Comparison of separation and detection techniques for human growth hormone releasing factor (hGRF) and the products derived from deamidation.

Separation of the deamidation products, Asp8 Leu27 hGRF(1-32)NH2 (MH+ = 3654) and isoAsp8 Leu27 hGRF(1-32)NH2 (MH+ = 3654), from the parent analogue Leu27 hGRF(1-32)NH2 (MH+ = 3653) was achieved by reversed-phase LC and CE, where the retention order was seen to change from tr isoAsp8 hGRF < tr Asn8 hGRF < tr Asp8 hGRF to tr Asn8 hGRF < tr Asp8 hGRF < tr isoAsp8 hGRF, respectively. Both reversed-phase LC and CE gave adequate separations, limits of detection and standard curves. However, CE was preferred due to shorter analysis time, better separation and a smaller demand for material. Packed capillary LC with ESI-MS was then compared with UV detection. On-line LC-MS was found to offer the most efficient approach to detection and identification of hGRF analogues within a single methodology. Identification of Asn8 hGRF from the isobaric deamidation products was achieved from analysis of the triply charged states, where the species were separated by 0.5 amu. LC-MS separation and identification of degradation products offers a viable alternative to fraction collection and subsequent sequencing or enzymatic identification methods. The method becomes increasingly useful for such cases as trace degradation product identification, minimal sample availability or instability of resulting degradation products.