Innovative Solutions - Large Molecule Bioanalysis

Therapeutic Peptide in Milk

Customer required low pg/mL quantification of a therapeutic peptide in rat, rabbit and human milk. Customer had previously had limited success in developing the milk method.


Milk is a complex matrix (an aqueous colloid of fat globules). Its main constituents are fat, protein, and carbohydrates. A combination of offline sample pretreatment and online SPE LC-MS/MS was used to overcome the complexity of the matrix and peptide instability.

Sample Pre-treatment: Proteins precipitated using acetonitrile/modifier and sonication. Proteins and lipids removed using protein lipid depletion plates.

Online SPE LC-MS/MS: Orthogonal chemistries used to achieve the required sensitivity and selectivity. Mixed mode anion exchange SPE followed by reverse phase chromatography. Precise ‘heart cutting’ was employed during the wash and elution step on the small particle SPE, to increase selectivity and to achieve the required high sensitivity.


Careful choice of instruments and techniques enabled a fully validated method to be developed for a high potency peptide in complex matrix. The low pg/mL LLOQ obtained ensured the generation of high quality PK data from early pre-clinical and clinical studies, enabling the rapid progression of both programmes.