International Journal of Biological Macromolecules 65 (2014) 65–71
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Development and validation of RP-HPLC method for quantification of glipizide in biological macromolecules Nihar Ranjan Pani a,∗ , Sujata Acharya b , Sradhanjali Patra b a b
Department of Pharmaceutics, Gayatri College of Pharmacy, Sambalpur, Odisha, India Department of Pharmaceutical Sciences, Utkal University, Bhubaneswar, Odisha, India
a r t i c l e
i n f o
Article history: Received 19 November 2013 Received in revised form 19 December 2013 Accepted 5 January 2014 Available online 10 January 2014 Keywords: Floating microspheres HPLC glipizide rabbit Pharmacokinetics
a b s t r a c t Glipizide (GPZ) has been widely used in the treatment of type-2 diabetics as insulin secretogague. Multiunit chitosan based GPZ floating microspheres was prepared by ionotropic gelation method for gastroretentive delivery using sodiumtripolyphosphate as cross-linking agent. Pharmacokinetic study of microspheres was done in rabbit and plasma samples were analyzed by a newly developed and validated high-performance liquid chromatographic method. Method was developed on Hypersil ODS18 column using a mobile phase of 10 mM phosphate buffer (pH, 3.5) and methanol (25:75, v/v). Elute was monitored at 230 nm with a flow rate of 1 mL/min. Calibration curve was linear over the concentration range of 25.38–2046.45 ng/mL. Retention times of GPZ and internal standard (gliclazide) were 7.32 and 9.02 min respectively. Maximum plasma drug concentration, area under the plasma drug concentration–time curve and elimination half life for GPZ floating microspheres were 2.88 ± 0.29 g mL−1 , 38.46 ± 2.26 g h mL−1 and 13.55 ± 1.36 h respectively. When the fraction of drug dissolved from microspheres in pH 7.4 was plotted against the fraction of drug absorbed, a linear corre— 0.991) was obtained in in vitro and in vivo correlation study. lation (R2 — © 2014 Elsevier B.V. All rights reserved.
1. Introduction Glipizide (GPZ), is a second-generation sulfonylurea that can acutely lower the blood glucose level in humans by stimulating the release of insulin from the pancreas and is typically prescribed to treat type II diabetes (non-insulin dependent diabetes mellitus) [1]. The chemical name of GPZ (Fig. 1) is l-cyclohexyl-3-[[p-[2-(5methyrpyrazine-carboxamido)ethyl]phenyl]sulfonyl]urea having molecular formula C21 H27 N5 O4 S and molecular weight 445.55 [1]. GPZ exhibits poor aqueous solubility and belongs to class II of the biopharmaceutics classification system (BCS) [2]. Being a weak acid (pKa — — 5.9), GPZ is better absorbed from basic medium; however, at very low pH levels, the solubility of glipizide is minimal [2,3]. High performance liquid chromatography (HPLC) with Photo diode array (PDA) detector is the most common technique used for routine analysis of drugs in industries and research based organization. Literature review assembled that a number of liquid chromatography methods have been reported for the determination of glipizide alone [4–6], with rosiglitazone [7,8], metformin [9,10] and other antidiabetic drugs [8] and glimepride alone [11], with rosiglitazone [12,13], pioglitazone [13–16], and metformin
∗ Corresponding author. Tel.: +91 8018544142. E-mail address: [email protected] (N.R. Pani). 0141-8130/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2014.01.007
[16,17]. A few bioassays for analysis of glipizide in plasma or serum have been reported. The determination of GPZ in plasma has been performed by radioimmunoassay technique. However, the selectivity of these methods has not been verified [7]. Gas Chromatography (GC) has also been used for determination of GPZ in plasma. However, GC requires a time consuming derivatization step to give volatile and thermally stable derivatives [5] described HPLC technique for determination of glipizide in human plasma and urine. It is limited by long elution time. The focus of this study described herein was to develop simple, precise, rapid, accurate, and economical HPLC method with PDA detection for the quantification of GPZ in biological macromolecule i.e. rabbit plasma. The developed method was applied to estimate the plasma drug concentration of GPZ in rabbit at predetermine time point after oral administration of controlled release floating microspheres and, those Pharmacokinetic parameters and in vitro–in vivo correlation were determined.
2. Materials and methods Glipizide and gliclazide were the generous gift from Glenmark Pharmaceuticals Ltd, Nashik and Macleods Pharmaceuticals Ltd Mumbai respectively. Chitosan was obtained from Thahira Chemicals, Kerela and sodium tripolyphosphate was obtained from Thomas Baker Chemicals Pvt. Ltd., India. HPLC grade acetonitrile,
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2.4. Chromatographic conditions HPLC analysis was performed using a Hypersil ODS C18, (average particle size 5 m) column (250 mm, 4.6 mm). The mobile phase consisted of 10 mM phosphate buffer (pH, 3.5) and methanol (25:75, v/v). Eluent was monitored with the PDA detector at 230 nm with a flow rate of 1 mL/min and sample size of 20 L was carried out at column oven temp 30 ± 2 ◦ C all over the study. Fig. 1. Chemical structure of glipizide.
2.5. Preparation of standard solution methanol and dichloromethane were procured from Merck, Mumbai. Potassium di-hydrogen phosphate (KH2 PO4 ) and orthophosphoric acid (H3 PO4 ) were purchased from S.D. Fine Chem Ltd., Mumbai, India). All other chemicals and solvents used were of analytical grade. Water used in the HPLC analysis was prepared by the water purifier (AriumR , 611UF, Sartorius, Germany). The mobile phase and all the solutions were filtered through a 0.45 m Ultipor® N66 ® membrane filter (Pall Life Sciences, USA) prior to use.
Stock solution of GPZ (100 g/mL) and gliclazide (internal standard, 100 g/mL) were prepared in methanol. Further dilution was carried out in mobile phase solvent for the preparation of working stock solution. Calibration standards were prepared freshly by spiking working GPZ solution into the control blank plasma to give the concentration of 25.38, 50.77, 101.53, 203.06, 406.12, 846.09, 1244.24, 1637.16, 2046.45 ng/mL. All the solutions were prepared once and analyzed daily over a period of 3 days for inter-day precision of the method. These solutions were stored at −20 ◦ C.
2.1. Instruments HPLC system (Waters, USA) consisting of quaternary pump (WaterTM 600), 7725i rheodyne manual injector and PDA detector and empower-II software were used for analysis. The plasma samples were processed by using of Micropipettes (Ependruff, USA), Spinix Vortexer (M37610-33, Barnstead International, USA), Biofuge Fresco Centrifuge (Heraeus, Germany), Ultra-Sonicator (Loba Chem, Mumbai), Nitrogen gas evaporator and Multi-Pulse Vortexer (Glas-COL, USA).
2.6. Quality control standards Lowest quality control standards, median quality control standards and highest quality control standards were prepared by spiking drug free plasma with GPZ to give solutions containing 101.53, 406.12 and 1244.24 ng/mL, respectively. They were stored at −20 ◦ C till analysis. 2.7. Sample preparation
2.2. Experimental animals Albino rabbits weighing 1.5–2.5 kg (housed in a temperature (22 ± 1 ◦ C) and relative humidity (55 ± 10%) controlled room were used in the experiments. The rabbits were maintained in accordance with the principles declared by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, Govt. of India). The rabbits were orally administered the floating microspheres containing 2.5 mg GPZ. 2.3. Preparation of floating microspheres Floating microspheres were prepared according to an ionotropic gelation method and physically cross-linking with sodium tripolyphosphate (sodium TPP) [17–19]. Briefly, GPZ (1% (w/v) was dispersed in a stirred solution of chitosan (1% (w/v)) prepared in 2% (v/v) acetic acid until a uniform dispersion was obtained. The microparticles were formed by dropping the bubble free dispersion (30 mL) through a 21G disposable syringe into 100 mL of mechanically agitated (300 rpm) solution of the cross-linking agent, sodium TPP (4% (w/v)) at the rate of 30 drops/min from 5 cm height. The microparticles formed were separated after a 30 min, washed with deionized water and then subsequently dried at room temperature for 3 h. The prepared microspheres were evaluated for yield value, drug entrapment efficiency, particle size measurement, in vitro bioadhesion study, in vitro buoyancy study, in vitro drug release and stability study [19]. The condition for in vitro drug release study was USP basket-type dissolution apparatus with basket rotation of 100 rpm, 900 mL of phosphate buffer (pH 7.4) as a dissolution medium and 5 mL of sample was withdrawn and replaced at predetermined time point. The fraction of drug dissolved (FRD) was calculated by dividing 100 in percentage of drug release at each time point.
An aliquot quantity of 180 L of rat plasma spiked with 10 L of calibration standard drug was taken in a 2 mL stopper centrifuge tube and mixed for 20 s. To this, 10 L of internal standard (IS) solution (100 g/mL) was added and mixed for 20 s. The drug was extracted by vortexing with 1.5 mL of selected extracting solvents among acetonitrile, methanol and di-chloromethane in a spinix vortexer for 10 min followed by centrifugation at 10,000 rpm for 5 min at 4 ◦ C. The supernatant was withdrawn and dried using nitrogen evaporator. The residue was reconstituted with 100 L of mobile phase and 20 L was injected onto the column. 2.8. Validation of method The validation of an analytical method confirms the characteristics of the method to satisfy the requirements of the application domain [20]. The method was validated following the ICH Guidelines [21,22] for specificity, recovery, linearity, precision and stability. Under the validation programme the following parameters were studied. 2.8.1. Specificity The specificity criterion demonstrates that the result of the method is not affected by the presence of interferences, i.e. whether the compound of interest elutes without interfering with other compounds and components of plasma. The specificity of the method was determined by comparing the chromatograms obtained from the aqueous samples of GPZ and IS with those obtained from blank plasma. Blank plasma samples from each of five rabbits were processed in presence of IS and another set of five samples processed with GPZ and IS to evaluate presence of interference around the peak of GPZ. The solutions containing 406.12 ng/mL of GPZ were injected into the column under the optimized chromatographic conditions to obtain the chromatographic
N.R. Pani et al. / International Journal of Biological Macromolecules 65 (2014) 65–71
peaks in plasma with IS so as to differentiate them from the interfering peaks of plasma components. 2.8.2. Selection of solvent for recovery of drug The recovery of an analyte is the extraction efficiency of an analytical process, reported as a percentage of the known amount of an analyte carried through the sample extraction and processing steps of the method. Different organic extraction solvents (acetonitrile, methanol and di-chloro methane) were tried in the experiment to recover GPZ from plasma samples. Quality control samples were prepared in triplicate at three levels of 101.53, 406.12 and 1244.24 ng/mL of GPZ and assayed by HPLC method as described as above. The extraction efficiency of GPZ was determined by comparing the peak areas obtained from extracted quality control samples with the peak area of aqueous working solution containing same concentration of GPZ at three levels. 2.8.3. Linearity Quantitative analytical results are highly influenced by the quality of the calibration curve [22,23]. Nine different concentrations of GPZ with fixed concentration of IS in blank plasma were processed and calibration curve was constructed in the specified concentration range (25.38, 50.77, 101.53, 203.06, 406.12, 846.09, 1244.24, 1637.16, 2046.45 ng/mL). The calibration curve was plotted between the ratio of peak areas of GPZ to IS and concentration of GPZ by replicate analysis (n — — 6) at all concentration levels and the linear relationship was evaluated using the least square method using Microsoft Excel® (Microsoft Corporation, USA) program. 2.8.4. Precision and accuracy Both repeatability (within a day precision) and reproducibility (between days precision) were determined as follows. Three quality control samples were subjected for the study. Five injections of each of the specified quality control samples at three levels were injected for analysis within the same day for repeatability, and over a period of 5 days for reproducibility. Mean and relative standard deviation were calculated and used to predict the accuracy and precision of the method. Accuracy was calculated as the percent of GPZ found in the intra-day and inter-day samples to that of the actual. 2.8.5. Stability studies The quality control standards containing 101.53, 406.12 and 1244.24 ng/mL (n — — 6) of GPZ were subjected for detection of stability of the drug in plasma. The initial assay of the samples was conducted. One set of five samples each was kept in poly propylene tubes and subjected to three freeze–thaw cycles each at −20 ◦ C for 24 h and room temperature for 24 h. The second set of five samples each was kept at room temperature for 24 h and the third set of five samples each were kept at room temperature for 1 month. All the samples were analyzed by standard chromatographic conditions to determine their peak areas. Samples were considered to be stable, when the final assay values of samples were found similar to that of the initial assay value of the drug. 2.9. Pharmacokinetic study in rabbits The method described above was applied to quantify the plasma concentration of GPZ in a single-dose pharmacokinetic study conducted on three white male albino rabbits. The protocol was approved by the Institutional Ethical Committee at the Gayatri College of Pharmacy, Odisha, India. The experiments were conducted as per CPCSEA (Committee for Prevention, Control and Supervision of Experimental Animals) guidelines. The rabbits weighing 1.5–2.5 kg were housed with free access to food and water, except for the final 12 h before experimentation. After a single oral administration of prepared floating microspheres containing 2.5 mg of
67
GPZ, 2 mL of blood samples were collected from the marginal ear vein at 0, 0.25, 0.5, 1, 2, 3, 4, 6, 9, 12 and 24 h time points into heparinized collection tubes. The blood was immediately centrifuged (1000 × g) for 10 min at an ambient temperature. The supernatant plasma layer was separated and stored at −20 ◦ C until analyzed. The plasma samples were analyzed for GPZ concentrations by RP-HPLC method. The first order elimination rate constant (kel ) was estimated by the least square regression of plasma concentration–time data points of the curves describing the terminal log-linear decaying phase. t1/2 was derived from kel (t1/2 = 0.693/ke1 ). The area under the plasma concentration–time curve from zero to the last measurable plasma concentration at time t (AUC0–t ) was calculated using the linear trapezoidal rule. The area was extrapolated to infinity (AUC0–∞ ) by addition of Ct /kel to AUC0–t , where Ct is the last detectable drug concentration. The absorption rate constant (ka ) was determined by residual method [24]. The maximum observed GPZ concentration (Cmax ) and the time at which Cmax was observed (Tmax ) were reported directly from the profile. The AUMC is the area under the plot of time vs. the product of time and concentration extrapolated to infinity and was calculated by trapezoidal rule. Volume of distribution (Vd ) and total clearance rate (TCR) were calculated using Eqs. (1) and (2) respectively. The mean residence time (MRT) was determined by AUMC divided by AUC. The clearance (Cl) was calculated as dose divided by AUC with extrapolation to infinity (AUC0–∞ ). All the pharmacokinetic parameters were calculated using Microsoft® Office Excel 2003 (Microsoft Corporation, USA) software application. Vd =
0 · AUMC) (DG
(1)
(AUC)2
TCR = kel · Vd =
Vd · 0 : 693 t1/2
(2)
The Wagner–Nelson method [25] was applied to deconvolute the percentage of the GPZ absorbed using Eq. (3). Ft = C(t) + kel AUC(0−t)
(3)
where Ft is the amount of drug absorbed. The fraction absorbed (FRA) was calculated using Eq. (4) and was plotted against the fraction of drug dissolved (FRD) at the same time and the linear regression analysis was used to examine the in vitro–in vivo relationship.
Fraction absorbed (FRA) =
C(t) + kel AUC(0–t)
[kel AUC0–∞ ]
(4)
2.10. Establishment of in vitro–in vivo correlation (IVIVC) An IVIVC refers to the predictive mathematical model describing the relationship between in vitro property of a controlled-release dosage form (usually the rate or extent of drug dissolution or release) and a relevant in vivo response, such as plasma drug concentration or amount of drug absorbed. A level A correlation is usually estimated by a two-stage procedure: deconvolution followed by comparison of the fraction of drug absorbed (FRA) to the fraction of drug dissolved (FRD). A correlation of this type represents a point-to-point relationship between in vitro dissolution and the in vivo input rate [26]. Accordingly, the deconvolution procedure was done on floating microspheres using mean GPZ plasma concentration vs. time profile using the method of Wagner–Nelson. The data of FRA (obtained by deconvolution) and FRD for each formulation were then plotted to develop an IVIVC model. The linear,
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Fig. 2. Chromatogram of the mixture of glipizide and Internal Standard in mobile phase.
quadratic, and cubic models (Eq. (5) bearing the following forms were tested to obtain the best fit:
of interferences. The best resolution and sensitivity of the method was obtained at 230 nm and 1 mL/min flow rate of mobile phase.
Y = a + bX, Y = a + bX + cX 2 , Y = a + bX + cX 2 + dX 3
3.3. Validation of methods
(5)
where a, b, c, d represent regression parameters associated with each function, Y is the FRA in vivo and X is the FRD in vitro. The coefficients of determination (r2 ) for all developed models were determined. The highest r2 value equation was considered as the best fitting model. 3. Results and discussion 3.1. Preparation of floating microspheres The microspheres were prepared by the interaction of sodium TPP, a polyanion (P3 O10 5− ) with the positively charged amino group of chitosan by electrostatic forces [19]. The prepared spherical microspheres were evaluated for yield value (82.61 ± 4.37%), entrapment efficiency (64.86 ± 2.68%), average particle size (711 m) and floating lag time (0). More than 80% of microspheres were floated on 0.1 N HCl for at least 12 h. The bioadhession of microspheres on agar plate after 8 h was 76 ± 2.44%. The in vitro drug release studies showed that 87.48 ± 2.73% of GPZ were released from microspheres in a controlled manner within 12 h. 3.2. Selection and development of chromatographic method Normal phase chromatography can be used for the separation of non-ionic and non-polar substances, while reversed-phase chromatography (C8 and C18 column) can be used for the separation of non-ionic as well as ionic non-polar to semi polar substances. Thus, GPZ (an ionisable semi polar weak acid) can satisfactorily separated by reversed phase chromatography. Octylsilane (C8) columns are similar to octadecylsilane (C18). However, octylsilane columns are less retentive as compared to octadecylsilane. Majority of the ionizable pharmaceutical compounds can be very well separated on octadecylsilane reversed phase columns [21]. Hence, octadecylsilane column was selected for GPZ. A mixture of methanol and phosphate buffer (pH 3.5) at flow rate 1 mL/min was used as mobile phase for the analysis of GPZ [21]. The optimum ratio of methanol to phosphate buffer (pH 3.5) used in the current investigation was 75:25, which was selected on the basis of resolution and absence
3.3.1. Specificity Typical chromatogram (Fig. 2) of mixture of GPZ and IS revealed that they are well separated under HPLC conditions applied. A chromatogram of blank plasma sample is shown in Fig. 3. Retention time was 7.32 min for GPZ and 09.02 min for IS. In the comparison of chromatogram of GPZ and IS (Fig. 2) with the chromatogram of blank plasma (Fig. 3), absence of interference of plasma components is observed around the zone of retention time of GPZ and IS. The chromatogram of medium level quality control sample i.e. 406.12 ng/mL of GPZ (Fig. 4) showed a good resolution peaks for GPZ and IS which are well differentiated from the peaks of plasma components. 3.3.2. Selection of extracting solvent for recovery of drug The solvent extracted maximum amount of drug from plasma sample was selected for processing of the samples. The recovery of dug was found to be 66. 22 ± 0.84, 78.62 ± 1.54 and 47.66 ± 1.68% in acetonitrile, methanol and di-chloromethane respectively at all three concentration levels which confirm the extraction efficiency of the solvents. Among three solvents, methanol showed maximum amount of drug recovered, hence methanol was selected as the extracting solvent. 3.3.3. Linearity range The ratio of peaks area of GPZ to IS at various concentrations of GPZ in plasma was shown in Table 1. A calibration curve was plotted between peak area ratios of GPZ to IS versus plasma GPZ concentrations. The chromatographic responses (ratio of peaks area of GPZ to IS vs. GPZ concentration) were found to be linear over an analytical range of 25.38–2046.45 ng/mL with regression coefficient (r2 ) value 0.999 which showed reproducibility. The linear regression equation obtained was Y = 0.001X − 0.002. 3.3.4. Precision and accuracy The accuracy of the measurements was determined by using three quality control samples and the results are reported in Table 2. The relative standard deviation (RSD) of intra-day assay of the drug was
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Development and validation of RP-HPLC method for quantification of glipizide in biological macromolecules Nihar Ranjan Pani a,∗ , Sujata Acharya b , Sradhanjali Patra b a b
Department of Pharmaceutics, Gayatri College of Pharmacy, Sambalpur, Odisha, India Department of Pharmaceutical Sciences, Utkal University, Bhubaneswar, Odisha, India
a r t i c l e
i n f o
Article history: Received 19 November 2013 Received in revised form 19 December 2013 Accepted 5 January 2014 Available online 10 January 2014 Keywords: Floating microspheres HPLC glipizide rabbit Pharmacokinetics
a b s t r a c t Glipizide (GPZ) has been widely used in the treatment of type-2 diabetics as insulin secretogague. Multiunit chitosan based GPZ floating microspheres was prepared by ionotropic gelation method for gastroretentive delivery using sodiumtripolyphosphate as cross-linking agent. Pharmacokinetic study of microspheres was done in rabbit and plasma samples were analyzed by a newly developed and validated high-performance liquid chromatographic method. Method was developed on Hypersil ODS18 column using a mobile phase of 10 mM phosphate buffer (pH, 3.5) and methanol (25:75, v/v). Elute was monitored at 230 nm with a flow rate of 1 mL/min. Calibration curve was linear over the concentration range of 25.38–2046.45 ng/mL. Retention times of GPZ and internal standard (gliclazide) were 7.32 and 9.02 min respectively. Maximum plasma drug concentration, area under the plasma drug concentration–time curve and elimination half life for GPZ floating microspheres were 2.88 ± 0.29 g mL−1 , 38.46 ± 2.26 g h mL−1 and 13.55 ± 1.36 h respectively. When the fraction of drug dissolved from microspheres in pH 7.4 was plotted against the fraction of drug absorbed, a linear corre— 0.991) was obtained in in vitro and in vivo correlation study. lation (R2 — © 2014 Elsevier B.V. All rights reserved.
1. Introduction Glipizide (GPZ), is a second-generation sulfonylurea that can acutely lower the blood glucose level in humans by stimulating the release of insulin from the pancreas and is typically prescribed to treat type II diabetes (non-insulin dependent diabetes mellitus) [1]. The chemical name of GPZ (Fig. 1) is l-cyclohexyl-3-[[p-[2-(5methyrpyrazine-carboxamido)ethyl]phenyl]sulfonyl]urea having molecular formula C21 H27 N5 O4 S and molecular weight 445.55 [1]. GPZ exhibits poor aqueous solubility and belongs to class II of the biopharmaceutics classification system (BCS) [2]. Being a weak acid (pKa — — 5.9), GPZ is better absorbed from basic medium; however, at very low pH levels, the solubility of glipizide is minimal [2,3]. High performance liquid chromatography (HPLC) with Photo diode array (PDA) detector is the most common technique used for routine analysis of drugs in industries and research based organization. Literature review assembled that a number of liquid chromatography methods have been reported for the determination of glipizide alone [4–6], with rosiglitazone [7,8], metformin [9,10] and other antidiabetic drugs [8] and glimepride alone [11], with rosiglitazone [12,13], pioglitazone [13–16], and metformin
∗ Corresponding author. Tel.: +91 8018544142. E-mail address: [email protected] (N.R. Pani). 0141-8130/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2014.01.007
[16,17]. A few bioassays for analysis of glipizide in plasma or serum have been reported. The determination of GPZ in plasma has been performed by radioimmunoassay technique. However, the selectivity of these methods has not been verified [7]. Gas Chromatography (GC) has also been used for determination of GPZ in plasma. However, GC requires a time consuming derivatization step to give volatile and thermally stable derivatives [5] described HPLC technique for determination of glipizide in human plasma and urine. It is limited by long elution time. The focus of this study described herein was to develop simple, precise, rapid, accurate, and economical HPLC method with PDA detection for the quantification of GPZ in biological macromolecule i.e. rabbit plasma. The developed method was applied to estimate the plasma drug concentration of GPZ in rabbit at predetermine time point after oral administration of controlled release floating microspheres and, those Pharmacokinetic parameters and in vitro–in vivo correlation were determined.
2. Materials and methods Glipizide and gliclazide were the generous gift from Glenmark Pharmaceuticals Ltd, Nashik and Macleods Pharmaceuticals Ltd Mumbai respectively. Chitosan was obtained from Thahira Chemicals, Kerela and sodium tripolyphosphate was obtained from Thomas Baker Chemicals Pvt. Ltd., India. HPLC grade acetonitrile,
66
N.R. Pani et al. / International Journal of Biological Macromolecules 65 (2014) 65–71
2.4. Chromatographic conditions HPLC analysis was performed using a Hypersil ODS C18, (average particle size 5 m) column (250 mm, 4.6 mm). The mobile phase consisted of 10 mM phosphate buffer (pH, 3.5) and methanol (25:75, v/v). Eluent was monitored with the PDA detector at 230 nm with a flow rate of 1 mL/min and sample size of 20 L was carried out at column oven temp 30 ± 2 ◦ C all over the study. Fig. 1. Chemical structure of glipizide.
2.5. Preparation of standard solution methanol and dichloromethane were procured from Merck, Mumbai. Potassium di-hydrogen phosphate (KH2 PO4 ) and orthophosphoric acid (H3 PO4 ) were purchased from S.D. Fine Chem Ltd., Mumbai, India). All other chemicals and solvents used were of analytical grade. Water used in the HPLC analysis was prepared by the water purifier (AriumR , 611UF, Sartorius, Germany). The mobile phase and all the solutions were filtered through a 0.45 m Ultipor® N66 ® membrane filter (Pall Life Sciences, USA) prior to use.
Stock solution of GPZ (100 g/mL) and gliclazide (internal standard, 100 g/mL) were prepared in methanol. Further dilution was carried out in mobile phase solvent for the preparation of working stock solution. Calibration standards were prepared freshly by spiking working GPZ solution into the control blank plasma to give the concentration of 25.38, 50.77, 101.53, 203.06, 406.12, 846.09, 1244.24, 1637.16, 2046.45 ng/mL. All the solutions were prepared once and analyzed daily over a period of 3 days for inter-day precision of the method. These solutions were stored at −20 ◦ C.
2.1. Instruments HPLC system (Waters, USA) consisting of quaternary pump (WaterTM 600), 7725i rheodyne manual injector and PDA detector and empower-II software were used for analysis. The plasma samples were processed by using of Micropipettes (Ependruff, USA), Spinix Vortexer (M37610-33, Barnstead International, USA), Biofuge Fresco Centrifuge (Heraeus, Germany), Ultra-Sonicator (Loba Chem, Mumbai), Nitrogen gas evaporator and Multi-Pulse Vortexer (Glas-COL, USA).
2.6. Quality control standards Lowest quality control standards, median quality control standards and highest quality control standards were prepared by spiking drug free plasma with GPZ to give solutions containing 101.53, 406.12 and 1244.24 ng/mL, respectively. They were stored at −20 ◦ C till analysis. 2.7. Sample preparation
2.2. Experimental animals Albino rabbits weighing 1.5–2.5 kg (housed in a temperature (22 ± 1 ◦ C) and relative humidity (55 ± 10%) controlled room were used in the experiments. The rabbits were maintained in accordance with the principles declared by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, Govt. of India). The rabbits were orally administered the floating microspheres containing 2.5 mg GPZ. 2.3. Preparation of floating microspheres Floating microspheres were prepared according to an ionotropic gelation method and physically cross-linking with sodium tripolyphosphate (sodium TPP) [17–19]. Briefly, GPZ (1% (w/v) was dispersed in a stirred solution of chitosan (1% (w/v)) prepared in 2% (v/v) acetic acid until a uniform dispersion was obtained. The microparticles were formed by dropping the bubble free dispersion (30 mL) through a 21G disposable syringe into 100 mL of mechanically agitated (300 rpm) solution of the cross-linking agent, sodium TPP (4% (w/v)) at the rate of 30 drops/min from 5 cm height. The microparticles formed were separated after a 30 min, washed with deionized water and then subsequently dried at room temperature for 3 h. The prepared microspheres were evaluated for yield value, drug entrapment efficiency, particle size measurement, in vitro bioadhesion study, in vitro buoyancy study, in vitro drug release and stability study [19]. The condition for in vitro drug release study was USP basket-type dissolution apparatus with basket rotation of 100 rpm, 900 mL of phosphate buffer (pH 7.4) as a dissolution medium and 5 mL of sample was withdrawn and replaced at predetermined time point. The fraction of drug dissolved (FRD) was calculated by dividing 100 in percentage of drug release at each time point.
An aliquot quantity of 180 L of rat plasma spiked with 10 L of calibration standard drug was taken in a 2 mL stopper centrifuge tube and mixed for 20 s. To this, 10 L of internal standard (IS) solution (100 g/mL) was added and mixed for 20 s. The drug was extracted by vortexing with 1.5 mL of selected extracting solvents among acetonitrile, methanol and di-chloromethane in a spinix vortexer for 10 min followed by centrifugation at 10,000 rpm for 5 min at 4 ◦ C. The supernatant was withdrawn and dried using nitrogen evaporator. The residue was reconstituted with 100 L of mobile phase and 20 L was injected onto the column. 2.8. Validation of method The validation of an analytical method confirms the characteristics of the method to satisfy the requirements of the application domain [20]. The method was validated following the ICH Guidelines [21,22] for specificity, recovery, linearity, precision and stability. Under the validation programme the following parameters were studied. 2.8.1. Specificity The specificity criterion demonstrates that the result of the method is not affected by the presence of interferences, i.e. whether the compound of interest elutes without interfering with other compounds and components of plasma. The specificity of the method was determined by comparing the chromatograms obtained from the aqueous samples of GPZ and IS with those obtained from blank plasma. Blank plasma samples from each of five rabbits were processed in presence of IS and another set of five samples processed with GPZ and IS to evaluate presence of interference around the peak of GPZ. The solutions containing 406.12 ng/mL of GPZ were injected into the column under the optimized chromatographic conditions to obtain the chromatographic
N.R. Pani et al. / International Journal of Biological Macromolecules 65 (2014) 65–71
peaks in plasma with IS so as to differentiate them from the interfering peaks of plasma components. 2.8.2. Selection of solvent for recovery of drug The recovery of an analyte is the extraction efficiency of an analytical process, reported as a percentage of the known amount of an analyte carried through the sample extraction and processing steps of the method. Different organic extraction solvents (acetonitrile, methanol and di-chloro methane) were tried in the experiment to recover GPZ from plasma samples. Quality control samples were prepared in triplicate at three levels of 101.53, 406.12 and 1244.24 ng/mL of GPZ and assayed by HPLC method as described as above. The extraction efficiency of GPZ was determined by comparing the peak areas obtained from extracted quality control samples with the peak area of aqueous working solution containing same concentration of GPZ at three levels. 2.8.3. Linearity Quantitative analytical results are highly influenced by the quality of the calibration curve [22,23]. Nine different concentrations of GPZ with fixed concentration of IS in blank plasma were processed and calibration curve was constructed in the specified concentration range (25.38, 50.77, 101.53, 203.06, 406.12, 846.09, 1244.24, 1637.16, 2046.45 ng/mL). The calibration curve was plotted between the ratio of peak areas of GPZ to IS and concentration of GPZ by replicate analysis (n — — 6) at all concentration levels and the linear relationship was evaluated using the least square method using Microsoft Excel® (Microsoft Corporation, USA) program. 2.8.4. Precision and accuracy Both repeatability (within a day precision) and reproducibility (between days precision) were determined as follows. Three quality control samples were subjected for the study. Five injections of each of the specified quality control samples at three levels were injected for analysis within the same day for repeatability, and over a period of 5 days for reproducibility. Mean and relative standard deviation were calculated and used to predict the accuracy and precision of the method. Accuracy was calculated as the percent of GPZ found in the intra-day and inter-day samples to that of the actual. 2.8.5. Stability studies The quality control standards containing 101.53, 406.12 and 1244.24 ng/mL (n — — 6) of GPZ were subjected for detection of stability of the drug in plasma. The initial assay of the samples was conducted. One set of five samples each was kept in poly propylene tubes and subjected to three freeze–thaw cycles each at −20 ◦ C for 24 h and room temperature for 24 h. The second set of five samples each was kept at room temperature for 24 h and the third set of five samples each were kept at room temperature for 1 month. All the samples were analyzed by standard chromatographic conditions to determine their peak areas. Samples were considered to be stable, when the final assay values of samples were found similar to that of the initial assay value of the drug. 2.9. Pharmacokinetic study in rabbits The method described above was applied to quantify the plasma concentration of GPZ in a single-dose pharmacokinetic study conducted on three white male albino rabbits. The protocol was approved by the Institutional Ethical Committee at the Gayatri College of Pharmacy, Odisha, India. The experiments were conducted as per CPCSEA (Committee for Prevention, Control and Supervision of Experimental Animals) guidelines. The rabbits weighing 1.5–2.5 kg were housed with free access to food and water, except for the final 12 h before experimentation. After a single oral administration of prepared floating microspheres containing 2.5 mg of
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GPZ, 2 mL of blood samples were collected from the marginal ear vein at 0, 0.25, 0.5, 1, 2, 3, 4, 6, 9, 12 and 24 h time points into heparinized collection tubes. The blood was immediately centrifuged (1000 × g) for 10 min at an ambient temperature. The supernatant plasma layer was separated and stored at −20 ◦ C until analyzed. The plasma samples were analyzed for GPZ concentrations by RP-HPLC method. The first order elimination rate constant (kel ) was estimated by the least square regression of plasma concentration–time data points of the curves describing the terminal log-linear decaying phase. t1/2 was derived from kel (t1/2 = 0.693/ke1 ). The area under the plasma concentration–time curve from zero to the last measurable plasma concentration at time t (AUC0–t ) was calculated using the linear trapezoidal rule. The area was extrapolated to infinity (AUC0–∞ ) by addition of Ct /kel to AUC0–t , where Ct is the last detectable drug concentration. The absorption rate constant (ka ) was determined by residual method [24]. The maximum observed GPZ concentration (Cmax ) and the time at which Cmax was observed (Tmax ) were reported directly from the profile. The AUMC is the area under the plot of time vs. the product of time and concentration extrapolated to infinity and was calculated by trapezoidal rule. Volume of distribution (Vd ) and total clearance rate (TCR) were calculated using Eqs. (1) and (2) respectively. The mean residence time (MRT) was determined by AUMC divided by AUC. The clearance (Cl) was calculated as dose divided by AUC with extrapolation to infinity (AUC0–∞ ). All the pharmacokinetic parameters were calculated using Microsoft® Office Excel 2003 (Microsoft Corporation, USA) software application. Vd =
0 · AUMC) (DG
(1)
(AUC)2
TCR = kel · Vd =
Vd · 0 : 693 t1/2
(2)
The Wagner–Nelson method [25] was applied to deconvolute the percentage of the GPZ absorbed using Eq. (3). Ft = C(t) + kel AUC(0−t)
(3)
where Ft is the amount of drug absorbed. The fraction absorbed (FRA) was calculated using Eq. (4) and was plotted against the fraction of drug dissolved (FRD) at the same time and the linear regression analysis was used to examine the in vitro–in vivo relationship.
Fraction absorbed (FRA) =
C(t) + kel AUC(0–t)
[kel AUC0–∞ ]
(4)
2.10. Establishment of in vitro–in vivo correlation (IVIVC) An IVIVC refers to the predictive mathematical model describing the relationship between in vitro property of a controlled-release dosage form (usually the rate or extent of drug dissolution or release) and a relevant in vivo response, such as plasma drug concentration or amount of drug absorbed. A level A correlation is usually estimated by a two-stage procedure: deconvolution followed by comparison of the fraction of drug absorbed (FRA) to the fraction of drug dissolved (FRD). A correlation of this type represents a point-to-point relationship between in vitro dissolution and the in vivo input rate [26]. Accordingly, the deconvolution procedure was done on floating microspheres using mean GPZ plasma concentration vs. time profile using the method of Wagner–Nelson. The data of FRA (obtained by deconvolution) and FRD for each formulation were then plotted to develop an IVIVC model. The linear,
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Fig. 2. Chromatogram of the mixture of glipizide and Internal Standard in mobile phase.
quadratic, and cubic models (Eq. (5) bearing the following forms were tested to obtain the best fit:
of interferences. The best resolution and sensitivity of the method was obtained at 230 nm and 1 mL/min flow rate of mobile phase.
Y = a + bX, Y = a + bX + cX 2 , Y = a + bX + cX 2 + dX 3
3.3. Validation of methods
(5)
where a, b, c, d represent regression parameters associated with each function, Y is the FRA in vivo and X is the FRD in vitro. The coefficients of determination (r2 ) for all developed models were determined. The highest r2 value equation was considered as the best fitting model. 3. Results and discussion 3.1. Preparation of floating microspheres The microspheres were prepared by the interaction of sodium TPP, a polyanion (P3 O10 5− ) with the positively charged amino group of chitosan by electrostatic forces [19]. The prepared spherical microspheres were evaluated for yield value (82.61 ± 4.37%), entrapment efficiency (64.86 ± 2.68%), average particle size (711 m) and floating lag time (0). More than 80% of microspheres were floated on 0.1 N HCl for at least 12 h. The bioadhession of microspheres on agar plate after 8 h was 76 ± 2.44%. The in vitro drug release studies showed that 87.48 ± 2.73% of GPZ were released from microspheres in a controlled manner within 12 h. 3.2. Selection and development of chromatographic method Normal phase chromatography can be used for the separation of non-ionic and non-polar substances, while reversed-phase chromatography (C8 and C18 column) can be used for the separation of non-ionic as well as ionic non-polar to semi polar substances. Thus, GPZ (an ionisable semi polar weak acid) can satisfactorily separated by reversed phase chromatography. Octylsilane (C8) columns are similar to octadecylsilane (C18). However, octylsilane columns are less retentive as compared to octadecylsilane. Majority of the ionizable pharmaceutical compounds can be very well separated on octadecylsilane reversed phase columns [21]. Hence, octadecylsilane column was selected for GPZ. A mixture of methanol and phosphate buffer (pH 3.5) at flow rate 1 mL/min was used as mobile phase for the analysis of GPZ [21]. The optimum ratio of methanol to phosphate buffer (pH 3.5) used in the current investigation was 75:25, which was selected on the basis of resolution and absence
3.3.1. Specificity Typical chromatogram (Fig. 2) of mixture of GPZ and IS revealed that they are well separated under HPLC conditions applied. A chromatogram of blank plasma sample is shown in Fig. 3. Retention time was 7.32 min for GPZ and 09.02 min for IS. In the comparison of chromatogram of GPZ and IS (Fig. 2) with the chromatogram of blank plasma (Fig. 3), absence of interference of plasma components is observed around the zone of retention time of GPZ and IS. The chromatogram of medium level quality control sample i.e. 406.12 ng/mL of GPZ (Fig. 4) showed a good resolution peaks for GPZ and IS which are well differentiated from the peaks of plasma components. 3.3.2. Selection of extracting solvent for recovery of drug The solvent extracted maximum amount of drug from plasma sample was selected for processing of the samples. The recovery of dug was found to be 66. 22 ± 0.84, 78.62 ± 1.54 and 47.66 ± 1.68% in acetonitrile, methanol and di-chloromethane respectively at all three concentration levels which confirm the extraction efficiency of the solvents. Among three solvents, methanol showed maximum amount of drug recovered, hence methanol was selected as the extracting solvent. 3.3.3. Linearity range The ratio of peaks area of GPZ to IS at various concentrations of GPZ in plasma was shown in Table 1. A calibration curve was plotted between peak area ratios of GPZ to IS versus plasma GPZ concentrations. The chromatographic responses (ratio of peaks area of GPZ to IS vs. GPZ concentration) were found to be linear over an analytical range of 25.38–2046.45 ng/mL with regression coefficient (r2 ) value 0.999 which showed reproducibility. The linear regression equation obtained was Y = 0.001X − 0.002. 3.3.4. Precision and accuracy The accuracy of the measurements was determined by using three quality control samples and the results are reported in Table 2. The relative standard deviation (RSD) of intra-day assay of the drug was
