Project Requirement:
To develop an LC-MS/MS method for quantification of a highly polar, and rarely encountered peptide,for a biotech company’s discovery process.
The Peptide:
- 40 amino acids, ~4915 da
- All D-amino acids
- 10 Arginines & 5 Lysines
- C-terminal acid and N-terminal amine
- Highly polar positively charged peptide
The challenges and Aurigene’s solutions:
Here are the trial results towards the optimization of LC parameters –
Trial 01
SPE-HLB; SB Phenyl 4.6 X 150 mm 3.5 µm; Isocratic; 70:30 Formic ā in H2O + Formic ā in MeOH
Good peak shape and response. Analyte eluted in void volume in 50 & 150 mm columns
Trial 02
SPE-HLB; SB Phenyl 4.6 X 150 mm 3.5 µm; Isocratic; 50:50 Formic ā in H2O + Formic ā in MeOH
Split peak observed
Trial 03
SPE-HLB; SB Phenyl 4.6 X 150 mm 3.5 µm; Gradient; Formic ā in H2O + Formic ā in MeOH
Poor peak shape and low response. Tailing observed
Trial 04
SPE-HLB; Waters Xbridge BEH; 4.6*50mm; 3.5µm; Isocratic; Formic ā in H2O + Formic ā in ACN
Good peak shape and response.
Analyte eluted in void volume.
Trial 05
SPE-HLB; Waters Xbridge BEH; 4.6*50mm; 3.5µm; Gradient; Formic ā in H2O + Formic ā in ACN
Split peak observed
Trial 06
SPE-HLB; Waters Xbridge BEH; 4.6*50mm; 3.5µm; Gradient; Formic ā in H2O + Formic ā in MeOH
Good peak shape and response
Analyte retained; LLOQ 20 ng/mL
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