A Reproducible Method for the Quantification of Pioglitazone and Metabolites in Human Plasma Using the ACQUITY UPLC H-Class System and Xevo TQD MS with UNIFI

In this application note, we report the development of a highly sensitive solid phase extraction, and LC-MS/MS assay using the Xevo TQD for the analysis of pioglitazone and the two active metabolites in human plasma with an assay sensitivity of 10 pg/mL.

Benefits

A high sensitivity method was developed for the analysis of pioglitazone and its two active metabolites, keto pioglitazone and hydroxy pioglitazone, in human plasma. The extremely low carryover exhibited by the ACQUITY UPLC H-Class System allows the full sensitivity of the Xevo TQD Mass Spectrometer to be utilized.

Pioglitazone is part of the thiazolidinedione class of drugs used in the treatment of diabetes through hypoglycemic action. It selectively stimulates the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-γ) to modulate the transcription of the insulin-sensitive genes involved in the control of glucose and lipid metabolism.¹

Following oral administration ranging from 15 to 45 mg, the dosed compound undergoes hepatic metabolism with CYP2C8, and to a lesser degree CYP3A4, to give rise to the following two active metabolites: keto pioglitazone and hydroxy pioglitazone. Both metabolites are present at higher systemic concentrations than the parent compound at steady state, reached seven days after dosing. At steady state, in patients with type 2 diabetes, pioglitazone comprises approximately 30% to 50% of the peak total pioglitazone serum concentrations (pioglitazone plus active metabolites) and 20% to 25% of the total area under the curve (AUC). 

In this application note, we report the development of a highly sensitive solid phase extraction, and LC-MS/MS assay using the Xevo TQD for the analysis of pioglitazone and the two active metabolites in human plasma with an assay sensitivity of 10 pg/mL.

Sample Description

Samples were prepared using an Oasis HLB μElution solid phase extraction plate. The plasma samples, measuring 300 μL, were mixed with 20 μL of internal standard solution (deuturated analogues of all three compounds) and 300 μL of 2% phosphoric acid. The samples were applied to the solid phase extraction plate, which was previously conditioned and equilibrated with methanol (200 μL) and water (200 μL). The sample was washed with a 5% methanol/water solution, and then eluted with a 50 μL and subsequently 25 μL aliquot of methanol. Samples were further diluted with 75 μL of water prior to injection.

Method Conditions

The analysis was performed on an ACQUITY UPLC H-Class System. A 10-μL aliquot of the sample was injected onto an ACQUITY UPLC BEH C₁₈ 2.1 x 50 mm, 1.7-μm Column. The column was operated under gradient conditions over 2 min at a flow rate of 600 μL/min. The mobile phases used were 0.1% ammonium hydroxide and methanol. The column effluent was monitored using a Xevo TQD Mass Spectrometer operated in multiple reaction monitoring (MRM) positive ion electrospray mode.

The transitions monitored included the following:

Pioglitazone: 357 > 134

Keto pioglitazone: 371 > 148

Hydroxy pioglitazone: 373 > 150

d₄-pioglitazone: 361 > 138

d₄-keto pioglitazone: 375 > 152

d₅-hydroxy pioglitazone: 378 > 154

Data integration and calculation software

UNIFI Scientific Information System

Pioglitazone, keto pioglitazone, and hydroxy pioglitazone eluted with retention times of 1.59, 1.35, and 1.34 minutes, respectively, as shown in Figure 2. This data reveals very symmetrical peaks produced by the chromatography system with a width at the base of approximately 3 s for all three compounds. The narrow peak width and the symmetrical nature allow for efficient processing and peak integration. The data displayed in Figure 2 illustrates the injection of an extracted plasma blank injection, immediately following analysis of the 1000 pg/mL standard. This data demonstrates that there is no discernible carryover in the blank chromatogram (the baseline has been magnified) for any of the compounds. The extremely low carryover exhibited by the ACQUITY UPLC H-Class System allows the full sensitivity of the Xevo TQD Mass Spectrometer to be exploited.

The lower limit of quantification (LLOQ) for the assay was 10 pg/mL for all three analytes. Calculated signal-to-noise values for the LLOQ were 44:1 for pioglitazone, 21:1 for keto pioglitazone, and 58:1 for hydroxy pioglitazone, as shown in Figure 3. A typical calibration curve obtained for the assay of pioglitazone is shown in Figure 4. The correlation coefficient ranged between 0.997 and 0.999 for the three compounds using a 1/x weighting linear regression. The single day accuracy and precision data are displayed in Tables 1 through 3 for quality control (QC) samples prepared at four levels spanning the calibration range, and extracted in replicates of five. The validation data shows that the coefficient of variation for the parent and two metabolites ranged from 8.4% to 13.4% for the 10 pg/mL LLOQ with a bias between -5.7% and 1.8%. For the high QC (800 pg/mL), the coefficient of variation ranged from 1.1% to 4.4% with a bias between -0.1% to 0.5%.

• A high sensitivity method has been developed for the analysis of pioglitazone and its two active metabolites in human plasma using the Xevo TQD.
  
• The assay showed excellent intra-day accuracy and precision for QCs prepared at four concentration levels.
  
• The lower limit of quantification was determined to be 10 pg/mL with a %CV and bias, both well below the required +/- 20% required for assay validation.
  
• The carryover was determined to be significantly less than 20% of the LLOQ in an extracted blank, following the injection of a high concentration standard.

1. Baughman TM, Graham RA, Wells-Knecht K, Silver IS, Tyler LO, Wells-Knecht M, Zhao Z. Metabolic activation of pioglitazone identified from rat and human liver microsomes and freshly isolated hepatocytes. Drug Metabolism and Disposition. 2005; 33: 733-738.