Ultra high-performance liquid chromatography of porphyrins in clinical materials: column and mobile phase selection and optimisation.
Ultra high-performance liquid chromatographic (UHPLC) systems on columns packed with materials ranging from 1.9 to 2.7 µm average particle size were assessed for the fast and sensitive analysis of porphyrins in clinical materials. The fastest separation was achieved on an Agilent Poroshell C(18) column (2.7 µm particle size, 50 × 4.6 mm i.d.), followed by a Thermo Hypersil Gold C(18) column (1.9 µm particle size, 50 × 2.1 mm i.d.) and the Thermo Hypersil BDS C(18) column (2.4 µm particle size, 100 × 2.1 mm i.d.). All columns required a mobile phase containing 1 m ammonium acetate buffer, pH 5.16, with a mixture of acetonitrile and methanol as the organic modifiers for optimum resolution of the type I and III isomers, particularly for uroporphyrin I and III isomers. All UHPLC columns were suitable and superior to conventional HPLC columns packed with 5 µm average particle size materials for clinical sample analysis.
Clinical Biochemistry, King's College Hospital, Denmark Hill, London, SE5 9RS, UK.
Christopher M
CM
Benton
Chang Kee
CK
Lim
Caje
C
Moniz
Donald J L
DJ
Jones
D000085:Acetates / Q000737:chemistry; D000097:Acetonitriles / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation* / Q000379:methods*; D005243:Feces / Q000737:chemistry; D006801:Humans; D000432:Methanol / Q000737:chemistry; D008863:Microspheres; D010316:Particle Size; D017119:Porphyria Cutanea Tarda; D011166:Porphyrins / Q000032:analysis* / Q000737:chemistry / Q000652:urine; D052616:Solid Phase Extraction
D000085:Acetates; D000097:Acetonitriles; D011166:Porphyrins; C018824:ammonium acetate; D000432:Methanol; C032159:acetonitrile