Bio-Analytical SPE Method: Tirzepatide
Quantitative Estimation of Tirzepatide from Human K2 EDTA plasma samples
Tirzepatide
Tirzepatide is an Antidiabetic medication used for the treatment of type 2 diabetes and an anti-obesity medication used for long-term weight management. It is a peptide similar to the hormone glucagon-like peptide-1 (GLP-1), modified with a side chain. It can be administered by subcutaneous injection or taken orally (under development, Oral bioavailability is 1%). There is continued need for their robust, sensitive, and selective sample preparation and liquid chromatography mass spectrometry (LC-MS) analysis.
However, sensitive and robust quantification of large peptides, like Tirzepatide (MWT 4813), are often difficult to analyze by LC-MS/MS, as MS sensitivity is low due to poor transfer of the peptide into the gas phase, dilution of signal across charge states, and poor fragmentation. In addition, the Tirzepatide suffers from significant non-specific binding and poor solubility, making LC and sample preparation method development challenging.
The work described herein takes advantage of a selective reversed-phase anion exchange solid-phase-extraction (SPE), optimal MS settings and MRM transitions, and a high-efficiency UPLC separation using a column packed with C18 particles containing a low-level positive surface charge to provide excellent chromatography peak shape, and analyte recoveries, ultimately facilitating highly sensitive, and robust, low-ng/mL quantification of Tirzepatide from plasma.
Results Summary
Table-1 Results
| Experiments | Results |
|---|---|
| S/N | >10:1 for LLOQ 0.25 ng/ml |
| Selectivity | Interference <20% of LLOQ in Normal, haemolytic and lipemic plasma |
| Recovery | 73, 75 and 73% at LQC, MQC and HQC levels |
| Precision | -1.02 to 5.27% for LLQC, LQC, MQC and HQC levels |
| Accuracy | 3.19 to 5.18% for LLQC, LQC, MQC and HQC levels |
| Stability | All matrix stabilities are within specifications |
Table-2 Parameters- Sample Preparation
| Step | Procedure |
|---|---|
| PPT | 200 μL Blank Plasma, Add SILIS + Add 500 μL Methanol |
| SPE | GS Affinity MAX Pro, 30 mg, 1CC |
| Condition | 1 ml Methanol, Add 30-50% Ammonia in 500 μL of PPT supernatant |
| Equilibrate | 1 ml 2-5% Ammonia solution |
| Sample Load | Load sample |
| Wash-1 | 1 ml 2-5% Ammonia |
| Wash-2 | 1 ml water |
| Wash-3 | 1 ml Methanol two times |
| Elution | 500 μL 0.5 - 1% FA in Methanol (Lower% is FA is better if recovery doesn't improve with increasing FA%) |
| Evaporate | 40°C |
| Reconstitute | 400 μL with 0.2% FA in 400:600 Water: Methanol |
Table-3 Parameters- Sample Analysis
| Parameters | Details |
|---|---|
| Mass Spec | API-5500 |
| HPLC | Nexera UPLC system |
| Concentration range | 0.25 to 250 ng/mL |
| Mass Transition Tirzepatide | 1204.3/396.3 (Mol Wt 4813.5), m/4 state |
| Mass Transition Tirzepatide d8 (SILIS) | 1206.3/396.3 (Mol Wt 4821.5), m/4 state |
| Sample Extraction | Protein Precipitation followed by SPE |
| SPE | GS Affinity MAX Pro, 30 mg, 1CC |
| Column | ACQUITY UPLC Peptide CSH C18 300 Å, 1.7 um 2.1 mm x 50 mm |
| Mobile Phase A | 0.2% Formic acid in ACN |
| Mobile Phase B | 0.2% Formic acid in Water |
| Flow Gradient Programme | - |
| Run time | 8 Minutes |
| Injection Volume | 20 μL |
Conclusion
The combination of a selective PPT-reversed-phase Anion Exchange SPE cleanup, proper MS fragment choice, and chromatographic separation using the Peptide CSH C18 UPLC Column, enabled sensitive, accurate, and robust Tirzepatide quantification from plasma.