Rapid quantitative analysis of 8-iso-prostaglandin-F2α using liquid chromatography–tandem mass spectrometry and comparison with an enzyme immunoassay method

Abstract

A rapid liquid chromatography-tandem mass spectrometry (LC-MS-MS) assay was developed for the measurement of urinary 8-iso prostaglandin F2α (8-iso-PGF2α), a biomarker of lipid peroxidation. Since urine contains numerous F2 prostaglandin isomers, each of identical mass and similar mass spectrometric fragmentation patterns, chromatographic separation of 8-iso-PGF2α from its isomers is necessary for its quantitative analysis using tandem mass spectrometry. We were able to achieve this separation using an isocratic LC method with a run time under nine minutes which is at least three-fold faster than previous methods—while maintaining sensitivity, accuracy, precision and reliability. The limits of detection and quantitation were 53 and 178 pg/mL urine, respectively. We compared our method with a commercially available affinity purification and enzyme immunoassay kit and found both assays in agreement. Despite the high sensitivity of the enzyme immunoassay method, it is more expensive and has a narrower dynamic range than LC-MS-MS. Our method was optimized for rapid measurement of 8-iso-PGF2α in urine, and it is ideally suited for clinical sample analysis.

Competitive Enzymatic Interactions Determine the Relative Amounts of Prostaglandins E2 and D2

“…The collision energy (Ϫ24 to Ϫ30 V) was optimized for each PG to maximize the formation of product ions for detection using selected reaction monitoring (SRM). Isomeric PGE 2 , PGD 2 , and PGH 2 were measured using a SRM transition of m/z 351 to m/z 271, and the SRM transition of m/z 353 to m/z 193 was selected for PGF 2␣ (Dahl and van Breemen, 2010). The SRM transition of m/z 369 to m/z 163 was used for 6-keto-PGF 1␣ , and the transition of m/z 369 to m/z 169 was used for the measurement of TXB 2 .…”

Nano-LC-MS/MS for the quantitation of prostanoids in immune cells

“…In the last decade, LC-MS/MS has more and more evolved into the method of choice for prostanoid quantitation as it provides high sensitivity combined with a large number of analytes that can be determined from one sample in a single run without prior derivatization [12]. A lot of LC-MS/MS methods have been developed for various prostanoids, other eicosanoids, and their metabolites in diverse biological matrices, including plasma or serum [13,14], urine [15][16][17], tissue [18][19][20], or cell culture supernatants [21,22]. Hellman et al as well as Cao et al quantified PGE 2 and PGD 2 in supernatants collected from RAW macrophages which are a murine macrophage cell line [23,24].…”

A simple and robust UPLC-SRM/MS method to quantify urinary eicosanoids

“…In fact, we achieved the lowest LODs and LOQs for these analytes described until now ( 24,31,34,36,42,44 ). In our view, this might be primarily due to three reasons: i ) a very sensitive instrument was used for LC-SRM/MS analysis; ii ) analyte separation was carried out on an UPLC column with a 1.7 µm particle size, leading to sharper peaks and thus to improved S/N ratios; and iii ) LLE has been applied for sample preparation.…”

“…To extract all analytes of interest with one common sample preparation approach, different extraction procedures ( 29,31,(36)(37)(38)(39). Therefore, we tested sample preparation using C18 RP SPE and polymeric RP SPE.…”

“…Extraction was performed for both SPE cartridges as described by Dahl and colleges ( 31 ). The sorbents were rinsed with 5 ml methanol containing 2% formic acid and 5 ml water.…”