Original Article 399
Bioanalytical LC-MS/MS Method Development and Validation of Novel Antidiabetic Candidate S007-1261 in Rat Plasma and its Application to Pharmacokinetic and Oral Bioavailability Studies
Affiliations
Key words ▶ antidiabetic ● ▶ LC-MS/MS ● ▶ S007-1261 ● ▶ pharmacokinetics ● ▶ rat plasma ●
received 12.09.2013 accepted 24.10.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1358739 Published online: November 20, 2013 Drug Res 2014; 64: 399–405 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence Dr. S. K. Singh, PhD Senior Principal Scientist and In-Charge Pharmacokinetics and Metabolism Division CSIR-Central Drug Research Institute Sector 10 Jankipuram Extension Sitapur Road Lucknow 226031 India Tel.: + 91/522/2771 940 + 91/522/2771 960 Extn.: 4840, 2474 Fax: + 91/522/2771 941 [email protected]
A. Misra1, H. N. Kushwaha1, N. Gautam1, B. Singh1, P. C. Verma2, R. Pratap2, S. K. Singh1 1 2
Pharmacokinetics and Metabolism Division, CSIR-Central Drug Research Institute, Lucknow, India Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow, India
Abstract
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A sensitive and selective LC-MS/MS method has been developed and validated for CDRI antidiabetic candidate S007-1261 in rat plasma using 16-dehydropregnenolone as an internal standard. The API 4000 triple quadrupole LC-MS/MS system was operated under multiple reaction monitoring mode using electrospray ionization technique in positive mode. The sample processing method involves 2-step liquid-liquid extraction using n-hexane as an extracting solvent. The analyte was chromatographed on RP 18, waters column (3.5 μm, 2.1 mm i.d. × 30 mm) with guard using acetonitrile and ammonium acetate buffer (pH 5.0, 10 mM) in 90:10 (v/v) composition at a flow rate of 0.40 mL min − 1. The chromatographic
Introduction
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Type II diabetes generally arises due to insufficient production of insulin by pancreas or it may also arise when body cannot effectively use the insulin it produces. Since, insulin is a hormone responsible for regulation of blood sugar level, its uncontrolled action will lead to hyperglycemia which results in type II diabetes [1, 2]. With exception to insulin injection, treatment of diabetes mellitus is possible by oral administration of anti-hyperglycemic agents which help to reduce blood glucose levels. There are different classes of antidiabetic drugs depending upon their mode of action. Alpha–glucosidase inhibitors works by interfering the action of alpha-glucosidase which results in reduction of glucose uptake and its absorption [3]. Sulfonylureas are another class of antidiabetic drugs which function by releasing endogenous pancreatic insulin [4]. Meglitinides also help pancreas to produce insulin [5]. Biguanides works by increasing glucose uptake by skeletal muscles and hence reduces hepatic glucose
run time was 5.30 min. Calibration curve shows linearity over concentration range 1.56–200 ng mL − 1. The lower limit of detection was 0.39 ng mL − 1 and lower limit of quantitation was 1.56 ng mL − 1. The inter- and intra-day accuracy and precision were found to be within the assay variability limits as per US FDA guidelines. The absolute recovery of S007-1261 was found to be > 90 %. S007-1261 does not show any stability problems as it was stable at room temperature for 8 h. S007-1261 was also stable up to 3 freeze-thaw cycles and can be stored up to 30 days at − 60 °C. The assay was successfully applied to both oral (40 mg kg − 1) and intravenous (10 mg kg − 1) pharmacokinetic studies in male Sprague-Dawley rats. The oral bioavailability of S007-1261 was found to be 33.61 %.
output [6]. Thiazolinediones reduces insulin resistance by activating PPAR in fat and muscle [7]. However, all classes of antidiabetic drugs have more or less side effects. In search to develop compound with good efficacy, low toxicity and more safety and affordable for the treatment of diabetes and diabetes related complications, Medicinal and Process Chemistry Division of CSIR-Central Drug Research Institute (CDRI), Lucknow, India, developed a new antidiabetic mol▶ Fig. 1). S007-1261 ecule coded as S007-1261 (● is a novel pregnane-oximino-aminoalkylethers and showed promising anti-hyperglycemic activity in streptozotocin (STZ) diabetic rat model. Type II diabetes mellitus (DM) increases the risk of cardiovascular diseases. S007-1261 also possesses significant antidyslipidemic activity and does not show primary toxic effect (Patent filed: 0193DEL2013, filing date 24 January 2013), therefore, it is of interest to study this compound in greater detail. Preclinical analytical method development and validation is prime requisite for quantitative determination of drug in different biological
Misra A et al. Pharmacokinetics Study of S007-1261 … Drug Res 2014; 64: 399–405
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Authors
400 Original Article
matrices, for generating reproducible and reliable data [8]. Hence, analytical method development and validation are essential for interpretation of bioavailability, bioequivalence and pharmacokinetics. In this study, we developed and validated a sensitive and selective LC-MS/MS method for S007-1261 in Sprague-Dawley (SD) rat plasma. Analytical quality criteria including method specificity, accuracy, precision and recovery were also taken in to account in this study. The developed and validated method of S007-1261 has been applied for pharmacokinetics studies in male SD rat after oral and intravenous administration.
Materials and Methods
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Drugs and chemicals Both CDRI candidate S007-1261 ( > 99 % pure) and 16-dehydropregnenolone (16-DHP) as an internal standard (IS) were obtained from Medicinal & Process Chemistry division of CSIRCDRI (Lucknow, India). Acetonitrile of HPLC grade was purchased from Sigma Aldrich. For preparation of buffer, analytical grade ammonium acetate and glacial acetic acid were purchased from E. Merck Limited. Triple distilled water of 18.2 MΩ cm was obtained from Milli-Q Ultrapure Water Purification system. HPLC grade n-hexane for extraction procedure was obtained from E. Merck Limited. Heparin sodium injection I.P. (1 000 IU mL − 1) was obtained from Troikaa Pharmaceutical Limited. For drug free plasma, male SD rats were provided by National Laboratory Animal Centre (NLAC), CSIR-CDRI, Lucknow. Drug free plasma was obtained from heparinized whole blood of rat by centrifugation at 1 500 × g for 10 min at 4 °C. The supernatant obtained was collected and stored at − 60 °C until use. This blank plasma was used for preparation of both calibration standard (CS) and quality control (QC) samples.
LC-MS/MS instrumentation and analytical conditions A series 200 HPLC system consisting of quaternary low pressure gradient pumps, online degasser and auto sampler with temperature controlled peltier tray (Perkin-Elmer Instruments, Norwalk, CT, USA) was used. Separation of analyte was achieved on RP 18, waters column (3.5 μm, 2.1 mm i.d. × 30 mm) using acetonitrile and ammonium acetate buffer (pH 5.0, 10 mM) at a flow rate of 0.40 mL min − 1. Buffer was filtered through 0.22 μm Millipore filter (Billeria, USA) before use. The ratio of acetonitrile to ammonium acetate buffer was 90:10 (v/v). The run time of the method was 5.30 min and injection volume was optimized to 20 μL. The rinsing solution used to rinse needle was a mixture of acetonitrile and triple distilled water (90:10 v/v). The analysis was carried out at ambient temperature. An API 4000 LC-MS/MS system (Applied Biosystem, MDS Sciex, Toronto, Canada) with Analyst 1.4 software was used for detection. The mass spectrometer was operated with electrospray ion
Misra A et al. Pharmacokinetics Study of S007-1261 … Drug Res 2014; 64: 399–405
[ESI] source in positive mode. Multiple reaction monitoring (MRM) mode was used to quantify S007-1261 and IS. Zero air was used as a source gas while nitrogen was used both as curtain gas and collision gas. Both MS and MS/MS condition of S007-1261 was optimized by continuous infusion at 10 μL min − 1 using syringe pump (model ‘11’, Harvard Apparatus, Holliston, MA, USA). MS and MS/MS of IS was also optimized by the same procedure. The precursor (hydrogen adduct of S007-1261, [M+H]+) to product ion transitions monitored at m/z 443.4 →112.3 for S007-1261 and m/z 315.1→137.5 for IS were used for quantification. Declustering potential (DP), collision energy (CE), entrance potential (EP) and collision cell exit potential (CXP) were also optimized for both S007-1261 and IS.
Preparation of calibration standard and quality control samples Stock solution of 1 mg mL − 1 of S007-1261 was prepared in acetonitrile. Working stocks having concentration range 2 000, 1 000, 500, 250, 125, 62.5, 31.25 and 15.6 ng mL − 1 of S0071261were also made in acetonitrile. CS of S007-1261 was prepared by spiking 10 μL of working stock into 100 μL of rat plasma over a concentration range of 1.56–200 ng mL − 1. Stock solution of 1 mg mL − 1 of IS was prepared in acetonitrile. Working stock of IS (10 000 ng mL − 1) was also prepared in acetonitrile. 10 μL of working stock of IS was also spiked in plasma to get final concentration of 1 000 ng mL − 1. QC samples were prepared in 5 replicates at low1 (1.56 ng mL − 1), low2 (3.12 ng mL − 1), medium (25 ng mL − 1) and high (200 ng mL − 1) concentration respectively. Low1 is also the lower limit of quantitation (LLOQ). These QC samples were prepared independent of CS. Concentration of test samples and QC samples were obtained from CS of S007-1261.
Sample preparation CS, QC and test samples were prepared from 2 step liquid-liquid extraction procedure using n-hexane as an extracting solvent. The volume of plasma was fixed at 100 μL and 10 μL of IS was added to each sample to get final concentration of 1 000 ng mL − 1 and vortexed for 60 s. After vortex-mixed, 2 mL of n-hexane was added and again vortexed for 3 min and centrifuged at 2 000 × g for 10 min at 4 °C. Thereafter, organic layer was transferred into clean tube by snap freezing of aqueous layer in liquid nitrogen. The organic layer in tube was allowed to evaporate till dryness in speed vac-concentrator. The remaining plasma of the above step was re-extracted in similar manner. The dry-residue was reconstituted in 200 μL of acetonitrile and centrifuged at 2 000 × g for 5 min at 4 °C and then injected for analysis.
Method Validation The bioanalytical method was validated in terms of specificity, sensitivity, accuracy, precision, calibration curve and reproducibility. The method specificity was determined by preparing and analyzing 6 individual as well as pooled rat blank plasma. Chromatogram obtained from spiked plasma at LLOQ was compared to chromatogram of blank plasma. This was done to check the specificity of the method. Each blank plasma samples was also tested for visible interference. Matrix effect is common, when mass spectrometer used as detector, since there may be possible competition between ionization of analyte and co-eluent residual matrix component from plasma [9]. To find out the possible matrix effect, peak area of the analyte dissolved in supernatant of the processed blank
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Fig. 1 Chemical structure of S007-1261.
plasma should be compare to standard solution at the same concentration. Matrix effect of S007-1261 was analyzed at 4 concentrations (1.56, 31.25, 25 and 200 ng mL − 1) in 5 replicates. Same procedure was also applied for determination of matrix effect of IS. The lower limit of detection (LOD) is the amount of drug in plasma after sample clean up corresponding to 3 times the baseline noise ratio (S/N > 3). LLOQ is the analyte concentration that can be quantified with ± 20 % accuracy and precision. Its signal should be at least 10 times as compared to blank (S/N > 10). Linearity of CS was anticipated for 5 days with concentration range of 1.56–200 ng mL − 1. Analytical standard (AS) and CS curve were drawn between the peak area ratio of analyte to IS vs. concentration of the analyte using weighted (1/x2) leastsquare linear regression. Accuracy was determined for both inter- and intra-day by calculating the % bias [ % bias = (Observed concentration − Nominal concentration)/ Nominal concentration × 100] of QC samples. The inter- and intra-day precision was calculated by one way analysis of variance (ANOVA) in terms of relative standard deviation (RSD). The extraction recoveries of S007-1261 were determined at 4 concentrations viz. 1.56, 3.12, 25 and 200 ng mL − 1 (QC low1, low2, medium and high) respectively. These were determined by comparing the peak area of extracted samples spiked with a known amount of the analyte with peak area of AS at the same concentration. Recovery of IS was determined in same manner at concentration of 1 000 ng mL − 1 in 10 replicates.
Stability studies Stability studies of S007-1261 were performed to evaluate the stability of this compound in stock solution and in plasma at different processing conditions like temperature and timing. To find out the effect of freeze-thaw cycle, spiked plasma QC samples were prepared at 1.56, 3.12, 25 and 200 ng mL − 1 (QC low1, low2, medium and high) concentrations with 5 replicates. One set was analyzed without freeze-thaw cycle and referred as control. Experimental set was analyzed after 3 freeze-thaw cycles. The bench-top temperature stability was determined by analyzing QC samples, which were kept at ambient temperature for a period of 8 h and compared with freshly prepared QC samples. Dry-residual stability studies was performed by storage of dryresidue after extraction for 4 and 7 days respectively at − 60 °C and compared its standard deviation from control set which was prepared and extracted at the day of analysis. The long-term stability was evaluated by determining QC plasma samples, kept at − 60 °C for period of 7, 15 and 30 days respectively and its standard deviation was calculated from control set which was prepared at the day of analysis. The autosampler stability was measured by reanalyzing extracted QC samples kept in the HPLC autosampler at 4 °C for 12 h. The working stock stability of S0071261 and IS were assessed at room temperature for 8 h.
Pharmacokinetic studies Healthy and young male SD rats weighing 250 ± 25 g were procured from NLAC, CSIR-CDRI, India. They were housed in wellventilated cages and kept at room temperature (24 ± 2 °C) on a regular 12 h light-dark cycle. The rats were acclimatized to this environment for at least 2 days before the initiation of experiment. They were fasted overnight (8–12 h) prior to experiment, however, water was freely provided. Animals were cared in accordance with the guidelines laid by the animal lab (Depart-
ment of Health Education and Welfare, no. [NIH] 85-32). Studies were approved by Institutional Animal Ethics Committee (Ethical approval 58/08/PKM/IAEC). For oral pharmacokinetic study, 0.5 % aqueous suspension of methyl cellulose was used and was administered by oral feeding needle at dose 40 mg kg − 1. The strength of the formulation was 20 mg mL − 1 and the volume administered was 500 μL. Blood samples were collected under light anesthesia at pre-dose and then at 0.083, 0.33, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72, 84, 96, 108 and 120 h post oral dose administration. All the samples were collected through orbital sinus vein puncture into microcentrifuge tubes containing heparin (22 IU heparin mL − 1 of blood). For intravenous pharmacokinetic study, formulation of S0071261 was prepared of absolute alcohol and propylene glycol (1:4 v/v) [10, 11]. This formulation was filtered through 0.22 μm filter before use. 500 μL of 10 mg kg − 1 dose was administered to rats through tail vein after dilation with xylene. Blood samples were collected under light anesthesia at pre-dose and then at 0.083, 0.33, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72, 84 and 96 h post intravenous dose administration. Samples of intravenous pharmacokinetic study were also collected through orbital sinus vein puncture into micro-centrifuge tubes containing heparin. For both oral and intravenous studies, sparse sampling technique was applied for sample collection. In this technique, animals were divided in to 2 groups viz. Group A and Group B. Both the groups had 4 rats. Thus, total 8 rats were there in a study (8 rats for oral and 8 rats for intravenous study). Predose sample (0 min) and post dose sample at 0.083, 0.75, 1.5, 4, 12, 36, 60, 84 and 108 h were collected from group A, while sample of 0.33, 1, 2, 6, 8, 24, 48, 72, 96 and 120 h were collected from group B. So, for both oral and intravenous pharmacokinetic studies, the volume withdrawn within 24 h was less than 10 % of the total blood volume of the rats. For both the studies, plasma was separated by centrifugation of blood at 1 500 × g for 10 min at 4 °C and stored at − 60 °C until analysis. WinNonlin version 5.1 (Pharsight Corp, USA) software was used to determine the pharmacokinetic parameters for both the studies.
Results and Discussion
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Condition optimization of the LC-MS/MS method Flow injection analysis of S007-1261 and IS were used to obtain full-scan mass spectra of parent and product ions. Mass spectra (MS) of S007-1261 and IS showed peak at 443.4 and 315.1 respectively of the parent compound. S007-1261 exists as protonated molecular ion [M + H]+. Continuous flow injection for MS/ MS analysis was carried out to obtain the product ion spectra of both the analyte and IS. The product ion for S007-1261 was opti▶ Fig. 2). The product ion for IS was optimized mized to 112.3 (● to 137.5. For quantification, m/z 443.4 parent ion transitions to m/z 112.3 product ions were taken for S007-1261. Similarly, m/z 315.1 parent ion transitions to m/z 137.5 product ions was used for quantification of IS. The dwell time was optimized and fixed at 300 ms. DP optimization was carried out using full scan acquisition over a mass range of 100–500 Da by flow injection analysis. Optimization of CE includes most intense fragments of the S007-1261 and IS. Optimized parameters like DP, CE, EP and CXP ▶ Table 1. The curtain gas for S007-1261 and IS are presented in ● (CUR) and collision gas (CAD) were optimized to 15 and 3 psi respectively. The ion source gas 1 (GS1) and ion source gas 2 Misra A et al. Pharmacokinetics Study of S007-1261 … Drug Res 2014; 64: 399–405
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402 Original Article
Table 1 Optimized LC-MS/MS condition for S007-1261and IS parent to product ion transition. Analyte S007-1261 16-DHP (IS)
Precursor
Product
DP
CE
EP
ion (m/z)
ion (m/z)
(V)
(V)
(V)
CXP (V)
443.4 315.1
112.3 137.5
160 90
46 41
10 10
10 10
(GS2) were optimized to 30 and 45 psi respectively. The ion source potential was kept at 5 500 v and source gas temperature was fixed to 200 °C. After optimization of all the parameters, MRM method was made for quantification of analyte and IS to obtain specific and selective detection. Using these transitions for analyte and IS, no interference in plasma was observed. Buffers of different pH and molarity were used to asses best conditions for peak intensity and shape. Finally, 10 mM ammonium acetate buffer of pH 5.0 was chosen along with acetonitrile as mobile phase, at a flow rate of 0.40 mL min − 1 because peak shape, intensity and elution time of analyte and IS was best under these conditions. Column of different dimensions were used for optimization of peak, of which RP 18 waters column (3.5 μm, 2.1 mm i.d. × 30 mm) with guard was found to give optimum results. Optimization of the injection volume gave best results at 20 μL. For sample clean-up, different non-polar solvents were tested. Pure n-hexane (2 × 2 mL) gave good recovery as compared to other extracting solvents. Different compounds were screened to find a suitable IS. Generally structural analog are used, which having almost similar retention time (RT) and extracting conditions. Finally, 16-DHP was selected as an IS, because both S0071261 and 16-DHP contain steroidal moeity and having same extracting conditions. No significant ionization suppression between S007-1261 and IS was observed throughout the LC-MS/ MS study.
Assay performance and validation The parameter used for validation and assessing the bioanalytes assay performance were selectivity, sensitivity, linearity, accuracy, precision, recovery and stability [12–17]. Plasma was checked from endogenous interference in the region of analyte and IS elution. There was no significant endogenous interference Misra A et al. Pharmacokinetics Study of S007-1261 … Drug Res 2014; 64: 399–405
at the RT of analyte and IS in blank rat plasma. Representative chromatogram of blank plasma, plasma spiked with S007-1261, spiked plasma containing IS and test plasma sample after intra▶ Fig. 3. venous dosing of S007-1261 are shown in ● On the basis of peak area ratio of analyte and IS, S007-1261 was linear over concentration range 1.56–200 ng mL − 1. S007-1261 was also reliable and reproducible over this calibration range. For well-designed and interpret calibration curve, weighing factor was used to improve residual points and to counter variance, since variance was not found constant across the calibration standard. Linear equation Y = mX + c with 1/x2 weighing factor showed the best fit for calibration curve. The correlation coefficient (r2) was > 99.9 for calibration curve was used. % RSD of RT for both analyte and IS were within the acceptable limits of 5 %. LOD (S/N > 3) was 0.39 ng mL − 1 and LLOQ was 1.56 ng mL − 1 respectively for S007-1261. Recovery was > 90 % for S007-1261 and it was evaluated from the spiked plasma at 1.56, 3.12, 25 and 200 ng mL − 1. No significant matrix effect was found since peak area ratio of the analyte dissolved in the supernatant of processed blank plasma and the standard solution of S007-1261 at the same concentration level were between 90 % and 110 %. Accuracy in terms of % bias and precision in terms % RSD was calculated at concentrations 1.56, 3.12, 25 and 200 ng ml − 1 with 5 determinants per concentration for 5 days in rat plasma. Accuracy and precision was calculated for both inter- and intra-day ▶ Table 2). The concentration of each sample was calculated by (● standard curve prepared and analyzed on same day. Bioanalytical method is accurate and precise over the concentration range 1.56–200 ng mL − 1 as % bias were within the acceptance limits and precision in terms of % RSD were never exceeds 15 %.
Stability studies in rat plasma Freeze-thaw stability and all other stability studies in rat plasma were carried out at 4 concentrations viz. 1.56, 3.12, 25 and 200 ng mL-1 in 5 replicates. The percentage deviation after 3 freeze-thaw cycles for S007–1261 at concentrations 1.56, 3.12, 25 and 200 ng mL − 1 were found to be 0.80 %, 0.05 %, − 0.06 % and ▶ Table 3). So, percentage deviation was 1.79 % respectively (● non-significant, when compared with freshly prepared QC samples without subject to freeze-thaw cycle. For bench-top stabil-
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Fig. 2 MS/MS spectra of S007-1261 with proposed fragmentation of selected product ion.
Original Article 403
Table 2 Inter-day and intra-day accuracy ( % bias) and precision ( % RSD) of S007-1261 in rat plasma (n = 5). Nominal
Type of
Accuracy
Precision
concentration
QC
( % bias)
( % RSD)
(ng mL − 1) Inter-day 1.56 3.12 25 200
Low1 Low2 Medium High
2.92 − 11.19 − 10.22 8.37
Intra-day − 0.37 − 3.55 − 2.32 5.44
Inter-day
Intra-day
3.0 11.6 11.3 9.1
1.8 0.6 4.2 2.3
ity, there was also non-significant deviation between sample ▶ Table 3). So, analyte is stable at room temkept at 0 h and 8 h (● perature up to 8 h for sample handling and processing during experiments. Dry-residue samples of analyte were stable up to 7 days when kept at − 60 °C after double extraction with n-hexane. The percentage deviation was non-significant when compared ▶ Table 4). The long-term with immediately prepared samples (● samples kept at − 60 °C for 7, 15 and 30 days also showed nonsignificant deviation when compared to freshly prepared sam▶ Table 4). Samples were, therefore, stable and can be kept ples (●
up to 30 days at − 60 °C. In auto sampler stability, repeated injection of same set of QC samples was given to check reproducibility at every 3 h up to 12 h, shows deviation were less than 10 % (data not shown).
Application of developed and validated method toward pharmacokinetic studies The plasma concentration-time profile of S007-1261 after single oral dose administration at 40 mg kg − 1 has been depicted ▶ Fig. 4. As seen from the figure, plasma concentration-time in ● profile is irregular having multiple peaks which could not be explained by any of the compartmental model. Hence, data was subjected to non-compartmental modeling. After oral dose, samples were collected up to 120 h and S007-1261 could be detected up to 60 h in rat plasma. The peak plasma concentration (Cmax) and time at which peak plasma concentration attained (Tmax) were 40.35 ± 7.03 ng mL − 1 and 10 ± 2.3 h respectively. Both these factors were also visually examined from raw data. After oral dose of S007-1261, it seems that absorption was slow, because plasma concentration peaks at 10 h post dose. The elimination half life (T1/2) and mean residence time (MRT) of S007-1261 were found to be 14.27 ± 0.95 h and 23.23 ± 1.31 h. Misra A et al. Pharmacokinetics Study of S007-1261 … Drug Res 2014; 64: 399–405
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Fig. 3 Representative MRM chromatogram of blank rat plasma a, plasma spiked with S0071261at 1.56 ng mL − 1 b, blank rat plasma c, plasma spiked with IS at 1 000 ng mL − 1 d and test sample after intravenous administration of S007-1261 at 2 h e.
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Table 3 Summary of freeze-thaw and bench-top stability studies of S007-1261 in rat plasma (n = 5). Storage condition
Nominal concentration
Type of QC
(ng mL − 1) After 3 freeze-thaw cycles
1.56 3.12 25 200 1.56 3.12 25 200
Bench-top for 8 h at ambient temperature
Concentration at time t = 0
Concentration recovered
Percent ( %)
(mean ± SD; ng mL − 1)
(mean ± SD; ng mL − 1)
deviation
1.65 ± 0.10 3.27 ± 0.22 27.26 ± 0.37 204 ± 20.64 1.39 ± 0.03 2.79 ± 0.04 22.76 ± 0.92 182 ± 1.0
Low1 Low2 Medium High Low1 Low2 Medium High
1.66 ± 0.02 3.27 ± 0.19 27.25 ± 0.91 208.33 ± 24.66 1.40 ± 0.04 2.83 ± 0.06 24.53 ± 2.07 195 ± 15.52
0.80 0.05 − 0.06 1.79 1.07 1.16 7.75 7.14
*t = 0 stands for concentration of QCs at the time of storage *Concentration recovered means concentration of QCs after storage at defined conditions
Dry-residue at − 60 °C
Long-term at − 60 °C
Nominal concentration
Type of
(ng mL − 1)
QC
1.56
Low1
3.12
Low2
25
Medium
200
High
1.56
Low1
3.12
Low2
25
Medium
200
High
Days
Concentration recovered
Percent ( %)
(mean ± SD; ng mL − 1)
deviation
4 7 4 7 4 7 4 7 7 15 30 7 15 30 7 15 30 7 15 30
Fig. 4 Plasma concentration-time profile of S007-1261 after oral administration at 40 mg kg − 1.
The volume of distribution was found to be 738.69 ± 87.13 L kg − 1. This high value of volume of distribution indicates that S007-1261 gets well distributed outside the vascular compartment. Clearance of S007-1261 was 36.19 ± 4.55 L h − 1 kg − 1 which suggested that extra-hepatic tissues are significantly involved in Misra A et al. Pharmacokinetics Study of S007-1261 … Drug Res 2014; 64: 399–405
1.54 ± 0.18 1.65 ± 0.03 3.23 ± 0.41 3.33 ± 0.20 22.22 ± 0.69 22.67 ± 1.34 182 ± 13.24 177 ± 7.43 1.50 ± 0.02 1.49 ± 0.12 1.41 ± 0.02 2.78 ± 0.06 2.84 ± 0.16 2.82 ± 0.13 26.13 ± 1.41 24.57 ± 2.30 22.7 ± 0.21 208.5 ± 20.76 216.66 ± 3.21 207.25 ± 13.81
− 4.93 2.05 − 3.59 − 0.62 − 3.99 − 2.05 − 0.81 − 3.54 6.99 6.22 0.53 − 0.62 1.42 0.80 8.88 2.39 − 5.41 5.56 9.70 4.93
Table 4 Summary of dryresidual and long-term stability studies of S007-1261 in rat plasma (n = 5).
the elimination of the drug. Both volume of distribution and clearance values were higher after oral administration as compared to intravenous administration. The value of area under curve (AUC0–∞) was 1 209.48 ± 166.47 ng h mL − 1. The plasma concentration-time profile after intravenous administration of S007-1261 at 10 mg kg − 1 also depicted multiple ▶ Fig. 5). So, data after intravenous administration were peaks (● again subjected to non-compartmental modeling. After intravenous administration, samples were collected up to 96 h and S007-1261 could be detected up to 84 h in rat plasma. The initial concentration (CO) was 166.28 ± 90.25 ng mL − 1. The elimination half life (T1/2) and mean residence time (MRT) of S007-1261 after intravenous administration were found to be 15.42 ± 1.25 h and 25.29 ± 1.99 h. High MRT value after intravenous administration may be due to S007-1261 is retained in the system for longer period of time as its elimination is slow from the body. The volume of distribution and clearance were found to be 267.52 ± 11.33 L kg − 1 and 12.34 ± 1.37 L h − 1 kg − 1 respectively. The value of area under curve (AUC0–∞) was 825.58 ± 88.71 ng h mL − 1. The bioavailability was found to be 33.61 % when compared AUC after oral and intravenous dosing of S007-1261. This means 33.61 % of S007-1261 dose reaches the systemic circulation. All the pharmacokinetics parameters, both after oral and intrave▶ Table 5. nous administration, are summarized in ●
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Storage condition
Original Article 405 Acknowledgements
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We are thankful to Director, CSIR-CDRI, for providing facilities and infrastructure for the study. We also acknowledge Council of Scientific and Industrial Research (CSIR) and University Grant Commission (UGC) New Delhi, India, for providing funds and fellowships. This is CDRI communication no. 8555.
Conflict of Interest
▼
There is no conflict of interest to be disclosed.
Fig. 5 Concentration-time profile of S007-1261 after intravenous administration at 10 mg kg − 1.
Table 5 Pharmacokinetic parameters for S007-1261in male rats plasma following 40 mg kg − 1 oral and 10 mg kg − 1 intravenous dose (Mean ± SEM, n = 4). PK parameters Tmax (h) Cmax (ng mL − 1) Co (ng mL − 1) T1/2 (h) AUC0–∞ (ng h mL − 1) Vz (L kg − 1) Cl (L h − 1 kg − 1) MRT (h)
Oral
Intravenous
10 ± 2.3 40.35 ± 7.03 * 14.27 ± 0.95 1 209.48 ± 166.47 738.69 ± 87.13 36.19 ± 4.55 23.23 ± 1.31
* * 166.28 ± 90.25 15.42 ± 1.25 825.58 ± 88.71 267.52 ± 11.33 12.34 ± 1.37 25.29 ± 1.99
*not applicable
Multiple peaks after both oral and intravenous administration of S007-1261 may arise due to non-aqueous solubility that might have resulted in precipitation of dose in the intestine and did not redissolved in the absorptive region of the GIT [18]. Other reasons may be formation of depot on intestinal walls, enterohepatic recirculation of S007-1261 and its variable rates along the gastrointestinal tract.
Conclusion
▼
The LC-MS/MS method developed and validated for antidiabetic S007-1261 shows good sensitivity, specificity, accuracy and precision. The LLOQ was established at 1.56 ng mL − 1. Calibration curve in the range of 1.56–200 ng mL − 1 shows good linearity in rat plasma. For samples preparation, 2 step liquid-liquid extraction procedure was used with n-hexane as an extracting solvent which shows the recovery > 90 %. S007-1261 did not show any stability problem during storage and sample processing. This developed and validated method was successfully applied to both single oral and intravenous pharmacokinetic studies. The oral bioavailability of S007-1261 was found to be 33.61 %. This newly developed and validated method may be applied in future for both pre-clinical and clinical studies, toxicity studies, drug interaction studies and multiple dose pharmacokinetic studies.
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References
Bioanalytical LC-MS/MS Method Development and Validation of Novel Antidiabetic Candidate S007-1261 in Rat Plasma and its Application to Pharmacokinetic and Oral Bioavailability Studies
Affiliations
Key words ▶ antidiabetic ● ▶ LC-MS/MS ● ▶ S007-1261 ● ▶ pharmacokinetics ● ▶ rat plasma ●
received 12.09.2013 accepted 24.10.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1358739 Published online: November 20, 2013 Drug Res 2014; 64: 399–405 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence Dr. S. K. Singh, PhD Senior Principal Scientist and In-Charge Pharmacokinetics and Metabolism Division CSIR-Central Drug Research Institute Sector 10 Jankipuram Extension Sitapur Road Lucknow 226031 India Tel.: + 91/522/2771 940 + 91/522/2771 960 Extn.: 4840, 2474 Fax: + 91/522/2771 941 [email protected]
A. Misra1, H. N. Kushwaha1, N. Gautam1, B. Singh1, P. C. Verma2, R. Pratap2, S. K. Singh1 1 2
Pharmacokinetics and Metabolism Division, CSIR-Central Drug Research Institute, Lucknow, India Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow, India
Abstract
▼
A sensitive and selective LC-MS/MS method has been developed and validated for CDRI antidiabetic candidate S007-1261 in rat plasma using 16-dehydropregnenolone as an internal standard. The API 4000 triple quadrupole LC-MS/MS system was operated under multiple reaction monitoring mode using electrospray ionization technique in positive mode. The sample processing method involves 2-step liquid-liquid extraction using n-hexane as an extracting solvent. The analyte was chromatographed on RP 18, waters column (3.5 μm, 2.1 mm i.d. × 30 mm) with guard using acetonitrile and ammonium acetate buffer (pH 5.0, 10 mM) in 90:10 (v/v) composition at a flow rate of 0.40 mL min − 1. The chromatographic
Introduction
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Type II diabetes generally arises due to insufficient production of insulin by pancreas or it may also arise when body cannot effectively use the insulin it produces. Since, insulin is a hormone responsible for regulation of blood sugar level, its uncontrolled action will lead to hyperglycemia which results in type II diabetes [1, 2]. With exception to insulin injection, treatment of diabetes mellitus is possible by oral administration of anti-hyperglycemic agents which help to reduce blood glucose levels. There are different classes of antidiabetic drugs depending upon their mode of action. Alpha–glucosidase inhibitors works by interfering the action of alpha-glucosidase which results in reduction of glucose uptake and its absorption [3]. Sulfonylureas are another class of antidiabetic drugs which function by releasing endogenous pancreatic insulin [4]. Meglitinides also help pancreas to produce insulin [5]. Biguanides works by increasing glucose uptake by skeletal muscles and hence reduces hepatic glucose
run time was 5.30 min. Calibration curve shows linearity over concentration range 1.56–200 ng mL − 1. The lower limit of detection was 0.39 ng mL − 1 and lower limit of quantitation was 1.56 ng mL − 1. The inter- and intra-day accuracy and precision were found to be within the assay variability limits as per US FDA guidelines. The absolute recovery of S007-1261 was found to be > 90 %. S007-1261 does not show any stability problems as it was stable at room temperature for 8 h. S007-1261 was also stable up to 3 freeze-thaw cycles and can be stored up to 30 days at − 60 °C. The assay was successfully applied to both oral (40 mg kg − 1) and intravenous (10 mg kg − 1) pharmacokinetic studies in male Sprague-Dawley rats. The oral bioavailability of S007-1261 was found to be 33.61 %.
output [6]. Thiazolinediones reduces insulin resistance by activating PPAR in fat and muscle [7]. However, all classes of antidiabetic drugs have more or less side effects. In search to develop compound with good efficacy, low toxicity and more safety and affordable for the treatment of diabetes and diabetes related complications, Medicinal and Process Chemistry Division of CSIR-Central Drug Research Institute (CDRI), Lucknow, India, developed a new antidiabetic mol▶ Fig. 1). S007-1261 ecule coded as S007-1261 (● is a novel pregnane-oximino-aminoalkylethers and showed promising anti-hyperglycemic activity in streptozotocin (STZ) diabetic rat model. Type II diabetes mellitus (DM) increases the risk of cardiovascular diseases. S007-1261 also possesses significant antidyslipidemic activity and does not show primary toxic effect (Patent filed: 0193DEL2013, filing date 24 January 2013), therefore, it is of interest to study this compound in greater detail. Preclinical analytical method development and validation is prime requisite for quantitative determination of drug in different biological
Misra A et al. Pharmacokinetics Study of S007-1261 … Drug Res 2014; 64: 399–405
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Authors
400 Original Article
matrices, for generating reproducible and reliable data [8]. Hence, analytical method development and validation are essential for interpretation of bioavailability, bioequivalence and pharmacokinetics. In this study, we developed and validated a sensitive and selective LC-MS/MS method for S007-1261 in Sprague-Dawley (SD) rat plasma. Analytical quality criteria including method specificity, accuracy, precision and recovery were also taken in to account in this study. The developed and validated method of S007-1261 has been applied for pharmacokinetics studies in male SD rat after oral and intravenous administration.
Materials and Methods
▼
Drugs and chemicals Both CDRI candidate S007-1261 ( > 99 % pure) and 16-dehydropregnenolone (16-DHP) as an internal standard (IS) were obtained from Medicinal & Process Chemistry division of CSIRCDRI (Lucknow, India). Acetonitrile of HPLC grade was purchased from Sigma Aldrich. For preparation of buffer, analytical grade ammonium acetate and glacial acetic acid were purchased from E. Merck Limited. Triple distilled water of 18.2 MΩ cm was obtained from Milli-Q Ultrapure Water Purification system. HPLC grade n-hexane for extraction procedure was obtained from E. Merck Limited. Heparin sodium injection I.P. (1 000 IU mL − 1) was obtained from Troikaa Pharmaceutical Limited. For drug free plasma, male SD rats were provided by National Laboratory Animal Centre (NLAC), CSIR-CDRI, Lucknow. Drug free plasma was obtained from heparinized whole blood of rat by centrifugation at 1 500 × g for 10 min at 4 °C. The supernatant obtained was collected and stored at − 60 °C until use. This blank plasma was used for preparation of both calibration standard (CS) and quality control (QC) samples.
LC-MS/MS instrumentation and analytical conditions A series 200 HPLC system consisting of quaternary low pressure gradient pumps, online degasser and auto sampler with temperature controlled peltier tray (Perkin-Elmer Instruments, Norwalk, CT, USA) was used. Separation of analyte was achieved on RP 18, waters column (3.5 μm, 2.1 mm i.d. × 30 mm) using acetonitrile and ammonium acetate buffer (pH 5.0, 10 mM) at a flow rate of 0.40 mL min − 1. Buffer was filtered through 0.22 μm Millipore filter (Billeria, USA) before use. The ratio of acetonitrile to ammonium acetate buffer was 90:10 (v/v). The run time of the method was 5.30 min and injection volume was optimized to 20 μL. The rinsing solution used to rinse needle was a mixture of acetonitrile and triple distilled water (90:10 v/v). The analysis was carried out at ambient temperature. An API 4000 LC-MS/MS system (Applied Biosystem, MDS Sciex, Toronto, Canada) with Analyst 1.4 software was used for detection. The mass spectrometer was operated with electrospray ion
Misra A et al. Pharmacokinetics Study of S007-1261 … Drug Res 2014; 64: 399–405
[ESI] source in positive mode. Multiple reaction monitoring (MRM) mode was used to quantify S007-1261 and IS. Zero air was used as a source gas while nitrogen was used both as curtain gas and collision gas. Both MS and MS/MS condition of S007-1261 was optimized by continuous infusion at 10 μL min − 1 using syringe pump (model ‘11’, Harvard Apparatus, Holliston, MA, USA). MS and MS/MS of IS was also optimized by the same procedure. The precursor (hydrogen adduct of S007-1261, [M+H]+) to product ion transitions monitored at m/z 443.4 →112.3 for S007-1261 and m/z 315.1→137.5 for IS were used for quantification. Declustering potential (DP), collision energy (CE), entrance potential (EP) and collision cell exit potential (CXP) were also optimized for both S007-1261 and IS.
Preparation of calibration standard and quality control samples Stock solution of 1 mg mL − 1 of S007-1261 was prepared in acetonitrile. Working stocks having concentration range 2 000, 1 000, 500, 250, 125, 62.5, 31.25 and 15.6 ng mL − 1 of S0071261were also made in acetonitrile. CS of S007-1261 was prepared by spiking 10 μL of working stock into 100 μL of rat plasma over a concentration range of 1.56–200 ng mL − 1. Stock solution of 1 mg mL − 1 of IS was prepared in acetonitrile. Working stock of IS (10 000 ng mL − 1) was also prepared in acetonitrile. 10 μL of working stock of IS was also spiked in plasma to get final concentration of 1 000 ng mL − 1. QC samples were prepared in 5 replicates at low1 (1.56 ng mL − 1), low2 (3.12 ng mL − 1), medium (25 ng mL − 1) and high (200 ng mL − 1) concentration respectively. Low1 is also the lower limit of quantitation (LLOQ). These QC samples were prepared independent of CS. Concentration of test samples and QC samples were obtained from CS of S007-1261.
Sample preparation CS, QC and test samples were prepared from 2 step liquid-liquid extraction procedure using n-hexane as an extracting solvent. The volume of plasma was fixed at 100 μL and 10 μL of IS was added to each sample to get final concentration of 1 000 ng mL − 1 and vortexed for 60 s. After vortex-mixed, 2 mL of n-hexane was added and again vortexed for 3 min and centrifuged at 2 000 × g for 10 min at 4 °C. Thereafter, organic layer was transferred into clean tube by snap freezing of aqueous layer in liquid nitrogen. The organic layer in tube was allowed to evaporate till dryness in speed vac-concentrator. The remaining plasma of the above step was re-extracted in similar manner. The dry-residue was reconstituted in 200 μL of acetonitrile and centrifuged at 2 000 × g for 5 min at 4 °C and then injected for analysis.
Method Validation The bioanalytical method was validated in terms of specificity, sensitivity, accuracy, precision, calibration curve and reproducibility. The method specificity was determined by preparing and analyzing 6 individual as well as pooled rat blank plasma. Chromatogram obtained from spiked plasma at LLOQ was compared to chromatogram of blank plasma. This was done to check the specificity of the method. Each blank plasma samples was also tested for visible interference. Matrix effect is common, when mass spectrometer used as detector, since there may be possible competition between ionization of analyte and co-eluent residual matrix component from plasma [9]. To find out the possible matrix effect, peak area of the analyte dissolved in supernatant of the processed blank
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Fig. 1 Chemical structure of S007-1261.
plasma should be compare to standard solution at the same concentration. Matrix effect of S007-1261 was analyzed at 4 concentrations (1.56, 31.25, 25 and 200 ng mL − 1) in 5 replicates. Same procedure was also applied for determination of matrix effect of IS. The lower limit of detection (LOD) is the amount of drug in plasma after sample clean up corresponding to 3 times the baseline noise ratio (S/N > 3). LLOQ is the analyte concentration that can be quantified with ± 20 % accuracy and precision. Its signal should be at least 10 times as compared to blank (S/N > 10). Linearity of CS was anticipated for 5 days with concentration range of 1.56–200 ng mL − 1. Analytical standard (AS) and CS curve were drawn between the peak area ratio of analyte to IS vs. concentration of the analyte using weighted (1/x2) leastsquare linear regression. Accuracy was determined for both inter- and intra-day by calculating the % bias [ % bias = (Observed concentration − Nominal concentration)/ Nominal concentration × 100] of QC samples. The inter- and intra-day precision was calculated by one way analysis of variance (ANOVA) in terms of relative standard deviation (RSD). The extraction recoveries of S007-1261 were determined at 4 concentrations viz. 1.56, 3.12, 25 and 200 ng mL − 1 (QC low1, low2, medium and high) respectively. These were determined by comparing the peak area of extracted samples spiked with a known amount of the analyte with peak area of AS at the same concentration. Recovery of IS was determined in same manner at concentration of 1 000 ng mL − 1 in 10 replicates.
Stability studies Stability studies of S007-1261 were performed to evaluate the stability of this compound in stock solution and in plasma at different processing conditions like temperature and timing. To find out the effect of freeze-thaw cycle, spiked plasma QC samples were prepared at 1.56, 3.12, 25 and 200 ng mL − 1 (QC low1, low2, medium and high) concentrations with 5 replicates. One set was analyzed without freeze-thaw cycle and referred as control. Experimental set was analyzed after 3 freeze-thaw cycles. The bench-top temperature stability was determined by analyzing QC samples, which were kept at ambient temperature for a period of 8 h and compared with freshly prepared QC samples. Dry-residual stability studies was performed by storage of dryresidue after extraction for 4 and 7 days respectively at − 60 °C and compared its standard deviation from control set which was prepared and extracted at the day of analysis. The long-term stability was evaluated by determining QC plasma samples, kept at − 60 °C for period of 7, 15 and 30 days respectively and its standard deviation was calculated from control set which was prepared at the day of analysis. The autosampler stability was measured by reanalyzing extracted QC samples kept in the HPLC autosampler at 4 °C for 12 h. The working stock stability of S0071261 and IS were assessed at room temperature for 8 h.
Pharmacokinetic studies Healthy and young male SD rats weighing 250 ± 25 g were procured from NLAC, CSIR-CDRI, India. They were housed in wellventilated cages and kept at room temperature (24 ± 2 °C) on a regular 12 h light-dark cycle. The rats were acclimatized to this environment for at least 2 days before the initiation of experiment. They were fasted overnight (8–12 h) prior to experiment, however, water was freely provided. Animals were cared in accordance with the guidelines laid by the animal lab (Depart-
ment of Health Education and Welfare, no. [NIH] 85-32). Studies were approved by Institutional Animal Ethics Committee (Ethical approval 58/08/PKM/IAEC). For oral pharmacokinetic study, 0.5 % aqueous suspension of methyl cellulose was used and was administered by oral feeding needle at dose 40 mg kg − 1. The strength of the formulation was 20 mg mL − 1 and the volume administered was 500 μL. Blood samples were collected under light anesthesia at pre-dose and then at 0.083, 0.33, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72, 84, 96, 108 and 120 h post oral dose administration. All the samples were collected through orbital sinus vein puncture into microcentrifuge tubes containing heparin (22 IU heparin mL − 1 of blood). For intravenous pharmacokinetic study, formulation of S0071261 was prepared of absolute alcohol and propylene glycol (1:4 v/v) [10, 11]. This formulation was filtered through 0.22 μm filter before use. 500 μL of 10 mg kg − 1 dose was administered to rats through tail vein after dilation with xylene. Blood samples were collected under light anesthesia at pre-dose and then at 0.083, 0.33, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72, 84 and 96 h post intravenous dose administration. Samples of intravenous pharmacokinetic study were also collected through orbital sinus vein puncture into micro-centrifuge tubes containing heparin. For both oral and intravenous studies, sparse sampling technique was applied for sample collection. In this technique, animals were divided in to 2 groups viz. Group A and Group B. Both the groups had 4 rats. Thus, total 8 rats were there in a study (8 rats for oral and 8 rats for intravenous study). Predose sample (0 min) and post dose sample at 0.083, 0.75, 1.5, 4, 12, 36, 60, 84 and 108 h were collected from group A, while sample of 0.33, 1, 2, 6, 8, 24, 48, 72, 96 and 120 h were collected from group B. So, for both oral and intravenous pharmacokinetic studies, the volume withdrawn within 24 h was less than 10 % of the total blood volume of the rats. For both the studies, plasma was separated by centrifugation of blood at 1 500 × g for 10 min at 4 °C and stored at − 60 °C until analysis. WinNonlin version 5.1 (Pharsight Corp, USA) software was used to determine the pharmacokinetic parameters for both the studies.
Results and Discussion
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Condition optimization of the LC-MS/MS method Flow injection analysis of S007-1261 and IS were used to obtain full-scan mass spectra of parent and product ions. Mass spectra (MS) of S007-1261 and IS showed peak at 443.4 and 315.1 respectively of the parent compound. S007-1261 exists as protonated molecular ion [M + H]+. Continuous flow injection for MS/ MS analysis was carried out to obtain the product ion spectra of both the analyte and IS. The product ion for S007-1261 was opti▶ Fig. 2). The product ion for IS was optimized mized to 112.3 (● to 137.5. For quantification, m/z 443.4 parent ion transitions to m/z 112.3 product ions were taken for S007-1261. Similarly, m/z 315.1 parent ion transitions to m/z 137.5 product ions was used for quantification of IS. The dwell time was optimized and fixed at 300 ms. DP optimization was carried out using full scan acquisition over a mass range of 100–500 Da by flow injection analysis. Optimization of CE includes most intense fragments of the S007-1261 and IS. Optimized parameters like DP, CE, EP and CXP ▶ Table 1. The curtain gas for S007-1261 and IS are presented in ● (CUR) and collision gas (CAD) were optimized to 15 and 3 psi respectively. The ion source gas 1 (GS1) and ion source gas 2 Misra A et al. Pharmacokinetics Study of S007-1261 … Drug Res 2014; 64: 399–405
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402 Original Article
Table 1 Optimized LC-MS/MS condition for S007-1261and IS parent to product ion transition. Analyte S007-1261 16-DHP (IS)
Precursor
Product
DP
CE
EP
ion (m/z)
ion (m/z)
(V)
(V)
(V)
CXP (V)
443.4 315.1
112.3 137.5
160 90
46 41
10 10
10 10
(GS2) were optimized to 30 and 45 psi respectively. The ion source potential was kept at 5 500 v and source gas temperature was fixed to 200 °C. After optimization of all the parameters, MRM method was made for quantification of analyte and IS to obtain specific and selective detection. Using these transitions for analyte and IS, no interference in plasma was observed. Buffers of different pH and molarity were used to asses best conditions for peak intensity and shape. Finally, 10 mM ammonium acetate buffer of pH 5.0 was chosen along with acetonitrile as mobile phase, at a flow rate of 0.40 mL min − 1 because peak shape, intensity and elution time of analyte and IS was best under these conditions. Column of different dimensions were used for optimization of peak, of which RP 18 waters column (3.5 μm, 2.1 mm i.d. × 30 mm) with guard was found to give optimum results. Optimization of the injection volume gave best results at 20 μL. For sample clean-up, different non-polar solvents were tested. Pure n-hexane (2 × 2 mL) gave good recovery as compared to other extracting solvents. Different compounds were screened to find a suitable IS. Generally structural analog are used, which having almost similar retention time (RT) and extracting conditions. Finally, 16-DHP was selected as an IS, because both S0071261 and 16-DHP contain steroidal moeity and having same extracting conditions. No significant ionization suppression between S007-1261 and IS was observed throughout the LC-MS/ MS study.
Assay performance and validation The parameter used for validation and assessing the bioanalytes assay performance were selectivity, sensitivity, linearity, accuracy, precision, recovery and stability [12–17]. Plasma was checked from endogenous interference in the region of analyte and IS elution. There was no significant endogenous interference Misra A et al. Pharmacokinetics Study of S007-1261 … Drug Res 2014; 64: 399–405
at the RT of analyte and IS in blank rat plasma. Representative chromatogram of blank plasma, plasma spiked with S007-1261, spiked plasma containing IS and test plasma sample after intra▶ Fig. 3. venous dosing of S007-1261 are shown in ● On the basis of peak area ratio of analyte and IS, S007-1261 was linear over concentration range 1.56–200 ng mL − 1. S007-1261 was also reliable and reproducible over this calibration range. For well-designed and interpret calibration curve, weighing factor was used to improve residual points and to counter variance, since variance was not found constant across the calibration standard. Linear equation Y = mX + c with 1/x2 weighing factor showed the best fit for calibration curve. The correlation coefficient (r2) was > 99.9 for calibration curve was used. % RSD of RT for both analyte and IS were within the acceptable limits of 5 %. LOD (S/N > 3) was 0.39 ng mL − 1 and LLOQ was 1.56 ng mL − 1 respectively for S007-1261. Recovery was > 90 % for S007-1261 and it was evaluated from the spiked plasma at 1.56, 3.12, 25 and 200 ng mL − 1. No significant matrix effect was found since peak area ratio of the analyte dissolved in the supernatant of processed blank plasma and the standard solution of S007-1261 at the same concentration level were between 90 % and 110 %. Accuracy in terms of % bias and precision in terms % RSD was calculated at concentrations 1.56, 3.12, 25 and 200 ng ml − 1 with 5 determinants per concentration for 5 days in rat plasma. Accuracy and precision was calculated for both inter- and intra-day ▶ Table 2). The concentration of each sample was calculated by (● standard curve prepared and analyzed on same day. Bioanalytical method is accurate and precise over the concentration range 1.56–200 ng mL − 1 as % bias were within the acceptance limits and precision in terms of % RSD were never exceeds 15 %.
Stability studies in rat plasma Freeze-thaw stability and all other stability studies in rat plasma were carried out at 4 concentrations viz. 1.56, 3.12, 25 and 200 ng mL-1 in 5 replicates. The percentage deviation after 3 freeze-thaw cycles for S007–1261 at concentrations 1.56, 3.12, 25 and 200 ng mL − 1 were found to be 0.80 %, 0.05 %, − 0.06 % and ▶ Table 3). So, percentage deviation was 1.79 % respectively (● non-significant, when compared with freshly prepared QC samples without subject to freeze-thaw cycle. For bench-top stabil-
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Fig. 2 MS/MS spectra of S007-1261 with proposed fragmentation of selected product ion.
Original Article 403
Table 2 Inter-day and intra-day accuracy ( % bias) and precision ( % RSD) of S007-1261 in rat plasma (n = 5). Nominal
Type of
Accuracy
Precision
concentration
QC
( % bias)
( % RSD)
(ng mL − 1) Inter-day 1.56 3.12 25 200
Low1 Low2 Medium High
2.92 − 11.19 − 10.22 8.37
Intra-day − 0.37 − 3.55 − 2.32 5.44
Inter-day
Intra-day
3.0 11.6 11.3 9.1
1.8 0.6 4.2 2.3
ity, there was also non-significant deviation between sample ▶ Table 3). So, analyte is stable at room temkept at 0 h and 8 h (● perature up to 8 h for sample handling and processing during experiments. Dry-residue samples of analyte were stable up to 7 days when kept at − 60 °C after double extraction with n-hexane. The percentage deviation was non-significant when compared ▶ Table 4). The long-term with immediately prepared samples (● samples kept at − 60 °C for 7, 15 and 30 days also showed nonsignificant deviation when compared to freshly prepared sam▶ Table 4). Samples were, therefore, stable and can be kept ples (●
up to 30 days at − 60 °C. In auto sampler stability, repeated injection of same set of QC samples was given to check reproducibility at every 3 h up to 12 h, shows deviation were less than 10 % (data not shown).
Application of developed and validated method toward pharmacokinetic studies The plasma concentration-time profile of S007-1261 after single oral dose administration at 40 mg kg − 1 has been depicted ▶ Fig. 4. As seen from the figure, plasma concentration-time in ● profile is irregular having multiple peaks which could not be explained by any of the compartmental model. Hence, data was subjected to non-compartmental modeling. After oral dose, samples were collected up to 120 h and S007-1261 could be detected up to 60 h in rat plasma. The peak plasma concentration (Cmax) and time at which peak plasma concentration attained (Tmax) were 40.35 ± 7.03 ng mL − 1 and 10 ± 2.3 h respectively. Both these factors were also visually examined from raw data. After oral dose of S007-1261, it seems that absorption was slow, because plasma concentration peaks at 10 h post dose. The elimination half life (T1/2) and mean residence time (MRT) of S007-1261 were found to be 14.27 ± 0.95 h and 23.23 ± 1.31 h. Misra A et al. Pharmacokinetics Study of S007-1261 … Drug Res 2014; 64: 399–405
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Fig. 3 Representative MRM chromatogram of blank rat plasma a, plasma spiked with S0071261at 1.56 ng mL − 1 b, blank rat plasma c, plasma spiked with IS at 1 000 ng mL − 1 d and test sample after intravenous administration of S007-1261 at 2 h e.
404 Original Article
Table 3 Summary of freeze-thaw and bench-top stability studies of S007-1261 in rat plasma (n = 5). Storage condition
Nominal concentration
Type of QC
(ng mL − 1) After 3 freeze-thaw cycles
1.56 3.12 25 200 1.56 3.12 25 200
Bench-top for 8 h at ambient temperature
Concentration at time t = 0
Concentration recovered
Percent ( %)
(mean ± SD; ng mL − 1)
(mean ± SD; ng mL − 1)
deviation
1.65 ± 0.10 3.27 ± 0.22 27.26 ± 0.37 204 ± 20.64 1.39 ± 0.03 2.79 ± 0.04 22.76 ± 0.92 182 ± 1.0
Low1 Low2 Medium High Low1 Low2 Medium High
1.66 ± 0.02 3.27 ± 0.19 27.25 ± 0.91 208.33 ± 24.66 1.40 ± 0.04 2.83 ± 0.06 24.53 ± 2.07 195 ± 15.52
0.80 0.05 − 0.06 1.79 1.07 1.16 7.75 7.14
*t = 0 stands for concentration of QCs at the time of storage *Concentration recovered means concentration of QCs after storage at defined conditions
Dry-residue at − 60 °C
Long-term at − 60 °C
Nominal concentration
Type of
(ng mL − 1)
QC
1.56
Low1
3.12
Low2
25
Medium
200
High
1.56
Low1
3.12
Low2
25
Medium
200
High
Days
Concentration recovered
Percent ( %)
(mean ± SD; ng mL − 1)
deviation
4 7 4 7 4 7 4 7 7 15 30 7 15 30 7 15 30 7 15 30
Fig. 4 Plasma concentration-time profile of S007-1261 after oral administration at 40 mg kg − 1.
The volume of distribution was found to be 738.69 ± 87.13 L kg − 1. This high value of volume of distribution indicates that S007-1261 gets well distributed outside the vascular compartment. Clearance of S007-1261 was 36.19 ± 4.55 L h − 1 kg − 1 which suggested that extra-hepatic tissues are significantly involved in Misra A et al. Pharmacokinetics Study of S007-1261 … Drug Res 2014; 64: 399–405
1.54 ± 0.18 1.65 ± 0.03 3.23 ± 0.41 3.33 ± 0.20 22.22 ± 0.69 22.67 ± 1.34 182 ± 13.24 177 ± 7.43 1.50 ± 0.02 1.49 ± 0.12 1.41 ± 0.02 2.78 ± 0.06 2.84 ± 0.16 2.82 ± 0.13 26.13 ± 1.41 24.57 ± 2.30 22.7 ± 0.21 208.5 ± 20.76 216.66 ± 3.21 207.25 ± 13.81
− 4.93 2.05 − 3.59 − 0.62 − 3.99 − 2.05 − 0.81 − 3.54 6.99 6.22 0.53 − 0.62 1.42 0.80 8.88 2.39 − 5.41 5.56 9.70 4.93
Table 4 Summary of dryresidual and long-term stability studies of S007-1261 in rat plasma (n = 5).
the elimination of the drug. Both volume of distribution and clearance values were higher after oral administration as compared to intravenous administration. The value of area under curve (AUC0–∞) was 1 209.48 ± 166.47 ng h mL − 1. The plasma concentration-time profile after intravenous administration of S007-1261 at 10 mg kg − 1 also depicted multiple ▶ Fig. 5). So, data after intravenous administration were peaks (● again subjected to non-compartmental modeling. After intravenous administration, samples were collected up to 96 h and S007-1261 could be detected up to 84 h in rat plasma. The initial concentration (CO) was 166.28 ± 90.25 ng mL − 1. The elimination half life (T1/2) and mean residence time (MRT) of S007-1261 after intravenous administration were found to be 15.42 ± 1.25 h and 25.29 ± 1.99 h. High MRT value after intravenous administration may be due to S007-1261 is retained in the system for longer period of time as its elimination is slow from the body. The volume of distribution and clearance were found to be 267.52 ± 11.33 L kg − 1 and 12.34 ± 1.37 L h − 1 kg − 1 respectively. The value of area under curve (AUC0–∞) was 825.58 ± 88.71 ng h mL − 1. The bioavailability was found to be 33.61 % when compared AUC after oral and intravenous dosing of S007-1261. This means 33.61 % of S007-1261 dose reaches the systemic circulation. All the pharmacokinetics parameters, both after oral and intrave▶ Table 5. nous administration, are summarized in ●
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Storage condition
Original Article 405 Acknowledgements
▼
We are thankful to Director, CSIR-CDRI, for providing facilities and infrastructure for the study. We also acknowledge Council of Scientific and Industrial Research (CSIR) and University Grant Commission (UGC) New Delhi, India, for providing funds and fellowships. This is CDRI communication no. 8555.
Conflict of Interest
▼
There is no conflict of interest to be disclosed.
Fig. 5 Concentration-time profile of S007-1261 after intravenous administration at 10 mg kg − 1.
Table 5 Pharmacokinetic parameters for S007-1261in male rats plasma following 40 mg kg − 1 oral and 10 mg kg − 1 intravenous dose (Mean ± SEM, n = 4). PK parameters Tmax (h) Cmax (ng mL − 1) Co (ng mL − 1) T1/2 (h) AUC0–∞ (ng h mL − 1) Vz (L kg − 1) Cl (L h − 1 kg − 1) MRT (h)
Oral
Intravenous
10 ± 2.3 40.35 ± 7.03 * 14.27 ± 0.95 1 209.48 ± 166.47 738.69 ± 87.13 36.19 ± 4.55 23.23 ± 1.31
* * 166.28 ± 90.25 15.42 ± 1.25 825.58 ± 88.71 267.52 ± 11.33 12.34 ± 1.37 25.29 ± 1.99
*not applicable
Multiple peaks after both oral and intravenous administration of S007-1261 may arise due to non-aqueous solubility that might have resulted in precipitation of dose in the intestine and did not redissolved in the absorptive region of the GIT [18]. Other reasons may be formation of depot on intestinal walls, enterohepatic recirculation of S007-1261 and its variable rates along the gastrointestinal tract.
Conclusion
▼
The LC-MS/MS method developed and validated for antidiabetic S007-1261 shows good sensitivity, specificity, accuracy and precision. The LLOQ was established at 1.56 ng mL − 1. Calibration curve in the range of 1.56–200 ng mL − 1 shows good linearity in rat plasma. For samples preparation, 2 step liquid-liquid extraction procedure was used with n-hexane as an extracting solvent which shows the recovery > 90 %. S007-1261 did not show any stability problem during storage and sample processing. This developed and validated method was successfully applied to both single oral and intravenous pharmacokinetic studies. The oral bioavailability of S007-1261 was found to be 33.61 %. This newly developed and validated method may be applied in future for both pre-clinical and clinical studies, toxicity studies, drug interaction studies and multiple dose pharmacokinetic studies.
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