Ultra high‐performance liquid chromatography of porphyrins

Abstract

An ultra high-performance liquid chromatographic (UHPLC) system was developed and optimized for the separation of porphyrins of clinical interest. Optimum conditions for the simultaneous separation of uroporphyrin, hepta-, hexa-, penta-carboxylic acid porphyrins and coproporphyrin and their type I and III isomers on a Thermo Hypersil BDS C18 column (2.4 µm particle size, 100 × 2.1 mm i.d.) using a gradient elution with 10% (v/v) acetonitrile in 1.0 m ammonium acetate buffer (pH 5.16) and 10% (v/v) acetonitrile in methanol at a flow-rate of 0.4 mL/min. The effect of mobile phase buffer molarity on the sensitivity of fluorescence detection and resolution of porphyrin isomers was investigated. The method was successfully applied to the analysis of porphyrins extracted from the urine and faeces of patients with various human porphyrias.

Travelling wave ion mobility mass spectrometry of 5‐aminolaevulinic acid, porphobilinogen and porphyrins

“…The separation of the type I and III isomers of porphyrins has not been achieved by IMS. However, the separation of these isomers can be easily achieved by HPLC or UHPLC . This additional separation step can be easily added in future studies.…”

“…ALA and PBG are usually determined separately by spectrophotometry following ion‐exchange purification . Porphyrins are separated and determined by high‐performance liquid chromatography (HPLC), ultra‐high‐performance liquid chromatography (UHPLC) and HPLC/mass spectrometry (HPLC/MS) . A method for the simultaneous separation, detection and identification of ALA, PBG and porphyrins would allow the metabolites of the entire haem biosynthetic pathway to be analysed in a single run, greatly facilitating the screening and diagnosis of porphyrias.…”

Porphyrinogen fragmentation profiles by ultra‐high‐performance liquid chromatography/electrospray ionisation tandem mass spectrometry

“…A reversed‐phase elution system, using a mixture of acetonitrile/methanol and 1 mol/L aqueous ammonium acetate/acetic acid buffer (pH 5.16), effectively separated the porphyrinogens and their type I and III isomers. Ammonium acetate buffer, which is an excellent mobile phase additive for the HPLC of porphyrinogens and porphyrins, is also recommended for the UHPLC/MS analysis of porphyrinogens.…”

Liquid chromatography and mass spectrometry of haem biosynthetic intermediates: a review

“…The method by Lim et al . () has been modified using columns with porous 2.4 and 1.9 µm size particles, 2.7 µm size superficially porous particles (Benton et al ., , ) and a monolithic stationary phase (Macours and Cotton, ). All columns required a mobile phase containing 1 mol/L 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.…”

“…The I and III isomers of all porphyrins were easily resolved; however, the III isomers of heptacarboxylic acid porphyrin were unresolvable and uroporphyrin III and IV have only been partially resolved using a C 18 column with 4% acetonitrile in 0.01 mol/L phosphate buffer, pH 6.95 (Wayne et al ., ). Coproporphyrin I and III isomers, with four carboxylic groups, were more easily resolved by reversed‐phase HPLC than highly carboxylated porphyrins and methods have been described using 0.1 mol/L ammonium acetate (pH 5.16) and methanol–acetonitrile (Benton et al ., ), 0.015 mol/L sodium acetate–acetonitrile (Respaud et al ., ) and ion‐pairing agents (Jacob et al ., ).…”