Pharmacokinetic Study of Oxime Prodrug of Gliclazide by LC-MS/MS Method in Rabbit Plasma

 

Surendran Vijayaraj¹*, Anoop Singh², Kokilam Perumal Sampathkumar³

¹Department of Pharmaceutical Sciences, NIMS University, Jaipur, Rajasthan, India

²Department of Pharmaceutical Chemistry, NIMS Institute of Pharmacy, NIMS University, Jaipur, Rajasthan, India

³Department of Pharmaceutical Sciences, Coimbatore Medical College, Coimbatore, Tamilnadu, India.

*Corresponding Author E-mail: vijaysurender@yahoo.co.in

Oxime prodrug of gliclazide is a water soluble and biologically inactive derivative of gliclazide, a sulphonyl urea analogue used to treat type II diabetes mellitus. A rapid liquid chromatography tandem mass spectrometry LC-MS-MS method has been optimized for analysis of oxime prodrug of gliclazide in rabbit plasma using clopidogrel as internal standard. Following turboionspray ionization, the analytes were quantified on a triple–quadrupole mass spectrometer in multiple-reaction-monitoring (MRM) positive ion mode. Sample preparation involved a simple one-step protein precipitation with methanol, followed by centrifugation and evaporation of the organic solvent. The residue was redissolved in mobile phase and analyzed by LC–MS/MS. A Symmetry C18, 50x4.6, 5µ, a mobile phase composed of Acetonitrile: 25mM Potassium dihydrogen orthophosphate (pH-6.5) (50:50 v/v), and a flow rate of 0.6 mL/min were employed, and the total run time was 3.0 min. The method was validated for accuracy, precision, linearity, selectivity, lower limit of quantification (LLOQ), recovery and matrix effect. The method was found to be linear in the range of 150 to 6000ng/mL. LLOQ was found to be 27ng/mL. All validation parameters met the acceptance criteria according to regulatory guidelines. This method was successfully applied to pharmacokinetic study of the prodrug in rabbit through oral administration.

 

 

1. INTRODUCTION:

Oxime prodrug of gliclazide, chemically (E)-N-(N-(hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-N'-hydroxycarbamimidoyl)-4-methylbenzenesulfonamide is a synthetic derivative of gliclazide fig. 1.

 

Fig.1: Oxime prodrug of gliclazide

 

Gliclazide is a second generation sulphonyl urea which acts as a hypoglycemic agent to treat diabetes¹. Oxime prodrug of gliclazide is an inactive prodrug of gliclazide which shows better solubility compared to existing drug. In the literature, methods have been reported for the quantification of gliclazide from biological matrix by spectrophotometric and spectrofluorimetric method^2,3. Methods are also reported for estimation of gliclazide in low volume human plasma using liquid chromatography/tandem mass spectroscopy. Other methods were reported with estimation of gliclazide in plasma by HPLC methods^4-6, LC ESI-MS^7-9. As oxime prodrug of gliclazide is a newly reported prodrug of gliclazide so far no bioanalytical methods were developed to estimate it. Hence the current research paper describes rapid and sensitive analytical method to determine oxime prodrug of gliclazide concentrations in rabbit plasma. The plasma assay utilized protein precipitation for sample preparation.LC/MS/MS was employed to separate further and detect the analytes in the positive ionization mode using electrospray ionization (ESI) and monitoring their precursor–product ion combination in the multiple-reaction monitoring (MRM) mode. The method could be applied to pharmacokinetic study of the prodrugs.

 

2. MATERIALS AND METHODS:

2.1 Chemicals and reagents:

Oxime prodrug of gliclazide was synthesized and purified, clopidogrel (internal standard) was procured from Yarrow Chem laboratories, Mumbai. HPLC grade acetonitrile, methanol and water are purchased from Merck India Pvt. Ltd. All the other chemical used for the study were of analytical grade.

 

2.2 LC-MS/MS conditions:

Chromatographic separation was performed on a Symmetry C18, (50x4.6, 5 µ) column using Shimadzu LC system equipped with degasser, binary pump along with auto sampler. An isocratic mobile phase of Acetonitrile: 25mM Potassium dihydrogen Orthophosphate (pH-6.5) (50:50 v/v) was applied at a flow rate of 0.6 mL/min. The column temperature was kept at 30^◦C. The total analytical run time was 3 min for each sample. The eluent flow was led into the MS/MS starting 0.5 min after injection by switching the MS inlet valve.

 

The mass spectrometer was run in positive ion ESI-APCI combined mode equipped with turboionspray using multiple reaction monitoring (MRM) to monitor the mass transitions. The MRM transitions were chosen to be m/z 324.4→ m/z 239.4 for gliclazide, m/z 337.1 → m/z 274.0 for oxime prodrug of gliclazide and m/z 322.1 → m/z 274.0 for clopidogrel (IS). Instrument parameters optimized were collision activated dissociation gas (CAD): 5 psi; curtain (CUR) gas: 15 psi; nebulizer gas (gas1): 65 psi; heater gas (gas 2): 40 psi; ion spray voltage: 4500 V; source temperature: 500◦C. Compound dependent parameters collision energy (CE) was set at 15 V for gliclazide and oxime prodrug of gliclazide  and 20 V for IS. Declustering potential (DP), entrance potential (EP) and cell exit potential (CXP) for gliclazide, oxime prodrug of gliclazide  and IS were kept at 50, 10and 6 V, respectively. Quadrupole 1 and quadrupole 3 were maintained at unit resolution and dwell time was set at 100 ms. System control and data analysis were performed by AB Sciex Analyst soft-ware (version 1.5.2).

 

2.3 Synthesis of Oxime prodrug of gliclazide:

Weighed quantity of 1.5 g of gliclizide was dissolved in 8 mL of 95% ethanol. To the above solution 0.6 g of hydroxylamine hydrochloride (97 % pure), along with 25 mL of deionized water was added. 10 % by weight aqueous solution of sodium hydroxide (15mL) was added. The solution was refluxed at 100 °C. Completion of the reaction is marked by the formation of large crystals that float on the surface of the reaction mixture. The mixture was cooled in an ice/water bath, collected and then the recrystallised by using 95 % ethanol ¹⁰.

 

2.4 Animals:

Albino Rabbits (Male and Female) weighing 2.5 kg were used for oral bioavailability studies. All animal experiments were approved by Institutional Animal Ethical Committee, (CPCSEA No: 930/PO/a/2006/CPCSEA). All the rabbits were fasted for 12 h before the experiments but had free access to water.

 

2.5 Preparation of stock solutions:

2.5.1           Preparation of standard stock solution of oxime prodrug of gliclazide (GZ1):

10mg of oxime prodrug of gliclazide was transferred into a 10mL volumetric flask and the volume was made up to the mark with mobile phase [Acetonitrile: 25mM Potassium dihydrogen Orthophosphate (pH-6.5) (50:50 v/v)] to give 1mg/mL (1000µg/mL) solution. From this stock solution, 10mL of 100µg/mL solution was prepared.

 

2.5.2           Preparation of standard stock solution of gliclazide (GZ):

10mg of gliclazide was transferred into a 10mL volumetric flask and the volume was made up to the mark with mobile phase [Acetonitrile: 25mM Potassium dihydrogen Orthophosphate (pH-6.5) (50:50 v/v)] to give 1mg/mL (1000µg/mL) solution. From this stock solution, 10mL of 100µg/mL solution was prepared.

 

2.5.3           Preparation of blank plasma:

Blank plasma (0.5 mL) was transferred into 2.0 ml centrifuge tube and 0.1 mL of mobile phase and 0.3 mL of precipitating agent (10% perchloric acid) were added. The resulting solution was vortexed for 5 minutes and centrifuged at 4000 rpm for 4 minutes. The supernatant layer was separated and analyzed.

 

2.6. Drug administration and plasma sample collection:

In the pharmacokinetic study 18 rabbits were randomly divided into three groups (n =6 per group). According to the principle of parallelism, gliclazide and oxime prodrug of gliclazide were dissolved in 0.3% w/v CMC-Na. The rabbits of the first group were treated with blank, the rabbits of the 2nd groups were orally administered with the dose of 22 mg (calculated based on human dose of 320 mg, conversion factor = 0.07) of gliclazide, the rabbits of the third group were orally administered with the dose of 22 mg of oxime prodrug of gliclazide.  0.5 mL of blood samples were collected by marginal ear vein puncture at 0, 1, 2 4, 6, 7, 8, 9.5, 10, 10.5, 12, 18 and 24 hours after dose administration. Blood samples were collected into eppendorf tubes containing 0.3 mL of anticoagulant (citrate) solution and centrifuged at 4000rpm for 4 minutes. The plasma was separated and stored at -20^oC until further analysis.

 

2.7.   Preparation of plasma samples:

Plasma samples (0.5 mL) obtained from study subjects was transferred into 2.0 mL centrifuge tube and 0.3 mL of precipitating agent (10% perchloric acid) was added. The resulting solution was vortexed for 5 minutes and centrifuged at 4000 rpm for 4 minutes. The supernatant layer was then evaporated to dryness at 40◦C under nitrogen. The resulting extract was dissolved in 1mL of mobile phase and vortex mixed for 3 min. After centrifugation at 4000 rpm for 10 min, an aliquot of 10 µL sample was then injected into the LC–MS.

 

2.8.   Method validation:

Linearity was analysed by regression equation obtained by plotting peak area ratios of gliclazide and oxime prodrug of gliclazide to IS versus concentrations. The accuracy and precision were determined by analyzing plasma samples at three concentration levels on three validation run in three different days. Within-run and between-run precision was expressed as coefficient of variation (CV, %) and accuracy was estimated (expressed as coefficient of variation, CV (%)) for the same samples by comparing concentration measured with the nominal value. The assay accuracy was calculated as (observed concentration − spiked concentration)/ (spiked concentration) × 100%.

 

Sensitivity of the method was determined by limit of quantitation by analyzing six replicates of LLOQ that can be measured with acceptable accuracy and precision. Analyte stability was evaluated at two levels (LQC and HQC) in matrix and in sample extracts placed in autosampler (8^◦C). For room temperature stability, long-term stability (≤ 15^◦C) and five freeze-thaw cycles stability test, the concentration of each analyte after storage was compared to its nominal concentration. To evaluate the matrix effect in the experiment, chromatographic peak areas of each analyte from the spike after extraction samples, at low, medium and high concentration levels, were compared to those for the clean standard solutions at the same concentrations. Short term, post preparative and freeze thaw stability studies were performed ^11-14.

 

2.9. Application to pharmacokinetic study:

The LC–MS/MS procedure developed here was used to investigate the plasma profiles of gliclazide and oxime prodrug of gliclazide after oral administration. All data were subsequently processed by the Analyst 1.4.1 software and PK1 & PK2 kinetic software. The non-compartmental pharmacokinetic parameters of C_max, T_max, half-life (T1/2), area under the plasma concentration time curve (AUC_0-t and AUC_0-∞) and elimination rate constant (K_e), were calculated based on moment methods. Data were shown as mean ± standard deviation (mean ± SD).

 

3. RESULTS AND DISCUSSION:

3.1. Method validation:

3.1.1. Linearity of calibration curves and lower limit of quantification (LLOQ):

The standard calibration curve for spiked rabbit plasma containing gliclazide and oxime prodrug of gliclazide were linear over the range 150–6000 ng/mL with a correlation coefficient r² > 0.998, 0.999 respectively. The results indicated that the calibration curve was linear, accurate, and precise over the range of the method fig. 2,3.

 

Fig. 2: Calibration graph for gliclazide

 

Fig. 3: Calibration graph for oxime prodrug of gliclazide (GZ1)

 

3.1.2. Sensitivity:

The developed method was sensitive. The lowest reliable detection was set at the concentration of LOD and the lowest reliable quantification was set at the concentration of LLOQ for all the analytes Table 1.

 

Table 1: LLOD and LLOQ datas of gliclazide and oxime prodrug of gliclazide

S.No	Analytes	LLOD (ng/ml)	LLOQ (ng/ml)
1	Gliclazide (GZ)	10	30
2	GZ1	9	27

 

3.1.3. Assay precision and accuracy:

The intra-day and inter-day precision and accuracy were shown in Table 2, 3. The accuracy and precision of within-run and between-run should be within 15% of the nominal values, except for the LLOQ which should be within 20% of the nominal value. The obtained value complies with the limit. This indicated that the method was accurate and precise over the range of the assay fig. 4-7.

 

Table 2: Precision studies of gliclazide and oxime prodrug of gliclazide

S.No	Drugs	Concentration (ng/mL)	Intraday precision			Inter day precision
			Mean	SD	% C.V	Mean	SD	% C.V
1	Gliclazide (n=6)	150	149.87	0.40	0.26	148.56	0.33	0.22
		2100	2098.87	0.90	0.04	2084.52	1.16	0.05
		6000	6058.14	1.97	0.03	6012.23	1.59	0.02
2.	GZ1  (n=6)	150	148.95	0.27	0.18	149.52	0.14	0.09
		2100	2085.42	0.61	0.02	2096.32	1.06	0.05
		6000	6013.52	1.52	0.02	6011.21	1.57	0.02

 

Table 3: Accuracy studies of gliclazide and oxime prodrug of gliclazide

Drug	Concentration (ng/mL)		Measured concentration (ng/mL)	% Nominal	Mean	SD (n=3)	% C.V
	Actual	Added
Gliclazide	150	50	198.87	99.44	99.53	0.33	0.331
	2100	50	2134.12	99.26
	6000	50	6043.98	99.90
GZ1	150	50	199.41	99.71	99.73	0.27	0.271
	2100	50	2147.15	99.86
	6000	50	6027.18	99.62

 

Fig. 4: LC-MS-MS chromatogram of blank plasma

 

Fig. 5: LC-MS-MS chromatogram of gliclazide

 

Fig. 6: LC-MS-MS chromatogram of oxime prodrug of gliclazide

 

 

Fig. 7: Representative Chromatogram of oxime prodrug of gliclazide and internal standard

 

 

Table 4: Stability of gliclazide and oxime prodrug of gliclazide under a variety of storage conditions

Conditions	Gliclazide			Oxime prodrug of glicllazide
	Accuracy (average ± SD; ng/mL)			Accuracy (average ± SD; ng/mL)
	150ng/mL	1800ng/mL	6000ng/mL	150ng/mL	1800ng/mL	6000ng/mL
Short-term stability (at ambient condition for 2h)	149.87±0.11	1799.91±0.14	5999.98±0.13	148.91±0.16	1799.86±0.18	5999.14±0.10
Post preparative stability (in the auto sampler at ambient condition for 12h)	148.92±0.11	1789.82±0.19	5998.88±0.12	150.04±0.17	1789.96±0.13	6001.04±0.09
Freeze–thaw stability (three cycles)	151.08±0.21	1778.88±0.13	5987.88±0.14	149.92±0.13	179.76±0.11	5986.84±0.10

3.1.4. Analyte stability:

The freeze–thaw stability was determined after three freeze–thaw cycles at -20^◦C with a minimal interval of 24h. The short-term stability was assessed after keeping the samples at ambient condition for 2h. Post-preparative stability was evaluated by analyzing samples kept in the auto sampler at ambient condition for 12 h. The results of the stability study are presented in Table 4, which confirm the high stability of gliclazide and oxime prodrug of gliclazide throughout the determination.

 

3.2.Application of the analytical method to pharmacokinetic studies:

The LC–MS/MS method was successfully applied to the determination of gliclazide and oxime prodrug of gliclazide plasma concentration levels in rabbits following oral administration of a single dose. In the study we compared the pharmacokinetic parameters of rabbits which were administered gliclazide and oxime prodrug of gliclazide. The main pharmacokinetic parameters calculated using non-compartmental analysis was shown in Table 5. Mean plasma concentration–time curves (n = 6) were presented in figs. 8, 9. Significant differences of Tmax, Cmax, AUC(0–t), AUC(0–∞) and K_e were observed between gliclazide and oxime prodrug of gliclazide treated groups  after administration of  equal dose.

 

Table 5: Pharmacokinetic parameters of gliclazide and oxime prodrug of gliclazide

Parameters	Gliclazide	Oxime prodrug of gliclazide
Cmax (µg/mL)	1194.898±7.470	4881.71± 17.13
tmax (hr)	7	6
auc 0-t	11742.01±54.37	30219.52±  634.19
t1/2 (hr)	7.557704±0.174501	4.6766 ± 0.35
Ke	0.091714±0.002115	0.147898 ± 0.0095
auc 0-inf	14546.29±178.5796	31998.55 ± 295.25

 

 

Fig. 8: Mean Plasma Concentration Vs time graph for gliclazide

 

Fig.9: Mean Plasma Concentration Vs time graph for oxime prodrug of gliclazide (GZ1)

 

4. CONCLUSION:

The developed LC–MS/MS method for gliclazide and oxime prodrug of gliclazide is selective, rapid and suitable for routine analysis of subject samples. This method has significant advantages in terms of simple one step protein precipitation procedure and a shorter chromatographic run time (3.0 min). The validation was performed according to regulatory guidelines. In addition, to our knowledge, this is the first validated LC–MS/MS method for estimation of oxime prodrug of gliclazide in rabbit plasma. The established LLOQ is sufficient to conduct a pharmacokinetic study for the oxime prodrug of gliclazide. Increment in C_max value of oxime prodrug of gliclazide was 4.08 folds compared to that of gliclazide which revealed the possibility of enhancement in bioavailability of synthesized prodrugs. This increase may be due to enhancement in solubility of oxime prodrug of gliclazide. The results suggest synthesized oxime prodrug is better than existing drug in terms of solubility and bioavailability.

 

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Received on 29.03.2015         Modified on 09.04.2015

Accepted on 22.04.2015         © AJRC All right reserved