title: Determination of proline in human serum by a robust LC-MS/MS method: application to identification of human metabolites as candidate biomarkers for esophageal cancer early detection and risk stratification.
abstract: Altered serum proline levels are related to cancer metabolism. This study developed and validated a LC-MS/MS method to analyze proline in human serum. Surrogate blank serum, coupled with stable isotope l-proline-(13) C5 ,(15)  N as internal standard, was used for generating standard curves ranging from 2.5 to 100 μg/mL. Proline was extracted from serum samples using methanol. A Phenomenex Lux 5u Cellulose-1 column (250 × 4.6 mm) was used for chromatographic separation with 40% methanol in 0.05% formic acid aqueous solution as a mobile phase. Mass detection was performed under positive ionization electrospray. Intra- and inter-day accuracy and precision were <10%. The extraction recovery and matrix factor were 99.17 and 1.47%, respectively. Our study showed that the chiral column had high specificity and selectivity for separating proline from serum components. The assay was successfully applied for the quantification of human serum proline levels from 30 esophageal cancer patients and 30 healthy volunteers. Statistical analyses showed significantly lower levels of serum proline in the patients as compared with the healthy volunteers (p-value = 0.011). We report here a simple, specific and reproducible LC-MS/MS method for the quantification of proline in human serum as a potential screening biomarker for esophageal cancer.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Texas Southern University, Houston, TX, 77004, USA.
authors.forename: Su
authors.initials: S
authors.lastname: Liang
authors.forename: Beatriz
authors.initials: B
authors.lastname: Sanchez-Espiridion
authors.forename: Huan
authors.initials: H
authors.lastname: Xie
authors.forename: Jing
authors.initials: J
authors.lastname: Ma
authors.forename: Xifeng
authors.initials: X
authors.lastname: Wu
authors.forename: Dong
authors.initials: D
authors.lastname: Liang
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000097:blood* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D055088:Early Detection of Cancer; D004938:Esophageal Neoplasms / Q000097:blood / Q000175:diagnosis* / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D011392:Proline / Q000097:blood*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D015415:Biomarkers; D011392:Proline
keywords: LC-MS/MS; biomarker; esophageal cancer; proline; serum