Abstract
Although phototherapy (PT) is a standard treatment for neonatal jaundice, no validated clinical methods for determination of bilirubin phototherapy products are available. Thus, the aim of our study was to establish a such method for clinical use. To achieve this aim, a LC-MS/MS assay for simultaneous determination of Z-lumirubin (LR) and unconjugated bilirubin (UCB) was conducted. LR was purified after irradiation of UCB at 460 nm. The assay was tested on human sera from PT-treated neonates. Samples were separated on a HPLC system with a triple quadrupole mass spectrometer detector. The instrument response was linear up to 5.8 and 23.4 mg/dL for LR and UCB, respectively, with submicromolar limits of detection and validity parameters relevant for use in clinical medicine. Exposure of newborns to PT raised serum LR concentrations three-fold (p < 0.01), but the absolute concentrations were low (0.37 ± 0.16 mg/dL), despite a dramatic decrease of serum UCB concentrations (13.6 ± 2.2 vs. 10.3 ± 3.3 mg/dL, p < 0.01). A LC-MS/MS method for the simultaneous determination of LR and UCB in human serum was established and validated for clinical use. This method should help to monitor neonates on PT, as well as to improve our understanding of both the kinetics and biology of bilirubin phototherapy products.
Highlights
Neonatal jaundice is a very prevalent condition during the newborn period
The first one was published by McDonagh and co-workers[16] in 1982; but this method was not quantitative with only limited resolution of the separated bilirubin photoisomers. The latter was based on the correction of the HPLC chromatogram peak areas according to the different relative molar absorption coefficients of bilirubin photoisomers, but this method was not tested on clinical samples[17]
Typical chromatograms of LR and unconjugated bilirubin (UCB) in the serum are given in Fig. 2. (A-B for LR; C-D for UCB; and E-F for internal standard (ISTD))
Summary
Neonatal jaundice is a very prevalent condition during the newborn period. virtually all newborn infants develop some degree of hyperbilirubinemia (>1 mg/dL, 17 μmol/L) during the first week of life. A significant increased risk of late-onset solid tumors in childhood infants treated with PT in the neonatal period has been reported recently[10], a phenomenon likely related to DNA damage caused by blue-green light therapy PT11 Based on this data, there is a need for a robust, accurate, and sensitive quantitative analytical method for the determination of bilirubin photoisomers; as well as photo-rearrangement and photooxidation products formed during PT. The first one was published by McDonagh and co-workers[16] in 1982; but this method was not quantitative with only limited resolution of the separated bilirubin photoisomers The latter was based on the correction of the HPLC chromatogram peak areas according to the different relative molar absorption coefficients of bilirubin photoisomers, but this method was not tested on clinical samples[17]. The goal of our study was to establish and validate a robust analytical method for the LR quantification in samples of clinical neonatal serum
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