treatment. We also performed a study concerning the simultaneous determination of these compounds in human serum samples using HPLC technique. Three methods were developed and validated in this work: a first derivative spectrophotometric of the ratio-spectra
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and gradient HPLC methods for the simultaneous determination of lamivudine and zidovudine. The methods had sufficiently good accuracy, precision and permitted a simple, time- and money-saving assay of these compounds in mixtures. 2. Experimental 2.1. Apparatus and conditions The Hewlett-Packard liquid chromatographic system consisted of a gradient Quat pump Model G 1311 A (HP, Avondale, PA, USA) connected with a HP G 1314 A UV–VIS detector (HP, Avondale, PA, USA) operating at 265 nm, a G 1328 A (Cotati, CA) injection valve, with a 20 l loop. The chromatographic data was collected and analyzed using HP Chem Station for LC and LC/MS system (Hewlett-Packard, Avondale, PA, USA). The chromatographic separation was performed at ambient temperature (20–22 ◦ C) using an analytical column, Spherisorb® , 5 m, 4.6 × 150 mm i.d., (Waters, Milford, MA, USA). The reversed mobile phase was obtained by mixture of methanol:water:acetonitrile (70:20:10 (v/v/v)). The flow rate was 0.9 ml/min. Finasteride was used as an internal standard. An amount of 20 l of each solution was injected and chromatograms were recorded. A Shimadzu 1601 PC double beam spectrophotometer equipped with 1.0 cm quartz cells with a fixed slit width (2 nm) was used, coupled an IBM-PC computer running spectrophotometric software Schimadzu UVPC software. 2.2. Chemicals and reagents All chemicals and solvents were of analytical-reagent grade. Lamivudine and zidovudine and its pharmaceutical dosage form were kindly provided by the Glaxo-Smith-Kline Pharm. Ind. (Istanbul, Turkey), finasteride used, as the internal standard was kindly supplied from Nobel Pharm. Ind. (Istanbul, Turkey). HPLC grade methanol (Merck, Darmstadt, Germany) and doubly distilled water was used for preparing mobile phase solutions. For spectrophotometric studies, methanol was purchased from Merck (Darmstadt, Germany).
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2.3. Preparation of standard solutions and calibration Stock standard solutions of each of lamivudine and zidovudine were prepared separately by dissolving 10 mg of each drug in 10 ml methanol. The standard solutions were prepared individually by dilution of the stock solutions with methanol for spectrophotometric experiments and with mobile phase for chromatographic experiments to reach concentration range of 1–50 g/ml for lamivudine and 2–100 g/ml for zidovudine and 0.025–50 g/ml for lamivudine and 0.15–50 g/ml for zidovudine, respectively. 2.3.1. For derivative spectrophotometric method (D1 ) The values of the D1 amplitudes were measured at 265.6 nm (zero-crossing of zidovudine) and 271.6 nm (zero-crossing of lamivudine) for the determination of lamivudine and zidovudine, respectively. 2.3.2. For first derivative of the ratio spectrophotometric method (DD1 ) According to the theory of the ratio-spectra derivative method [35,36], for lamivudine; the stored UV absorption spectra of standard solutions of lamivudine were divided wavelength-by-wavelength by a standard spectrum of zidovudine (20 g/ml). The first derivative was calculated for the obtained spectra with λ = 4 nm. The amplitudes at 239.5 and 245.3 nm were measured and found to be linear to the concentration of lamivudine. For zidovudine, the stored UV absorption spectra of standard solutions of zidovudine were divided wavelength-by-wavelength by a standard spectrum of lamivudine (15 g/ml). The first derivative was calculated for the obtained spectra with λ = 4 nm. The amplitudes at 225.1 and 251.5 nm were measured and found to be linear to the concentration of zidovudine. 2.3.3. For high-performance liquid chromatographic method Standard solutions were prepared separately with mobile phase by varying concentrations of lamivudine and zidovudine in the range of 0.025–50 and 0.15–50 g/ml, respectively. Maintaining concentration of finasteride (IS) at a constant level of 25 g/ml.
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Table 1 Statistical data for the calibration graphs of lamivudine (LMV) and zidovudine (ZDV) by D1 , DD1 and HPLC methods LMV
Linearity range (g/ml) Slope Intercept Correlation coefficient
HPLC
D1
DD1
265.6 nm
239.5 nm
245.3 nm
1–50 6.8 × 10−4 −2.3 × 10−4 0.9989
1–50 0.011 −0.0034 0.9996
1–50 0.007 −0.0006 0.9993
0.025–50 0.617 0.107 0.9999
ZDV
HPLC
D1
DD1
271.6 nm
225.1 nm
251.5 nm
2–100 5.3 × 10−4 −9.1 × 10−4 0.9994
2–100 0.003 −0.0014 0.9998
2–100 0.001 −0.0015 0.9990
0.15–50 0.178 0.038 0.9998
R.S.D. of the slope R.S.D. of the intercept
0.39 0.66
0.38 0.03
0.71 0.26
0.08 0.55
0.09 0.06
0.51 0.18
0.50 0.10
0.17 0.85
LOD (g/ml) LOQ (g/ml)
0.25 0.85
0.026 0.087
0.05 0.17
0.00098 0.0033
0.56 1.89
0.13 0.44
0.26 0.86
0.0049 0.016
Repeatabilitya (R.S.D.; %) Reproducibilitya (R.S.D.; %)
0.17 0.47
0.62 0.83
0.51 0.74
0.70 0.83
0.20 0.61
0.32 0.45
0.48 0.61
0.93 0.96
a
Each value is obtained from four experiments.
Triplicate 10 l injections were made for each solution and peak area ratio of each concentration to the internal standard was plotted against the corresponding concentration to obtain the calibration graph. All of the proposed methods were validated as to precision (reported as the relative standard deviation, R.S.D. (%)), linearity (evaluated by regression equations), detection and determination limits and accuracy. The limit of detection (LOD) and limit of quantitation (LOQ) of the procedure are also as shown in Table 1, which were calculated according to the 3 s/m and 10 s/m criterions, respectively, where s, is the standard deviation of the absorbance (n = 4) of the sample and m is the slope of the corresponding calibration curve. The ruggedness and precision were checked in the same and different days. The R.S.D. (%) was calculated to check the ruggedness and precision of the methods. Accuracy was determined by recovery studies. 2.4. Procedure for tablets For all methods, 10 tablets labeled to contain 150.0 mg of lamivudine and 300.0 mg of zidovudine and excipients were weighed and finely powdered. An accurate weight of the powder equivalent to one tablet content was accurately weighed, transferred into a 100 ml calibrated flask, diluted with methanol, stirred for about 10 min and then completed to volume with
the same solution. This solution was filtered to separate any insoluble matter. The filtrate was collected in a clean flask. After filtration, appropriate solutions were prepared by taking suitable aliquots of clear filtrate (for HPLC study, adding of the constant amount of internal standard) and diluting with selected solvent in order to obtain a final solution. The assay was completed as described under Section 2.3. The contents amount of lamivudine and zidovudine were calculated from the corresponding regression equations. 2.5. Percent recovery study To study the accuracy of the proposed methods, and to check the interference from excipients used in the dosage forms, recovery experiments were carried out by the standard addition method. This study was performed by addition of known amounts of lamivudine and zidovudine to a known concentration of the commercial tablets. The resulting mixtures were analyzed as described under Section 2.3. 2.6. Recovery studies in human serum by HPLC Serum sample, obtained from healthy individuals (after obtaining their written consent), were stored frozen until assay. After gentle, thawing aliquots of serum were spiked with lamivudine and zidovudine dissolved in methanol to achieve final concentration
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of 500 and 1000 g/ml, respectively and treated with 500 l acetonitrile as serum protein precipitating agent, then the volume was completed to 2 ml with the same serum sample. The tubes were vortexed for 5 min at 1500 g and then centrifuged for 10 min at 5000 × g for getting rid of protein residues. The supernatant was taken carefully, serum samples including various concentrations of lamivudine and zidovudine and constant amount of internal standard were injected into the column.
3. Results and discussion 3.1. D1 method As shown in Fig. 2, the zero-order spectra of pure drugs were found to be overlapping, making simultaneous determination difficult. It can be seen that the absorption spectra of lamivudine and zidovudine are very overlapped and as a result, the determination of the two drugs cannot be possible for reliable direct absorbance measurements. In contrast, the D1 spectra of each pure drug was found to show zero-crossing points (Fig. 3) and assisted in their simultaneous estimation. In practice, the measurement selected is that which exhibits the best linear response, gives a zero or near zero intercept on the coordinate of the calibration graph, and is less affected by the concentration of any other component. The shape of the first derivative
Fig. 3. First-derivative spectra of lamivudine (1) 15 g/ml; (2) 20 g/ml; (3) 30 g/ml (continuous lines) and zidovudine (1) 30 g/ml; (2) 50 g/ml; (3) 75 g/ml (dashed lines) in methanol. The arrows indicate the working wavelengths.
spectra is adequate for determining lamivudine in the presence of zidovudine and vice versa. Lamivudine was determined by measurement of its D1 amplitude at the zero-crossing point of zidovudine (at 265.6 nm). While zidovudine was determined by measurement of its D1 at the zero-crossing point of lamivudine (at 271.6 nm). Linear relationships between derivative amplitude and drug concentration were obtained over the concentration range of 1–50 g/ml for lamivudine and 2–100 g/ml for zidovudine. The linear regression equations together with correlation coefficients, slope and intercept, R.S.D. of slope and intercept repeatability (withinday) and reproducibility (between-day) obtained for each drug are as shown in Table 1. Thus, it was evident from the values obtained that the intercept values did not differ significantly from zero in each case. 3.2. DD1 method
Fig. 2. Absorbtion spectra of: (a) lamivudine (15 g/ml), (b) zidovudine (30 g/ml), and (c) a mixture of lamivudine and zidovudine in methanol.
To optimize the simultaneous determination of lamivudine and zidovudine by using DD1 method, it is necessary to test the influence of the variables: divisor standard concentration, λ and smoothing function. All these variables were studied. This influence of λ for obtaining the first derivative of the ratio-spectra was tested and λ = 4 nm was selected as optimum value. An accurate choice of divisor standard concentration is fundamental for several reasons [35,37], hence, we tested the methods with various
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divisor concentrations. The results of all the tests are not shown for the sake of brevity and because these do not add to the scientific value of the work. A standard spectrum of 20 g/ml of zidovudine was considered as suitable for the lamivudine determination and a standard spectrum of 15 g/ml of lamivudine as divisor was considered as suitable for the zidovudine determination. In Fig. 4a, two series of the ratio-spectra of lamivudine/zidovudine and zidovudine/lamivudine was shown. In Fig. 4b, the corresponding first derivative of the ratio-spectra of Fig. 4a was shown. For calibration graph, the wavelengths were selected which exhibited the best linear response to the analyte concentration, i.e. in the first derivative mode 239.5 and 245.3 nm for lamivudine and 225.1 and 251.5 nm for
zidovudine. The calibration graphs of each drug at both wavelengths were achieved by plotting the values of the first derivative of the ratio-spectra lamivudine/zidovudine and zidovudine/lamivudine, with variable concentrations of lamivudine and zidovudine. The proposed method is applicable over the ranges 1–50 g/ml for lamivudine and 2–100 g/ml for zidovudine. The characteristic parameters and necessary statistical data of the regression equations, LOD and LOQ values, repeatability and reproducibility data are compiled in Table 1. The LOD and LOQ values were calculated as described in the previous section. Repeatability and reproducibility variabilities were characterized by R.S.D. (%) and by the difference between theoretical and measured concentrations. There was no significant difference for the assay, which was tested within-day (repeatability) and between-days (reproducibility). In order to demonstrate the validity and applicability of the proposed D1 and DD1 methods, recovery studies were performed by analyzing in synthetic mixtures of lamivudine and zidovudine, which reproduced different composition ratios (Table 2). 3.3. High-performance liquid chromatographic method
Fig. 4. (a) Ratio-spectra and (b) first derivative of the ratio-spectra for different concentrations of lamivudine (15, 20, 30 g/ml; continuous lines, 1–3; divisor zidovudine, 20 g/ml) and zidovudine (30, 50, 75 g/ml; dashed lines, 1–3; divisor lamivudine, 15 g/ml). The arrow indicate the working wavelengths.
Drug analysis is undertaken during various phases of pharmaceutical development, such as formulation and stability studies, quality control and pharmacological testing in animals and humans. All these investigations require reliable and validated analytical methods in order to measure drugs in pharmaceutical formulations and biological samples. In order to effect the simultaneous elution of lamivudine and zidovudine peaks under gradient conditions, the mixtures of methanol, acetonitrile and water in different combinations at various flow rates were assayed. The optimum wavelength for detection was 265 nm at which much better detector responses for both drugs were obtained. The mixture of methanol: water:acetonitrile (70:20:10 (v/v/v)) at 0.9 ml/min flow rate, proved to be better than the other mixtures and flow rates for the separation, since the chromatographic peaks were better defined, resolved and free from tailing. As shown in Fig. 5, the retention times were 2.06 min for lamivudine, 3.36 min for zidovudine and 4.32 min for finasteride (IS).
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Table 2 Determination of lamivudine (LMV) and zidovudine (ZDV) in laboratory prepared mixtures using first derivative (D1 ) and first derivative of the ratio-spectra (DD1 ) Added (g/ml) LMV
ZDV
Found 5 15 25 30 50
(g/ml) 20 20 20 20 20
D1 LMV (265.6 nm)
DD1 ZDV (271.6 nm)
LMV (239.5 nm)
LMV (245.3 nm)
ZDV (225.1 nm)
ZDV (251.5 nm)
4.92 14.97 24.96 30.01 49.85
29.50 29.68 29.75 30.00 29.90
4.90 14.90 25.01 29.85 49.80
4.95 14.95 25.00 29.90 49.90
19.85 19.85 19.85 19.85 19.90
20.00 19.90 19.90 19.90 19.70
98.40 99.80 99.80 100.03 99.70
98.33 98.93 99.16 100.00 99.67
98.00 99.30 100.04 99.50 99.60
99.00 99.70 100.00 99.70 99.80
99.25 99.25 99.25 99.25 99.50
100.00 99.50 99.50 99.50 98.50
Mean recovery (%) R.S.D. (%)
99.55 0.65
99.22 0.66
99.29 0.78
99.64 0.38
99.30 0.11
99.40 0.55
Found 15 15 15 15 15
15.04 15.04 14.75 14.90 15.04
4.99 10.01 29.99 50.00 74.90
15.00 15.01 14.95 14.90 15.01
15.01 14.95 14.90 14.95 15.00
4.95 9.83 29.50 49.40 74.60
4.97 9.85 29.73 49.00 74.80
100.30 100.30 98.33 99.33 100.30
99.8 100.10 99.97 100.00 99.87
100.00 100.07 99.67 99.33 100.07
100.07 99.67 99.33 99.67 100.00
99.00 98.30 98.33 98.80 99.47
99.40 98.50 99.10 98.00 99.73
99.71 0.88
99.95 0.12
99.83 0.33
99.75 0.30
98.78 0.50
98.95 0.71
Recovery (%) 5 20 15 20 25 20 30 20 50 20
(g/ml) 5 10 30 50 75
Recovery (%) 15 5 15 10 15 30 15 50 15 75 Mean recovery (%) R.S.D. (%)
In HPLC methods, precision and accuracy can often be enhanced by the use of an appropriate internal standard, which also serves to correct for fluctuations in the detector response. Ideally, an internal standard should display similar physico-chemical properties to the analytes. But it did not obtained good resolution and peak shape with the similar compounds. The structure of finasteride is not similar to lamivudine and zidovudine. However, it was chosen as the internal standard because it showed a shorter retention time with better peak shapes and better resolution, compared to other potential internal standards. System suitability tests are an integral part of a liquid chromatographic method. System suitability tests are used to verify that the resolution and reproduci-
bility of the chromatographic system are adequate for the analysis to be done. System suitability tests were carried out according to USP 24, method 621 [38] on the chromatogram of freshly prepared standard solutions to check various parameters, such as resolution, selectivity, tailing and capacity factors. Resolution and selectivity factors for this system were found 2.17 and 2.48, respectively. The tailing and capacity factors were obtained as 1.11 and 0.75 for lamivudine and 1.33 and 1.85 for zidovudine, respectively. The variation in retention times among six replicate injections of lamivudine and zidovudine standard solutions was very low, rendering a R.S.D. of 0.98 and 0.62%, respectively. The results obtained from system suitability tests are in agreement with the USP requirements.
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B. Uslu, S.A. Özkan / Analytica Chimica Acta 466 (2002) 175–185 Table 3 Resolution of lamivudine (LMV) and zidovudine (ZDV) laboratory-made mixtures using by the HPLC technique Added (g/ml)
Found (g/ml)
Recovery (%)
LMV
ZDV
LMV
ZDV
LMV
ZDV
1 2.5 5 10 25
10 10 10 10 10
0.99 2.5 5.02 9.95 24.92
10.04 10.00 10.00 10.14 10.03
99.00 100.00 100.40 99.50 99.68
100.40 100.00 100.00 101.40 100.30
99.72 0.53
100.42 0.57
100.00 99.00 99.00 98.00 99.00
100.40 100.40 100.30 99.32 99.62
99.00 0.71
100.01 0.50
Mean recovery (%) R.S.D. (%) 1 1 1 1 1
2.5 5 10 25 50
1.00 0.99 0.99 0.98 0.99
Mean recovery (%) R.S.D. (%)
2.51 5.02 10.03 24.83 49.81
carried out by analyzing in the synthetic mixtures of lamivudine and zidovudine, which reproduced different composition ratios (Table 3). Fig. 5. Chromatogram obtained from tablet dosage forms containing: (1) 2.5 g/ml lamivudine, (2) 5.0 g/ml zidovudine, and (3) 25 g/ml finasteride (IS).
Peak area ratios (Asample /AIS ) were plotted against corresponding concentrations in the range of 0.025– 50 g/ml for lamivudine and 0.15–50 g/ml for zidovudine. Linear regression parameters of the peak area ratios versus concentrations of lamivudine and zidovudine are presented in Table 1. The results showed highly reproducible calibration curves with correlation coefficients of >0.999. Necessary statistical data of the regression equations, such as LOD value, LOQ value, repeatability and reproducibility data was also as shown in Table 1. The LOD and LOQ values were calculated as described in Section 3.1. Repeatability and reproducibility variabilities were characterized by R.S.D. (%) (Table 1). According to this results, there was no significant difference for the assay, which was tested within-day (repeatability) and between-days (reproducibility; Table 1). In order to demonstrate the validity and applicability of the proposed HPLC method, recovery tests were
3.4. Analysis of tablets When working on synthetic mixture, results encourage the use of the all proposed methods described for the assay of lamivudine and zidovudine in commercial tablet dosage forms. The three proposed methods could be used for the simultaneous determination of lamivudine and zidovudine in the presence of each other and without prior separation of the excipients. Each film-coated tablet contains 150 mg of lamivudine, 300 mg of zidovudine, and the inactive ingredients colloidal silicon dioxide, magnesium stearate, microcrystalline cellulose and sodium starch glycolate. The film-coating solution contains Opadry YS-1-7706-G White and purified water. Fig. 5 shows a typical chromatogram obtained follow by analysis of lamivudine and zidovudine in tablets. As shown in Fig. 5, the substances were eluted, forming well shaped, symmetrical single peaks, well separated from the solvent front. No interfering peaks were obtained in the chromatogram due to tablet excipients. The utility of all of the proposed methods was verified by means of replicate estimations of
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Table 4 Results of the determination and the recovery analysis of lamivudine (LMV) and zidovudine (ZDV) in commercial tablet dosage forms using first derivative (D1 ), first derivative of the ratio-spectra (DD1 ) and HPLC methods HPLC
Labelled claim (mg) Amount found (mg)a R.S.D. (%) Calculated t Calculated F Recoveryc (%) R.S.D. (%)
D1
DD1
LMV
ZDV
LMV (265.6 nm)
ZDV (271.6 nm)
LMV (239.5 nm)
LMV (245.3 nm)
ZDV (225.1 nm)
ZDV (251.5 nm)
150.00 149.81 0.16 2.31b 2.60b 99.54 0.31
300.00 299.90 0.25 2.31b 2.60b 99.63 0.08
150.00 148.98 0.69 0.79 0.45 99.22 0.17
300.00 299.10 0.57 0.34 0.14 99.87 0.18
150.00 148.04 0.72 0.20 0.49 99.38 0.07
150.00 149.50 0.20 0.69 0.008 99.88 0.12
300.00 298.60 0.37 0.004 0.28 99.56 0.04
300.00 297.30 0.44 0.0005 0.48 99.24 0.26
a
Mean value of the five determinations. Theoretical values for t- and F-tests. c Mean value of the four determinations. b
pharmaceutical preparations and results obtained were evaluated statistically. Table 4 shows the results obtained in the analysis of tablets for spectrophotometric and HPLC methods. Results obtained from proposed methods of the analysis of both drugs in tablets indicate that the proposed techniques can be used for simultaneous quantitation and routine quality control analysis of this binary mixture in pharmaceuticals. A comparison with an official reference determination method has not been possible in any pharmacopoeias and literature, because so far no other procedure for the quantitation of lamivudine and zidovudine from pharmaceutical formulations has been reported. Proposed HPLC results were compared with the spectrophotometric results. According to the student’s t-test and variance ratio F-test, the calculated t- and F-values were less than the theoretical values in either test at the 95% confidence level. This indicates that there is no significant difference between the performance all of the proposed methods as regards to mean values and standard deviations (Table 4). Recovery studies were realized from the tablets for accuracy and precision of the proposed techniques. The recovery of the procedure was carried out by spiking the already analyzed samples of tablets with the known concentrations of standard solutions of lamivudine and zidovudine. The results of the recovery analysis for all techniques are tabulated as shown in Table 4. It is concluded that the proposed methods are sufficiently accurate and precise in order
to be applied to pharmaceutical dosage forms. High percentage recovery data shows that all of the proposed methods are free from the interferences of the excipients used in the formulations. 3.5. Application to the serum samples In order to check the applicability of the proposed HPLC method to biological materials, the recovery studies were performed in human serum samples. Analysis of drugs from serum by HPLC usually requires extensive time-consuming sample preparation, use of expensive organic solvent and other chemicals [23–25]. In our proposed method, the serum proteins are precipitated by the addition of acetonitrile, which is centrifuged at 5000 g, and the supernatant is diluted, directly injected and analyzed. Fig. 6 shows the typical chromatogram obtained the serum spiked with lamivudine, zidovudine and IS (a) and blank serum (b), which indicate no interference from the endogenous substances present in serum. Serum samples were spiked with two different concentrations of lamivudine and zidovudine and constant level of IS. The determined results and recoveries of known amounts of lamivudine and zidovudine added to serum samples are given in Table 5. The proposed method gives reproducible results, is easy to perform and is sensitive enough to simultaneous determination of lamivudine and zidovudine in human serum samples (Table 5).
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Fig. 6. Chromatogram of: (a) serum spiked with (1) 2.0 g/ml of lamivudine, (2) 10.0 g/ml of zidovudine, (3) 25 g/ml finasteride (IS) and (b) blank serum.
Table 5 Results obtained for lamivudine (LMV) and zidovudine (ZDV) analysis from human serum using HPLC method LMV Added (g/ml) n Found (g/ml) R.S.D. (%) Average recovery (%)
2.00 4 1.98 1.04 99.20
ZDV 10.00 4 9.81 0.7 98.07
10.00 4 9.78 0.84 97.80
30.00 4 29.38 0.94 97.93
4. Conclusion The D1 and DD1 and HPLC methods enable the quantitation or mixtures of lamivudine and zidovudine with good accuracy and precision, either in laboratory made samples or in pharmaceutical dosage forms. From a comparison of the results obtained with the three methods, they do not appear to have sub-
stantial differences. A little superiority of the HPLC method over the spectrophotometric techniques in the simultaneous determination of lamivudine and zidovudine, results from the limits of detection, the limits of determination and correlation coefficients in Table 1. All of these procedures are rapid, precise and work without solving equations or separation steps. The D1 method is more rapid and simple than the DD1 method, while the proposed DD1 method has greater sensitivity and accuracy. The advantages of the ratio-spectra method proposed over the zero-crossing derivative method, is the possibility of performing measurements in correspondence of peaks, hence, a potentially greater sensitivity and accuracy. Another advantages of the DD1 assay are easy measurement on the separate peaks, higher values of analytical signals and no need to work only at zero-crossing point in comparison with the derivative spectrophotometric method. In this study, proposed HPLC method was especially used as a versatile reference method and could be also satisfactory for biological fluids since its high separation power. The proposed HPLC method gives a good resolution between lamivudine, zidovudine and internal standard within a short analysis time. The present HPLC study purposes a rapid, simple, sufficiently precise and accurate method for the simultaneous determination of lamivudine and zidovudine in raw material, pharmaceutical formulations and human serum. The proposed method used a simple serum deproteination step instead of extraction. No interferences from endogenous substances were observed in biological samples. The three proposed methods are suitable for quality control laboratories, where economy and time are essential. High percentage recovery shows that the methods are free from the interferences of the commonly used excipients and additives in the formulations of drugs.
Acknowledgements The authors greatfully acknowledge Glaxo-SmithKline Pharm. Ind. for supplying lamivudine, zidovudine and pharmaceutical preparations.
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Determination of lamivudine and zidovudine in binary mixtures using first derivative spectrophotometric, first derivative of the ratio-spectra and high-performance liquid chromatography–UV methods Bengi Uslu, Sibel A. Özkan∗ Department of Analytical Chemistry, Faculty of Pharmacy, Ankara University, 06100 Ankara, Turkey Received 20 February 2002; received in revised form 22 May 2002; accepted 10 June 2002
Abstract Three methods are presented for the simultaneous determination of lamivudine and zidovudine. The first method depends on first derivative UV spectrophotometry, with zero-crossing and peak-to-base measurement. The first derivative amplitudes at 265.6 and 271.6 nm were selected for the assay of lamivudine and zidovudine, respectively. The second method depends on first derivative of the ratio-spectra by measurements of the amplitudes at 239.5 and 245.3 nm for lamivudine and 225.1 and 251.5 nm for zidovudine. Calibration graphs were established for 1–50 g/ml for lamivudine and 2–100 g/ml for zidovudine. In the third method (HPLC), a reversed-phase column with a mobile phase of methanol:water:acetonitrile (70:20:10 (v/v/v)) at 0.9 ml/min flow rate was used to separate both compounds with a detection of 265.0 nm. Linearity was obtained in the concentration range of 0.025–50 g/ml for lamivudine and 0.15–50 g/ml for zidovudine. All of the proposed methods have been extensively validated. These methods allow a number of cost and time saving benefits. The described methods can be readily utilized for analysis of pharmaceutical formulations. There was no significant difference between the performance of all of the proposed methods regarding the mean values and standard deviations. The described HPLC method showed to be appropriate for simultaneous determination of lamivudine and zidovudine in human serum samples. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Lamivudine; Zidovudine; First derivative spectrophotometry; Ratio derivative spectrophotometry; HPLC; Pharmaceuticals; Human serum
1. Introduction Lamivudine and zidovudine are synthetic nucleoside analogues with activity against human immunodeficiency virus (HIV). Lamivudine was initially developed for the treatment of HIV infection. The chemical name of lamivudine is (2R,cis)-4-amino-1-
∗ Corresponding author. Fax: +90-312-2238243. E-mail address: [email protected] (S.A. Özkan).
(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one. Lamivudine the (−) enantiomer of 2 deoxy-3 -thiacytidine, is a nucleoside analog in which the 3 carbon of the ribose of zalcitabine has been replaced by sulfur. The (−) enantiomer of the racemic mixture shows much less cytotoxicity than the positive enantiomer. Although, generally less potent than zidovudine in inhibiting HIV-1 and -2 replication in vitro, lamivudine has very low cellular cytotoxicity. It is rapidly absorbed with bioavailability of approximately 80% [1,2].
0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 0 5 4 5 - 7
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Fig. 1. The structural formulae of lamivudine and zidovudine.
Zidovudine is a nucleoside analogue structurally similar to thymidine. It is used in the management of AIDS and AIDS-related complex and may be given to symptomatic patients with early HIV infection or to symptomatic patients [3]. It is rapidly absorbed from the gastro-intestinal tract with a bioavailability of about 60–70%. It crosses the blood–brain barrier. The plasma half-life is about 1 h. In HIV-1 infected MT-4 cells, lamivudine in combination with zidovudine had synergistic antiretroviral activity (Fig. 1). There have been several high-performance liquid chromatographic (HPLC) method reports of the determination of zidovudine in biological samples [4–17]. Very few methods appeared in the literature for the determination of lamivudine based on HPLC in biological fluids [18–22]. Only three HPLC-tandem mass spectrometric methods have been described in the literature for the simultaneous determination of lamivudine and zidovudine in biological samples [23–25]. The reported methods require solid-phase extraction or expensive equipments, which are not economically feasible for routine use in pharmacokinetic and pharmaceutical studies, where numerous samples should be analyzed. No references were found to the simultaneous determination of lamivudine and zidovudine using spectrophotometric methods. Also, no method has been reported in literatures so far for the determination of lamivudine and zidovudine in drug dosage forms. In recent years, derivative spectrophotometry has been found to be a useful method in the determination of mixtures with two or more components having
overlapping spectra and in eliminating interference from formulation matrix by using the zero-crossing technique [26–28]. Furthermore, ratio-spectra derivative spectrophotometric method [29–31] has also been found to be useful in the estimation of drugs from their mixtures. The use of the zero-crossing method in derivative spectrophotometry for resolving a mixture of compounds with overlapped spectra produces a considerable loss of accuracy and sensitivity. This problem may be due to the fact that the measurements are taken at a very critical wavelength, the localization of which is sometimes very difficult and whose value is sometimes very small for obtaining a good analysis. Fortunately, in the present case, the above circumstances did not occur. A spectrophotometric procedure for resolving mixture, named “ratio-spectra” derivative spectrophotometry has recently been developed [32,33]. This method permits the determination of a component in their mixture at the wavelengths corresponding to a maximum or minimum and also the use of the peak-topeak between consecutive maximum and minimum. The main advantage of derivative of the ratio-spectra method may be the chance of doing easy measurements in correspondence of peaks so it permits the use of the wavelength of highest value of analytical signals (maximum or minimum). Moreover, the presence of a lot of maxima and minima is another advantage by the fact that these wavelengths give an opportunity for the determination of active compounds in the presence of other active compounds and excipients which possibly interfere the analysis. HPLC methods are useful in the determination of drugs in pharmaceutical dosage forms and biological sample. Owing to the widespread use of HPLC in routine analysis, it is important that good HPLC methods are developed and that these are thoroughly validated [34–36]. The purpose of the present study was to investigate the utility of derivative and ratio derivative spectrophotometry and HPLC in the assay of lamivudine and zidovudine in combination in the pharmaceutical preparations without the necessity of sample pre-treatment. We also performed a study concerning the simultaneous determination of these compounds in human serum samples using HPLC technique. Three methods were developed and validated in this work: a first derivative spectrophotometric of the ratio-spectra
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and gradient HPLC methods for the simultaneous determination of lamivudine and zidovudine. The methods had sufficiently good accuracy, precision and permitted a simple, time- and money-saving assay of these compounds in mixtures. 2. Experimental 2.1. Apparatus and conditions The Hewlett-Packard liquid chromatographic system consisted of a gradient Quat pump Model G 1311 A (HP, Avondale, PA, USA) connected with a HP G 1314 A UV–VIS detector (HP, Avondale, PA, USA) operating at 265 nm, a G 1328 A (Cotati, CA) injection valve, with a 20 l loop. The chromatographic data was collected and analyzed using HP Chem Station for LC and LC/MS system (Hewlett-Packard, Avondale, PA, USA). The chromatographic separation was performed at ambient temperature (20–22 ◦ C) using an analytical column, Spherisorb® , 5 m, 4.6 × 150 mm i.d., (Waters, Milford, MA, USA). The reversed mobile phase was obtained by mixture of methanol:water:acetonitrile (70:20:10 (v/v/v)). The flow rate was 0.9 ml/min. Finasteride was used as an internal standard. An amount of 20 l of each solution was injected and chromatograms were recorded. A Shimadzu 1601 PC double beam spectrophotometer equipped with 1.0 cm quartz cells with a fixed slit width (2 nm) was used, coupled an IBM-PC computer running spectrophotometric software Schimadzu UVPC software. 2.2. Chemicals and reagents All chemicals and solvents were of analytical-reagent grade. Lamivudine and zidovudine and its pharmaceutical dosage form were kindly provided by the Glaxo-Smith-Kline Pharm. Ind. (Istanbul, Turkey), finasteride used, as the internal standard was kindly supplied from Nobel Pharm. Ind. (Istanbul, Turkey). HPLC grade methanol (Merck, Darmstadt, Germany) and doubly distilled water was used for preparing mobile phase solutions. For spectrophotometric studies, methanol was purchased from Merck (Darmstadt, Germany).
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2.3. Preparation of standard solutions and calibration Stock standard solutions of each of lamivudine and zidovudine were prepared separately by dissolving 10 mg of each drug in 10 ml methanol. The standard solutions were prepared individually by dilution of the stock solutions with methanol for spectrophotometric experiments and with mobile phase for chromatographic experiments to reach concentration range of 1–50 g/ml for lamivudine and 2–100 g/ml for zidovudine and 0.025–50 g/ml for lamivudine and 0.15–50 g/ml for zidovudine, respectively. 2.3.1. For derivative spectrophotometric method (D1 ) The values of the D1 amplitudes were measured at 265.6 nm (zero-crossing of zidovudine) and 271.6 nm (zero-crossing of lamivudine) for the determination of lamivudine and zidovudine, respectively. 2.3.2. For first derivative of the ratio spectrophotometric method (DD1 ) According to the theory of the ratio-spectra derivative method [35,36], for lamivudine; the stored UV absorption spectra of standard solutions of lamivudine were divided wavelength-by-wavelength by a standard spectrum of zidovudine (20 g/ml). The first derivative was calculated for the obtained spectra with λ = 4 nm. The amplitudes at 239.5 and 245.3 nm were measured and found to be linear to the concentration of lamivudine. For zidovudine, the stored UV absorption spectra of standard solutions of zidovudine were divided wavelength-by-wavelength by a standard spectrum of lamivudine (15 g/ml). The first derivative was calculated for the obtained spectra with λ = 4 nm. The amplitudes at 225.1 and 251.5 nm were measured and found to be linear to the concentration of zidovudine. 2.3.3. For high-performance liquid chromatographic method Standard solutions were prepared separately with mobile phase by varying concentrations of lamivudine and zidovudine in the range of 0.025–50 and 0.15–50 g/ml, respectively. Maintaining concentration of finasteride (IS) at a constant level of 25 g/ml.
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Table 1 Statistical data for the calibration graphs of lamivudine (LMV) and zidovudine (ZDV) by D1 , DD1 and HPLC methods LMV
Linearity range (g/ml) Slope Intercept Correlation coefficient
HPLC
D1
DD1
265.6 nm
239.5 nm
245.3 nm
1–50 6.8 × 10−4 −2.3 × 10−4 0.9989
1–50 0.011 −0.0034 0.9996
1–50 0.007 −0.0006 0.9993
0.025–50 0.617 0.107 0.9999
ZDV
HPLC
D1
DD1
271.6 nm
225.1 nm
251.5 nm
2–100 5.3 × 10−4 −9.1 × 10−4 0.9994
2–100 0.003 −0.0014 0.9998
2–100 0.001 −0.0015 0.9990
0.15–50 0.178 0.038 0.9998
R.S.D. of the slope R.S.D. of the intercept
0.39 0.66
0.38 0.03
0.71 0.26
0.08 0.55
0.09 0.06
0.51 0.18
0.50 0.10
0.17 0.85
LOD (g/ml) LOQ (g/ml)
0.25 0.85
0.026 0.087
0.05 0.17
0.00098 0.0033
0.56 1.89
0.13 0.44
0.26 0.86
0.0049 0.016
Repeatabilitya (R.S.D.; %) Reproducibilitya (R.S.D.; %)
0.17 0.47
0.62 0.83
0.51 0.74
0.70 0.83
0.20 0.61
0.32 0.45
0.48 0.61
0.93 0.96
a
Each value is obtained from four experiments.
Triplicate 10 l injections were made for each solution and peak area ratio of each concentration to the internal standard was plotted against the corresponding concentration to obtain the calibration graph. All of the proposed methods were validated as to precision (reported as the relative standard deviation, R.S.D. (%)), linearity (evaluated by regression equations), detection and determination limits and accuracy. The limit of detection (LOD) and limit of quantitation (LOQ) of the procedure are also as shown in Table 1, which were calculated according to the 3 s/m and 10 s/m criterions, respectively, where s, is the standard deviation of the absorbance (n = 4) of the sample and m is the slope of the corresponding calibration curve. The ruggedness and precision were checked in the same and different days. The R.S.D. (%) was calculated to check the ruggedness and precision of the methods. Accuracy was determined by recovery studies. 2.4. Procedure for tablets For all methods, 10 tablets labeled to contain 150.0 mg of lamivudine and 300.0 mg of zidovudine and excipients were weighed and finely powdered. An accurate weight of the powder equivalent to one tablet content was accurately weighed, transferred into a 100 ml calibrated flask, diluted with methanol, stirred for about 10 min and then completed to volume with
the same solution. This solution was filtered to separate any insoluble matter. The filtrate was collected in a clean flask. After filtration, appropriate solutions were prepared by taking suitable aliquots of clear filtrate (for HPLC study, adding of the constant amount of internal standard) and diluting with selected solvent in order to obtain a final solution. The assay was completed as described under Section 2.3. The contents amount of lamivudine and zidovudine were calculated from the corresponding regression equations. 2.5. Percent recovery study To study the accuracy of the proposed methods, and to check the interference from excipients used in the dosage forms, recovery experiments were carried out by the standard addition method. This study was performed by addition of known amounts of lamivudine and zidovudine to a known concentration of the commercial tablets. The resulting mixtures were analyzed as described under Section 2.3. 2.6. Recovery studies in human serum by HPLC Serum sample, obtained from healthy individuals (after obtaining their written consent), were stored frozen until assay. After gentle, thawing aliquots of serum were spiked with lamivudine and zidovudine dissolved in methanol to achieve final concentration
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of 500 and 1000 g/ml, respectively and treated with 500 l acetonitrile as serum protein precipitating agent, then the volume was completed to 2 ml with the same serum sample. The tubes were vortexed for 5 min at 1500 g and then centrifuged for 10 min at 5000 × g for getting rid of protein residues. The supernatant was taken carefully, serum samples including various concentrations of lamivudine and zidovudine and constant amount of internal standard were injected into the column.
3. Results and discussion 3.1. D1 method As shown in Fig. 2, the zero-order spectra of pure drugs were found to be overlapping, making simultaneous determination difficult. It can be seen that the absorption spectra of lamivudine and zidovudine are very overlapped and as a result, the determination of the two drugs cannot be possible for reliable direct absorbance measurements. In contrast, the D1 spectra of each pure drug was found to show zero-crossing points (Fig. 3) and assisted in their simultaneous estimation. In practice, the measurement selected is that which exhibits the best linear response, gives a zero or near zero intercept on the coordinate of the calibration graph, and is less affected by the concentration of any other component. The shape of the first derivative
Fig. 3. First-derivative spectra of lamivudine (1) 15 g/ml; (2) 20 g/ml; (3) 30 g/ml (continuous lines) and zidovudine (1) 30 g/ml; (2) 50 g/ml; (3) 75 g/ml (dashed lines) in methanol. The arrows indicate the working wavelengths.
spectra is adequate for determining lamivudine in the presence of zidovudine and vice versa. Lamivudine was determined by measurement of its D1 amplitude at the zero-crossing point of zidovudine (at 265.6 nm). While zidovudine was determined by measurement of its D1 at the zero-crossing point of lamivudine (at 271.6 nm). Linear relationships between derivative amplitude and drug concentration were obtained over the concentration range of 1–50 g/ml for lamivudine and 2–100 g/ml for zidovudine. The linear regression equations together with correlation coefficients, slope and intercept, R.S.D. of slope and intercept repeatability (withinday) and reproducibility (between-day) obtained for each drug are as shown in Table 1. Thus, it was evident from the values obtained that the intercept values did not differ significantly from zero in each case. 3.2. DD1 method
Fig. 2. Absorbtion spectra of: (a) lamivudine (15 g/ml), (b) zidovudine (30 g/ml), and (c) a mixture of lamivudine and zidovudine in methanol.
To optimize the simultaneous determination of lamivudine and zidovudine by using DD1 method, it is necessary to test the influence of the variables: divisor standard concentration, λ and smoothing function. All these variables were studied. This influence of λ for obtaining the first derivative of the ratio-spectra was tested and λ = 4 nm was selected as optimum value. An accurate choice of divisor standard concentration is fundamental for several reasons [35,37], hence, we tested the methods with various
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divisor concentrations. The results of all the tests are not shown for the sake of brevity and because these do not add to the scientific value of the work. A standard spectrum of 20 g/ml of zidovudine was considered as suitable for the lamivudine determination and a standard spectrum of 15 g/ml of lamivudine as divisor was considered as suitable for the zidovudine determination. In Fig. 4a, two series of the ratio-spectra of lamivudine/zidovudine and zidovudine/lamivudine was shown. In Fig. 4b, the corresponding first derivative of the ratio-spectra of Fig. 4a was shown. For calibration graph, the wavelengths were selected which exhibited the best linear response to the analyte concentration, i.e. in the first derivative mode 239.5 and 245.3 nm for lamivudine and 225.1 and 251.5 nm for
zidovudine. The calibration graphs of each drug at both wavelengths were achieved by plotting the values of the first derivative of the ratio-spectra lamivudine/zidovudine and zidovudine/lamivudine, with variable concentrations of lamivudine and zidovudine. The proposed method is applicable over the ranges 1–50 g/ml for lamivudine and 2–100 g/ml for zidovudine. The characteristic parameters and necessary statistical data of the regression equations, LOD and LOQ values, repeatability and reproducibility data are compiled in Table 1. The LOD and LOQ values were calculated as described in the previous section. Repeatability and reproducibility variabilities were characterized by R.S.D. (%) and by the difference between theoretical and measured concentrations. There was no significant difference for the assay, which was tested within-day (repeatability) and between-days (reproducibility). In order to demonstrate the validity and applicability of the proposed D1 and DD1 methods, recovery studies were performed by analyzing in synthetic mixtures of lamivudine and zidovudine, which reproduced different composition ratios (Table 2). 3.3. High-performance liquid chromatographic method
Fig. 4. (a) Ratio-spectra and (b) first derivative of the ratio-spectra for different concentrations of lamivudine (15, 20, 30 g/ml; continuous lines, 1–3; divisor zidovudine, 20 g/ml) and zidovudine (30, 50, 75 g/ml; dashed lines, 1–3; divisor lamivudine, 15 g/ml). The arrow indicate the working wavelengths.
Drug analysis is undertaken during various phases of pharmaceutical development, such as formulation and stability studies, quality control and pharmacological testing in animals and humans. All these investigations require reliable and validated analytical methods in order to measure drugs in pharmaceutical formulations and biological samples. In order to effect the simultaneous elution of lamivudine and zidovudine peaks under gradient conditions, the mixtures of methanol, acetonitrile and water in different combinations at various flow rates were assayed. The optimum wavelength for detection was 265 nm at which much better detector responses for both drugs were obtained. The mixture of methanol: water:acetonitrile (70:20:10 (v/v/v)) at 0.9 ml/min flow rate, proved to be better than the other mixtures and flow rates for the separation, since the chromatographic peaks were better defined, resolved and free from tailing. As shown in Fig. 5, the retention times were 2.06 min for lamivudine, 3.36 min for zidovudine and 4.32 min for finasteride (IS).
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Table 2 Determination of lamivudine (LMV) and zidovudine (ZDV) in laboratory prepared mixtures using first derivative (D1 ) and first derivative of the ratio-spectra (DD1 ) Added (g/ml) LMV
ZDV
Found 5 15 25 30 50
(g/ml) 20 20 20 20 20
D1 LMV (265.6 nm)
DD1 ZDV (271.6 nm)
LMV (239.5 nm)
LMV (245.3 nm)
ZDV (225.1 nm)
ZDV (251.5 nm)
4.92 14.97 24.96 30.01 49.85
29.50 29.68 29.75 30.00 29.90
4.90 14.90 25.01 29.85 49.80
4.95 14.95 25.00 29.90 49.90
19.85 19.85 19.85 19.85 19.90
20.00 19.90 19.90 19.90 19.70
98.40 99.80 99.80 100.03 99.70
98.33 98.93 99.16 100.00 99.67
98.00 99.30 100.04 99.50 99.60
99.00 99.70 100.00 99.70 99.80
99.25 99.25 99.25 99.25 99.50
100.00 99.50 99.50 99.50 98.50
Mean recovery (%) R.S.D. (%)
99.55 0.65
99.22 0.66
99.29 0.78
99.64 0.38
99.30 0.11
99.40 0.55
Found 15 15 15 15 15
15.04 15.04 14.75 14.90 15.04
4.99 10.01 29.99 50.00 74.90
15.00 15.01 14.95 14.90 15.01
15.01 14.95 14.90 14.95 15.00
4.95 9.83 29.50 49.40 74.60
4.97 9.85 29.73 49.00 74.80
100.30 100.30 98.33 99.33 100.30
99.8 100.10 99.97 100.00 99.87
100.00 100.07 99.67 99.33 100.07
100.07 99.67 99.33 99.67 100.00
99.00 98.30 98.33 98.80 99.47
99.40 98.50 99.10 98.00 99.73
99.71 0.88
99.95 0.12
99.83 0.33
99.75 0.30
98.78 0.50
98.95 0.71
Recovery (%) 5 20 15 20 25 20 30 20 50 20
(g/ml) 5 10 30 50 75
Recovery (%) 15 5 15 10 15 30 15 50 15 75 Mean recovery (%) R.S.D. (%)
In HPLC methods, precision and accuracy can often be enhanced by the use of an appropriate internal standard, which also serves to correct for fluctuations in the detector response. Ideally, an internal standard should display similar physico-chemical properties to the analytes. But it did not obtained good resolution and peak shape with the similar compounds. The structure of finasteride is not similar to lamivudine and zidovudine. However, it was chosen as the internal standard because it showed a shorter retention time with better peak shapes and better resolution, compared to other potential internal standards. System suitability tests are an integral part of a liquid chromatographic method. System suitability tests are used to verify that the resolution and reproduci-
bility of the chromatographic system are adequate for the analysis to be done. System suitability tests were carried out according to USP 24, method 621 [38] on the chromatogram of freshly prepared standard solutions to check various parameters, such as resolution, selectivity, tailing and capacity factors. Resolution and selectivity factors for this system were found 2.17 and 2.48, respectively. The tailing and capacity factors were obtained as 1.11 and 0.75 for lamivudine and 1.33 and 1.85 for zidovudine, respectively. The variation in retention times among six replicate injections of lamivudine and zidovudine standard solutions was very low, rendering a R.S.D. of 0.98 and 0.62%, respectively. The results obtained from system suitability tests are in agreement with the USP requirements.
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B. Uslu, S.A. Özkan / Analytica Chimica Acta 466 (2002) 175–185 Table 3 Resolution of lamivudine (LMV) and zidovudine (ZDV) laboratory-made mixtures using by the HPLC technique Added (g/ml)
Found (g/ml)
Recovery (%)
LMV
ZDV
LMV
ZDV
LMV
ZDV
1 2.5 5 10 25
10 10 10 10 10
0.99 2.5 5.02 9.95 24.92
10.04 10.00 10.00 10.14 10.03
99.00 100.00 100.40 99.50 99.68
100.40 100.00 100.00 101.40 100.30
99.72 0.53
100.42 0.57
100.00 99.00 99.00 98.00 99.00
100.40 100.40 100.30 99.32 99.62
99.00 0.71
100.01 0.50
Mean recovery (%) R.S.D. (%) 1 1 1 1 1
2.5 5 10 25 50
1.00 0.99 0.99 0.98 0.99
Mean recovery (%) R.S.D. (%)
2.51 5.02 10.03 24.83 49.81
carried out by analyzing in the synthetic mixtures of lamivudine and zidovudine, which reproduced different composition ratios (Table 3). Fig. 5. Chromatogram obtained from tablet dosage forms containing: (1) 2.5 g/ml lamivudine, (2) 5.0 g/ml zidovudine, and (3) 25 g/ml finasteride (IS).
Peak area ratios (Asample /AIS ) were plotted against corresponding concentrations in the range of 0.025– 50 g/ml for lamivudine and 0.15–50 g/ml for zidovudine. Linear regression parameters of the peak area ratios versus concentrations of lamivudine and zidovudine are presented in Table 1. The results showed highly reproducible calibration curves with correlation coefficients of >0.999. Necessary statistical data of the regression equations, such as LOD value, LOQ value, repeatability and reproducibility data was also as shown in Table 1. The LOD and LOQ values were calculated as described in Section 3.1. Repeatability and reproducibility variabilities were characterized by R.S.D. (%) (Table 1). According to this results, there was no significant difference for the assay, which was tested within-day (repeatability) and between-days (reproducibility; Table 1). In order to demonstrate the validity and applicability of the proposed HPLC method, recovery tests were
3.4. Analysis of tablets When working on synthetic mixture, results encourage the use of the all proposed methods described for the assay of lamivudine and zidovudine in commercial tablet dosage forms. The three proposed methods could be used for the simultaneous determination of lamivudine and zidovudine in the presence of each other and without prior separation of the excipients. Each film-coated tablet contains 150 mg of lamivudine, 300 mg of zidovudine, and the inactive ingredients colloidal silicon dioxide, magnesium stearate, microcrystalline cellulose and sodium starch glycolate. The film-coating solution contains Opadry YS-1-7706-G White and purified water. Fig. 5 shows a typical chromatogram obtained follow by analysis of lamivudine and zidovudine in tablets. As shown in Fig. 5, the substances were eluted, forming well shaped, symmetrical single peaks, well separated from the solvent front. No interfering peaks were obtained in the chromatogram due to tablet excipients. The utility of all of the proposed methods was verified by means of replicate estimations of
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Table 4 Results of the determination and the recovery analysis of lamivudine (LMV) and zidovudine (ZDV) in commercial tablet dosage forms using first derivative (D1 ), first derivative of the ratio-spectra (DD1 ) and HPLC methods HPLC
Labelled claim (mg) Amount found (mg)a R.S.D. (%) Calculated t Calculated F Recoveryc (%) R.S.D. (%)
D1
DD1
LMV
ZDV
LMV (265.6 nm)
ZDV (271.6 nm)
LMV (239.5 nm)
LMV (245.3 nm)
ZDV (225.1 nm)
ZDV (251.5 nm)
150.00 149.81 0.16 2.31b 2.60b 99.54 0.31
300.00 299.90 0.25 2.31b 2.60b 99.63 0.08
150.00 148.98 0.69 0.79 0.45 99.22 0.17
300.00 299.10 0.57 0.34 0.14 99.87 0.18
150.00 148.04 0.72 0.20 0.49 99.38 0.07
150.00 149.50 0.20 0.69 0.008 99.88 0.12
300.00 298.60 0.37 0.004 0.28 99.56 0.04
300.00 297.30 0.44 0.0005 0.48 99.24 0.26
a
Mean value of the five determinations. Theoretical values for t- and F-tests. c Mean value of the four determinations. b
pharmaceutical preparations and results obtained were evaluated statistically. Table 4 shows the results obtained in the analysis of tablets for spectrophotometric and HPLC methods. Results obtained from proposed methods of the analysis of both drugs in tablets indicate that the proposed techniques can be used for simultaneous quantitation and routine quality control analysis of this binary mixture in pharmaceuticals. A comparison with an official reference determination method has not been possible in any pharmacopoeias and literature, because so far no other procedure for the quantitation of lamivudine and zidovudine from pharmaceutical formulations has been reported. Proposed HPLC results were compared with the spectrophotometric results. According to the student’s t-test and variance ratio F-test, the calculated t- and F-values were less than the theoretical values in either test at the 95% confidence level. This indicates that there is no significant difference between the performance all of the proposed methods as regards to mean values and standard deviations (Table 4). Recovery studies were realized from the tablets for accuracy and precision of the proposed techniques. The recovery of the procedure was carried out by spiking the already analyzed samples of tablets with the known concentrations of standard solutions of lamivudine and zidovudine. The results of the recovery analysis for all techniques are tabulated as shown in Table 4. It is concluded that the proposed methods are sufficiently accurate and precise in order
to be applied to pharmaceutical dosage forms. High percentage recovery data shows that all of the proposed methods are free from the interferences of the excipients used in the formulations. 3.5. Application to the serum samples In order to check the applicability of the proposed HPLC method to biological materials, the recovery studies were performed in human serum samples. Analysis of drugs from serum by HPLC usually requires extensive time-consuming sample preparation, use of expensive organic solvent and other chemicals [23–25]. In our proposed method, the serum proteins are precipitated by the addition of acetonitrile, which is centrifuged at 5000 g, and the supernatant is diluted, directly injected and analyzed. Fig. 6 shows the typical chromatogram obtained the serum spiked with lamivudine, zidovudine and IS (a) and blank serum (b), which indicate no interference from the endogenous substances present in serum. Serum samples were spiked with two different concentrations of lamivudine and zidovudine and constant level of IS. The determined results and recoveries of known amounts of lamivudine and zidovudine added to serum samples are given in Table 5. The proposed method gives reproducible results, is easy to perform and is sensitive enough to simultaneous determination of lamivudine and zidovudine in human serum samples (Table 5).
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Fig. 6. Chromatogram of: (a) serum spiked with (1) 2.0 g/ml of lamivudine, (2) 10.0 g/ml of zidovudine, (3) 25 g/ml finasteride (IS) and (b) blank serum.
Table 5 Results obtained for lamivudine (LMV) and zidovudine (ZDV) analysis from human serum using HPLC method LMV Added (g/ml) n Found (g/ml) R.S.D. (%) Average recovery (%)
2.00 4 1.98 1.04 99.20
ZDV 10.00 4 9.81 0.7 98.07
10.00 4 9.78 0.84 97.80
30.00 4 29.38 0.94 97.93
4. Conclusion The D1 and DD1 and HPLC methods enable the quantitation or mixtures of lamivudine and zidovudine with good accuracy and precision, either in laboratory made samples or in pharmaceutical dosage forms. From a comparison of the results obtained with the three methods, they do not appear to have sub-
stantial differences. A little superiority of the HPLC method over the spectrophotometric techniques in the simultaneous determination of lamivudine and zidovudine, results from the limits of detection, the limits of determination and correlation coefficients in Table 1. All of these procedures are rapid, precise and work without solving equations or separation steps. The D1 method is more rapid and simple than the DD1 method, while the proposed DD1 method has greater sensitivity and accuracy. The advantages of the ratio-spectra method proposed over the zero-crossing derivative method, is the possibility of performing measurements in correspondence of peaks, hence, a potentially greater sensitivity and accuracy. Another advantages of the DD1 assay are easy measurement on the separate peaks, higher values of analytical signals and no need to work only at zero-crossing point in comparison with the derivative spectrophotometric method. In this study, proposed HPLC method was especially used as a versatile reference method and could be also satisfactory for biological fluids since its high separation power. The proposed HPLC method gives a good resolution between lamivudine, zidovudine and internal standard within a short analysis time. The present HPLC study purposes a rapid, simple, sufficiently precise and accurate method for the simultaneous determination of lamivudine and zidovudine in raw material, pharmaceutical formulations and human serum. The proposed method used a simple serum deproteination step instead of extraction. No interferences from endogenous substances were observed in biological samples. The three proposed methods are suitable for quality control laboratories, where economy and time are essential. High percentage recovery shows that the methods are free from the interferences of the commonly used excipients and additives in the formulations of drugs.
Acknowledgements The authors greatfully acknowledge Glaxo-SmithKline Pharm. Ind. for supplying lamivudine, zidovudine and pharmaceutical preparations.
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