A validated LC-MS/MS method for the pharmacokinetic study of alogliptin in healthy rabbits

Apparatus and software

The LC-MS/MS used for the study consist of Shimadzu LC-20ADvp (Shimadzu, Japan) coupled with Applied Biosystem sampler Sciex (MDS Sciex, Canada) API 4000 Tandem mass spectrometer and the autosampler was SIL-HTC from Shimadzu, Japan. The system was operated by Analyst software (version: 1.4.2; Applied Biosystems).

Chemicals and reagents

Alogliptin and Alogliptin D3 (IS) were procured by Hetero Drugs Ltd., Hyderabad, India, formic acid was purchased from Merck Specialities Pvt. Ltd, Mumbai, India. The water for chromatography was produced from water purification systems (Milli Q, Milli Pore, USA) installed in the laboratory. The HPLC grade methanol and acetonitrile were supplied by Baker, USA. Hyderabad. The rabbit plasma was prepared by freeze centrifugation. The study was approved by Institutional Ethical committee no: VCP/IAEC/2016-44.

Calibration standard solutions

Stock solutions (1 mg/ml) of Alogliptin and Alogliptin D3 internal standard (IS) were prepared in methanol further the eight calibration standards were prepared from stock by diluting with 70% methanol subsequently they are spiked with drug free plasma to give the concentrations of 4.00, 8.00, 25.00, 50.0, 100.0, 150, 300, and 600 ng/ml.

Quality control standards

Lowest quality control (LQC), median quality control (MQC) and highest quality control (HQC) standards were prepared from stock by spiking drug-free plasma with Alogliptin to give a solution containing 8, 250, and 500 ng/ml respectively. They were stored at −20°C till the time analyzed.

Chromatographic conditions

Chromatographic separation was achieved on Inertsil ODS-C18 (50 × 4,6 mm, 5 μm) with 0.1% formic acid-acetonitrile-methanol (30: 56: 14 v/v). as an isocratic mobile phase with a flow rate of 1.0 ml/min. Injection volume was 5 μl. The total chromatographic run time was fixed to 2.00 minutes and column oven and autosampler temperature was set to 40°C and 5°C, respectively.

Mass spectrometric conditions

The LC eluent was split (75%), and approximately 0.25 ml/min was introduced into LC and the Quantitation was achieved through negative ion mode by employing turbo ion spray interface at 400°C. The 5500 V voltage for ion spray and the pressure 8, 10 and 6 psi for a nebulizer, the curtain was set at 5500 V. The source parameters viz., the nebulizer, curtain and CAD gas were set at 8, 10, and 6 psi, respectively. The compound parameters viz. the declustering potential (DP), collision energy (CE), entrance potential (EP) and collision cell exit potential (CXP) for alogliptin and alogliptin-D3 were similar and are set at 18, 10, 10, and 10 V. The multiple-reaction monitoring mode (MRM) was employed for detecting and monitoring the transition pairs of m/z 340.2 → 116.0 for alogliptin, and m/z 343.3 → 196.2 for IS.

Study design

Six male albino Rabbits (weighing about 2.5 kg) were selected as the animal model. The age of the rabbits was 8–12 weeks. The rabbits selected for the study had no medication for two weeks prior to the study. Twelve hours before drug administration, food was withdrawn from the rabbits until 24 hr post-dosing, while, water was available for rabbits throughout the study. The tablet with the dose of 0.59 mg based on the animal surface area was administered to rabbits using a balling gun. The study was approved by Institutional Ethical committee no: P50/VCP/IAEC/2016/09/DBP/AE12/Rabbits. Blood samples (0.6 ml) were withdrawn from the marginal ear vein before dosing (zero time) and at time intervals of 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 12, 16, 24, 36, and 48 hours after administration. For each animal, the total number of blood samples drawn during the study was 16. EDTA disodium salt was used as an anticoagulant. Plasma was separated by centrifugation at 5000 rpm for 10 min and the resulting plasma sample from each blood sample was divided into two aliquots and stored in suitably labeled polypropylene tubes at −20°C until used. All the plasma samples were analyzed under the construction of standard calibration curve of alogliptin in rabbit’s plasma. The alogliptin concentrations in the rabbit plasma samples was calculated using the calibration curve, obtained after linear regression of the peak area ratio (alogliptin/alogliptin-D3) versus the concentration of alogliptin.

Sample preparation method

The samples were prepared by mixing 50 μl of alogliptin D3 (50 ng/ml) and 100 μl buffer (0.05 M NaOH, pH: 4) to 250 μl of plasma. The drug was extracted by employing LLE method using 2.5 ml of ethyl acetate followed by centrifugation at 2000 rpm/min for 15 min at 4°C. The supernatant was withdrawn and dried using lyophilizer. The residue was reconstituted with 300 μl of the mobile phase.

Pharmacokinetic analysis

The pharmacokinetic parameters for single oral dose non-compartmental study were calculated through PK Solver tool. The C_max (maximum plasma concentration) and T_max (time to maximum plasma concentration) were obtained directly from the plasma time profile curve. The linear trapezoidal rule was used to estimate all other pharmacokinetic parameters. i.e. AUC 0-t, AUC 0-∞, t_1/2 and ke.

Specificity

The specificity of the method was determined by injecting 4.0 ng/ml concentration under optimized chromatographic conditions to the LC system to show the separation of alogliptin from impurities and plasma. The specificity of the method was checked for the interference from plasma.

Linearity

The linearity of the method was determined by plotting eight-point calibration curves (4 ng/ml to 600 ng/ml) between actual and calculated concentrations of samples spiked with plasma.

Recovery studies

Two sets of six HQC, MQC, and LQC samples were prepared, one is with plasma and another one is with 70% methanol. The % mean recoveries were determined by measuring the responses of the plasma quality control samples against quality control samples prepared with 70% methanol.

Precision and accuracy

Intraday precision and accuracy was obtained by analyzing the quality control standards containing concentrations 8, 250, and 500 ng/ml and LLOQ Quality control standard containing concentration 4.00 ng/ml five times a day randomly, interday precision and accuracy was achieved by determining each quality control standards (8, 250, and 500 ng/ml) and LLOQ Quality Control standards (4.00 ng/mL) once on each of five different days.

Matrix effect

The matrix effect for the established method was determined by using six lots of rabbit plasma and is used to prepare the concentrations equivalent to LQC and HQC of alogliptin the samples prepared were injected on to the column.

Stability studies

The stability of alogliptin was determined by measuring concentration change in control samples overtime under set conditions. Freeze-thaw stability study (−80°C) of alogliptin was carried out by subjecting samples to three freeze and thaw cycles. Samples before the study and after study were analyzed by developed method. Similarly stock solution stability study of alogliptin (Stability after 6 Hours), Long-term stability (−80°C, 30 days), benchtop stability study of alogliptin (at ambient temperature, 6 h), Dry residue stability (4°C, 48 h) and Auto-sample stability (4°C, 24 h) of alogliptin were carried out by subjecting samples to study conditions.

Results of method validation

The chromatograms obtained during method validation was acceptable and the representative chromatograms of standard blank, HQC, MQC, and LQC samples are shown in (Figures 1 to 4).

The linearity, accuracy & precision, and stability of the method were established as per ICH guidance (ICH guidelines Q1A(R2), 2005; ICH guidelines Q2(R1), 2005).

Fig. 1: blank chromatograms of alogliptin and IS in blank Plasma. [Click here to view]

Fig. 2: HQC-chromatograms of alogliptin in plasma with internal standard. [Click here to view]

Fig. 3: MQC-chromatograms of alogliptin & its internal standard. [Click here to view]

Fig. 4: Chromatograms of alogliptin & its internal standard at LQC Level. [Click here to view]

Linearity

The calibration curve (calculated concentrations Vs actual Concentration) obtained (n = 3) were linear over working range of 4 ng/ml to 600 ng/ml (Figure 5 and Table 1). The linear regression equation for the analysis was 0.994x-2.692 with coefficient of correction (r²) = 0.999.

Fig. 5: Calibration curve. [Click here to view]

Table 1: Summary of Linearity Standards for Alogliptin. [Click here to view]

Table 2: The % Mean Recovery of alogliptin for LQC, MQC, and HQC. [Click here to view]

Recovery

The % mean recovery of the method for LQC(8 ng/ml), MQC (250 ng/ml) and HQC (500 ng/ml) was found to be 112.2%, 109% and 106% respectively (Table 2).

Intraday and Inter-day Precision

The mean intraday and inter-day precision of the method for alogliptin was found to be 1.26 to 4.97% (≤15%) for LQC, MQC, HQC, and LLOQC (Table 3).

Table 3: Intra-day and Inter-day quality control samples for alogliptin. [Click here to view]

Matrix effect

After the analysis of HQC and LQC from six different plasma lots the % CV was observed 0.1% and 2.8% (≤15%) respectively (Table 4).

Table 4: Matrix effect obtained with six different lots of plasma. [Click here to view]

Stability studies

The quality control samples (HQC and LQC) were used for stability studies. The % mean stability for HQC and LQC were presented in table 5 and are within the acceptance limits of 85 to 115%. (Table 5).