RPL Peptide
Official Technical Documentation
Analytical Methods
Analytical Methods — Tirzepatide¶
Method Overview¶
| Method | Purpose | Specification |
|---|---|---|
| HPLC | Purity Analysis | ≥99.0% |
| LC-MS | Identity Confirmation | ±0.5 Da |
| Karl Fischer | Water Content | <5.0% |
| LAL Test | Endotoxin | <5 EU/mg |
| UPLC | Chromatographic Profiling | Complimentary |
| Visual Inspection | Appearance | White to off-white powder |
1. High-Performance Liquid Chromatography (HPLC)¶
Purpose¶
Determination of peptide purity by reversed-phase HPLC with UV detection at 214 nm.
Method Parameters¶
| Parameter | Specification |
|---|---|
| Instrument | HPLC system with UV/Vis detector |
| Column | C18, 4.6 × 250 mm, 5 μm particle size |
| Detection | UV at 214 nm |
| Mobile Phase A | 0.1% TFA in Water (v/v) |
| Mobile Phase B | 0.1% TFA in Acetonitrile (v/v) |
| Gradient Program | |
| 0–5 min | 20% B (isocratic) |
| 5–30 min | 20–60% B (linear gradient) |
| 30–35 min | 60–80% B (wash) |
| 35–40 min | 80–20% B (re-equilibration) |
| Flow Rate | 1.0 mL/min |
| Injection Volume | 20 μL (1 mg/mL solution) |
| Column Temperature | 25°C |
| Run Time | 40 minutes |
System Suitability¶
| Parameter | Acceptance Criteria |
|---|---|
| Theoretical Plates | ≥10,000 |
| Tailing Factor | 0.8–1.5 |
| Resolution | ≥2.0 (between main peak and closest impurity) |
| % RSD (Retention Time) | ≤1.0% (6 injections) |
| % RSD (Peak Area) | ≤2.0% (6 injections) |
Calculation¶
Purity (%) = (Main Peak Area / Total Peak Area) × 100
2. Liquid Chromatography-Mass Spectrometry (LC-MS)¶
Purpose¶
Confirmation of peptide identity through molecular weight determination.
Method Parameters¶
| Parameter | Specification |
|---|---|
| Instrument | LC-MS system with ESI source |
| Ionization Mode | Electrospray Ionization (ESI), Positive |
| Mass Analyzer | Quadrupole or Q-TOF |
| Scan Range | m/z 500–2500 |
| Resolution | ≥20,000 (FWHM) |
| Mass Accuracy | ±0.5 Da |
| LC Conditions | Same as HPLC method above |
Acceptance Criteria¶
- Observed mass within ±0.5 Da of theoretical monoisotopic mass
- Charge state envelope consistent with peptide molecular weight
- Deconvoluted mass matches calculated molecular weight
3. Karl Fischer (KF) Moisture Analysis¶
Purpose¶
Determination of residual water content in lyophilized peptide.
Method Parameters¶
| Parameter | Specification |
|---|---|
| Method | Coulometric Karl Fischer Titration |
| Sample Size | 5–10 mg |
| Reagent | Hydranal-Coulomat AG |
| Acceptance Criteria | <5.0% water content |
4. LAL Endotoxin Testing¶
Purpose¶
Quantitative determination of bacterial endotoxin levels.
Method Parameters¶
| Parameter | Specification |
|---|---|
| Method | Limulus Amebocyte Lysate (LAL) — Kinetic Turbidimetric |
| Standard | Control Standard Endotoxin (CSE) |
| Acceptance Criteria | <5 EU/mg |
5. UPLC Chromatographic Profiling¶
Purpose¶
Complementary high-resolution chromatographic analysis.
Method Parameters¶
| Parameter | Specification |
|---|---|
| Column | C18, 2.1 × 100 mm, 1.7 μm |
| Detection | UV at 214 nm and 280 nm |
| Flow Rate | 0.3 mL/min |
| Run Time | 15 minutes |
6. Visual Inspection¶
Purpose¶
Qualitative assessment of physical appearance.
Acceptance Criteria¶
- White to off-white lyophilized powder
- Free from visible contamination
- Intact cake structure
Document Control¶
| Version | Date | Author | Changes |
|---|---|---|---|
| 1.0 | July 2026 | RPL Peptide Quality | Initial release |
© 2026 RPL Peptide
Official Technical Documentation
rplpeptides.com