Simultaneous Quantification of Pioglitazone and Omarigliptin in Rat Plasma by UHPLC-MS/MS and Its Application to Pharmacokinetic Study after Coadministration of the Two Drugs

Combination therapy is a common approach for clinical treatment of type 2 diabetes mellitus, especially for patients with poor monotherapy. Meta-analysis suggested that omarigliptin, a long-acting DPP-4 inhibitor, combined with pioglitazone might improve the side effects of pioglitazone. However, little is known about the pharmacokinetic properties after a coadministration. In this study, a rapid and reliable method for the simultaneous determination of the pioglitazone and omarigliptin in rat plasma by UHPLC-MS/MS was established and validated for the first time. An exsil mono C18 column (2.0 × 50 mm, 3 μm) was used to separate the analytes and the column temperature was kept at 30°C. Sitagliptin was selected as the internal standard. 0.02% formic acid aqueous solution (A) and methanol-acetonitrile (B) were used as mobile phases with gradient elution at a flow rate of 0.3 mL/min. The elution procedure was as follows: 20%B (0–0.1 min), 80%B (0.1–0.3 min), 80%B (0.3–2.0 min), and 20%B (2.1–3.0 min). A multiple reaction monitor (MRM) was used under positive ionization mode with electrospray ion source to detect pioglitazone (357.1 ⟶ 134.1), omarigliptin (399.2 ⟶ 153.0), and sitagliptin (408.2 ⟶ 235.0). The linear ranges of pioglitazone and omarigliptin were 5–2000 ng/mL and 10–4000 ng/mL, respectively. Good linear relationships were exhibited in the corresponding linear ranges (r ≥ 0.9944). The bioanalytical method was validated, and the selectivity, linearity, sensitivity, accuracy, precision, stability, recovery, and matrix effect were acceptable. The validated method was then successfully applied to pharmacokinetic study of pioglitazone combined with omarigliptin in rats. Results suggested that the combination of the two drugs had little effect on the pharmacokinetic parameters of each other in rats.


Introduction
Type 2 diabetes mellitus (T2DM) is one of the most common metabolic diseases around the whole world. It is mainly caused by pancreatic ß cell dysfunction and insulin resistance, leading to continuous increase in blood glucose [1]. Statistics demonstrated that more than 415 million adults suffered from T2DM [2,3]. ere are many treatments for T2DM, such as exercise, surgery, and traditional Chinese medicine and drug therapy. Among them, oral hypoglycemic drug treatment is the most convenient and effective method.
Pioglitazone, a member of the thiazolidinediones, was an oral antidiabetic drug widely used in China [4]. It is an insulin sensitizer that can reduce insulin resistance in peripheral tissues and liver, and meanwhile increase the uptake of peripheral glucose by activating peroxisome proliferatoractivated receptor gamma (PPAR c).
erefore, blood glucose could be controlled but the endogenous secretion of insulin has not been increased yet [5]. Studies have shown that pioglitazone might reduce major adverse cardiovascular events, correct multiple components of metabolic syndrome, and improve nonalcoholic fatty liver disease [6,7]. However, side effects such as weight gain, fluid retention, or even fracture could not be ignored either [7,8].
Dipeptidyl peptidase-4 (DPP-4) inhibitors could improve glycemic control in patients with T2DM and have gained extensive interest because of long-term efficacy and better glycemic control. DPP-4 inhibitors exerted a hypoglycemic effect by inhibiting the inactivation of glucagonlike peptide-1 (GLP-1), promoting the release of insulin from pancreatic ß cells and inhibiting the secretion of glucagon from pancreatic α cells [9,10]. Furthermore, there were some other advantages for DPP-4 inhibitors like not increasing the weight, decreasing the risk of hypoglycemia, repairing the function of pancreatic islet, and protecting the cardiovascular system [11]. More importantly, recent studies have found that DPP-4 inhibitors may exhibit potent neurorestorative effects [12]. Consequently, DPP-4 inhibitors have been gradually used in clinical practice.
When the blood glucose could not be controlled by monotherapy any more, the combination should be considered. Studies have suggested that the combination of DPP-4 inhibitors and thiazolidinediones could simultaneously solve insulin resistance and islet dysfunction, two major defects of T2DM [13]. In addition, DPP-4 inhibitors could enhance the excretion of sodium and fluid in kidney, thus alleviating the possible side effects of pioglitazone [14]. Moreover, the combination therapy could significantly improve the function of pancreatic ß cells and had a better effect on blood glucose control [15]. Meta-analysis showed that, compared with pioglitazone alone, the combination could remarkably reduce hemoglobin A1c (HbA1c) levels and fasting blood glucose [16]. erefore, the combination therapy is considered more suitable for patients with poor curative effect by monotherapy. Omarigliptin, a weekly DPP-4 inhibitor, which has been approved in Japan since 2015, showed more effective than sitagliptin [17]. Compared with the once-daily administered DPP-4 inhibitors available in market, omarigliptin significantly improved the compliance of patients. Although a micelle-enhanced spectrofluorimetric method was developed for determination of omarigliptin [18], a HPLC method was used to investigate the degradation kinetics of omarigliptin in the oxidative and photolytic medium [19], and the literature has revealed pharmacokinetics of omarigliptin [20], there is no study to elucidate the pharmacokinetic or pharmacodynamic characteristics of the combination of omarigliptin and pioglitazone. e aim of this study was to investigate the pharmacokinetic characteristics of the omarigliptin combined with and pioglitazone in rats. For this purpose, a simple and sensitive method for the simultaneous quantification of pioglitazone and omarigliptin in rat plasma needed to be established first. en, the validated method was successfully applied to the pharmacokinetic study of pioglitazone combined with omarigliptin in rats. To the best of our knowledge, there was no similar study before, and the results may give hints for the combined administration of pioglitazone and omarigliptin.  Figure 1. Formic acid (FA) was bought from Fluka Chemie (Buchs, Switzerland). Methanol and acetonitrile were HPLC grade and purchased from Fisher Scientific (Pittsburgh, USA). Deionized water was manufactured by Milli-Q purification system (Bedford, USA).

Instrument and Conditions.
Dionex Ultimate 3000 UHPLC system including two RS pumps, an RS autosampler, and an RS column compartment was employed in this study ( ermo Scientific, Massachusetts, USA). An Exsil Mono C18 column (2.0 × 50 mm, 3 μm, Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) was used to achieve the chromatographic separation. Deionized water containing 0.02% (v/v) formic acid was labelled as mobile phase A and methanol: acetonitrile (v/v, 1 : 1) was labelled as mobile phase B. Gradient elution program is listed in Table 1. e column temperature was held at 30°C and the autosampler temperature was kept at 10°C. e injection volume was 2 μL.
TSQ Quantiva triple quadrupole mass spectrometer with Xcalibur ( ermo Scientific, Massachusetts, USA) was adopted for precise quantification. Data were acquired under positive ionization mode. Individual parameters for the analytes and IS such as ion pairs, collision energies, and RF lens are displayed in Table 2. Other mass spectrometry parameters were set as follows: positive ion, 3500 V; ion transfer tube temperature, 350°C; vaporizer temperature, 300°C; sheath gas, 35 Arb; aux gas, 15 Arb; sweep gas, 2 Arb; CID gas, 1.5 mTorr. Dwell times were 100 msec for all compounds.

Preparation of Standard Calibration Samples and Quality
Control Samples. All compounds were dissolved and diluted with methanol to prepare stock solutions and working solutions. All solutions were stored at 4°C. Different concentrations of working solutions were diluted 20 times with blank rat plasma to prepare corresponding calibration samples. Final concentrations of calibration samples were 5, 25, 50, 100, 250, 500, 1000, and 2000 ng/mL for pioglitazone and 10, 50, 100, 200, 500, 1000, 2000, and 4000 ng/mL for omarigliptin. Quality control (QC) samples (5, 15, 400, 1500 ng/mL for pioglitazone and 10, 30, 800, 3000 ng/mL for omarigliptin) were prepared as the same way. All plasma samples were stored at −20°C until analysis.

Extraction of Plasma Samples.
Whole blood was centrifuged at 8000 rpm for 5 min to obtain plasma samples, which were then extracted by simple protein precipitation.
In brief, 25 μL of thawed plasma was added to 800 μL IS solution (sitagliptin, 50 ng/mL) and was whirled for 1 min to mix well. Centrifugation was performed at 12000 rpm for 5 min and then supernatant fluid was obtained for analysis.

Method Validation.
e method was validated according to the US Food and Drug Administration (FDA) guidelines [21].

Selectivity.
e selectivity was evaluated by comparing blank plasma from six different rats, blank plasma spiked with analytes and IS, and practical plasma samples. e intensity of blank samples should be less than 20% of LLOQ samples.

Linearity and Sensitivity.
e calibration curve was fitted by plotting the peak area ratio of the analyte to IS against the nominal concentration of the analyte in plasma, and the weighting coefficient was set to 1/x 2 . e minimum concentration of the standard curve was taken as the lower limit of quantification (LLOQ). e precision and accuracy of LLOQ should be 80-120%.

Precision and Accuracy.
e intraday precision and accuracy of the method were evaluated by analyzing QC samples at low, medium, and high concentrations (n � 6) on the same day. Results of three consecutive days were used to assess the interday precision and accuracy. Precision and accuracy were characterized by relative standard deviation (RSD, %) and relative error (RE, %), respectively. RE (%) and RSD (%) should be within ±15%.

Recovery and Matrix Effect.
In order to calculate the extraction recovery and matrix effect, blank plasma samples were collected from six different rats and processed as follows. (A) Blank plasma samples were spiked with analytes and then were precipitated with methanol containing IS. (B) e blank plasma was precipitated with pure methanol to obtain supernatant, and then analytes and IS with the same concentration as group A were added to the supernatant. (C) e working solution was added to an equal amount of deionized water instead of blank plasma and then were extracted with methanol containing IS as described in Section 2.4. e recovery was calculated by dividing the peak area of group A by that of group B, while the matrix effect was calculated by dividing the peak area of group B by that of group C.
2.5.5. Stability. QC samples with low, medium, and high concentrations were analyzed under different conditions, such as kept at room temperature for 6 h, frozen and thawed for three cycles, and frozen at −20°C for 7 days. e   e animal certification number was 3707262011004044 and the production license number was SCXK (Lu) 20190003. Before the formal experiment, all rats were adaptively fed for one week at a temperature of 24 ± 2°C and a humidity of 55 ± 5% and then were randomly divided into three groups. In order to guarantee animal welfare, Animal Experiments Guidelines of Yantai Institute of Materia Medica (Yantai, China) was enforced.
Pioglitazone and omarigliptin were suspended into 0.5% carboxymethyl cellulose sodium salt (CMC-Na) for intragastric administration. e dosage for rats was calculated based on the clinical dose. Rats were fasted for at least 12 h before the experiment to avoid possible effects of food and then were administrated with pioglitazone (2.6 mg/kg), omarigliptin (2.6 mg/kg), or a combination of the two compounds, respectively. About 200 μL whole blood was collected into the heparinized tube at 0 min (predose), 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h and centrifugated immediately to collect plasma. All plasma samples were frozen at −20°C until analysis. Samples were processed and analyzed by the validated method.

Data Analysis.
Data were acquired and analyzed by Xcalibur 4.1 software ( ermo Fisher Scientific, Massachusetts, USA). Pharmacokinetic parameters were calculated based on noncompartmental model using Phoenix WinNonlin 7.0 (Pharsight, Mountain View, CA). All data were shown as mean ± SD.

Method Development and Optimization. Full mass scan
was performed under positive mode and [M + H] + were formed for these three compounds probably due to the presence of amino groups. e corresponding product ions were then confirmed and relevant mass parameters such as collision energy and RF lens were optimized for higher response (Figure 2, Table 2). A satisfactory resolution was obtained on an Exsil Mono C18 column (2.0 × 50 mm, 3 μm) with gradient elution. In order to increase the response, 0.02% formic acid was added to the aqueous phase.
Methanol: acetonitrile (v/v, 1 : 1) was used to improve the shape of chromatographic peak instead of pure methanol. e samples were extracted by protein precipitation. Compared with the liquid-liquid extraction methods reported in the literature to extract omarigliptin from biological samples [22,23], the protein precipitation was simple, cheap, and time-saving. Figure 3, there was no obvious endogenous interference and the retention times for pioglitazone, omarigliptin, and sitagliptin (IS) were 1.75 min, 1.54 min, and 1.62 min, respectively. e peak shape and resolution were good and the selectivity of this method was satisfactory.

Linearity and Sensitivity.
Linear regression was performed with the concentration as the abscissa and the peak area ratio of the analyte to IS as the ordinate. e standard curve equation was calculated by least square method with weighting factor (w �1/x 2 ). As shown in Figure 4, the pioglitazone and omarigliptin exhibited good linearity during their respective linear ranges. e sensitivity is shown in Table 3 and the results were acceptable. Table 3 indicates the precision and accuracy of the method. e intraday precision (RSD, %) of pioglitazone and omarigliptin was less than 7.6% and the accuracy (RE, %) was within ±8.7%. e interday precision (RSD, %) was less than 6.9% and accuracy (RE, %) was within ±8.4%. e method was accurate and reliable. Table 4, the recoveries of pioglitazone and omarigliptin at three concentrations were 86.7%-96.5% and 89.1%-94.8% and the recovery of IS was 107%. e recoveries were satisfactory. Meanwhile, the RSD of all QC samples prepared with biological matrix from different sources was less than 7.7% ( Table 4), indicating that the method was reproducible, so the matrix effect was tolerable.

Stability.
e results are shown in Table 5. RE and RSD were less than 15% under different conditions, demonstrating the stability of the method was acceptable.

Diluted Quality Control Samples.
Since the concentration of individual samples exceeded the upper limit of quantification, appropriate dilution was required before detection. So it was necessary to study diluted quality control samples. QC samples containing 3000 ng/mL pioglitazone and 6000 ng/mL omarigliptin were diluted twice with blank plasma and then precipitated and centrifuged for injection analysis. Table 6 shows that both RSD and RE were less than 15%, and the diluted QC samples were valid.

Pharmacokinetics Study.
e pharmacokinetic parameters after single or combined administration are displayed in Tables 7 and 8. Figure 5 shows the mean plasma concentration-time curves for pioglitazone and omarigliptin. Both pioglitazone and omarigliptin were absorbed rapidly after a single administration, with t max being 1.8 h             Figure 5: Mean plasma concentration-time curves after oral administration of pioglitazone (2.6 mg/kg), omarigliptin (2.6 mg/kg), or pioglitazone (2.6 mg/kg) plus omarigliptin (2.6 mg/kg) (mean ± SD, n � 5). and 1.5 h, respectively. e elimination of omarigliptin was much slower than that of pioglitazone, with t1/2 of omarigliptin being 10.9 h while t1/2 of pioglitazone was 2.8 h. e AUC (0-t) and AUC (0-∞) were 16887 and 16973 ngh/mL for pioglitazone alone, and there was a slight increase after combining with omarigliptin, but with no statistical significance (18988 and 19013 ngh/mL). On the contrary, after coadministration, the bioavailability of omarigliptin decreased gently (Table 8). In general, the combination had little effect on pharmacokinetic parameters compared with single administration. It was worth noting that the pharmacokinetic study was only investigated on normal rats in this paper; further studies should be carried out on diabetic models to explore the pharmacokinetic and pharmacodynamic characteristics to ensure the effectiveness and safety of the combination therapy.

Conclusion
Drug combination is commonly used clinically to treat patients with T2DM who are not sensitive to monotherapy. Pioglitazone combined with omarigliptin could simultaneously solve insulin resistance and islet dysfunction in patients with T2DM, which can be used as an option for treatment. In this study, the quantification and pharmacokinetics of the two drugs were reported. Results indicated that the pharmacokinetic behavior of pioglitazone and omarigliptin was almost unchanged after the combined administration. From the perspective of PK, there was no obvious interaction between the two drugs, suggesting that there may be no need to adjust the dose when used together. e research provided an experimental basis for evaluating the efficacy and safety of pioglitazone combined with omarigliptin in vivo and laid a foundation for wider clinical applications.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest.