Development and validation of a rapid LC–MS/MS method for determination of methylated nucleosides and nucleobases in urine

Raćkowska, Emilia; Bobrowska-Korczak, Barbara; Giebułtowicz, Joanna

doi:10.1016/j.jchromb.2019.121775

Cited by 30 publications

(17 citation statements)

References 40 publications

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“…The method was validated for selectivity, calibration curve, precision, accuracy, stability, matrix effect, dilution integrity and carryover according to the guidelines of the European Medicines Agency (EMA). The results were published previously [47]. The linearity of the calibration curve was evaluated via the R 2 regression coefficient >0.995.…”

Section: Validationmentioning

confidence: 99%

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LC-MS/MS Determination of Modified Nucleosides in The Urine of Parkinson’s Disease and Parkinsonian Syndromes Patients

et al. 2020

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Epigenetic modifications play a key role in gene regulation and expression and are involved in numerous cellular processes. Due to the limited research on nucleosides in Parkinson’s disease (PD), it is very important to consider epigenetic factors and their role in the development of PD. The aim of this study was to investigate and compare the levels of modified nucleosides, such as O-methylguanosine, N6-methyl-2′-deoxyadenosine, 1-methyladenosine, 1-methylguanine, 7-methylguanine, 3-methyladenine and 7-methylguanosine in the urine of Parkinson’s disease (PD) patients and the control group, and to verify that the results obtained differ in a subgroup of patients with parkinsonian syndromes. The study group comprised 18 patients with diagnosed idiopathic Parkinson’s disease and four parkinsonian syndromes. The control group consisted of 30 age- and sex-matched neurological patients without confirmation by neuroimaging brain damage and extrapyramidal symptoms. The levels of nucleosides were determined by validated liquid chromatography coupled with the mass spectrometry (LC-MS/MS) method using the multiple reaction monitoring (MRM) mode. Lower levels of O-methylguanosine, 3-methyladenine, 1-methylguanine, N6-methyl-2′-deoxyadenosine and a higher level of 7-methylguanine in the urine of 22 PD patients were observed. Moreover, elevated levels of 1-methyladenosine, 7-methylguanine, and O-methylguanosine were observed in the parkinsonian syndrome subgroup. These preliminary results may indicate that modified nucleosides describe metabolic disturbances in the metabolism of purine, which was the most severely affected pathway that mediated the detrimental effects of neuroinflammation on PD.

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Section: Validationmentioning

confidence: 99%

“…In this method, the relative matrix effect for all analytes in urine did not exceed 15%, as required by validation criteria. It was found that the quantification of nucleosides performed by LC-MS/MS using tubercidin as an internal standard was of acceptable accuracy and precision [47].…”

Section: Validationmentioning

confidence: 99%

LC-MS/MS Determination of Modified Nucleosides in The Urine of Parkinson’s Disease and Parkinsonian Syndromes Patients

et al. 2020

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“…To obtain the desired separation, such chromatographic factors need to be properly adjusted. HILIC methods are often developed using a typical univariate approach, − which can be very time-consuming and often leads to a non-optimized method without adequate comprehension of main and interaction effects of method parameters. In contrast, a multivariate approach via DoE tools emphasizes systematic method development through assessment of multiparameter interaction effects using the minimum number of experiments, while also enabling a better comprehension of the method performance.…”

Section: Prediction Of Retention In Hilicmentioning

confidence: 99%

Prediction of Analyte Retention Time in Liquid Chromatography

2020

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“…Compared with tissue DNA and RNA samples, urine sample analysis also obviates the needs of tedious processes of extraction or digestion of nucleic acids. Along this line, various modified nucleosides, e.g., alkylated and oxidized nucleosides, were detected in human urine. − As a novel epigenetic mark in eukaryotes, m 6 dA is present in human genome and its levels vary in different tissues, ,, and m 6 dA was also recently shown to be present in human urine . In addition, while previous studies revealed the presence of A m , m 6 A m , and m 6 2 A in human urine, , the levels of these modified nucleosides in urine samples have yet been accurately quantified.…”

Section: Introductionmentioning

confidence: 97%

HILIC-MS/MS for the Determination of Methylated Adenine Nucleosides in Human Urine

Guo

Cao

et al. 2021

Anal. Chem.

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N 6 -methyl-2′-deoxyadenosine (m 6 dA) is a newly discovered DNA epigenetic mark in mammals. N 6 -methyladenosine (m 6 A), 2′-O-methyladenosine (A m ), N 6 ,2′-O-dimethyladenosine (m 6 A m ), and N 6 ,N 6 -dimethyladenosine (m 6 2 A) are common RNA modifications. Previous studies illustrated the associations between the aberrations of these methylated adenosines in nucleic acids and cancer. Herein, we developed Fe 3 O 4 /graphene-based magnetic dispersive solid-phase extraction for the enrichment and hydrophilic interaction liquid chromatography−mass spectrometry (HILIC-MS/MS) for the measurements of m 6 dA, m 6 A, A m , m 6 A m , and m 6 2 A in human urine samples. We found that malic acid could improve the HILIC-based separation of these modified nucleosides and markedly enhance the sensitivity of their MS detection. With this method, we accurately quantified the contents of these modified adenine nucleosides in urine samples collected from gastric and colorectal cancer patients as well as healthy controls. We found that, relative to healthy controls, urinary m 6 dA and A m levels are significantly lower for gastric and colorectal cancer patients; while gastric cancer patients also exhibited lower levels of urinary m 6 A, the trend was opposite for colorectal cancer patients. Together, we developed a robust analytical method for simultaneous measurements of five methylated adenine nucleosides in human urine, and our results revealed an association between the levels of urinary methylated adenine nucleosides and the occurrence of gastric as well as colorectal cancers, suggesting the potential applications of these modified nucleosides as biomarkers for the early detection of these cancers.

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Development and validation of a rapid LC–MS/MS method for determination of methylated nucleosides and nucleobases in urine

Cited by 30 publications

References 40 publications

LC-MS/MS Determination of Modified Nucleosides in The Urine of Parkinson’s Disease and Parkinsonian Syndromes Patients

LC-MS/MS Determination of Modified Nucleosides in The Urine of Parkinson’s Disease and Parkinsonian Syndromes Patients

Prediction of Analyte Retention Time in Liquid Chromatography

HILIC-MS/MS for the Determination of Methylated Adenine Nucleosides in Human Urine

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