Validation Of Pioglitazone In Human Serum By Two Sample Introduction Methods: Ldtd-apci/ms/ms And Lc-esi/ms/ms

Michael Pugh, Don Gray, Rachel Sun, Brian J. Engel
BASi®, 2701 Kent Avenue, West Lafayette, IN 47906


  • Prior publications have described method development and validation for pioglitazone in human serum using traditional LC-MS/MS methodologies
  • We present and compare validation data from two sample introduction methods: LC-ESI/MS/MS and LDTDAPCI/MS/MS
  • LDTD-APCI/MS/MS analysis utilizes the same extraction technique as does LC analysis, but decreases instrumental analysis time by approximately an order of magnitude
  • Traditional LC-MS/MS - Validation sample (VS) precisions of 1.1% - 3.6%
  • LDTD-MS/MS - Validation sample (VS) precisions of 1.3% - 5.9%



Pioglitazone is a prescription drug of the class thiazolidinedione with hypoglycemic (antihyperglycemic, antidiabetic) action and is used for the treatment of diabetes mellitus type 2 in monotherapy. It is usually administered in combination with sulfonylurea, metformin, or insulin. Pioglitazone is typically assayed with traditional LC-MS/MS methodologies. Here we present validation data for pioglitazone assayed on an alternative system, LDTD-MS/MS and the notion that utilization of the LDTD™ source has the potential to reduce cost of analysis and method development while at the same time increasing throughput, analysis turnaround, and efficiency of instrumentation usage.


Sample Preparation

  • 100 L sample + 50 µL of IS + 600 µL of acidified water
  • Tomtec automated extraction procedure
  • Solid phase extraction with Phenomenex® Strata™ X (30mg/well)
  • Pioglitazone-d internal standard
  • Evaporated samples reconstituted in 250 µL of a water/acetonitrile/acetate buffer mixture
  • Method range is 25.0- 2500 ng/mL in human serum


  • Column: Imtakt Cadenza CD-C18 (2.0 x 50 mm, 3 micron)
  • Isocratic elution with water/acetonitrile/acetate buffer mobile phase
  • Injection volume: 10.0 L
  • Autosampler cycle time: 4.5 minutes


  • Sciex API3000™
  • Source: Electrospray ionization
  • Ion mode: Positive
  • Resolution (Q1/Q3): Unit/Unit
  • Ions monitored: Pioglitazone m/z 357.0/134.2; Pioglitazone-d m/z 361.0/138.2
  • Reconstituted samples injected via HPLC setup noted above


  • Sciex API4000™
  • Thermo TSQ Quantum Ultra™
  • Source: Phytronix Technologies, Inc LDTD™ ionization source (model S-960) using APCI
  • Ion mode: Positive
  • Resolution (Q1/Q3): Unit/Unit
  • Ions monitored: Pioglitazone m/z 357.0/134.2; Pioglitazone-d m/z 361.0/138.2
  • Reconstituted samples (3 L) spotted onto LazWell sample plate, evaporated to dryness at room temperature, and loaded into the Phytronix LDTD source


Table 1. LC-ESI/MS/MS (Sciex API3000) VS Precision and Accuracy

Table 2. LDTD-APCI/MS/MS (Sciex API4000) VS Precision and Accuracy

Table 3. LDTD-APCI/MS/MS (Thermo TSQ Quantum Ultra) VS Precision and Accuracy

Table 4. Validation Performance - LDTD versus HPLC sample introduction methods

Figure 1. Respresentative chromatogram- Pioglitazone LLOQ extract via LC-ESI/MS/MS

Figure 2. Respresentative desorption peak- Pioglitazone LLOQ extract via LDTD-APCI/MS/MS

Figure 3. Representative chromatogram- Pioglitazone blank serum extract via LC-ESI/MS/MS

Figure 4. Respresentative desorption peak- Pioglitazone blank Serum extract via LDTD-APCI/MS/MS

Figure 5. Representative calibration curve for LC-ESI/MS/MS

Figure 6. Representative calibration curve for LDTD-APCI/MS/MS


The comparison of two sample introduction methods for a model bioanalytical system demonstrates that accuracy, precision, and selectivity comparable to those achieved by traditional LC-MS can be obtained in complex biological matricies with the LDTD™ source. Validation results that were well within the recommended criteria for bioanalytical applications allude to promise for use of this system in daily bioanalytical applications. While the LDTD™ source eliminates chromatographic development with no loss in the quality of the results for selected applications, each compound must first be evaluated for compatibility with the LDTD™ source before this system should be deployed.

Although this sample introduction method is not appropriate for all test systems, the advantages offered for those cases where it is appropriate are significant. A potential decrease in the amount of method development time while significantly increasing sample analysis throughput, reduction in solvent use and disposal costs, front-end setup time, and instrument plus peripheral equipment maintenance and verifications are appealing to the end user as well as to clients who may reap the benefits of the rapid analysis turnaround times. Methods able to utilize the LDTD™ source will allow more analysis with less equipment and has the potential to help streamline resource efficiency while reducing costs and improving analysis turnaround times.