Ionene‐dynamically coated capillary for analysis of urinary and recombinant human erythropoietin by capillary electrophoresis and online electrospray ionization mass spectrometry

Abstract

In this article, a series of ionene polymers were synthesized and used to coat fused-silica capillaries for the separation of recombinant and urinary human erythropoietin (rhEPO and uEPO) standards by CE. The influence of the charge density of coatings on the separation of rhEPO and uEPO glycoforms was investigated. Then, we further studied the method for fast separation and detection of rhEPO and uEPO standards by CE-ESI-MS. The influence of several CE and MS operating parameters, such as the concentration of CE running buffer, applied external pressure, and the composition and flow rate of sheath liquid on CE-ESI-MS was studied. The results demonstrated that when the capillary was permanently coated with 6,6-ionene and the pH value of acetic acid-ammonium acetate running buffer was 4.80 and 5.50, respectively, a significantly reproducible separation was achieved for rhEPO and uEPO glycoforms. In the online CE-ESI-MS experiments, we not only achieved the online MS signal of uEPO, but also obtained baseline separation of three major rhEPO glycoforms successfully and reproducibly on the 6,6-ionene-coated capillaries. Furthermore, the standard mixture of rhEPO and uEPO was separated, and two incompletely resolved peaks that were identified to be rhEPO and uEPO by the unique MS "fingerprint" were obtained. Additionally, the molecular weight of rhEPO and uEPO were verified and compared to the results by MALDI-TOF-MS. It can be concluded that, in contrast to other indirect methods, the online CE-ESI-MS technique with the combination of the advantages of both CE and MS shows great potential for the separation and detection of rhEPO doping directly in competitive sports.

Carboxymethyl chitosan‐coated capillary and its application in CE of proteins

“…Furthermore, the CE separation conditions for rhEPO glycoforms were optimized with the CMC-coated capillary, in which nine peaks of rhEPO glycoforms were detected within 8 min. The result proved to be better than those of uncoated and chitosan-coated capillaries, and was also superior to the results of the capillaries coated with other polymers reported in the literature [33][34][35]. Therefore, CMC could be widely used as a new capillary coating for CE analyses of glycoprotein rhEPO and proteins due to its easy availability, stability, low cost, and simple procedure for coating capillary.…”

“…4), with which nine peaks of rhEPO glycoforms can be observed within 8 min. Furthermore, the result obtained with CMC-coated capillary is also better than that of other capillary coating, such as Polybrene and ionenes [33][34][35], and we can separate more bands with shorter time than that of previously reported, revealing that CMC-coated capillary was a preferable choice for the separation of rhEPO glycoforms.…”

Trends in CE‐MS 2005–2006

“…However, recent developments on polycations, like ionine or poly-LA 313, show, what coatings might not hinder the ESI if proper charge density and structure were chosen. The 6,6-ionine-coated capillary was used to differentiate between recombinant and urinary human erythropoietin by on-line CZE-MS coupling [18] (see Figs. 3C and D).…”

Recent advances in the applications of CE to forensic sciences (2005–2007)

“…A CE-ESI-MS method based on the use of a 6,6-ionene coating of the capillary wall and an acetic acid-ammonium acetate running buffer was developed by Yu et al [71] for the separation and on-line MS characterization of urinary EPO and of three major rhEPO glycoforms.…”