title: A high pressure liquid chromatography method for separation of prolactin forms.
abstract: BACKGROUND
  Prolactin has multiple forms and macroprolactin, which is thought not to be bioavailable, can cause a raised serum prolactin concentration. Gel filtration chromatography (GFC) is currently the gold standard method for separating macroprolactin, but is labour-intensive. Polyethylene glycol (PEG) precipitation is suitable for routine use but may not always be accurate. We developed a high pressure liquid chromatography (HPLC) assay for macroprolactin measurement.
  
  
  METHODS
  Chromatography was carried out using an Agilent Zorbax GF-250 (9.4 × 250 mm, 4 μm) size exclusion column and 50 mmol/L Tris buffer with 0.15 mmol/L NaCl at pH 7.2 as mobile phase, with a flow rate of 1 mL/min. Serum or plasma was diluted 1:1 with mobile phase and filtered and 100 μL injected. Fractions of 155 μL were collected for prolactin measurement and elution profile plotted. The area under the curve of each prolactin peak was calculated to quantify each prolactin form, and compared with GFC.
  
  
  RESULTS
  Clear separation of monomeric-, big- and macroprolactin forms was achieved. Quantification was comparable to GFC and precision was acceptable. Total time from injection to collection of the final fraction was 16 min.
  
  
  CONCLUSIONS
  We have developed an HPLC method for quantification of macroprolactin, which is rapid and easy to perform and therefore can be used for routine measurement.
authors.affiliation: Department of Core Clinical Pathology and Biochemistry, Royal Perth Hospital, University of Western Australia, Perth, WA 6000, Australia.
authors.forename: Damon A
authors.initials: DA
authors.lastname: Bell
authors.forename: Kirsten
authors.initials: K
authors.lastname: Hoad
authors.forename: Lillian
authors.initials: L
authors.lastname: Leong
authors.forename: Juwaini Abu
authors.initials: JA
authors.lastname: Bakar
authors.forename: Paul
authors.initials: P
authors.lastname: Sheehan
authors.forename: Samuel D
authors.initials: SD
authors.lastname: Vasikaran
mesh_terms: D019540:Area Under Curve; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D006966:Hyperprolactinemia / Q000097:blood / Q000175:diagnosis; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D011388:Prolactin / Q000097:blood*
chemical_list: C060678:prolactin, polymeric; D011388:Prolactin