High-performance liquid chromatographic determination of methotrexate, 7-hydroxymethotrexate, 5-methyltetrahydrofolic acid and folinic acid in serum and cerebrospinal fluid

Belz, Susanne; Frickel, Christine; Wolfrom, C.; Nau, Heinz; Henze, Günter

doi:10.1016/0378-4347(94)00328-9

Cited by 58 publications

(26 citation statements)

References 21 publications

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“…In order to measure MTX, 5-methyl-THF and calcium folinate in the CSF a method from Belz et al [31] based on reversed-phase HPLC with simultaneous UV and fluorescence detection was Pediatr Blood Cancer DOI 10.1002/pbc modified. In brief, the stationary phase was a Supelcosil TM column (15 cm Â 0.46 cm, 3 mm) and Supelguard TM precolumn (2 cm Â 0.46 cm, 5 mm) both from Supelco (Bellefonte, PA).…”

Section: Analysis Of Mtx and Reduced Folatesmentioning

confidence: 99%

Methotrexate‐associated alterations of the folate and methyl‐transfer pathway in the CSF of ALL patients with and without symptoms of neurotoxicity

Vezmar

Schüsseler

Becker

et al. 2008

Pediatric Blood & Cancer

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BackgroundSevere neurotoxicity has been observed after systemic high‐dose and intrathecal methotrexate (MTX) treatment. The role of biochemical MTX‐induced alterations of the folate and methyl‐transfer pathway in the development of neurotoxic symptoms is not yet fully elucidated.ProcedureMTX, 5‐methyltetrahydrofolate, calcium folinate, S‐adenosylmethionine, and S‐adenosylhomocysteine were measured in the cerebrospinal fluid (CSF) of 29 patients with acute lymphoblastic leukemia (ALL) who were treated with high‐dose MTX (5 g/m2) followed by calcium folinate rescue (3 × 15 mg/m2) and/or intrathecal (8–12 mg) MTX. Two patients developed subacute MTX‐associated neurotoxicity. CSF was obtained by lumbal puncture 1–3 weeks after administration of MTX and shortly after the occurrence of neurotoxicity. The analytes were measured using HPLC assays with UV and/or fluorescence detection.ResultsIn non‐toxic patients, CSF concentrations of 5‐methyltetrahydrofolate and S‐adenosylmethionine were in the normal range 2 weeks after administration of high‐dose and intrathecal MTX followed by rescue. In contrast, when these patients received intrathecal MTX without rescue, 5‐methyltetrahydrofolate concentrations were significantly decreased 12 days after the first MTX administration. S‐adenosylmethionine concentrations were significantly decreased up to 45 days. The two patients suffering from neurotoxicity had decreased levels of 5‐methyltetrahydrofolate and S‐adenosylmethionine during or following toxicity. S‐adenosylhomocysteine was determined in all samples of neurotoxic patients but was below the limit of quantification in most samples of non‐toxic patients. Calcium folinate was not detected; MTX was present only in samples obtained during its infusion.ConclusionIntrathecal MTX without folinate rescue as well as MTX‐associated neurotoxicity are likely to be associated with specific alterations of the folate and methyl‐transfer pathway. Pediatr Blood Cancer 2009;52:26–32. © 2008 Wiley‐Liss, Inc.

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Section: Analysis Of Mtx and Reduced Folatesmentioning

confidence: 99%

Methotrexate‐associated alterations of the folate and methyl‐transfer pathway in the CSF of ALL patients with and without symptoms of neurotoxicity

Vezmar

Schüsseler

Becker

et al. 2008

Pediatric Blood & Cancer

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“…Serum folate has been measured by various methods (15 ), including chromatographic methods based on HPLC (16,17 ), LC-MS (18 -21 ), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) (9,22 ). Despite efforts to establish reliable methods for determining folate status, large interassay and interlaboratory differences have been reported (23)(24)(25), and each laboratory must establish its own reference intervals.…”

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Liquid Chromatography–Tandem Mass Spectrometry Analysis of Folate and Folate Catabolites in Human Serum

2009

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“…There has been concern about the variation in the binding properties of folate-binding proteins from different sources and their different affinities for different folate species (11 ). Chromatographic methods measure the separate folate forms, including 5-methyltetrahydrofolate, and are based on HPLC (12,13 ), liquid chromatographymass spectrometry (LC-MS) (14 -17 ), or LC-MS/MS (18,19 ). Despite the efforts to establish reliable methods for evaluating folate status, large interassay and interlaboratory differences have been reported (20 -22 ).…”

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confidence: 99%

Measurement of Folate in Fresh and Archival Serum Samples as p-Aminobenzoylglutamate Equivalents

2008

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Background: The development of accurate and precise folate assays has been difficult, mainly because of folate instability. Large interassay and interlaboratory differences have been reported. We therefore developed a serum folate assay that measures folate and putative degradation products as p-aminobenzoylglutamate (pABG) equivalents following oxidation and acid hydrolysis. Methods: Serum was deproteinized with acid in the presence of 2 internal calibrators ([13C2]pABG and [13C5]5-methyltetrahydrofolate). 5-Methyltetrahydrofolate and other folate species in serum were converted to pABG by oxidation and mild acid hydrolysis. pABG and its internal calibrators were quantified by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Results: The limit of quantification was 0.25 nmol/L, and the assay was linear in the range 0.25–96 nmol/L, which includes the 99.75 percentile for serum folate concentrations in healthy blood donors. Within- and between-day imprecision was ≤5%. We detected no residual folate in serum samples after sample preparation. Folate concentrations in fresh serum samples obtained with the pABG assay and with a microbiologic assay showed good agreement (r = 0.96). In stored samples containing low folate concentrations due to folate degradation, the pABG assay yielded substantially higher folate concentrations than the microbiologic assay. Conclusions: The pABG assay combines automated sample preparation with LC-MS/MS analysis. It allows measurement of folate not only in fresh samples of serum/plasma but also in stored samples in which the folate has become oxidized and degraded to an extent that it cannot be assayed with traditional folate assays.

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High-performance liquid chromatographic determination of methotrexate, 7-hydroxymethotrexate, 5-methyltetrahydrofolic acid and folinic acid in serum and cerebrospinal fluid

Cited by 58 publications

References 21 publications

Methotrexate‐associated alterations of the folate and methyl‐transfer pathway in the CSF of ALL patients with and without symptoms of neurotoxicity

Methotrexate‐associated alterations of the folate and methyl‐transfer pathway in the CSF of ALL patients with and without symptoms of neurotoxicity

Liquid Chromatography–Tandem Mass Spectrometry Analysis of Folate and Folate Catabolites in Human Serum

Measurement of Folate in Fresh and Archival Serum Samples as p-Aminobenzoylglutamate Equivalents

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