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Rapid determination of lamivudine in human plasma by high-performance liquid chromatography
Journal of Chromatography B, 975 (2015) 40–44
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Rapid determination of lamivudine in human plasma by high-performance liquid chromatography Mahmoud Alebouyeh a , Hossein Amini b,∗ a b
Food and Drug Control Research Center, FDO, Ministry of Health, Tehran, Iran Department of Pharmacology, Neuroscience Research Center, Golestan University of Medical Science, P.O. Box: 49175-553, Gorgan, Iran
a r t i c l e
i n f o
Article history: Received 11 July 2014 Accepted 9 November 2014 Available online 18 November 2014 Keywords: Lamivudine Acyclovir HPLC Plasma Bioequivalence studies
a b s t r a c t A simple and rapid high-performance liquid chromatographic method with spectrophotometric detection was developed for the determination of lamivudine in human plasma. Sample preparation was accomplished through protein precipitation with acetonitrile followed by aqueous phase separation using dichloromethane. Lamivudine and the internal standard acyclovir were well separated from endogenous plasma peaks on a Chromolith RP-18e column under isocratic elution with 50 mM sodium dihydrogen phosphate–triethylamine (996:4, v/v), pH 3.2 at 20 ◦ C. Total run time at a flow-rate of 1.5 ml/min was less than 5 min. Detection was made at 278 nm. The method was specific and sensitive, with a lower quantification limit of 40 ng/ml and a detection limit of 10 ng/ml. The absolute recovery was 97.7%, while the within- and between-day coefficient of variation and percent error values of the assay method were all less than 7%. The linearity was assessed in the range of 40–2560 in plasma, with a correlation coefficient of greater than 0.999. The method was successfully applied to a bioequivalence study in healthy volunteers. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Lamivudine (Fig. 1) is a synthetic nucleoside analogue with activity against human immunodeficiency (HIV) and hepatitis B viruses (HBV). It is phosphorylated inside the cell to its active 5 triphosphate metabolite and then incorporated into viral DNA by HIV reverse transcriptase and HBV polymerase, resulting in DNA chain termination [1,2]. The pharmacokinetics of lamivudine is similar in patients with HIV or HBV infection and healthy volunteers. The drug is rapidly absorbed after oral administration, with maximum serum concentrations usually attained 0.5–1.5 h after the dose. The absolute bioavailability is approximately 82 and 68% in adults and children, respectively. Lamivudine systemic exposure is not altered when it is administered with food. It is widely distributed into total body fluid, the mean apparent volume of distribution being approximately 1.3 L/kg following intravenous administration. The wide distribution of lamivudine may be partly related to its relatively low molecular weight (229D) and low plasma protein binding (generally <36%) [3]. Many analytical methods including high performance liquid chromatography (HPLC) with UV detection [4–13] and mass
∗ Corresponding author. Tel.: +98 17 32525972; fax: +98 17 34533554. E-mail address: [email protected] (H. Amini). http://dx.doi.org/10.1016/j.jchromb.2014.11.004 1570-0232/© 2014 Elsevier B.V. All rights reserved.
spectrometry [14,15] has been reported for determination of lamivudine in human plasma. Due to highly polar nature of lamivudine, efficient extraction from plasma could be problematic. Thereby, in spite of many available solid-phase [4,6–8,12,15] and liquid–liquid [11,13] extraction techniques, the most simple and fast method for sample preparation could be achieved by plasma protein precipitation with acids [5,9], or acetonitrile [10]. However, the stability of the analytical column over the injection of strongly acidic samples is not satisfactory. Moreover, acetonitrile or acidtreated plasma produced interferences in the chromatogram that complicated separation and elongated run times [5,9,10]. This paper describes a simple and fast assay method for the determination of lamivudine in human plasma samples using a new protein precipitation procedure that does not suffer from the above mentioned limitations. The method was promising for the bioequivalence studies where a large number of plasma samples should be assayed in a short time. 2. Experimental 2.1. Reagents Lamivudine and the internal standard acyclovir (Fig. 1) were purchase from Sigma (St. Louis, MO, USA). HPLC grade methanol, acetonitrile and analytical grade triethylamine (TEA), sodium dihydrogen phosphate monohydrate, phosphoric acid,
M. Alebouyeh, H. Amini / J. Chromatogr. B 975 (2015) 40–44
A)
O NH2
Retention factor
Acyclovir 2.50
O N
H2N
N
N O
2.00
O HO
Lamivudine
3.00
N
HN
N
41
S
A) Lamivudine
OH
1.50
B) Acyclovir 1.00
Fig. 1. Chemical structure of (A) lamivudine and (B) the internal standard acyclovir.
0
10
20
30
40
50
Temperature (°°C)
2.2. Instrumentation
B)
3.00
Retention factor
dichloromethane were obtained from E. Merck (Darmstadt, Germany).
2.50
The chromatographic system equipped with a Smartline 1000 solvent delivery pump, Smartline 2500 ultraviolet detector (operated at 278 nm), Rheodyne 7725i loop injector, Jet stream column heater/cooler and ChromGate HPLC software (Knauer, Berlin, Germany). A Chromolith RP-18e column (100 mm × 4.6 mm) with an RP-18e guard column (5 mm × 4.6 mm), both from Merck (Darmstadt, Germany) were used for the chromatographic separation. The mobile phase comprised of 50 mM sodium dihydrogen phosphate–TEA (996:4, v/v), adjusted to pH 3.2 with concentrated phosphoric acid. Analyses were run at a flow rate of 1.5 ml/min at 20 ◦ C. 2.3. Standard solutions Stock solution of lamivudine and acyclovir were prepared in methanol–water (1:1, v/v) to make concentrations of 0.1 mg/ml and stored at −20 ◦ C. Working standard solutions were prepared from stock solutions by dilution with methanol–water (1:1, v/v). 2.4. Calibration curve and quantitation Seven-point standard calibration curves were prepared by spiking the blank plasma with appropriate amount of lamivudine. The plasma standards ranged from 40 to 2560 ng/ml. Calibration curves were constructed by plotting peak height ratio (y) of lamivudine to the internal standard versus lamivudine concentrations (x). A linear regression was used for quantitation. It should be noted that the integration of the peak by area was also possible. 2.5. Extraction procedure All the processes were performed at room temperature (25 ◦ C). A volume of 250 l of plasma was transferred to a 1.5-ml polypropylene microcentrifuge tube. The internal standard (20 l, equal to 500 ng of acyclovir) and 500 l of acetonitrile were added, and followed by shaking for 30 s. After centrifugation at 12,000 × g for 2 min (Microfuge® 18 from Beckman Coulter, Germany), 500 l of the supernatant was transferred into another tube and 1 ml of dichloromethane was added. The mixture was vortex-mixed for 30 s and centrifuged at 12,000 × g for 2 min. Finally, 50 l of the aqueous supernatant was transferred into another tube and a 10-l aliquot was injected onto the HPLC system.
2.00
1.50
1.00 0
10
20
30
40
50
Temperature (°°C) Fig. 2. The effects of temperature on the retention factor of lamivudine and acyclovir. The chromatographic conditions were as follows: Column, Chromolith RP 18e (100 mm × 4.6 mm) with guard; mobile phase, (A) 0.4% or (B) 0.6% triethylamine in 50 mM NaH2 PO4 at pH 3.3; flow rate, 1.5 ml/min; detection, 278 nm.
2.6. Assay validation Obtained blank plasma samples from 24 healthy volunteers were assessed by the procedure as described above and compared with spiked and real plasma samples from pharmacokinetic study to evaluate selectivity of the method. The precision and accuracy of the method were examined by adding known amounts of lamivudine to pool plasma. These quality control samples (40, 160, 640 and 2560 ng/ml) were made from a stock solution separate from that used to prepare plasma standards and were not used for constructing calibration curves. For intra-day precision and accuracy five replicate quality control samples at each concentration were assayed on the same day. The inter-day precision and accuracy were evaluated on five different days within 2 weeks along analyzing plasma samples of volunteers. The absolute recoveries (n = 5) was calculated by comparing peak heights obtained from prepared sample extracts with those found by direct injection of drug solution made in 0.1% acetic acid at the same concentration. The lower limit of quantification (LLOQ) was estimated by analyzing lamivudine at low concentrations of the calibration curve. The LLOQ was defined as a concentration level where accuracy and precision were still better than 10%. To determine the limit of detection (LOD), lower plasma concentrations than the lower end of the calibration curve were used. The LOD was then defined as the concentration which caused a signal three times the noise (S/N = 3/1).
42
Lamivudine
4
3
Acyclovir (IS)
M. Alebouyeh, H. Amini / J. Chromatogr. B 975 (2015) 40–44
mAU
2
1
0
C) B)
-1
A) -2 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Minutes Fig. 3. Representative chromatograms of (A) a blank plasma; (B) plasma spiked with 40 ng/ml lamivudine; (C) a volunteer plasma sample, 2 h after taking 150 mg tablet of lamivudine (1682 ng/ml).
2.7. Application The assay was used for a comparative bioavailability study of two tablets preparations containing 150 mg lamivudine. The Institutional Review Board of Golestan University of Medical University (Gorgan, Iran) was dedicated to endorse the ethical conduct of the study and to approve the protocol (the authorization number: A880507). The board is constituted and operates in accordance with the principles and requirements described in the Guidelines on Research Involving Human Subjects. Twenty four healthy volunteers participated in the study. The study was conducted using a two-way crossover design, as a single dose, randomized trial. The two formulations were administrated on 2 treatment days, separated by a washout period of 7 days, to fasted subjects who received one of the study medications. Food and drinks were not allowed until 3 h after ingestion of the tablets. Multiple blood samples (3 ml) were collected before and 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8 and 10 h post-dosing. The plasma was immediately separated by centrifugation and frozen at −20 ◦ C until analysis. 3. Results and discussion 3.1. Sample preparation Lamivudine with a pKa value of 4.3 is classified by WHO as “soluble” in water [16]. Review of the literature reveals that liquid–liquid extraction (LLE) of highly polar drugs like lamivudine is a big challenge. According to LLE-based reports [11,13], we first tried
several solvent mixtures at different pH values to extract lamivudine, which all resulted in low and irreproducible recoveries. The problem may be in part due to the low pKa value of lamivudine. We have already reported that metformin, another highly hydrophilic compound with a pKa value of 12.4, could be effectively extracted by a suitable organic solvent under strong alkaline condition [17]. Solid-phase extraction (SPE) procedure [4,6–8,12,15] has been reported. However, the technique is expensive and timeconsuming. Removal of proteins from plasma samples using acid [5,9] or by acetonitrile followed by evaporation of the supernatant [10] has been proposed as an alternative to LLE and SPE methods. However, the problem of potential endogenous interferences in the chromatogram limits their usefulness and further purification of sample seems to be mandatory. In the present work, we postulated that after protein removal by acetonitrile, the separation of aqueous phase could concentrate lamivudine while will leave many plasma interferences in the acetonitrile layer. Acetonitrile does not associate strongly with water and thus, as opposed to methanol, acetonitrile–water mixtures remain binary in character. Although water and acetonitrile are practically immiscible at 0–4 ◦ C [18], the separation could be efficiently take place at room temperature by adding a kosmotrope such as ammonium sulfate [19] or a water-immiscible organic solvent like dichloromethane with high solubility for acetonitrile [18,20]. In the present work, acetonitrile acts as an efficient protein precipitation agent and then, dichloromethane eliminates neutral lipophilic substances as well as acetonitrile used for protein precipitation and lamivudine remains undiluted in the aqueous layer. The aqueous layer could be
Table 1 The accuracy, within- and between-day precision and recovery data for the measurement of lamivudine in human plasma (n = 5). Nominal concentration (ng/ml)
Recovery (%)
40 160 640 2560
99.1 96.7 98.1 96.8
Intra-day
Inter-day
Mean ± SD ± ± ± ±
7.2 4.4 3.2 1.5
40.81 162.4 636.9 2572.2
± ± ± ±
1.58 7.72 27.84 72.62
Precision (%)
Accuracy (%)
Mean ± SD
3.9 4.7 4.4 2.8
2 1.5 −0.5 0.5
38.52 164.62 645.42 2543.2
± ± ± ±
2.62 8.92 31.32 93.14
Precision (%)
Accuracy (%)
6.8 5.4 4.8 3.7
−3.7 2.3 0.8 −0.6
M. Alebouyeh, H. Amini / J. Chromatogr. B 975 (2015) 40–44
stability of reversed-phase columns when operated with totally aqueous mobile phases. The monolithic column showed suitable stability of performance as reflected in constant lamivudine peak asymmetry and plate number throughout the study.
4000
Lamivudine plasma concentration (ng/ml)
A)
Lamivudine-Exir-T
3500 3000 2500 2000 1500 1000 500 0 0
Lamivudine plasma concentration (ng/ml)
B
2
4
6
8
10
Time (h) 4000
EPIVIR-gsk-R
3500 3000 2500 2000 1500 1000 500 0 0
2
4
6
8
43
10
3.3.2. Mobile phase The separation of lamivudine under varying mobile phase composition was investigated. A mobile phase containing a very low amount of TEA in an acidic phosphate buffer gave the best separation of lamivudine from the plasma interferences. It should be noted that increasing the TEA amount in the mobile phase decrease the retention times and simultaneously improves the separation of lamivudine and acyclovir peaks due to greater susceptibility of lamivudine to TEA, but then the separation from plasma interferences will be a major concern. Therefore, the amount of TEA in the mobile phase was carefully optimized to achieve a suitable separation between lamivudine, internal standard and endogenous plasma peaks. 3.3.3. Chromatographic temperature Temperature had a critical role in the separation of lamivudine from acyclovir. As presented in Fig. 2, the effects of chromatographic temperature on the chromatographic behavior of lamivudine and acyclovir were evaluated using 0.4 and 0.6% TEA in phosphate buffer as mobile phase. It was observed that the resolution between two peaks is lost at unsuitable temperatures. Interestingly, temperature also affects the elution order of lamivudine and acyclovir in the chromatogram. It could be probably concluded that raising the temperature increases the solubility of these compounds in the mobile phases, but the rate is higher for acyclovir in comparison to lamivudine.
Time (h) Fig. 4. Plasma concentration-time profiles of lamivudine following oral administration of test or reference tablets at a dose of 150 mg in 24 healthy volunteers in a crossover study.
completely separated from organic layer and it was not necessary to heat the obtained aqueous layer in order to remove the trace amount of organic solvents. 3.2. Selection of internal standard The internal standard, acyclovir was selected based on its comparable solubility and recovery, and stability properties to lamivudine during storage and sample preparation step. By changing the wavelength to 254 nm, the present method was also successfully used for acyclovir assay in plasma in another bioequivalence study using lamivudine as internal standard (data not shown). 3.3. Chromatographic separation 3.3.1. Chromatographic column In comparison to several tested particulate HPLC columns, Chromolith RP-18e column showed superior separation efficiency with less plasma endogenous peaks. Another critical point was the
3.4. Assay validation Representative chromatograms of drug-free plasma, plasma spiked with lamivudine and a volunteer sample collected after oral dosing with lamivudine are shown in Fig. 3. The retention times for lamivudine and the internal standard were 2.7 and 3.1 min, respectively. The injection of blank plasma samples of 24 volunteers showed no interfering peaks close to the retention times of lamivudine or the internal standard. Although the last endogenous peak was eluted at 6 min, but a new injection was done in every 5 min placing that peak in the fronting area of the next chromatogram. Lipemic and hemolysate plasma samples were spiked with different concentrations of lamivudine. The obtained concentrations were in the acceptable range of ±5%. Over 700 plasma samples were analyzed by this method and the asymmetry value of 1.2 and theoretical plate number of 6000 for both lamivudine and acyclovir, and the resolution factor of 1.8 between them were constant during the entire course of the study. The calibration curves were linear over the concentration range of 40–2560 ng/ml in human plasma, with a correlation coefficient greater than 0.999. The LLOQ was 40 ng/ml and the LOD was 10 ng/ml. The results of the method intra- and inter-day accuracy and precision are presented in Table 1. All values for accuracy and precision were within the recommended limits. Intra-day precision ranged between 2.8 and 4.7% whereas the interday precision was between 3.7 and 6.8%. The intra-day mean error
Table 2 Phamacokinetic parameters (mean ± SEM) for lamivudine following oral administration of test or reference tablets at a dose of 150 mg in 24 healthy volunteers. Group
Cmax (ng/ml)
tmax (h)
AUC0–t (ng/ml h)
AUC0–∞ (ng/ml h)
t1/2 (h)
T product Epivir-R
2213.1 (121.3) 2171.0 (115.8)
1.06 (0.09) 1.23 (0.09)
6753.2 (213.0) 6736.4 (247.5)
7152.7 (238.3) 7148.7 (274.0)
2.25 (0.05) 2.26 (0.05)
Cmax = maximum plasma concentration; tmax = time to reach Cmax ; AUC0–∞ = area under the concentration-time curve (AUC) from time zero to infinity; AUC0–t = AUC from time zero to the last measurable concentration; t1/2 = elimination plasma half-life.
44
M. Alebouyeh, H. Amini / J. Chromatogr. B 975 (2015) 40–44
was between −0.5 and 2% whereas the inter-day mean error was between −3.7 and 2.3%. The mean absolute recoveries for lamivudine and internal standard using the present extraction procedure were 97.7 and 99.5%, respectively. 3.5. Pharmacokinetic results The proposed method was applied to the determination of lamivudine in plasma samples for the purpose of the bioequivalence study. The plasma lamivudine profiles for volunteers after taking two products are shown in Fig. 4. Pharmacokinetic parameters obtained from two preparations are summarized in Table 2. The extrapolated fraction of the AUC0–∞ accounted only for 5–6%, which indicates the suitability of the present analytical method for pharmacokinetic studies. The test/reference geometric ratio for Cmax (90% CI) was 1.02 (95.3, 108.7). The test/reference geometric ratios for AUC0–t (90% CI) and AUC0–∞ (90% CI) were 1.01 (97.3, 103.9) and 1.00 (97.2, 103.7), respectively. The 90% CIs of Cmax , AUC0–t and AUC0–∞ were well within the acceptable range of 80 and 125 suggested by the US FDA bioequivalence guideline. It was concluded that the two products were bioequivalent and therefore, could be used interchangeably. 4. Conclusion In the present study, a simple and reliable HPLC method was developed and validated. The new strategy for sample preparation entails protein precipitation by acetonitrile followed by acetonitrile removal using dichloromethane. This very simple and quick sample preparation procedure offered a complete and reproducible recovery of lamivudine from plasma. Moreover, use of a simply available and structurally related internal standard, a short chromatographic run time, and the performance stability of analytical
column and the mobile phase propose the current method as a suitable choice for application to bioequivalence studies where a large number of plasma samples should be assayed in a short time. We demonstrated usefulness of the current method by its application to a bioequivalence study of lamivudine tablets in 24 healthy volunteers. References [1] J.A.V. Coates, N. Cammack, H.J. Jenkinson, A.J. Jowett, M.I. Jowett, B.A. Pearson, C.R. Penn, P.L. Rouse, K. Claire Viner, J.M. Cameron, Antimicrob. Agents Chemother. 36 (1992) 733–739. [2] G.A. Balint, Pharmacol. Ther. 89 (2001) 17–27. [3] M.A. Johnson, K.H.P. Moore, G.J. Yuen, A. Bye, G.E. Pakes, Clin. Pharmacokinet. 36 (1999) 41–66. [4] A.J. Harker, G.L. Evans, A.E. Hawley, D.M. Morris, J. Chromatogr. B 657 (1994) 227–232. [5] X.J. Zhou, J.P. Sommadossi, J. Chromatogr. B 691 (1997) 417–424. [6] R.M.W. Hoetelmans, M. Profijt, P.L. Meenhorst, J.W. Mulder, J.H. Beijnen, J. Chromatogr. B 713 (1998) 387–394. [7] J.J. Zheng, S.T. Wu, T.A. Emm, J. Chromatogr. B 761 (2001) 195–201. [8] B. Fan, J.T. Stewart, J. Pharm. Biomed. Anal. 28 (2002) 903–908. [9] Y. Alnouti, C.A. White, M.G. Bartlett, J. Chromatogr. B 803 (2004) 279–284. [10] E.K. Kano, C.H.R. Serra, E.E.M. Koono, S.S. Andrade, V. Porta, Int. J. Pharm. 297 (2005) 73–79. [11] G. Bahrami, S. Mirzaeei, A. Kiani, B. Mohammadi, J. Chromatogr. B 823 (2005) 213–217. [12] M. Shekarchi, M. Pourfarzib, B. Akbari-Adergani, A. Mehramizi, M. Javanbakht, R. Dinarvand, J. Chromatogr. B 931 (2013) 50–55. [13] U. Nandi, A. Das, B. Roy, H. Choudhury, B. Gorain, T.K. Pal, Drug Test. Anal. 5 (2013) 485–491. [14] K.B. Kenny, S.A. Wring, R.M. Carr, G.N. Wells, J.A. Dunn, J. Pharm. Biomed. Anal. 22 (2000) 967–983. [15] H.N. Mistri, A.G. Jangid, A. Pudage, N. Gomes, M. Sanyal, P. Shrivastav, J. Chromatogr. B 853 (2007) 320–332. [16] S. Strauch, E. Jantratid, J.B. Dressman, H.E. Junginger, S. Kopp, K.K. Midha, V.P. Shah, S. Stavchansky, D.M. Barends, J. Pharm. Sci. 100 (2011) 2054–2063. [17] H. Amini, A. Ahmadiani, P. Gazerani, J. Chromatogr. B 824 (2005) 319–322. [18] S.T. Forgue, K.A. Pittman, R.H. Barbhaiya, J. Chromatogr. 414 (1987) 343–353. [19] A. Rahman, N.E. Hoffman, J. Chromatogr. 433 (1988) 159–166. [20] F. Kees, H. Grobecker, K.G. Naber, J. Chromatogr. 305 (1984) 363–371.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Rapid determination of lamivudine in human plasma by high-performance liquid chromatography Mahmoud Alebouyeh a , Hossein Amini b,∗ a b
Food and Drug Control Research Center, FDO, Ministry of Health, Tehran, Iran Department of Pharmacology, Neuroscience Research Center, Golestan University of Medical Science, P.O. Box: 49175-553, Gorgan, Iran
a r t i c l e
i n f o
Article history: Received 11 July 2014 Accepted 9 November 2014 Available online 18 November 2014 Keywords: Lamivudine Acyclovir HPLC Plasma Bioequivalence studies
a b s t r a c t A simple and rapid high-performance liquid chromatographic method with spectrophotometric detection was developed for the determination of lamivudine in human plasma. Sample preparation was accomplished through protein precipitation with acetonitrile followed by aqueous phase separation using dichloromethane. Lamivudine and the internal standard acyclovir were well separated from endogenous plasma peaks on a Chromolith RP-18e column under isocratic elution with 50 mM sodium dihydrogen phosphate–triethylamine (996:4, v/v), pH 3.2 at 20 ◦ C. Total run time at a flow-rate of 1.5 ml/min was less than 5 min. Detection was made at 278 nm. The method was specific and sensitive, with a lower quantification limit of 40 ng/ml and a detection limit of 10 ng/ml. The absolute recovery was 97.7%, while the within- and between-day coefficient of variation and percent error values of the assay method were all less than 7%. The linearity was assessed in the range of 40–2560 in plasma, with a correlation coefficient of greater than 0.999. The method was successfully applied to a bioequivalence study in healthy volunteers. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Lamivudine (Fig. 1) is a synthetic nucleoside analogue with activity against human immunodeficiency (HIV) and hepatitis B viruses (HBV). It is phosphorylated inside the cell to its active 5 triphosphate metabolite and then incorporated into viral DNA by HIV reverse transcriptase and HBV polymerase, resulting in DNA chain termination [1,2]. The pharmacokinetics of lamivudine is similar in patients with HIV or HBV infection and healthy volunteers. The drug is rapidly absorbed after oral administration, with maximum serum concentrations usually attained 0.5–1.5 h after the dose. The absolute bioavailability is approximately 82 and 68% in adults and children, respectively. Lamivudine systemic exposure is not altered when it is administered with food. It is widely distributed into total body fluid, the mean apparent volume of distribution being approximately 1.3 L/kg following intravenous administration. The wide distribution of lamivudine may be partly related to its relatively low molecular weight (229D) and low plasma protein binding (generally <36%) [3]. Many analytical methods including high performance liquid chromatography (HPLC) with UV detection [4–13] and mass
∗ Corresponding author. Tel.: +98 17 32525972; fax: +98 17 34533554. E-mail address: [email protected] (H. Amini). http://dx.doi.org/10.1016/j.jchromb.2014.11.004 1570-0232/© 2014 Elsevier B.V. All rights reserved.
spectrometry [14,15] has been reported for determination of lamivudine in human plasma. Due to highly polar nature of lamivudine, efficient extraction from plasma could be problematic. Thereby, in spite of many available solid-phase [4,6–8,12,15] and liquid–liquid [11,13] extraction techniques, the most simple and fast method for sample preparation could be achieved by plasma protein precipitation with acids [5,9], or acetonitrile [10]. However, the stability of the analytical column over the injection of strongly acidic samples is not satisfactory. Moreover, acetonitrile or acidtreated plasma produced interferences in the chromatogram that complicated separation and elongated run times [5,9,10]. This paper describes a simple and fast assay method for the determination of lamivudine in human plasma samples using a new protein precipitation procedure that does not suffer from the above mentioned limitations. The method was promising for the bioequivalence studies where a large number of plasma samples should be assayed in a short time. 2. Experimental 2.1. Reagents Lamivudine and the internal standard acyclovir (Fig. 1) were purchase from Sigma (St. Louis, MO, USA). HPLC grade methanol, acetonitrile and analytical grade triethylamine (TEA), sodium dihydrogen phosphate monohydrate, phosphoric acid,
M. Alebouyeh, H. Amini / J. Chromatogr. B 975 (2015) 40–44
A)
O NH2
Retention factor
Acyclovir 2.50
O N
H2N
N
N O
2.00
O HO
Lamivudine
3.00
N
HN
N
41
S
A) Lamivudine
OH
1.50
B) Acyclovir 1.00
Fig. 1. Chemical structure of (A) lamivudine and (B) the internal standard acyclovir.
0
10
20
30
40
50
Temperature (°°C)
2.2. Instrumentation
B)
3.00
Retention factor
dichloromethane were obtained from E. Merck (Darmstadt, Germany).
2.50
The chromatographic system equipped with a Smartline 1000 solvent delivery pump, Smartline 2500 ultraviolet detector (operated at 278 nm), Rheodyne 7725i loop injector, Jet stream column heater/cooler and ChromGate HPLC software (Knauer, Berlin, Germany). A Chromolith RP-18e column (100 mm × 4.6 mm) with an RP-18e guard column (5 mm × 4.6 mm), both from Merck (Darmstadt, Germany) were used for the chromatographic separation. The mobile phase comprised of 50 mM sodium dihydrogen phosphate–TEA (996:4, v/v), adjusted to pH 3.2 with concentrated phosphoric acid. Analyses were run at a flow rate of 1.5 ml/min at 20 ◦ C. 2.3. Standard solutions Stock solution of lamivudine and acyclovir were prepared in methanol–water (1:1, v/v) to make concentrations of 0.1 mg/ml and stored at −20 ◦ C. Working standard solutions were prepared from stock solutions by dilution with methanol–water (1:1, v/v). 2.4. Calibration curve and quantitation Seven-point standard calibration curves were prepared by spiking the blank plasma with appropriate amount of lamivudine. The plasma standards ranged from 40 to 2560 ng/ml. Calibration curves were constructed by plotting peak height ratio (y) of lamivudine to the internal standard versus lamivudine concentrations (x). A linear regression was used for quantitation. It should be noted that the integration of the peak by area was also possible. 2.5. Extraction procedure All the processes were performed at room temperature (25 ◦ C). A volume of 250 l of plasma was transferred to a 1.5-ml polypropylene microcentrifuge tube. The internal standard (20 l, equal to 500 ng of acyclovir) and 500 l of acetonitrile were added, and followed by shaking for 30 s. After centrifugation at 12,000 × g for 2 min (Microfuge® 18 from Beckman Coulter, Germany), 500 l of the supernatant was transferred into another tube and 1 ml of dichloromethane was added. The mixture was vortex-mixed for 30 s and centrifuged at 12,000 × g for 2 min. Finally, 50 l of the aqueous supernatant was transferred into another tube and a 10-l aliquot was injected onto the HPLC system.
2.00
1.50
1.00 0
10
20
30
40
50
Temperature (°°C) Fig. 2. The effects of temperature on the retention factor of lamivudine and acyclovir. The chromatographic conditions were as follows: Column, Chromolith RP 18e (100 mm × 4.6 mm) with guard; mobile phase, (A) 0.4% or (B) 0.6% triethylamine in 50 mM NaH2 PO4 at pH 3.3; flow rate, 1.5 ml/min; detection, 278 nm.
2.6. Assay validation Obtained blank plasma samples from 24 healthy volunteers were assessed by the procedure as described above and compared with spiked and real plasma samples from pharmacokinetic study to evaluate selectivity of the method. The precision and accuracy of the method were examined by adding known amounts of lamivudine to pool plasma. These quality control samples (40, 160, 640 and 2560 ng/ml) were made from a stock solution separate from that used to prepare plasma standards and were not used for constructing calibration curves. For intra-day precision and accuracy five replicate quality control samples at each concentration were assayed on the same day. The inter-day precision and accuracy were evaluated on five different days within 2 weeks along analyzing plasma samples of volunteers. The absolute recoveries (n = 5) was calculated by comparing peak heights obtained from prepared sample extracts with those found by direct injection of drug solution made in 0.1% acetic acid at the same concentration. The lower limit of quantification (LLOQ) was estimated by analyzing lamivudine at low concentrations of the calibration curve. The LLOQ was defined as a concentration level where accuracy and precision were still better than 10%. To determine the limit of detection (LOD), lower plasma concentrations than the lower end of the calibration curve were used. The LOD was then defined as the concentration which caused a signal three times the noise (S/N = 3/1).
42
Lamivudine
4
3
Acyclovir (IS)
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mAU
2
1
0
C) B)
-1
A) -2 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Minutes Fig. 3. Representative chromatograms of (A) a blank plasma; (B) plasma spiked with 40 ng/ml lamivudine; (C) a volunteer plasma sample, 2 h after taking 150 mg tablet of lamivudine (1682 ng/ml).
2.7. Application The assay was used for a comparative bioavailability study of two tablets preparations containing 150 mg lamivudine. The Institutional Review Board of Golestan University of Medical University (Gorgan, Iran) was dedicated to endorse the ethical conduct of the study and to approve the protocol (the authorization number: A880507). The board is constituted and operates in accordance with the principles and requirements described in the Guidelines on Research Involving Human Subjects. Twenty four healthy volunteers participated in the study. The study was conducted using a two-way crossover design, as a single dose, randomized trial. The two formulations were administrated on 2 treatment days, separated by a washout period of 7 days, to fasted subjects who received one of the study medications. Food and drinks were not allowed until 3 h after ingestion of the tablets. Multiple blood samples (3 ml) were collected before and 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8 and 10 h post-dosing. The plasma was immediately separated by centrifugation and frozen at −20 ◦ C until analysis. 3. Results and discussion 3.1. Sample preparation Lamivudine with a pKa value of 4.3 is classified by WHO as “soluble” in water [16]. Review of the literature reveals that liquid–liquid extraction (LLE) of highly polar drugs like lamivudine is a big challenge. According to LLE-based reports [11,13], we first tried
several solvent mixtures at different pH values to extract lamivudine, which all resulted in low and irreproducible recoveries. The problem may be in part due to the low pKa value of lamivudine. We have already reported that metformin, another highly hydrophilic compound with a pKa value of 12.4, could be effectively extracted by a suitable organic solvent under strong alkaline condition [17]. Solid-phase extraction (SPE) procedure [4,6–8,12,15] has been reported. However, the technique is expensive and timeconsuming. Removal of proteins from plasma samples using acid [5,9] or by acetonitrile followed by evaporation of the supernatant [10] has been proposed as an alternative to LLE and SPE methods. However, the problem of potential endogenous interferences in the chromatogram limits their usefulness and further purification of sample seems to be mandatory. In the present work, we postulated that after protein removal by acetonitrile, the separation of aqueous phase could concentrate lamivudine while will leave many plasma interferences in the acetonitrile layer. Acetonitrile does not associate strongly with water and thus, as opposed to methanol, acetonitrile–water mixtures remain binary in character. Although water and acetonitrile are practically immiscible at 0–4 ◦ C [18], the separation could be efficiently take place at room temperature by adding a kosmotrope such as ammonium sulfate [19] or a water-immiscible organic solvent like dichloromethane with high solubility for acetonitrile [18,20]. In the present work, acetonitrile acts as an efficient protein precipitation agent and then, dichloromethane eliminates neutral lipophilic substances as well as acetonitrile used for protein precipitation and lamivudine remains undiluted in the aqueous layer. The aqueous layer could be
Table 1 The accuracy, within- and between-day precision and recovery data for the measurement of lamivudine in human plasma (n = 5). Nominal concentration (ng/ml)
Recovery (%)
40 160 640 2560
99.1 96.7 98.1 96.8
Intra-day
Inter-day
Mean ± SD ± ± ± ±
7.2 4.4 3.2 1.5
40.81 162.4 636.9 2572.2
± ± ± ±
1.58 7.72 27.84 72.62
Precision (%)
Accuracy (%)
Mean ± SD
3.9 4.7 4.4 2.8
2 1.5 −0.5 0.5
38.52 164.62 645.42 2543.2
± ± ± ±
2.62 8.92 31.32 93.14
Precision (%)
Accuracy (%)
6.8 5.4 4.8 3.7
−3.7 2.3 0.8 −0.6
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stability of reversed-phase columns when operated with totally aqueous mobile phases. The monolithic column showed suitable stability of performance as reflected in constant lamivudine peak asymmetry and plate number throughout the study.
4000
Lamivudine plasma concentration (ng/ml)
A)
Lamivudine-Exir-T
3500 3000 2500 2000 1500 1000 500 0 0
Lamivudine plasma concentration (ng/ml)
B
2
4
6
8
10
Time (h) 4000
EPIVIR-gsk-R
3500 3000 2500 2000 1500 1000 500 0 0
2
4
6
8
43
10
3.3.2. Mobile phase The separation of lamivudine under varying mobile phase composition was investigated. A mobile phase containing a very low amount of TEA in an acidic phosphate buffer gave the best separation of lamivudine from the plasma interferences. It should be noted that increasing the TEA amount in the mobile phase decrease the retention times and simultaneously improves the separation of lamivudine and acyclovir peaks due to greater susceptibility of lamivudine to TEA, but then the separation from plasma interferences will be a major concern. Therefore, the amount of TEA in the mobile phase was carefully optimized to achieve a suitable separation between lamivudine, internal standard and endogenous plasma peaks. 3.3.3. Chromatographic temperature Temperature had a critical role in the separation of lamivudine from acyclovir. As presented in Fig. 2, the effects of chromatographic temperature on the chromatographic behavior of lamivudine and acyclovir were evaluated using 0.4 and 0.6% TEA in phosphate buffer as mobile phase. It was observed that the resolution between two peaks is lost at unsuitable temperatures. Interestingly, temperature also affects the elution order of lamivudine and acyclovir in the chromatogram. It could be probably concluded that raising the temperature increases the solubility of these compounds in the mobile phases, but the rate is higher for acyclovir in comparison to lamivudine.
Time (h) Fig. 4. Plasma concentration-time profiles of lamivudine following oral administration of test or reference tablets at a dose of 150 mg in 24 healthy volunteers in a crossover study.
completely separated from organic layer and it was not necessary to heat the obtained aqueous layer in order to remove the trace amount of organic solvents. 3.2. Selection of internal standard The internal standard, acyclovir was selected based on its comparable solubility and recovery, and stability properties to lamivudine during storage and sample preparation step. By changing the wavelength to 254 nm, the present method was also successfully used for acyclovir assay in plasma in another bioequivalence study using lamivudine as internal standard (data not shown). 3.3. Chromatographic separation 3.3.1. Chromatographic column In comparison to several tested particulate HPLC columns, Chromolith RP-18e column showed superior separation efficiency with less plasma endogenous peaks. Another critical point was the
3.4. Assay validation Representative chromatograms of drug-free plasma, plasma spiked with lamivudine and a volunteer sample collected after oral dosing with lamivudine are shown in Fig. 3. The retention times for lamivudine and the internal standard were 2.7 and 3.1 min, respectively. The injection of blank plasma samples of 24 volunteers showed no interfering peaks close to the retention times of lamivudine or the internal standard. Although the last endogenous peak was eluted at 6 min, but a new injection was done in every 5 min placing that peak in the fronting area of the next chromatogram. Lipemic and hemolysate plasma samples were spiked with different concentrations of lamivudine. The obtained concentrations were in the acceptable range of ±5%. Over 700 plasma samples were analyzed by this method and the asymmetry value of 1.2 and theoretical plate number of 6000 for both lamivudine and acyclovir, and the resolution factor of 1.8 between them were constant during the entire course of the study. The calibration curves were linear over the concentration range of 40–2560 ng/ml in human plasma, with a correlation coefficient greater than 0.999. The LLOQ was 40 ng/ml and the LOD was 10 ng/ml. The results of the method intra- and inter-day accuracy and precision are presented in Table 1. All values for accuracy and precision were within the recommended limits. Intra-day precision ranged between 2.8 and 4.7% whereas the interday precision was between 3.7 and 6.8%. The intra-day mean error
Table 2 Phamacokinetic parameters (mean ± SEM) for lamivudine following oral administration of test or reference tablets at a dose of 150 mg in 24 healthy volunteers. Group
Cmax (ng/ml)
tmax (h)
AUC0–t (ng/ml h)
AUC0–∞ (ng/ml h)
t1/2 (h)
T product Epivir-R
2213.1 (121.3) 2171.0 (115.8)
1.06 (0.09) 1.23 (0.09)
6753.2 (213.0) 6736.4 (247.5)
7152.7 (238.3) 7148.7 (274.0)
2.25 (0.05) 2.26 (0.05)
Cmax = maximum plasma concentration; tmax = time to reach Cmax ; AUC0–∞ = area under the concentration-time curve (AUC) from time zero to infinity; AUC0–t = AUC from time zero to the last measurable concentration; t1/2 = elimination plasma half-life.
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was between −0.5 and 2% whereas the inter-day mean error was between −3.7 and 2.3%. The mean absolute recoveries for lamivudine and internal standard using the present extraction procedure were 97.7 and 99.5%, respectively. 3.5. Pharmacokinetic results The proposed method was applied to the determination of lamivudine in plasma samples for the purpose of the bioequivalence study. The plasma lamivudine profiles for volunteers after taking two products are shown in Fig. 4. Pharmacokinetic parameters obtained from two preparations are summarized in Table 2. The extrapolated fraction of the AUC0–∞ accounted only for 5–6%, which indicates the suitability of the present analytical method for pharmacokinetic studies. The test/reference geometric ratio for Cmax (90% CI) was 1.02 (95.3, 108.7). The test/reference geometric ratios for AUC0–t (90% CI) and AUC0–∞ (90% CI) were 1.01 (97.3, 103.9) and 1.00 (97.2, 103.7), respectively. The 90% CIs of Cmax , AUC0–t and AUC0–∞ were well within the acceptable range of 80 and 125 suggested by the US FDA bioequivalence guideline. It was concluded that the two products were bioequivalent and therefore, could be used interchangeably. 4. Conclusion In the present study, a simple and reliable HPLC method was developed and validated. The new strategy for sample preparation entails protein precipitation by acetonitrile followed by acetonitrile removal using dichloromethane. This very simple and quick sample preparation procedure offered a complete and reproducible recovery of lamivudine from plasma. Moreover, use of a simply available and structurally related internal standard, a short chromatographic run time, and the performance stability of analytical
column and the mobile phase propose the current method as a suitable choice for application to bioequivalence studies where a large number of plasma samples should be assayed in a short time. We demonstrated usefulness of the current method by its application to a bioequivalence study of lamivudine tablets in 24 healthy volunteers. References [1] J.A.V. Coates, N. Cammack, H.J. Jenkinson, A.J. Jowett, M.I. Jowett, B.A. Pearson, C.R. Penn, P.L. Rouse, K. Claire Viner, J.M. Cameron, Antimicrob. Agents Chemother. 36 (1992) 733–739. [2] G.A. Balint, Pharmacol. Ther. 89 (2001) 17–27. [3] M.A. Johnson, K.H.P. Moore, G.J. Yuen, A. Bye, G.E. Pakes, Clin. Pharmacokinet. 36 (1999) 41–66. [4] A.J. Harker, G.L. Evans, A.E. Hawley, D.M. Morris, J. Chromatogr. B 657 (1994) 227–232. [5] X.J. Zhou, J.P. Sommadossi, J. Chromatogr. B 691 (1997) 417–424. [6] R.M.W. Hoetelmans, M. Profijt, P.L. Meenhorst, J.W. Mulder, J.H. Beijnen, J. Chromatogr. B 713 (1998) 387–394. [7] J.J. Zheng, S.T. Wu, T.A. Emm, J. Chromatogr. B 761 (2001) 195–201. [8] B. Fan, J.T. Stewart, J. Pharm. Biomed. Anal. 28 (2002) 903–908. [9] Y. Alnouti, C.A. White, M.G. Bartlett, J. Chromatogr. B 803 (2004) 279–284. [10] E.K. Kano, C.H.R. Serra, E.E.M. Koono, S.S. Andrade, V. Porta, Int. J. Pharm. 297 (2005) 73–79. [11] G. Bahrami, S. Mirzaeei, A. Kiani, B. Mohammadi, J. Chromatogr. B 823 (2005) 213–217. [12] M. Shekarchi, M. Pourfarzib, B. Akbari-Adergani, A. Mehramizi, M. Javanbakht, R. Dinarvand, J. Chromatogr. B 931 (2013) 50–55. [13] U. Nandi, A. Das, B. Roy, H. Choudhury, B. Gorain, T.K. Pal, Drug Test. Anal. 5 (2013) 485–491. [14] K.B. Kenny, S.A. Wring, R.M. Carr, G.N. Wells, J.A. Dunn, J. Pharm. Biomed. Anal. 22 (2000) 967–983. [15] H.N. Mistri, A.G. Jangid, A. Pudage, N. Gomes, M. Sanyal, P. Shrivastav, J. Chromatogr. B 853 (2007) 320–332. [16] S. Strauch, E. Jantratid, J.B. Dressman, H.E. Junginger, S. Kopp, K.K. Midha, V.P. Shah, S. Stavchansky, D.M. Barends, J. Pharm. Sci. 100 (2011) 2054–2063. [17] H. Amini, A. Ahmadiani, P. Gazerani, J. Chromatogr. B 824 (2005) 319–322. [18] S.T. Forgue, K.A. Pittman, R.H. Barbhaiya, J. Chromatogr. 414 (1987) 343–353. [19] A. Rahman, N.E. Hoffman, J. Chromatogr. 433 (1988) 159–166. [20] F. Kees, H. Grobecker, K.G. Naber, J. Chromatogr. 305 (1984) 363–371.