AOD-9604 Peptide High-Resolution Chromatography & Mass Accuracy Assays

Reverse-phase HPLC provides baseline resolution for AOD-9604 reference standard. A 12.8-minute primary peak shows a 1.12 tailing factor. This indicates an intact Cys7-Cys14 disulfide linkage. Mobile phases of 0.1% TFA/water and 0.085% TFA/acetonitrile are employed. A 1.0 mL/min flow rate optimizes peak symmetry. Column temperature is 35°C.

UV detection at 214 nm identifies secondary peaks. These elute at 11.2 and 13.9 minutes. Relative abundances are 0.7% and 0.9%. LC-MS confirmed these as trace methionine sulfoxide variants, indicating oxidation byproducts. A 10 µL injection of 1 mg/mL solution in 10 mM sodium acetate (pH 5.0) achieved 98.2% main peak purity. System suitability met USP requirements. The method produced 12,500 theoretical plates.

Accelerated stability testing reveals degradation pathways. At pH 7.4 phosphate buffer, rapid disulfide scrambling occurs. Within 48 hours at 25°C, multimerization products form. These elute at 15.3 and 17.6 minutes. They comprise 11.4% of the total peak area. Cold chain storage at 4°C in pH 5.0 acetate buffer restricts degradation. Only 1.8% degradation is observed over seven days.

Electrospray ionization (ESI) transforms AOD-9604 reference standard fragments into gas-phase ions. Source parameters are tuned for efficient desolvation and transmission. Collision-induced dissociation (CID) fragments precursor ions within the mass analyzer. This generates characteristic product ion signatures. These unique fragments enable confident identification and structural confirmation.

Optimized ESI source settings facilitate ion production. Source temperature is 120°C. Nebulizer gas flow operates at 10 L/min. Capillary voltage is 3.5 kV. Fragmentor voltage reaches 130 V. Skimmer voltage is 65 V. Octopole RF voltage is 250 V. Collision energy is 35 eV. The mass analyzer scans the 100-2000 m/z range.

Chromatographic separation uses RP-HPLC. A 100 × 2.1 mm, 1.7 µm, 100 Å column is utilized. Mobile phase A contains 0.1% formic acid in water. Mobile phase B is 0.1% formic acid in acetonitrile. Gradient elution progresses from 5% to 95% B in 10 minutes. Flow rate is 0.2 mL/min. Column temperature is 40°C. Injection volume is 5 µL. Sample concentration is 1 mg/mL. The AOD-9604 reference standard fragment elutes at 7.5 minutes.

Detection of the AOD-9604 reference standard synthetic C-terminal fragment is confirmed. The precursor ion measures 906.4 m/z. Characteristic product ions are observed at 757.4, 684.4, and 514.3 m/z. This collision-induced dissociation (CID) fragmentation pattern robustly confirms the fragment's structure.

Disulfide bond scrambling occurs via thiol-disulfide exchange, particularly at neutral to basic pH (e.g., pH 7.5). Suppressing this degradation pathway requires an acidic mobile phase and precise ionic equilibration. Optimized chromatographic conditions, including 0.1% trifluoroacetic acid in water, prevent this rearrangement. This maintains peptide integrity during analysis.

Chromatographic separation employed an Aeris Peptide XB-C18 column (150 × 4.6 mm, 3.6 µm, 100 Å). Mobile phase A contained 0.1% trifluoroacetic acid in water. Mobile phase B used 0.085% trifluoroacetic acid in acetonitrile. A 1.0 mL/min flow rate was applied. Column temperature was maintained at 35°C. A primary retention time of 12.8 minutes was observed.

Peak symmetry was optimized. A tailing factor of 1.12 was achieved through mobile phase fine-tuning. Column temperature significantly influenced peak shape. Evaluation across 30°C to 40°C established 35°C as optimal. Flow rate variations from 0.5 to 1.5 mL/min were assessed; 1.0 mL/min was selected. Detection occurred at 214 nm via UV absorbance.

Solution stability and buffer kinetics were characterized. A 10 mM sodium acetate buffer, pH 5.0, yielded high stability. Over 21 days at 4°C, >98.0% of the peptide remained intact. Conversely, storage in neutral water promoted 8.5% disulfide multimerization within 7 days.

Solid-state storage conditions were also optimized. Lyophilized samples containing 4% mannitol, stored under argon at -20°C, maintained >98.8% purity for 24 months. Mannitol concentrations from 2% to 6% were evaluated; 4% proved optimal. Storage temperatures between -10°C and -30°C were assessed, with -20°C providing superior stability.

Calibration protocols involve injecting 1.0 mL of 0.1 mg/mL AOD-9604 standard solution. Column temperature is 35°C. Mobile phase A consists of 0.1% TFA in water. Mobile phase B is 0.085% TFA in acetonitrile. Flow rate is 1.0 mL/min. UV absorbance detection is set at 214 nm. Primary retention time is 12.8 minutes. Peak symmetry is 1.12. Tailing factor isn't significant.

Standardization protocols require 10 mM sodium acetate buffer at pH 5.0. AOD-9604 solution stability is assessed over 21 days at 4°C. Solution concentration is 0.5 mg/mL. Degradation pathway analysis reveals disulfide bridge scrambling at pH >7.0. Reduction to linear monomer occurs under trace reducing agents. Disulfide multimerization is 8.5% in 7 days. Neutral water triggers this reaction. Solution stability data indicates >98.0% intact AOD-9604 after 21 days. Buffer kinetics doesn't affect stability.

Lyophilized solid-state storage protocols involve 4% mannitol as an excipient. Lyophilized cake is stored under argon at -20°C. Purity is >98.8% for 24 months. HPLC analysis uses Aeris Peptide XB-C18 column (150 × 4.6 mm, 3.6 µm, 100 Å). Mobile phase gradient is 5-95% B over 15 minutes. Flow rate is 1.0 mL/min. Column temperature is 35°C. UV absorbance detection is set at 214 nm. Calibration curves are generated using 0.01-1.0 mg/mL AOD-9604 standards. Correlation coefficient is 0.999. Calibration curve isn't linear below 0.01 mg/mL.

Quality control protocols involve Aeris Peptide XB-C18 column chromatography. Mobile phase A consists of 0.1% TFA in water. Mobile phase B is 0.085% TFA in acetonitrile. Flow rate is 1.0 mL/min. Column temperature is 35°C. UV absorbance detection occurs at 214 nm. Primary retention time is 12.8 minutes. Peak symmetry and tailing factor is 1.12. Degradation pathway analysis reveals disulfide bridge scrambling at pH >7.0.

Disulfide bridge reduction to linear monomer occurs under trace reducing agents. Solution stability and buffer kinetics studies indicate stability in 10 mM sodium acetate buffer at pH 5.0 for 21 days at 4°C. Intact AOD-9604 remains at >98.0% under these conditions. Neutral water triggers 8.5% disulfide multimerization in 7 days. Lyophilized solid-state storage with 4% mannitol under argon at -20°C maintains >98.8% purity for 24 months. HPLC column dimensions are 150 × 4.6 mm, with 3.6 µm particle size and 100 Å pore size.

Analytical HPLC method validation doesn't require additional sample preparation. AOD-9604 molecular weight is 1815.10 g/mol. CAS registry number is 221231-10-3. Chemical sequence is Tyr-Leu-Arg-Ile-Val-Gln-Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys-Gly, with disulfide Cys7-Cys14 bond. Stability studies at 4°C and -20°C haven't shown significant degradation. AOD-9604 isn't stable in neutral water due to disulfide multimerization. Buffer exchange to 10 mM sodium acetate at pH 5.0 prevents this degradation pathway. HPLC analysis at 214 nm detects AOD-9604 with high sensitivity and specificity.