Determination of methotrexate in human urine at trace levels by solid phase extraction and high‐performance liquid chromatography/tandem mass spectrometry

Laboratory of Environmental Hygiene and Industrial Toxicology, Salvatore Maugeri Foundation, via Alzaia 29, Pavia, Italy.
Rapid Communications in Mass Spectrometry (Impact Factor: 2.25). 02/2000; 14(3):173-9. DOI: 10.1002/(SICI)1097-0231(20000215)14:3<173::AID-RCM862>3.0.CO;2-K
Source: PubMed


For biological monitoring of hospital personnel occupationally exposed to antineoplastic agents, highly sensitive and specific methods are required. In order to detect trace MTX urinary concentrations, a precise and accurate high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) procedure, incorporating solid phase extraction, has been developed. Urine samples were purified by solid phase extraction (SPE) on octadecyl bonded, endcapped silica SPE columns. After eluting with methanol, the solvent was evaporated obtaining a 25-fold concentration of the analyte. This procedure was validated by using 7-OHMTX as internal standard. Calibration curves had correlation coefficients always higher than 0.999, and the limit of detection was assessed at 0.2 microg L(-1). High specificity of the HPLC-MS/MS technique assures that no interfering substances are detected rather than the analyte of interest.

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Available from: Christina Sottani
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    • "Among the separation methods, high-performance liquid chromatography (HPLC) using different detection modes, such as electrochemical [9] [10], fluorescence through pre or post-column oxidation [11], UV [12] [13] and mass spectrometry [14] [15] is widely used for the determination of MTX in plasma for clinical purposes. "
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    ABSTRACT: Due to its high toxicity, methotrexate (MTX) is an antineoplastic drug for which therapeutic drug monitoring is strictly conducted in the clinical practice. The chemometric optimization and validation of a high performance liquid chromatography (HPLC) method using core-shell particles is presented for the determination of MTX in plasma during therapeutic monitoring. Experimental design and response surface methodology (RSM) were applied for the optimization of the chromatographic system and the analyte extraction step. A Poroshell 120 EC-C18 (3.0 mm × 75 mm) column with 2.7 µm particle size was used to obtain a fast and efficient separation in a complete run time of 4 min. The optimum conditions for the chromatographic system resulted in a mobile phase consisting of acetic acid/sodium acetate buffer solution (85.0 mM, pH= 4.00) and 11.2% of acetonitrile at a flow rate of 0.4 mL/min. Selectivity, linearity, accuracy and precision were demonstrated in a range of 0.10–6.0 µM of MTX. The application of the optimized method required only 150 µL of patient plasma and the consumption of low volume of solvent to provide rapid results.
    Full-text · Article · Dec 2015 · Journal of Pharmaceutical Analysis
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    • "and with various purposes, ranging from quality control to clinical pharmacokinetics and therapeutic drug monitoring [1] [12] [13]. Among these methods, a variety of chromatographic conditions have been described, including acidic or neutral mobile phase and UV, fluorimetry or mass spectrometry detection [1] [14] [15]. Likewise, standard analytical methodologies based on LC with UV detection are described in the main Pharmacopoeias for MTX as raw material [16] [17] [18] or injectable preparation [17] [18]. "

    Full-text · Article · Jan 2015
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    • "It should be noted that these procedures do not require any derivatization process . In addition, the on-line chromatographic analysis with mass-selective detection allows trace determination of the analytes (Minoia et al., 1998; Sottani et al., 1998; Turci et al., 2000a,b). Urinary Table 1 Anticancer drugs classified according to the IARC (last update: "
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    ABSTRACT: A sensitive, specific, accurate and reproducible high-performance liquid chromatography (HPLC) analytical method was developed and validated for the quantification of the novel oral taxane analogue BAY59-8862 in mouse plasma and tissue samples. A fully automated solid-phase extraction procedure was applied to the plasma after internal standard (IS) addition, with only 0.2 mL volume of the sample loaded on a CN-Sep-pak cartridge. In the case of the tissues a very simple acetonitrile extraction was used to recover BAY59-8862 and its internal standard from liver. The procedure for the quantification of BAY59-8862 and its IS (IDN5127) is based on high-performance liquid chromatography/ion spray-tandem mass spectrometry, operating in selected ion monitoring mode. The retention times of BAY and IS were 7.21 and 10.36 min, respectively. In both plasma and tissue specimens the assay was linear in the range 50-5000 ng/mL (ng/g). The overall precision and accuracy were assessed on three different days. The results for plasma were within 6.1% (precision) and between 99 and 112% (accuracy), and for the liver samples within 7.3% and between 104 and 118%, respectively. The LOD was 5 ng/mL and 20 ng/g in the plasma and liver, respectively. In addition, the biliary excretion of the compound in rats was studied. The study showed that an oxidative chemical reaction was the preferred metabolic pathway for biliary excretion, and two sets of mono- and dihydroxylated metabolites were detected by LC/ISP-MS/MS experiments. With this method, BAY59-8862 pharmacokinetic was determined in mice. The combined results demonstrate that the methodology can be considered a valid approach to conduct pharmacokinetic and metabolic studies during preclinical and clinical investigations.
    Full-text · Article · Oct 2001 · Rapid Communications in Mass Spectrometry
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