title: Liquid chromatography-electrospray ionization mass spectrometry for the simultaneous quantitation of collagen and elastin crosslinks.
abstract: We have developed a novel chromatographic analytical method for the simultaneous quantitation of collagen crosslinks. Seven non-derivatised crosslinks could be separated on a Cogent Diamond Hydride HPLC column using either isocratic or gradient conditions then detected by mass spectrometry. The total run time was less than 10min which is significantly shorter than that previously reported. This is the first method in which histidinohydroxylysinonorleucine (HHL) and histidinohydroxymero-desmosine (HHMD) were separated and identified by mass spectrometry without the need for pre- or post-column derivatization. The CVs of the retention times of all seven crosslinks were less than 1% and the limit of detection (LOD) and the limits of quantitation (LOQ) were 0.07-0.13pmol/μL and 0.20-0.38pmol/μL, respectively. This novel method was used for the routine analysis and quantitation of crosslinks in different animal skins in which potential new collagen crosslinks were identified that are as yet undocumented.
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand. Electronic address: r.naffa@massey.ac.nz.
authors.forename: Rafea
authors.initials: R
authors.lastname: Naffa
authors.affiliation: Leather and Shoe Research Association, Palmerston North, New Zealand.
authors.forename: Geoff
authors.initials: G
authors.lastname: Holmes
authors.affiliation: Leather and Shoe Research Association, Palmerston North, New Zealand.
authors.forename: Meekyung
authors.initials: M
authors.lastname: Ahn
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.
authors.forename: David
authors.initials: D
authors.lastname: Harding
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.
authors.forename: Gillian
authors.initials: G
authors.lastname: Norris
mesh_terms: D000818:Animals; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002853:Chromatography, Liquid; D003094:Collagen / Q000032:analysis* / Q000737:chemistry; D003895:Desmosine / Q000302:isolation & purification; D004151:Dipeptides / Q000302:isolation & purification; D004549:Elastin / Q000032:analysis* / Q000737:chemistry; D006639:Histidine / Q000031:analogs & derivatives / Q000302:isolation & purification; D057230:Limit of Detection; D012867:Skin / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D004151:Dipeptides; C075793:histidinohydroxylysinonorleucine; D003895:Desmosine; D006639:Histidine; D003094:Collagen; D004549:Elastin
keywords: Collagen crosslink analysis; Mass spectrometry; Mature crosslinks; Silica hydride column