Novel LC-MS/MS Method for Measuring Methotrexate in High-Dose Therapy: A Comparative Study with Commercial EMIT and EIA Immunoassays

Novel LC-MS/MS Method for Measuring Methotrexate in High-Dose Therapy: A Comparative Study with Commercial EMIT and EIA Immunoassays | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Novel LC-MS/MS Method for Measuring Methotrexate in High-Dose Therapy: A Comparative Study with Commercial EMIT and EIA Immunoassays Agnieszka Czajkowska, Aleksandra Mikulska, Martyna Poniewierska, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7529927/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Pharmacological Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Methotrexate (MTX) is a widely used chemotherapeutic agent in pediatric oncology, where high-dose protocols (HDMTX; >500 mg/m²) are standard for treating hematological and central nervous system malignancies. Due to its narrow therapeutic index and potential for severe toxicity, therapeutic drug monitoring (TDM) of plasma MTX concentrations is essential to guide leucovorin rescue therapy and prevent adverse effects. The presented study aimed to compare the analytical performance of two immunoassays—enzyme-multiplied immunoassay technique (EMIT) and enzyme immunoassay (EIA)—against a newly developed and validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The LC-MS/MS assay demonstrated excellent linearity, sensitivity (LLOQ = 0.01 µmol/L), and precision, meeting ICH M10 regulatory guidelines. Clinical samples from pediatric patients receiving HDMTX were analyzed using all three methods. Results showed strong correlations (r > 0.93) between methods; however, immunoassays exhibited biases related to cross-reactivity with MTX metabolites such as DAMPA (2, 4-diamino-N(10)-methylpteroic acid) and 7-OH-MTX, which may lead to overestimation of MTX levels and unnecessary prolongation of leucovorin rescue. While immunoassays remain practical for routine monitoring due to their accessibility and speed, LC-MS/MS provides superior accuracy and should be the method of choice in critical clinical situations. These findings underscore the importance of selecting the appropriate assay in optimizing HDMTX therapy and ensuring patient safety. methotrexate LC-MS/MS EMIT EIA therapeutic drug monitoring pediatric oncology Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Methotrexate (MTX; amethopterin) is an antimetabolite structurally similar to folic acid. It inhibits the enzyme dihydrofolate reductase (DHFR), blocking purine nucleotide synthesis and disrupting DNA replication and cell proliferation. A schematic overview of MTX distribution is shown in Fig. 1 [1]. Clinically, MTX is administered in two dosing protocols: low-dose MTX (LDMTX), primarily used for inflammatory diseases, and high-dose MTX (HDMTX), used in oncology. HDMTX, defined as doses of ≥ 500 mg/m², is widely used to treat various cancers such as acute lymphoblastic leukemia, osteosarcoma, and primary central nervous system lymphoma [1–3]. While HDMTX provides significant therapeutic benefits, it also poses a risk of toxicity if not adequately monitored. Therapeutic drug monitoring (TDM) guides adjustments for rescue therapy with folinic acid (leucovorin) or glucarpidase to reduce MTX toxicity. Standard monitoring points are at 24, 48, and 72 hours after infusion starts. If MTX plasma levels drop below 0.1 µmol/L, rescue therapy can be safely stopped [3–5]. MTX has complex pharmacokinetics, with variability in metabolism and excretion. After IV infusion, the maximum serum or plasma concentration (C max ) occurs within 0.5 to 1 hour. Distribution involves binding to plasma proteins and cellular uptake, including erythrocytes. MTX elimination occurs in three phases; however, approximately 95% of free MTX is cleared within 24 hours. To prevent renal toxicity, partly caused by the metabolite 7-OH-MTX, urine alkalization with sodium bicarbonate infusion is essential. This, combined with forced diuresis, accelerates elimination, requiring renal function monitoring through serum creatinine and GFR [1–5]. Currently, there are a few bioanalytical approaches to monitoring MTX in TDM services, namely: immunoassays (IAs) and chromatography applications (especially LC-MS/MS). The latter is considered the gold standard due to its relatively high sensitivity and specificity (compared to IAs). On the other hand, IAs are characterized by cross-reactivity with metabolites and lower sensitivity [6–8]. The ARK™ Methotrexate EIA assay is based on in vitro competition between the analyzed drug and glucose-6-phosphate dehydrogenase (G6PDH) labelled with MTX for binding to the antibody reagent. The activity of the enzyme increases proportionally with the MTX concentration in the sample being tested. The activated enzyme converts the coenzyme nicotinamide adenine dinucleotide (NAD) to NADH, and the differences in absorbance, determined spectrophotometrically, are evaluated. Endogenous human G6PDH enzyme does not interfere during analysis because ARK™ kits are based on a specific bacterial enzyme. In the presented study, the VITROS 5600™ (Quidel Ortho, San Diego, CA, USA) immunochemical system, integrated with the described test, was utilized, with a precision of less than 8% (CV%) [6,7]. The Syva ® MTX assay is an enzyme-multiplied immunoassay technique (EMIT). The principles of the immunochemical reactions are similar to those of the ART™ Methotrexate Assay test. The Syva immunochemical kits were used for MTX assays with the Dimension EXL 200 immunochemistry system (Siemens Healthcare, Erlangen, Germany), exhibiting a precision of less than 10% (CV) [8]. The comprehensive features of IAs, including linearity, limit of quantification (LOQ), and cross-reactivity interferences, are shown in Table 1 . Detailed information, such as reagent specifications and validation results, can be found in the leaflets provided by the manufacturers [6–8]. Table 1 Brief characteristics of the most common immunoassays used clinically for methotrexate determination [6–8]. Brand name of the test Type of method Linearity [µmol/L] LOQ [µmol/L] Interferences 7-OH-MTX [%] DAMPA [%] ARK™ Assay homogenous immunoassay 0.04–1.20 0.04 (≤ 0.07) 64.30–100 ARK ™ II Assay homogenous immunoassay 0.03–1.30 0.03 (≤ 0.01) 18.80–57.50 Syva ® EMIT Assay enzyme-multiplied immunoassay technique 0.30–2.00 0.30 ND strong LOQ- limit of quantification; 7-OH-MTX- 7-hydroxy-methotrexate; DAMPA- 2,4-Diamino-N 10 -methylpteroic acid. Continuous monitoring of MTX levels and elimination rate during high-dose therapy is essential for determining the appropriate leucovorin (LV) dosage [9]. The most critical clinical situations include: When MTX concentrations are too high, intracellular transport of leucovorin is selectively inhibited, which reduces the effectiveness of the rescue treatment. In patients with delayed MTX elimination, toxic levels of MTX may persist longer than the standard duration of rescue therapy, necessitating immediate adjustment of the LV dose. In patients with regular MTX elimination, excessively high LV doses should be avoided, as they may decrease treatment efficacy. Although partial agreement exists between the immunoassay and LC-MS/MS methods, results obtained using immunoassays may not always be reliable. Notably, cross-reactivity with MTX metabolites—especially DAMPA—may cause overestimation of MTX concentrations, potentially leading to unnecessary prolongation of leucovorin rescue therapy. This study aimed to develop and validate a novel LC-MS/MS method for measuring methotrexate (MTX) in serum samples. Additionally, the validated LC-MS/MS method was compared with two independent immunoassay (IA) techniques: enzyme immunoassay (EIA) and enzyme-multiplied immunoassay technique (EMIT), using regression, correlation, and bias estimation statistical tools. To demonstrate the clinical interchangeability of the tested method, clinical serum samples from patients undergoing HDMTX therapy were analyzed using each assay. To the best of our knowledge, this is the first study to provide an analytical and clinical comparison of EIA, EMIT, and a reference LC-MS/MS method using pediatric clinical samples. 2. Materials and Methods 2.1. Chemicals, Reagents and Laboratory Accessories The solid standard of methotrexate (MTX; TRC-M260675, ≥ 95% chemical purity by HPLC) was obtained from Toronto Research Chemicals (TRC, Toronto, Canada). The stable isotope-labelled internal standard (SIL-IS), d₃-MTX (22378, ≥ 99% deuterated forms: d₁–d₃), was supplied by Cayman Chemical (Ann Arbour, MI, USA). LC-MS grade solvents—methanol (MeOH), acetonitrile (ACN), 2-propanol (IPA), and ultrapure water—were sourced from Merck (Darmstadt, Germany). Inorganic reagents, including zinc sulfate heptahydrate and 25% ammonium hydroxide, were purchased from Supelco (Bellefonte, PA, USA). Mobile phase modifiers with LC-MS purity, such as acetic acid and ammonium acetate, were provided by Fisher Chemical (Pittsburgh, PA, USA). Immunochemical reagents for EMIT and EIA assays were obtained from Siemens Healthcare Diagnostics (Newark, DE, USA) and ARK Diagnostics (Fremont, CA, USA), respectively. Drug-free plasma and serum samples were obtained from the Regional Blood Donation and Hemotherapy Centre in Warsaw (Warsaw Blood Bank, Poland). Quality control materials for immunoassays and LC-MS analysis, including IAS and Liquichek Therapeutic Drug Monitoring Control, were supplied by Bio-Rad Laboratories (Hercules, CA, USA). Basic laboratory equipment, including Eppendorf Reference 2 pipettes, plastic test tubes, and pipette tips, was provided by Eppendorf (Hamburg, Germany). 2.2. Stock Solutions of MTX and Internal Standard, Calibration Standards (CSs) and Quality Control Samples (QCs) Calibration standards (CSs) and quality control (QC) solutions were prepared using two different weighed amounts of the solid MTX standard. Simultaneously, the stable isotope-labelled internal standard (SIL-IS) was prepared using the same procedure. Both compounds were dissolved in 0.2 M ammonium hydroxide in methanol using ultrasound assistance. The MTX stock solutions were further diluted to prepare working solutions at concentrations of 0.100, 0.010, and 0.001 mg/mL. One set of working solutions was used to prepare calibration standards, and another set was used for QC samples. The LC-MS/MS method was validated over the range of 0.01–25.00 µmol/L. Accordingly, MTX working solutions were diluted with a methanol: water mixture (1:1, v/v) to obtain the following concentrations for CSs and QCs, using drug-free human serum as matrix (prepared identically to clinical samples; see Section 2.3 ): Final MTX concentrations for calibration standards (in µmol/L): 0.010 (LLOQ – lower limit of quantification); 0.025; 0.050; 0.100; 0.500; 1.000; 2.500; 5.000; 10.000; 15.000; 25.000 (ULOQ – upper limit of quantification). Quality control (QC) samples were prepared at the following MTX concentration levels: 0.035 µmol/L (LQC – lower quality control); 0.25 µmol/L (MQC1 – medium quality control 1); 7.50 µmol/L (MQC2 – medium quality control 2); 20.00 µmol/L (HQC – high quality control). To convert MTX concentrations from µmol/L to mg/L, values should be recalculated using the factor 2.20. The internal standard (IS) solution of d₃-MTX was prepared as described previously to yield a stock concentration of 100 µmol/L. This solution was subsequently diluted to achieve a final concentration of 2.50 µmol/L in the processed samples. All prepared solutions were stored under light-protected conditions in amber low-binding vials at − 20°C. 2.3. Sample Pretreatment Protocol After serum collection in non-gel separator tubes, the sample was centrifuged at room temperature for 5 mins at 13500 × g. The resulting serum was stored in amber Eppendorf tubes at -20°C until analysis via IAs or LC-MS/MS. Before analysis, 40 µL of thawed serum (blank, calibrator, control, or patient sample) was used for LC-MS/MS. To the matrix, 5 µL of SIL-IS solution and 55 µL of water were added. After vortexing for 30 secs, 200 µL of the precipitation mixture (0.1 M ZnSO 4 :ACN, 1:1, v/v) was pipetted into the sample. The samples were then vortexed for 15 minutes and kept on ice for an additional 15 minutes. Finally, the Eppendorf tube containing the sample was centrifuged for 5 minutes at 13500 × g, and the extracted sample was transferred to an amber glass chromatographic vial with an insert. Samples for EIA and EMIT analysis were prepared according to the manufacturer’s instructions, with no additional steps necessary in serum sample preparation. 2.4. Clinical Samples Serum samples were collected from pediatric patients at predetermined time points—24, 48, and 72 hours after starting methotrexate (MTX) infusion. The main clinical aim of monitoring MTX levels was to guide adjustments to leucovorin rescue therapy. In this study, residual serum samples obtained during routine diagnostic procedures at the Children’s Memorial Health Institute (CMHI) in Warsaw, Poland, were analyzed. Informed consent was received from all patients and/or their legal guardians at the start of treatment. Each serum sample was analyzed in triplicate using three separate methods: EMIT, EIA, and LC-MS/MS. The average values from these methods were used for clinical decision-making during MTX chemotherapy. The use of residual diagnostic material for the validation and comparison of analytical techniques was reported to the local Bioethical Committee. The agreement between CMHI and Altium Poland was registered under reference number 2377/2023. 2.5. Instrumentation: Chromatographic and Mass Spectrometry Parameters A Shimadzu 8050 triple quadrupole mass spectrometer coupled with a Nexera X2 UPLC system (Shimadzu, Kyoto, Japan) was employed for LC-MS/MS method validation and clinical sample analysis. Both methotrexate (MTX) and the stable isotope-labelled internal standard (SIL-IS) were monitored using a multiple reaction monitoring (MRM) approach in positive ionization mode, utilizing electrospray ionization (ESI). Detection was based on singly protonated molecular ions [M + H] + . Details of the MRM transitions for MTX and the internal standard are listed in Table 2. The optimized ion source and MS detection parameters are provided in Table 3 . Table 2. Mass spectrometry conditions: MRM pairs with collision energy for MTX and SIL-IS. MTX-1 pair (bolded) has been used for quantitative purposes. MRM pair 1 Q1 Q3 CE [eV] MTX-1 455.25 308.10 -18.0 MTX-2 455.25 175.10 -36.0 MTX-3 455.25 134.05 -33.0 d 3 -MTX (SIL-IS) 458.20 211.20 -18.0 1 Dwell time was set as 100 ms for each MRM pair. MRM – multiple reaction monitoring, CE – collision energy, SIL-IS – stable isotope labelled-internal standard. Table 3 Ion source and specific mass spectrometry parameters. Parameter (unit) Optimized value nebulizing gas flow [L/min] 2.8 drying gas flow [L/min] 10.0 heating gas flow [L/min] 10.0 interface temperature [°C] 300 desolvatation line temperature [°C] 250 heating block temperature [°C] 400 interface voltage [kV] 1.0 CID gas pressure [kPa] 270 1 CID – collision-induced dissociation. The chromatographic part of the platform consisted of: a gradient pump with a mobile phase mixer (30AD), a degassing unit (DGU-205AR), an autosampler with a cooler (SIL-30AC), and a column oven (CTO-20AC). The chromatographic separation was carried out under gradient mode using a Zorbax Eclipse Plus C 18 RRHD column (2.10 × 50 mm, 1.80 µm) supplied by Agilent Technologies, Inc. (Santa Clara, CA, USA). A guarded with a complementary C 18 precolumn (2.1 × 5 mm, 1.80 µm). Throughout the entire analytical run, the column was maintained at 40°C. For the generation of a gradient, two mobile phases have been applied: (A) LC-MS grade water with 0.1% acetic acid and 2 mM of ammonium acetate, (B) pure LC-MS grade acetonitrile. Gradient mode has been utilized in the following steps with a 0.5 mL/min flow rate: 0.01–0.75 min 5% of B phase, 0.75–1.20 min linear increasing to 75% of B phase, 1.21–2.50 min 75% of B phase (maintained), 2.51–5.00 min re-equilibration to 5% of phase B. The autosampler needle was purged in internal-external mode using a 1:1 (v/v) water/IPA mixture. The injection volume was set as 1 µL of supernatant after the extraction procedure. 2.6. LC-MS/MS Method Validation The presented bioanalytical method has been validated in accordance with the current regulatory requirements, as outlined in ICH M10, as provided by the EMA according to its updated guideline [9]. The following parameters were evaluated: specificity, selectivity, lower limit of quantification (LLOQ), limit of detection (LOD), linearity, accuracy, precision, recovery, dilution integrity, carry-over, matrix effect, incurred sample reanalysis (ISR), and stability. For cross- and clinical validation, some regulations proposed by IATDMCT were applied [12]. Specificity was assessed to ensure accurate differentiation between the analyte, IS, and potentially interfering substances, including metabolites and endogenous co-eluted compounds. Interference at the analyte’s retention time was acceptable if it did not exceed 20% of the LLOQ response, and ≤ 5% for the IS. The selectivity was evaluated using drug-free serum samples obtained from six independent donors. The method was considered selective if no interfering peaks were observed at the retention times of MTX or IS within the specified response thresholds. Method precision was confirmed under these conditions, even with two MRM transitions applied: one for quantification and the second for qualitative purposes. LOD in LC-MS/MS has been estimated using the signal-to-noise (S/N) approach, with a threshold of S/N ≥ 50 used to define the lowest detectable concentration [9]. The linearity of the methodology was evaluated for the applied concentration range of 0.01–25.00 µmol/L, which included double-blank and blank samples in the set. For calibration curve construction, the 1/x 2 weighting was used in ten independent analytical runs. The mean coefficient of determination (R²) was higher than 0.995, which was a confirmation of linearity. The accuracy and precision were evaluated using nine independent samples at the LLOQ, LQC, MQC 1 , MQC 2 , and HQC levels during intra- and interday experiments. The precision was expressed as the coefficient of variation (CV%), while accuracy was expressed as the percentage deviation from the nominal value. Acceptance criteria were ± 15% for both parameters at all levels, except ± 20% for LLOQ. The potential carry-over effect was evaluated by injecting a blank sample immediately after a ULOQ sample in a single analytical run. If the calculated peak area does not exceed 20% of the LLOQ for MTX or 5% for the IS, the acceptance criteria are fulfilled [9]. The matrix effect (ME), a characteristic of ESI-LC-MS/MS-based methods, was evaluated using the post-extraction approach, as described in protocols by Taylor et al. and Matuszewski et al. Matrix factor (MF), absolute recovery (AR), and process efficiency (PE) were calculated from six replicates at LQC and HQC levels [10,11]. Incurred sample reanalysis (ISR) was performed on clinical samples (minimum 10% of clinical samples) and was accepted if at least 67% of values were within ± 20% of the initial analyte concentration measured during the first analysis [9]. The stability, as an initial part of the validation process, has been tested using LQC and HQC samples during experiments, including extracts autosampler stability, freeze–thaw stability over three cycles, and short- and long-term stability of the analyte in the matrix. All stability results were accepted if measured concentrations remained within ± 15% of nominal values [9]. 2.7. Immunoassays – EMIT and EIA techniques The EMIT and EIA techniques are easy-to-implement procedures that are often coupled with many immunochemical analyzers in the routine practice of biochemical laboratories. Serum samples were centrifuged (13500 × g), transferred to a reaction cup and placed in Dimension ® EXL™ 200 or VITROS ® 5600 analyzers (EMIT Syva ® and EIA ARK™, respectively). The measurements were performed automatically by analyzers with principles characteristic of EMIT and EIA assays, as mentioned in the Introduction. The systems were regularly calibrated and controlled using commercial QC samples. Because manufacturers have delivered methodologies as ready-to-use kits, in-house validation was not performed (except for systematic calibration, control, and stability testing). The detailed parameters of reagent ingredients, methodology principles, limitations, and validation results are provided in fully available manufacturer’s leaflets [6–8]. 2.8. Statistical Analysis Statistical analyses, including Passing–Bablok regression, Bland–Altman plots, and calculation of descriptive statistics (mean, standard deviation), as well as validation parameters, were performed using MedCalc software (version 22.023; MedCalc Software Ltd., Ostend, Belgium). The Shapiro–Wilk test was applied to assess the normality of data distributions. A significance threshold of p < 0.05 was adopted for all tests to ensure statistical robustness and reliability of the results. 3. Results 3.1. LC-MS/MS Method Optimization The LC-MS/MS method for MTX determination has been optimized and validated at the Department of Drug Chemistry, Pharmaceutical and Biomedical Analysis, Medical University of Warsaw, Poland. In the presented study, a LC-MS/MS method for determining MTX in serum was developed and validated. The analyte extraction protocol, chromatographic conditions, and mass spectrometry ion source parameters were optimized experimentally. Various methods for MTX isolation from serum were tested. The application of a less time-consuming protein precipitation method using a zinc sulfate/acetonitrile mixture, compared to liquid-liquid extraction or solid-phase extraction, resulted in high recovery rates (> 90%). Perchloric acid at 16% was slightly more effective than the mixture mentioned above, but it is not fully compatible with LC-MS/MS systems due to its high oxidative potential. The addition of zinc sulfate to acetonitrile generated lower matrix interferences than using pure acetonitrile. The chromatographic parameters were optimized experimentally, using various mobile phases with different types and amounts of modifiers in gradient mode. Additionally, different chromatography columns were tested, including Kinetex C18 (50 × 2.1 mm; 1.7 µm), Synergy Fusion-RP (10 × 2.1 mm; 4.0 µm), Hypurity-C18 (50 × 2.10 mm; 3 µm) from Thermo Scientific, Bonus RP RRHD Zorbax (2.1 × 50 mm; 1.8 µm) from Agilent, Zorbax Eclipse RRHD C8 (2.1 × 50 mm; 1.8 µm), Zorbax Eclipse RRHD C18 (2.1 × 50 mm; 1.8 µm), Poroshell 120-EC-C18 (150 × 3.00 mm; 2.70 µm), Ascentis-C18 (100 × 3.00 mm; 3.00 µm), Ascentis-C8 Express (100 × 3.00 mm; 3.00 µm), and Nucleosil C18 (125 × 3.00 mm; 5.00 µm). The Zorbax Eclipse RRHD C18 column was selected as optimal due to the relatively short retention time (~ 2.10 min) of the analyte and the Gaussian shape of the chromatographic peaks. Representative chromatograms of the tested compound are shown in Fig. 2 . 3.2. Validation Results The validated method met the criteria according to selectivity, which was evaluated by analyzing the 10 replicates of double blank samples (processed without stable isotope-labelled internal standard and analyte) and zero calibrator samples (with SIL-IS addition only). MTX and MTX-d 3 responses were considered acceptable because the interferences do not exceed 20% and 5% of the LLOQ signal, respectively. The limit of detection (LOD) was determined based on the signal-to-noise ratio (S/N) of standard solutions prepared by diluting the lowest calibration solution (LLOQ). The LOD was established at 0.005 µmol/L (0.0023 mg/L, S/N = 10). The linearity of the method was assessed based on ten calibration curves in 0.01–25.0 µmol/L. Weighted linear regression with a weighting factor of 1/x was applied to favour the lower points of the curve. The mean R² was 0.9932, and the mean equation of the curve was: y = 0.51441x + 0.10239, where y is the MTX/SIL-IS peaks area ratio, and x is the nominal MTX concentration. Accuracy and precision were evaluated in nine repetitions for LLOQ, LQC, MQC 1 , MQC 2 and HQC during intra-run (intra-day) and between-run (between-day) experiments (Table 4 ). The acceptance criteria for all quality control levels were fulfilled according to ICH M10 guidelines [9]. Table 4 Accuracy and precision experiment results [n = 9]. LLOQ 0.001 µmol/L LQC 0.035 µmol/L MQC 1 0.25 µmol/L MQC 2 2.50 µmol/L HQC 20.00 µmol/L Intra-run (intra-day) accuracy and precision C MTX [µmol/L] 0.011 ± 0.005 0.033 ± 0.004 0.246 ± 0.028 2.474 ± 0.171 20.198 ± 0.673 Accuracy [%] 104.67 94.61 98.31 99.98 100.99 Precision [%] 12.38 11.23 9.83 6.92 3.33 Between-run (inter-day) accuracy and precision C MTX [µmol/L] 0.011 ± 0.002 0.035 ± 0.003 0.255 ± 0.022 2.334 ± 0.120 20.064 ± 0.475 Accuracy [%] 102.34 99.05 102.18 93.37 100.24 Precision [%] 9.97 9.81 8.54 5.14 2.30 HQC – higher quality control; LLOQ – lower limit of quantification; LQC – lower quality control; MQC – medium quality control; MTX – methotrexate. Carry-over was assessed experimentally. For this purpose, a sequence was applied in which an HQC sample was injected directly after a blank sample without the analyte. According to EMA guidelines, the acceptance criteria for MTX are less than 20% and less than 5% for IS. The carry-over effect obtained for 10 samples was negligible and met the acceptance criteria for MTX (0.6912 ± 0.2491) and IS (0.02682 ± 0.01933). Dilution integrity parameter was verified by preparing a sample to which a working solution of the analyte at a concentration of 50 µmol/L was added. The sample was then diluted in ratios of 1:10 and 1:5 using a blank matrix to achieve the target concentrations (5 and 10 µmol/L). Five samples were prepared for each dilution level, and it was verified whether the obtained concentrations aligned with the calibration curve. According to the guidelines, accuracy should be within ± 15%, and precision should not exceed 15%. For dilution 1:10, the mean concentration with SD was 4.6712 ± 0.2387 (mean accuracy and precision: 93.42% and 5.11%, respectively). In case of dilution 1:5, the mean concentration with SD was 10.0018 ± 0.6051 (mean accuracy and precision: 100.02% and 6.05%, respectively) [9]. Matrix effect (ME), process efficiency (PE), and absolute recovery (AR) were evaluated according to the well-known approaches of Taylor et al. and Matuszewski et al. for LQC and HQC samples [10,11]. The results for six independent repetitions were calculated for analyte and analyte to IS ratio for evaluation the compensation of matrix effect by using SIL-IS: LQC: -14.20% ± 3.01% (ME), 72.56% ± 2.98% (PE) and 68.15% ± 4.15% (AR); LQC/IS ratio: -1.02% ± 0.29 (ME), 98.06% ± 3.06% (PE) and 97.99% ± 5.18% (AR); HQC: -11.48% ± 4.09% (ME), 71.24% ± 6.26% (PE) and 63.81% ± 7.16% (AR); HQC/IS ratio: -0.95% ± 0.30 (ME), 99.15% ± 5.93% (PE) and 98.37% ± 6.18% (AR); The serum samples spiked with LQC/HQC MTX levels remained stable during a minimum of five freeze-thaw cycles; however, samples stored for extended periods must be protected from light exposure. Additionally, the stability of extracts in the autosampler was evaluated at 4°C after 24 hours, 2, 5, and 7 days of storage. The results for LQC ranged from 96.20–101.45%, while those for HQC ranged from 92.47–100.67%. Serum samples spiked with LQC/HQC levels remained stable during storage in amber test tubes for 6 months at -20°C, with 98.99% and 100.66% of the mean nominal MTX value, respectively (n = 3). Exposure to sunlight appeared to reduce sample stability, as indicated by three-day bench-top stability tests. The experiment was performed in triplicate, with mean results for light-exposed and light-protected samples as follows: 78.25% / 84.23% versus 79.87% / 90.45% for LQC and HQC, respectively. The ISR experiment was conducted on all clinical samples included in the study. All determined MTX concentrations met the criteria (see Section 2.6 ), with a mean difference of -2.15% ± 0.49% [9]. 3.3. Selected results of EMIT and EIA methods calibration, control and stability evaluation The systems have been calibrated and controlled (using QCs) at the beginning of each daily batch run. Mean interday/intraday precisions (calculated as CV%; n = 6) were 5.46%/8.34% and 4.63%/9.16% for EMIT and EIA, respectively. Mean calibration parameters (n = 6) were: EMIT: y = 1.009x-0.004 (R 2 = 0.998), EIA: y = 1.013x + 0.011 (R 2 = 0.998), for observed versus expected MTX levels. The carry-over effect, observed after aspiration of a blank sample following the highest commercial quality control, was not detected. The LOD and LLOQ values are shown in Table 1 . Manufacturers indicated dilution integrity, set at 1:6 and 1:10 (or multiples), for EMIT and EIA, respectively. Onboard stability of reagents was assessed as 7 weeks for EMIT and 5 weeks for EIA. 3.4. Results of MTX Quantification in Clinical Serum Samples In the clinical part of the presented study, 53 clinical samples from patients receiving MTX intravenously under the HDMTX protocol were analyzed using three methods: LC-MS/MS (reference method), EMIT, and EIA. The results of the assays are presented in the plot (Fig. 3 ) and in the Supplementary File. Mean results with ranges (min/max) were as follows: 1.404 (0.024–13.800) for LC-MS/MS, 1.322 (0.026–16.091) for EIA, and 1.291 (0.040–9.140) for EMIT. 3.4. Cross- and Clinical Validation During cross- and clinical validation, the three sets of paired results for MTX determination in clinical samples were evaluated, namely: MTX EMIT versus MTX LC-MS/MS , MTX EMIA versus MTX LC-MS/MS, and MTX EMIT versus MTX EIA . The results of regression studies (Passing-Bablok test), bias estimation (Bland-Altman plots) and other correlation studies are comprehensively presented in Fig. 4 and in Table 6 . Table 6 Results of cross- and clinical validation for determined results of MTX determination using EMIT, EIA and LC-MS/MS methods [n = 53 triplicated results]. Acceptance criteria based on literature [12–14]. Statistical tool Analyzed relationship MTX LC-MS/MS versus MTX EMIT MTX LC-MS/MS versus MTX EIA MTX EMIT versus MTX EIA Passing-Bablok equation MTX LC-MS/MS = 1.0726(MTX EMIT )-0.0084 MTX LC-MS/MS = 0.9914(MTX EIA )-0.0096 MTX EMIT = 0.9299(MTX EIA ) + 0.0041 Slope (95% CI) 1.0726 (0.9722 to 1.1789) 0.9914 (0.9108 to 1.1724) 0.9299 (0.8729 to 1.0069) Intercept (95% CI) -0.0084 (-0.0230 to 0.0093) -0.0096 (-0.0292 to 0.0106) 0.0041 (-0.0150 to 0.0161) Bland-Altman bias [%] 7.7328 (-1.8404 to 17.3061) -2.4510 (-14.1862 to 9.2842) -9.9995 (-18.3460 to -1.6529) % of samples in LoA (bias < 20%) 66.03 71.70 67.92 % of samples in LoC (bias < 15%) 54.72 59.40 56.60 Random differences (RSD) 0.4301 (-0.8430 to 0.8430) 0.5364 (-1.0514 to 1.0514) 0.8214 (-1.6100 to 1.6100) Spearman Correlation coefficient (p < 0.0001) 0.9300 (0.8820 to 0.9590) 0.9110 (0.8490 to 0.9480) 0.9530 (0.9190 to 0.9730) LoA – analytical limit of bias agreement (< 20%), LoC – clinical limit of bias agreement (< 15%), EMIT - Enzyme-Multiplied Immunoassay, EIA- Homogeneous Enzyme Immunoassay, LC-MS/MS - Liquid Chromatography – Tandem Mass Spectrometry. However, the Passing-Bablok regression analysis confirmed the interchangeability between paired results (slope differences < 10%, and 0/1 values within intercept/slope 95% CI) [12–14]. The clinical validation, based on clinical limits of agreement, did not confirm equivalence between the analyzed results (less than 67% of paired samples showed a bias < 15%). The analytical limit of bias agreement (mean bias < 20% for over 67% of paired results) was acceptable in almost all cases of the evaluated relationships (slightly lower in MTX LC-MS/MS compared to MTX EMIT ). Strong positive correlations were identified in all analyzed scenarios. 4. Discussion This study evaluated three methods—EMIT, EIA, and LC-MS/MS—for measuring methotrexate (MTX) levels in plasma from pediatric patients receiving high-dose methotrexate (HDMTX). Results indicated that LC-MS/MS was the most accurate and reliable technique, whereas immunoassays offered practical advantages for routine therapeutic drug monitoring. The LC-MS/MS method complied with the validation criteria outlined in the ICH M10 guidelines, confirming its suitability for clinical use. A limited number of LC-MS/MS studies have been published on the determination of MTX in plasma or serum [15–19]. Most required 100 µL or more of biological matrix for MTX measurement, while our method used only 40 µL. Reducing the sample volume is especially important in pediatric populations. Protein precipitation with an organic solvent, using MTX-d 3 as an internal standard, proved effective for serum sample preparation, ensuring sufficient sensitivity and addressing matrix effects and other interferences. Following guidelines, it is essential to use a stable isotope labelled internal standard (SIL-IS) whenever possible. In literature, both unrelated internal standards (URIS) and SIL-IS ( 13 C,D 3 -MTX and MTX-D 3 ) have been successfully applied. For instance, Tripathy et al. demonstrated the successful use of aminoacetophenone for MTX determination via LC-MS/MS, yielding satisfactory validation results and adequate compensation for matrix effects. Our investigation demonstrated strong correlations; however, it also revealed that immunoassays frequently failed to stay within the < 15% bias threshold essential for interchangeable use with LC-MS/MS, especially in pediatric oncology. Interestingly, in some triplicated results, the EIA method produced higher results compared to LC-MS/MS, whereas EMIT results for MTX determination were lower than those of LC-MS/MS. The ARK test for MTX showed less cross-interference with 7-OH-MTX than the Syva ® test and is characterized by a lower limit of quantification (10 times more sensitive than the Syva ® test). These differences may also contribute to the inaccuracies observed in the immunochemical applications presented in this study. It should be noted that during leucovorin/glucarpidase rescue treatment after MTX infusion, the actual levels of its metabolites in serum may increase. Conversely, higher metabolite levels may lead to an overestimation of results in immunoassays due to potential cross-reactivity. It appears that relying solely on immunoassay results, particularly in complex clinical situations, may unnecessarily prolong leucovorin/glucarpidase rescue therapy, thereby delaying chemotherapy and increasing healthcare costs [18–26]. The results of the presented study confirmed previous findings regarding LC-MS/MS as the gold standard, due to its superior specificity and sensitivity, especially in complex clinical situations where metabolites may interfere with immunoassay results. For example, Bouquié et al. demonstrated an excellent correlation between the LC-MS/MS method and the FPIA method (Abbott TDx™). Still, they concluded that none of the immunoassays could be used after glucarpidase administration. In such cases, LC-MS/MS appears to be more suitable because of its good selectivity, especially for MTX and its metabolites [23, 24]. Cross-reactivities are not characteristic only of DAMPA and 7-OH-MTX. It should be noted that MTX polyglutamates, mainly present intracellularly in RBC, can cause a high overestimation of results. Particular attention should be paid to hemolysis of the sample, as some MTXPGs are usually present in the blood plasma fraction [1, 25]. The study with high application potential in pediatric oncology, published recently by Opitz et al., described volumetric absorptive microsampling (VAMS) as an alternative to daily TDM service of MTX [26]. The application of VAMS strategies is not possible with immunoassays due to the low volume of capillary dried blood collected by the device (10, 20, and 30 µL). Therefore, LC-MS/MS remains the method of choice for this type of sample. In the same study, the authors concluded that the mean difference between the tested ARK™ immunoassay and LC-MS/MS was approximately − 27% (with SD of 41%) [26]. It was indicated that the ARK test significantly overestimates MTX concentration compared to other immunoassays and LC-MS/MS methods. These overestimations are more pronounced at lower MTX concentrations, as observed in the presented study. Consequently, Opitz et al. proposed revised dose regimens for MTX based on the assay type, whether immunoassay (IA) or LC-MS/MS [26]. For example, after 54 hours of infusion, the estimated MTX level in serum should be ≤ 0,25 µmol/l when measured using IA (ARK™ test) and < 0.12 µmol/l when measured using LC-MS/MS [26]. Our study also confirms the need to adjust MTX thresholds depending on the assay type for safe leucovorin dosing. The choice of analytical method for determining methotrexate depends not only on economic factors but also on the specific clinical scenario, such as implications for metabolite excretion, the patient’s clinical condition, or unusual drug distribution that may lead to toxicity. Examples of clinical situations, along with the proposed analytical technique, are presented in Table 7 . Table 7 The examples of high-dose methotrexate protocol and proposed analytical approaches Clinical specific situation Proposed method Justification Type of oncology disorder (typical dose): • ALL (1–5 g/m 2 ) • osteosarcoma (8–12 g/m 2 ) • PCNSL ( \(\:\ge\:\) 3 g/m 2 ) • NHL (3–8 g/m 2 ) • choriocarcinoma (0.5–1.5 g/m 2 ) EMIT/EIA EMIT/EIA EMIT/EIA EMIT/EIA LC-MS/MS The immunoassays enabled a rapid turnaround time for administering an appropriate dose of leucovorin rescue therapy in the high-dose MTX protocol. However, this type of therapy with MTX may produce high levels of metabolites, which can interfere within 24–48 hours after drug administration. Cross-reactivity is acceptable during the initial stage of renal clearance of MTX/metabolites (< 24 hours). In some clinical situations—such as delayed elimination, abnormal drug distribution, or renal impairment—the interference caused by metabolites may be observed; in these cases, LC-MS/MS is the preferred method. In lower doses (e.g., choriocarcinoma), the sensitivity of the immunoassay may be insufficient. If MTX is administered directly to the CNS (with CSF as the preferred matrix for MTX measurement), LC-MS/MS is also recommended. MTX concentration level: • 0.2–1.0 \(\:\mu\:\) M • < 0.2 \(\:\mu\:\) M EMIT/EIA LC-MS/MS Immunoanalyses are suitable for detecting higher concentrations but often overestimate results due to the presence of metabolites. Accurate measurement of low concentrations, especially those occurring more than 48 hours after MTX administration, is essential for patient safety and ongoing therapy with leucovorin/glucarpidase. Therefore, the higher costs are justified in clinical practice. Number of analytes simultaneously determined: • MTX only • MTX with other anticancer drugs EMIT/EIA LC-MS/MS Selectivity is a key advantage of LC-MS/MS. When multiple analytes need to be determined simultaneously, this technique remains the preferred method over immunochemical techniques. Type of laboratory: • in small hospital • in large oncology centre • reference laboratory EMIT/EIA LC-MS/MS or IAs LC-MS/MS Immunoassays are cost-effective, quick, and entirely adequate for daily practice. Additionally, their low unit cost and ease of integration with a biochemistry analyzer make them appealing for both small and larger TDM laboratories. Conversely, LC-MS/MS enables the simultaneous measurement of multiple drugs, thereby enhancing cost efficiency, particularly when testing large batches of samples. ALL – acute lymphoblastic leukaemia; CSF – cerebrospinal fluid; CNS – central nervous system; EMIT – enzyme-multiplied immunoassay technique; EIA – enzyme immunoassay; IA – immunoassay; LC-MS/MS – liquid chromatography–tandem mass spectrometry; NHL – Non-Hodgkin lymphoma; PCNSL – primary central nervous system lymphoma In daily laboratory practice, the calibration process and regular external proficiency testing are essential for ensuring reliable and high-quality assay results. In the presented study, international proficiency testing was employed to evaluate the tested method (Reference Institute for Bioanalytics, Bonn, Germany). The limited number of patient samples may restrict the ability to draw broad conclusions about the clinical equivalence of the tested methods for MTX determination (LC-MS/MS, EMIT and EIA). However, the sample size was adequate for meaningful cross-correlation and clinical analyses. Future research should include a larger number of samples from multiple TDM laboratories. 5. Conclusions This study successfully developed and validated a fast, sensitive LC-MS/MS method for measuring methotrexate (MTX) levels in plasma from oncology patients receiving high-dose therapy. It complied with rigorous international standards (ICH M10, EMA, IATDMCT) and showed excellent analytical performance. A comparative analysis indicated that while immunochemical assays (EMIT and EIA) provide acceptable accuracy for routine monitoring, they can cross-react with MTX metabolites, such as DAMPA, potentially leading to overestimated MTX levels and unnecessary extension of leucovorin rescue therapy. Although immunoassays are practical due to their ease, lower cost, and rapid results, LC-MS/MS remains the definitive method for MTX quantification in critical cases requiring maximum specificity and accuracy. In summary, LC-MS/MS should be the preferred method for monitoring MTX in high-dose treatments, especially when precise measurement is crucial for patient safety and effective therapy. Nonetheless, immunoassays remain an accessible and cost-effective alternative when LC-MS/MS is not available, underscoring their ongoing importance in clinical routine TDM services for MTX. Declarations Author Contributions: Conceptualization, A.C. and A.K.; methodology, A.C. and A.K.; software, A.C, M.P. and A.S.; validation, A.M., A.C. and A.K.; formal analysis, A.M., A.C. and M.S.; investigation, A.K.; resources, A.C.; data curation, A.K.; writing—original draft preparation, A.C., A.M., M.P., A.S. and M.S.; writing—review and editing, A.K.; visualization, A.K.; supervision, A.K.; project administration, A.C. and A.K.; funding acquisition, A.C., A.M. and A.K. All authors have read and agreed to the published version of the manuscript. Funding: The research (especially LC-MS/MS tests) was partially funded by the Medical University of Warsaw, grant number 3/F/MG/N/24. Institutional Review Board Statement: This study was conducted in accordance with the principles of the Declaration of Helsinki. The presented study presented results of routine diagnostic testing for methotrexate in patients treated at the Children’s Memorial Institute in Warsaw. Approval reference number: 2377/2023. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study as routine diagnostic process of MTX measurements in the Children’s Memorial Health Institute in Warsaw. Data Availability Statement: The d ata will be made available upon request. Acknowledgements: The authors thank Altium Poland Sp. z.o.o for the possibility of testing the VITROS 5600 system for MTX measurements based on the Altium and CMHI agreement: 2377/2023. The part of the presented study is Aleksandra Mikulska’s master’s thesis. Conflicts of Interest: The authors declare that they have no conflicts of interest. References Kocur A, Mikulska A, Moczulski M, Pawiński T. Therapeutic Drug Monitoring of Low Methotrexate Doses for Drug Exposure and Adherence Assessment—Pre-Analytical Variables, Bioanalytical Issues, and Current Clinical Applications. Int J Mol Sci. 2024;25:13430. Levêque D, Becker G, Toussaint E, Fornecker L-M, Paillard C. Clinical Pharmacokinetics of Methotrexate in Oncology. Int J Pharmacokinet. 2017;2:137–47. Mikulska A, Kocur A. Pharmacotherapy with high doses of methotrexate (HDMTX) in oncology—How should chemotherapy be conducted based on therapeutic drug monitoring? Prospect Pharm Sci. 2023;21:40–7. Bielack SS, Soussain C, Fox CP, Houillier C, Murciano T, Osborne W, Zinzani PL, Rizzari C, Schwartz S. A European consensus recommendation on the management of delayed methotrexate elimination: supportive measures, leucovorin rescue and glucarpidase treatment. 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European Medicines Agency (EMA). ICH Guideline M10 on Bioanalytical Method Validation. Available online: https://www.ema.europa.eu/en/documents/scientific-guideline/ich-guideline-m10-bioanalytical-method-validation-step-5_en.pdf (accessed on 05 July 2025). Taylor PJ. Matrix effects: The Achilles heel of quantitative high-performance liquid chromatography-electrospray-tandem mass spectrometry. Clin Biochem. 2005;38:328–34. Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the Assessment of Matrix Effect in Quantitative Bioanalytical Methods Based on HPLC-MS/MS. Anal Chem. 2003;75:3019–30. Seger C, Shipkova M, Christians U, Billaud EM, Wang P, Holt DW, Brunet M, Kunicki PK, Pawiński T, Langman LJ, et al. 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Head to Head Evaluation of the Analytical Performance of Two Commercial Methotrexate Immunoassays and Comparison with Liquid ChromatographyMass Spectrometry and the Former Fluorescence Polarization Immunoassay. Clin Chem Lab Med. 2016;54:823–31. Tripathy NK, Mishra SK, Nathan G, Srivastava S, Gupta A, Lingaiah R. A Rapid Method for Determination of Serum Methotrexate Using UltraHighPerformance Liquid Chromatography–Tandem Mass Spectrometry and Its Application in Therapeutic Drug Monitoring. J Lab Physicians. 2023;15:344–53. Feng Z, Gao J, Gao X, Hua L, Nie X, Sun Y, Wang MA, Validated. HPLCMS/MS Method for Quantification of Methotrexate and Application for Therapeutic Drug Monitoring in Children and Adults with Non-Hodgkin Lymphoma. Drug Des Dev Ther. 2021;15:4575–83. Descoeur J, Dupuy A-M, Bargnoux A-S, Cristol J-P, Mathieu O. Comparison of Four Immunoassays to an HPLC Method for the Therapeutic Drug Monitoring of Methotrexate: Influence of the Hydroxylated Metabolite Levels and Impact on Clinical Threshold. J Oncol Pharm Pract. 2022;28:55–63. Wu D, Wang Y, Sun Y, Ouyang N, Qian JA, Simple. Rapid and Reliable Liquid Chromatography–Mass Spectrometry Method for Determination of Methotrexate in Human Plasma and Its Application to Therapeutic Drug Monitoring. Biomed Chromatogr. 2015;29:1197–202. Nayak S, Patel S, Nanda R, Shah SK, Mohapatra E et al. Processing Validation Metrics of Syva EnzymeMultiplied Immunoassay Technique (EMIT) Methotrexate Assay for Beckman Coulter System. Cureus 2023, 15(1), e34025. Chavan P, Bhat V, Karmore A, Mohite R, Waykar S, Kadu H. Establishing Syva Methotrexate Assay on Siemens Dimension RxL Analyzer: Experience in a Tertiary Cancer Care Laboratory. J Lab Physicians. 2017;9:67–8. Kibby D, Trinkman H. Methotrexate Level Discrepancy PostGlucarpidase: A Pediatric Case Series and Review of Literature. Pediatr Blood Cancer. 2024;71:e30831. Bouquié R, Deslandes G, Nieto Bernáldez B, Renaud C, Dailly E, Jolliet P. A fast LCMS/MS assay for methotrexate monitoring in plasma: Validation, comparison to FPIA and application in the setting of carboxypeptidase therapy. Anal Methods. 2014;6:178–86. 10.1039/C3AY40815A . Bouquié R, Grégoire M, Hernando H, Azoulay C, Dailly E, Monteil-Ganière C, Pineau A, Deslandes G, Jolliet P. Evaluation of a Methotrexate Chemiluminescent Microparticle Immunoassay: Comparison to Fluorescence Polarization Immunoassay and Liquid Chromatography-Tandem Mass Spectrometry. Am J Clin Pathol. 2016;146:119–24. Hayashi H, Fujimaki C, Tsuboi S, Matsuyama T, Daimon T, Itoh K. Application of Fluorescence Polarization Immunoassay for Determination of MethotrexatePolyglutamates in Rheumatoid Arthritis Patients. Tohoku J Exp Med. 2008;215:95–101. Opitz P, Fobker M, Fabian J, Hempel G. Development and Validation of a Bioanalytical Method for the Quantification of Methotrexate from Serum and Capillary Blood Using Volumetric Absorptive Microsampling (VAMS) and OnLine Solid Phase Extraction LCMS. J Chromatogr A. 2024;1715:464610. Additional Declarations No competing interests reported. Supplementary Files TableS1.docx Cite Share Download PDF Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Pharmacological Reports → Version 1 posted Editorial decision: Revision requested 17 Oct, 2025 Reviews received at journal 22 Sep, 2025 Reviews received at journal 22 Sep, 2025 Reviewers agreed at journal 15 Sep, 2025 Reviewers agreed at journal 12 Sep, 2025 Reviewers agreed at journal 12 Sep, 2025 Reviewers agreed at journal 12 Sep, 2025 Reviewers invited by journal 10 Sep, 2025 Editor assigned by journal 05 Sep, 2025 Submission checks completed at journal 04 Sep, 2025 First submitted to journal 03 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7529927","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":516047003,"identity":"25710acf-f231-43ae-9a8c-4eec1243dbc9","order_by":0,"name":"Agnieszka Czajkowska","email":"","orcid":"","institution":"The Children’s Memorial Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Agnieszka","middleName":"","lastName":"Czajkowska","suffix":""},{"id":516047004,"identity":"ec6fb88f-8ec5-42dc-bb93-ed4c31aa7351","order_by":1,"name":"Aleksandra Mikulska","email":"","orcid":"","institution":"Medical University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Aleksandra","middleName":"","lastName":"Mikulska","suffix":""},{"id":516047005,"identity":"46b92d4f-b32c-4fd4-a9cf-48529fd6c99d","order_by":2,"name":"Martyna Poniewierska","email":"","orcid":"","institution":"Altium Sp. z.o.o.","correspondingAuthor":false,"prefix":"","firstName":"Martyna","middleName":"","lastName":"Poniewierska","suffix":""},{"id":516047006,"identity":"dbf54665-6157-4b93-8876-40e8895051a5","order_by":3,"name":"Agnieszka Suchan","email":"","orcid":"","institution":"Altium Sp. z.o.o.","correspondingAuthor":false,"prefix":"","firstName":"Agnieszka","middleName":"","lastName":"Suchan","suffix":""},{"id":516047007,"identity":"dd53f31c-e3a5-4265-bb9b-2379408e90aa","order_by":4,"name":"Maciej Sierakowski","email":"","orcid":"","institution":"Cardinal Stefan Wyszynski University","correspondingAuthor":false,"prefix":"","firstName":"Maciej","middleName":"","lastName":"Sierakowski","suffix":""},{"id":516047008,"identity":"1c01d30c-8af4-47f0-9693-d504a76939f7","order_by":5,"name":"Tomasz Pawiński","email":"","orcid":"","institution":"Medical University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Tomasz","middleName":"","lastName":"Pawiński","suffix":""},{"id":516047010,"identity":"d3a86056-0c9b-42e8-9424-ea6a5c77418c","order_by":6,"name":"Arkadiusz Kocur","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYFACNgYGHgMoAwjk4KIM7ERqMUZoYcanBa6KgSGxgZAW/tnHEj+8KThsL9/AlvjgZ9u99A03shMYPpQdZjDHoUXiXNphyTkGhxMbG9gOG/a2FeduuJG7gXHGucMMls043HWGvUGax+BwAjMDe5s0Y1tC7jagFmbetsMMBoex65A/w978G6jFng2qJd0MpOUvHi0GZ9iOgWxh7GEAMoBaEsBaGPFoMTzDlmY5xyA9cQYzW7Jhz7kEw/1n3m442HMunQeXX+TOsBnfePPH2l6+vc3wwY+yBHnJ9tyNQIa1nDl7Aw7/wwBykB5ggMUvaYAMLaNgFIyCUTA8AQDCK1iHWaWqkQAAAABJRU5ErkJggg==","orcid":"","institution":"Medical University of Warsaw","correspondingAuthor":true,"prefix":"","firstName":"Arkadiusz","middleName":"","lastName":"Kocur","suffix":""}],"badges":[],"createdAt":"2025-09-03 19:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7529927/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7529927/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s43440-025-00807-5","type":"published","date":"2025-11-19T15:59:10+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91526492,"identity":"12f956d9-be46-40c3-812b-637d954273eb","added_by":"auto","created_at":"2025-09-17 11:09:18","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2785568,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism of methotrexate action: metabolism and distribution in red blood cells. Created using Biorender.com under publishing rights.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7529927/v1/b4801f4df3b22e9f854ecf0b.jpeg"},{"id":91526491,"identity":"87341dfa-c2a7-4971-b6fd-3b3280b92609","added_by":"auto","created_at":"2025-09-17 11:09:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":79463,"visible":true,"origin":"","legend":"\u003cp\u003eThe representative chromatograms of tested compounds – MTX and MTX-d\u003csub\u003e3\u003c/sub\u003e. Panel (a) is a double blank sample (processed without MTX and corresponding IS), (b) is an LLOQ level spiked sample and (c) is a real clinical sample with 0.046 μmol/L of MTX.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7529927/v1/570e7b9183e2919d4ed8fec1.png"},{"id":91526496,"identity":"f2d5f800-1673-4674-8e31-0c4c76667af1","added_by":"auto","created_at":"2025-09-17 11:09:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":438694,"visible":true,"origin":"","legend":"\u003cp\u003ePlot with summarized results of EMIT, EIA and LC-MS/MS determination of MTX. Data presented for 53 triplicated results; the arithmetic mean for each method is marked with a larger circle and a black border. EMIT - Enzyme-Multiplied Immunoassay, EIA- Homogeneous Enzyme Immunoassay, LC-MS/MS- Liquid Chromatography – Tandem Mass Spectrometry.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7529927/v1/186ea8d47557bf4d8ae99412.png"},{"id":91526499,"identity":"be309683-1e1b-409a-b009-1b599f540054","added_by":"auto","created_at":"2025-09-17 11:09:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":151853,"visible":true,"origin":"","legend":"\u003cp\u003eThe results of cross- and clinical validation of triplicated results of MTX determination. The mean bias, evaluated using Bland-Altman plots, is presented for each relationship on panels (A), (B), and (C). The mean bias is presented on the plot as a percentage mean. The green lines represented a ±1.96SD\u003cstrong\u003e \u003c/strong\u003econfidence interval, the red solid line represented the \u003cstrong\u003e±\u003c/strong\u003e20% bias range, the purple lines represented the regression model, and the black solid line represented the mean percentage bias. Panels (D), (E), and (F) represent a comparison of results using Passing-Bablok regression, with the regression equation as legend. Compared methods are described on each plot axis.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7529927/v1/7a628fba283a78af345ef9b3.png"},{"id":96651002,"identity":"69ebad8e-3949-4856-81ba-a08de3bf5745","added_by":"auto","created_at":"2025-11-24 16:13:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4396821,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7529927/v1/778c6fc3-a5b0-4450-97bb-caa3dd7c0ae1.pdf"},{"id":91527359,"identity":"89959384-5e3d-4cc3-adcc-58f93f168157","added_by":"auto","created_at":"2025-09-17 11:17:18","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":24292,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7529927/v1/719b616a17736f54886be924.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Novel LC-MS/MS Method for Measuring Methotrexate in High-Dose Therapy: A Comparative Study with Commercial EMIT and EIA Immunoassays","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMethotrexate (MTX; amethopterin) is an antimetabolite structurally similar to folic acid. It inhibits the enzyme dihydrofolate reductase (DHFR), blocking purine nucleotide synthesis and disrupting DNA replication and cell proliferation. A schematic overview of MTX distribution is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e [1]. Clinically, MTX is administered in two dosing protocols: low-dose MTX (LDMTX), primarily used for inflammatory diseases, and high-dose MTX (HDMTX), used in oncology. HDMTX, defined as doses of \u0026ge;\u0026thinsp;500 mg/m\u0026sup2;, is widely used to treat various cancers such as acute lymphoblastic leukemia, osteosarcoma, and primary central nervous system lymphoma [1\u0026ndash;3]. While HDMTX provides significant therapeutic benefits, it also poses a risk of toxicity if not adequately monitored. Therapeutic drug monitoring (TDM) guides adjustments for rescue therapy with folinic acid (leucovorin) or glucarpidase to reduce MTX toxicity. Standard monitoring points are at 24, 48, and 72 hours after infusion starts. If MTX plasma levels drop below 0.1 \u0026micro;mol/L, rescue therapy can be safely stopped [3\u0026ndash;5]. MTX has complex pharmacokinetics, with variability in metabolism and excretion. After IV infusion, the maximum serum or plasma concentration (C\u003csub\u003emax\u003c/sub\u003e) occurs within 0.5 to 1 hour. Distribution involves binding to plasma proteins and cellular uptake, including erythrocytes. MTX elimination occurs in three phases; however, approximately 95% of free MTX is cleared within 24 hours. To prevent renal toxicity, partly caused by the metabolite 7-OH-MTX, urine alkalization with sodium bicarbonate infusion is essential. This, combined with forced diuresis, accelerates elimination, requiring renal function monitoring through serum creatinine and GFR [1\u0026ndash;5].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eCurrently, there are a few bioanalytical approaches to monitoring MTX in TDM services, namely: immunoassays (IAs) and chromatography applications (especially LC-MS/MS). The latter is considered the gold standard due to its relatively high sensitivity and specificity (compared to IAs). On the other hand, IAs are characterized by cross-reactivity with metabolites and lower sensitivity [6\u0026ndash;8].\u003c/p\u003e\u003cp\u003eThe ARK\u0026trade; Methotrexate EIA assay is based on in vitro competition between the analyzed drug and glucose-6-phosphate dehydrogenase (G6PDH) labelled with MTX for binding to the antibody reagent. The activity of the enzyme increases proportionally with the MTX concentration in the sample being tested. The activated enzyme converts the coenzyme nicotinamide adenine dinucleotide (NAD) to NADH, and the differences in absorbance, determined spectrophotometrically, are evaluated. Endogenous human G6PDH enzyme does not interfere during analysis because ARK\u0026trade; kits are based on a specific bacterial enzyme. In the presented study, the VITROS 5600\u0026trade; (Quidel Ortho, San Diego, CA, USA) immunochemical system, integrated with the described test, was utilized, with a precision of less than 8% (CV%) [6,7].\u003c/p\u003e\u003cp\u003eThe Syva\u003csup\u003e\u0026reg;\u003c/sup\u003e MTX assay is an enzyme-multiplied immunoassay technique (EMIT). The principles of the immunochemical reactions are similar to those of the ART\u0026trade; Methotrexate Assay test. The Syva immunochemical kits were used for MTX assays with the Dimension EXL 200 immunochemistry system (Siemens Healthcare, Erlangen, Germany), exhibiting a precision of less than 10% (CV) [8].\u003c/p\u003e\u003cp\u003eThe comprehensive features of IAs, including linearity, limit of quantification (LOQ), and cross-reactivity interferences, are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Detailed information, such as reagent specifications and validation results, can be found in the leaflets provided by the manufacturers [6\u0026ndash;8].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBrief characteristics of the most common immunoassays used clinically for methotrexate determination [6\u0026ndash;8].\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eBrand name\u003c/p\u003e\u003cp\u003eof the test\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eType of method\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eLinearity\u003c/p\u003e\u003cp\u003e[\u0026micro;mol/L]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eLOQ\u003c/p\u003e\u003cp\u003e[\u0026micro;mol/L]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eInterferences\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7-OH-MTX\u003c/p\u003e\u003cp\u003e[%]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eDAMPA\u003c/p\u003e\u003cp\u003e[%]\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eARK\u0026trade; Assay\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ehomogenous immunoassay\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.04\u0026ndash;1.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(\u0026le;\u0026thinsp;0.07)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e64.30\u0026ndash;100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eARK\u003csup\u003e\u0026trade;\u003c/sup\u003e II Assay\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ehomogenous immunoassay\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.03\u0026ndash;1.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(\u0026le;\u0026thinsp;0.01)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e18.80\u0026ndash;57.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSyva\u003csup\u003e\u0026reg;\u003c/sup\u003e EMIT Assay\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eenzyme-multiplied immunoassay technique\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.30\u0026ndash;2.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eND\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003estrong\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eLOQ- limit of quantification; 7-OH-MTX- 7-hydroxy-methotrexate; DAMPA- 2,4-Diamino-N\u003csup\u003e10\u003c/sup\u003e-methylpteroic acid.\u003c/p\u003e\u003cp\u003eContinuous monitoring of MTX levels and elimination rate during high-dose therapy is essential for determining the appropriate leucovorin (LV) dosage [9]. The most critical clinical situations include:\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eWhen MTX concentrations are too high, intracellular transport of leucovorin is selectively inhibited, which reduces the effectiveness of the rescue treatment.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eIn patients with delayed MTX elimination, toxic levels of MTX may persist longer than the standard duration of rescue therapy, necessitating immediate adjustment of the LV dose.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eIn patients with regular MTX elimination, excessively high LV doses should be avoided, as they may decrease treatment efficacy.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAlthough partial agreement exists between the immunoassay and LC-MS/MS methods, results obtained using immunoassays may not always be reliable. Notably, cross-reactivity with MTX metabolites\u0026mdash;especially DAMPA\u0026mdash;may cause overestimation of MTX concentrations, potentially leading to unnecessary prolongation of leucovorin rescue therapy.\u003c/p\u003e\u003cp\u003eThis study aimed to develop and validate a novel LC-MS/MS method for measuring methotrexate (MTX) in serum samples. Additionally, the validated LC-MS/MS method was compared with two independent immunoassay (IA) techniques: enzyme immunoassay (EIA) and enzyme-multiplied immunoassay technique (EMIT), using regression, correlation, and bias estimation statistical tools. To demonstrate the clinical interchangeability of the tested method, clinical serum samples from patients undergoing HDMTX therapy were analyzed using each assay. To the best of our knowledge, this is the first study to provide an analytical and clinical comparison of EIA, EMIT, and a reference LC-MS/MS method using pediatric clinical samples.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003e2.1. Chemicals, Reagents and Laboratory Accessories\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eThe solid standard of methotrexate (MTX; TRC-M260675, \u0026ge;\u0026thinsp;95% chemical purity by HPLC) was obtained from Toronto Research Chemicals (TRC, Toronto, Canada). The stable isotope-labelled internal standard (SIL-IS), d₃-MTX (22378, \u0026ge;\u0026thinsp;99% deuterated forms: d₁\u0026ndash;d₃), was supplied by Cayman Chemical (Ann Arbour, MI, USA). LC-MS grade solvents\u0026mdash;methanol (MeOH), acetonitrile (ACN), 2-propanol (IPA), and ultrapure water\u0026mdash;were sourced from Merck (Darmstadt, Germany). Inorganic reagents, including zinc sulfate heptahydrate and 25% ammonium hydroxide, were purchased from Supelco (Bellefonte, PA, USA). Mobile phase modifiers with LC-MS purity, such as acetic acid and ammonium acetate, were provided by Fisher Chemical (Pittsburgh, PA, USA). Immunochemical reagents for EMIT and EIA assays were obtained from Siemens Healthcare Diagnostics (Newark, DE, USA) and ARK Diagnostics (Fremont, CA, USA), respectively.\u003c/p\u003e\n\u003cp\u003eDrug-free plasma and serum samples were obtained from the Regional Blood Donation and Hemotherapy Centre in Warsaw (Warsaw Blood Bank, Poland). Quality control materials for immunoassays and LC-MS analysis, including IAS and Liquichek Therapeutic Drug Monitoring Control, were supplied by Bio-Rad Laboratories (Hercules, CA, USA). Basic laboratory equipment, including Eppendorf Reference 2 pipettes, plastic test tubes, and pipette tips, was provided by Eppendorf (Hamburg, Germany).\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003e2.2. Stock Solutions of MTX and Internal Standard, Calibration Standards (CSs) and Quality Control Samples (QCs)\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eCalibration standards (CSs) and quality control (QC) solutions were prepared using two different weighed amounts of the solid MTX standard. Simultaneously, the stable isotope-labelled internal standard (SIL-IS) was prepared using the same procedure. Both compounds were dissolved in 0.2 M ammonium hydroxide in methanol using ultrasound assistance. The MTX stock solutions were further diluted to prepare working solutions at concentrations of 0.100, 0.010, and 0.001 mg/mL.\u003c/p\u003e\n\u003cp\u003eOne set of working solutions was used to prepare calibration standards, and another set was used for QC samples. The LC-MS/MS method was validated over the range of 0.01\u0026ndash;25.00 \u0026micro;mol/L. Accordingly, MTX working solutions were diluted with a methanol: water mixture (1:1, v/v) to obtain the following concentrations for CSs and QCs, using drug-free human serum as matrix (prepared identically to clinical samples; see Section \u003cspan class=\"InternalRef\"\u003e2.3\u003c/span\u003e):\u003c/p\u003e\n\u003cp\u003eFinal MTX concentrations for calibration standards (in \u0026micro;mol/L): 0.010 (LLOQ \u0026ndash; lower limit of quantification); 0.025; 0.050; 0.100; 0.500; 1.000; 2.500; 5.000; 10.000; 15.000; 25.000 (ULOQ \u0026ndash; upper limit of quantification). Quality control (QC) samples were prepared at the following MTX concentration levels:\u003c/p\u003e\n\u003c/div\u003e\n\u003cul\u003e\n\u003cli\u003e\n\u003cp\u003e0.035 \u0026micro;mol/L (LQC \u0026ndash; lower quality control);\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003e0.25 \u0026micro;mol/L (MQC1 \u0026ndash; medium quality control 1);\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003e7.50 \u0026micro;mol/L (MQC2 \u0026ndash; medium quality control 2);\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003e20.00 \u0026micro;mol/L (HQC \u0026ndash; high quality control).\u003c/p\u003e\n\u003c/li\u003e\n\u003c/ul\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eTo convert MTX concentrations from \u0026micro;mol/L to mg/L, values should be recalculated using the factor 2.20.\u003c/p\u003e\n\u003cp\u003eThe internal standard (IS) solution of d₃-MTX was prepared as described previously to yield a stock concentration of 100 \u0026micro;mol/L. This solution was subsequently diluted to achieve a final concentration of 2.50 \u0026micro;mol/L in the processed samples.\u003c/p\u003e\n\u003cp\u003eAll prepared solutions were stored under light-protected conditions in amber low-binding vials at \u0026minus;\u0026thinsp;20\u0026deg;C.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003e2.3. Sample Pretreatment Protocol\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eAfter serum collection in non-gel separator tubes, the sample was centrifuged at room temperature for 5 mins at 13500 \u0026times; g. The resulting serum was stored in amber Eppendorf tubes at -20\u0026deg;C until analysis via IAs or LC-MS/MS. Before analysis, 40 \u0026micro;L of thawed serum (blank, calibrator, control, or patient sample) was used for LC-MS/MS. To the matrix, 5 \u0026micro;L of SIL-IS solution and 55 \u0026micro;L of water were added. After vortexing for 30 secs, 200 \u0026micro;L of the precipitation mixture (0.1 M ZnSO\u003csub\u003e4\u003c/sub\u003e:ACN, 1:1, v/v) was pipetted into the sample. The samples were then vortexed for 15 minutes and kept on ice for an additional 15 minutes. Finally, the Eppendorf tube containing the sample was centrifuged for 5 minutes at 13500 \u0026times; g, and the extracted sample was transferred to an amber glass chromatographic vial with an insert. Samples for EIA and EMIT analysis were prepared according to the manufacturer\u0026rsquo;s instructions, with no additional steps necessary in serum sample preparation.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003e2.4. Clinical Samples\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eSerum samples were collected from pediatric patients at predetermined time points\u0026mdash;24, 48, and 72 hours after starting methotrexate (MTX) infusion. The main clinical aim of monitoring MTX levels was to guide adjustments to leucovorin rescue therapy. In this study, residual serum samples obtained during routine diagnostic procedures at the Children\u0026rsquo;s Memorial Health Institute (CMHI) in Warsaw, Poland, were analyzed. Informed consent was received from all patients and/or their legal guardians at the start of treatment. Each serum sample was analyzed in triplicate using three separate methods: EMIT, EIA, and LC-MS/MS. The average values from these methods were used for clinical decision-making during MTX chemotherapy. The use of residual diagnostic material for the validation and comparison of analytical techniques was reported to the local Bioethical Committee. The agreement between CMHI and Altium Poland was registered under reference number 2377/2023.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003e2.5. Instrumentation: Chromatographic and Mass Spectrometry Parameters\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eA Shimadzu 8050 triple quadrupole mass spectrometer coupled with a Nexera X2 UPLC system (Shimadzu, Kyoto, Japan) was employed for LC-MS/MS method validation and clinical sample analysis. Both methotrexate (MTX) and the stable isotope-labelled internal standard (SIL-IS) were monitored using a multiple reaction monitoring (MRM) approach in positive ionization mode, utilizing electrospray ionization (ESI). Detection was based on singly protonated molecular ions [M\u0026thinsp;+\u0026thinsp;H] \u003csup\u003e+\u003c/sup\u003e. Details of the MRM transitions for MTX and the internal standard are listed in Table\u0026nbsp;2. The optimized ion source and MS detection parameters are provided in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;2.\u003c/strong\u003e Mass spectrometry conditions: MRM pairs with collision energy for MTX and SIL-IS. MTX-1 pair (bolded) has been used for quantitative purposes.\u003c/p\u003e\n\u003ctable id=\"Taba\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMRM pair\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eQ1\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eQ3\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCE [eV]\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMTX-1\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e455.25\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e308.10\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e-18.0\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMTX-2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e455.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e175.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e-36.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMTX-3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e455.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e134.05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e-33.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ed\u003csub\u003e3\u003c/sub\u003e-MTX (SIL-IS)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e458.20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e211.20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e-18.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Dwell time was set as 100 ms for each MRM pair. MRM \u0026ndash; multiple reaction monitoring, CE \u0026ndash; collision energy, SIL-IS \u0026ndash; stable isotope labelled-internal standard.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eIon source and specific mass spectrometry parameters.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eParameter (unit)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eOptimized value\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enebulizing gas flow [L/min]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003edrying gas flow [L/min]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eheating gas flow [L/min]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003einterface temperature [\u0026deg;C]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e300\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003edesolvatation line temperature [\u0026deg;C]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e250\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eheating block temperature [\u0026deg;C]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e400\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003einterface voltage [kV]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCID gas pressure [kPa]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e270\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e CID \u0026ndash; collision-induced dissociation.\u003c/p\u003e\n\u003cp\u003eThe chromatographic part of the platform consisted of: a gradient pump with a mobile phase mixer (30AD), a degassing unit (DGU-205AR), an autosampler with a cooler (SIL-30AC), and a column oven (CTO-20AC). The chromatographic separation was carried out under gradient mode using a Zorbax Eclipse Plus C\u003csub\u003e18\u003c/sub\u003e RRHD column (2.10 \u0026times; 50 mm, 1.80 \u0026micro;m) supplied by Agilent Technologies, Inc. (Santa Clara, CA, USA). A guarded with a complementary C\u003csub\u003e18\u003c/sub\u003e precolumn (2.1 \u0026times; 5 mm, 1.80 \u0026micro;m). Throughout the entire analytical run, the column was maintained at 40\u0026deg;C. For the generation of a gradient, two mobile phases have been applied:\u003c/p\u003e\n\u003cp\u003e(A) LC-MS grade water with 0.1% acetic acid and 2 mM of ammonium acetate,\u003c/p\u003e\n\u003cp\u003e(B) pure LC-MS grade acetonitrile.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eGradient mode has been utilized in the following steps with a 0.5 mL/min flow rate:\u003c/p\u003e\n\u003c/div\u003e\n\u003col\u003e\n\u003cli\u003e\n\u003cp\u003e0.01\u0026ndash;0.75 min 5% of B phase,\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003e0.75\u0026ndash;1.20 min linear increasing to 75% of B phase,\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003e1.21\u0026ndash;2.50 min 75% of B phase (maintained),\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003e2.51\u0026ndash;5.00 min re-equilibration to 5% of phase B.\u003c/p\u003e\n\u003c/li\u003e\n\u003c/ol\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eThe autosampler needle was purged in internal-external mode using a 1:1 (v/v) water/IPA mixture. The injection volume was set as 1 \u0026micro;L of supernatant after the extraction procedure.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003e2.6. LC-MS/MS Method Validation\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eThe presented bioanalytical method has been validated in accordance with the current regulatory requirements, as outlined in ICH M10, as provided by the EMA according to its updated guideline [9]. The following parameters were evaluated: specificity, selectivity, lower limit of quantification (LLOQ), limit of detection (LOD), linearity, accuracy, precision, recovery, dilution integrity, carry-over, matrix effect, incurred sample reanalysis (ISR), and stability. For cross- and clinical validation, some regulations proposed by IATDMCT were applied [12].\u003c/p\u003e\n\u003cp\u003eSpecificity was assessed to ensure accurate differentiation between the analyte, IS, and potentially interfering substances, including metabolites and endogenous co-eluted compounds. Interference at the analyte\u0026rsquo;s retention time was acceptable if it did not exceed 20% of the LLOQ response, and \u0026le;\u0026thinsp;5% for the IS. The selectivity was evaluated using drug-free serum samples obtained from six independent donors. The method was considered selective if no interfering peaks were observed at the retention times of MTX or IS within the specified response thresholds. Method precision was confirmed under these conditions, even with two MRM transitions applied: one for quantification and the second for qualitative purposes. LOD in LC-MS/MS has been estimated using the signal-to-noise (S/N) approach, with a threshold of S/N\u0026thinsp;\u0026ge;\u0026thinsp;50 used to define the lowest detectable concentration [9].\u003c/p\u003e\n\u003cp\u003eThe linearity of the methodology was evaluated for the applied concentration range of 0.01\u0026ndash;25.00 \u0026micro;mol/L, which included double-blank and blank samples in the set. For calibration curve construction, the 1/x\u003csup\u003e2\u003c/sup\u003e weighting was used in ten independent analytical runs. The mean coefficient of determination (R\u0026sup2;) was higher than 0.995, which was a confirmation of linearity. The accuracy and precision were evaluated using nine independent samples at the LLOQ, LQC, MQC\u003csub\u003e1\u003c/sub\u003e, MQC\u003csub\u003e2\u003c/sub\u003e, and HQC levels during intra- and interday experiments. The precision was expressed as the coefficient of variation (CV%), while accuracy was expressed as the percentage deviation from the nominal value. Acceptance criteria were \u0026plusmn;\u0026thinsp;15% for both parameters at all levels, except \u0026plusmn;\u0026thinsp;20% for LLOQ. The potential carry-over effect was evaluated by injecting a blank sample immediately after a ULOQ sample in a single analytical run. If the calculated peak area does not exceed 20% of the LLOQ for MTX or 5% for the IS, the acceptance criteria are fulfilled [9].\u003c/p\u003e\n\u003cp\u003eThe matrix effect (ME), a characteristic of ESI-LC-MS/MS-based methods, was evaluated using the post-extraction approach, as described in protocols by Taylor et al. and Matuszewski et al. Matrix factor (MF), absolute recovery (AR), and process efficiency (PE) were calculated from six replicates at LQC and HQC levels [10,11].\u003c/p\u003e\n\u003cp\u003eIncurred sample reanalysis (ISR) was performed on clinical samples (minimum 10% of clinical samples) and was accepted if at least 67% of values were within \u0026plusmn;\u0026thinsp;20% of the initial analyte concentration measured during the first analysis [9].\u003c/p\u003e\n\u003cp\u003eThe stability, as an initial part of the validation process, has been tested using LQC and HQC samples during experiments, including extracts autosampler stability, freeze\u0026ndash;thaw stability over three cycles, and short- and long-term stability of the analyte in the matrix. All stability results were accepted if measured concentrations remained within \u0026plusmn;\u0026thinsp;15% of nominal values [9].\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003e2.7. Immunoassays \u0026ndash; EMIT and EIA techniques\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eThe EMIT and EIA techniques are easy-to-implement procedures that are often coupled with many immunochemical analyzers in the routine practice of biochemical laboratories. Serum samples were centrifuged (13500 \u0026times; g), transferred to a reaction cup and placed in Dimension\u003csup\u003e\u0026reg;\u003c/sup\u003e EXL\u0026trade; 200 or VITROS\u003csup\u003e\u0026reg;\u003c/sup\u003e 5600 analyzers (EMIT Syva\u003csup\u003e\u0026reg;\u003c/sup\u003e and EIA ARK\u0026trade;, respectively). The measurements were performed automatically by analyzers with principles characteristic of EMIT and EIA assays, as mentioned in the Introduction. The systems were regularly calibrated and controlled using commercial QC samples. Because manufacturers have delivered methodologies as ready-to-use kits, in-house validation was not performed (except for systematic calibration, control, and stability testing). The detailed parameters of reagent ingredients, methodology principles, limitations, and validation results are provided in fully available manufacturer\u0026rsquo;s leaflets [6\u0026ndash;8].\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003e2.8. Statistical Analysis\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eStatistical analyses, including Passing\u0026ndash;Bablok regression, Bland\u0026ndash;Altman plots, and calculation of descriptive statistics (mean, standard deviation), as well as validation parameters, were performed using MedCalc software (version 22.023; MedCalc Software Ltd., Ostend, Belgium). The Shapiro\u0026ndash;Wilk test was applied to assess the normality of data distributions. A significance threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was adopted for all tests to ensure statistical robustness and reliability of the results.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1. LC-MS/MS Method Optimization\u003c/h2\u003e\n\u003cp\u003eThe LC-MS/MS method for MTX determination has been optimized and validated at the Department of Drug Chemistry, Pharmaceutical and Biomedical Analysis, Medical University of Warsaw, Poland. In the presented study, a LC-MS/MS method for determining MTX in serum was developed and validated. The analyte extraction protocol, chromatographic conditions, and mass spectrometry ion source parameters were optimized experimentally. Various methods for MTX isolation from serum were tested. The application of a less time-consuming protein precipitation method using a zinc sulfate/acetonitrile mixture, compared to liquid-liquid extraction or solid-phase extraction, resulted in high recovery rates (\u0026gt;\u0026thinsp;90%). Perchloric acid at 16% was slightly more effective than the mixture mentioned above, but it is not fully compatible with LC-MS/MS systems due to its high oxidative potential. The addition of zinc sulfate to acetonitrile generated lower matrix interferences than using pure acetonitrile. The chromatographic parameters were optimized experimentally, using various mobile phases with different types and amounts of modifiers in gradient mode. Additionally, different chromatography columns were tested, including Kinetex C18 (50 \u0026times; 2.1 mm; 1.7 \u0026micro;m), Synergy Fusion-RP (10 \u0026times; 2.1 mm; 4.0 \u0026micro;m), Hypurity-C18 (50 \u0026times; 2.10 mm; 3 \u0026micro;m) from Thermo Scientific, Bonus RP RRHD Zorbax (2.1 \u0026times; 50 mm; 1.8 \u0026micro;m) from Agilent, Zorbax Eclipse RRHD C8 (2.1 \u0026times; 50 mm; 1.8 \u0026micro;m), Zorbax Eclipse RRHD C18 (2.1 \u0026times; 50 mm; 1.8 \u0026micro;m), Poroshell 120-EC-C18 (150 \u0026times; 3.00 mm; 2.70 \u0026micro;m), Ascentis-C18 (100 \u0026times; 3.00 mm; 3.00 \u0026micro;m), Ascentis-C8 Express (100 \u0026times; 3.00 mm; 3.00 \u0026micro;m), and Nucleosil C18 (125 \u0026times; 3.00 mm; 5.00 \u0026micro;m). The Zorbax Eclipse RRHD C18 column was selected as optimal due to the relatively short retention time (~\u0026thinsp;2.10 min) of the analyte and the Gaussian shape of the chromatographic peaks. Representative chromatograms of the tested compound are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2. Validation Results\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eThe validated method met the criteria according to selectivity, which was evaluated by analyzing the 10 replicates of double blank samples (processed without stable isotope-labelled internal standard and analyte) and zero calibrator samples (with SIL-IS addition only). MTX and MTX-d\u003csub\u003e3\u003c/sub\u003e responses were considered acceptable because the interferences do not exceed 20% and 5% of the LLOQ signal, respectively.\u003c/p\u003e\n\u003cp\u003eThe limit of detection (LOD) was determined based on the signal-to-noise ratio (S/N) of standard solutions prepared by diluting the lowest calibration solution (LLOQ). The LOD was established at 0.005 \u0026micro;mol/L (0.0023 mg/L, S/N\u0026thinsp;=\u0026thinsp;10).\u003c/p\u003e\n\u003cp\u003eThe linearity of the method was assessed based on ten calibration curves in 0.01\u0026ndash;25.0 \u0026micro;mol/L. Weighted linear regression with a weighting factor of 1/x was applied to favour the lower points of the curve. The mean R\u0026sup2; was 0.9932, and the mean equation of the curve was: y\u0026thinsp;=\u0026thinsp;0.51441x\u0026thinsp;+\u0026thinsp;0.10239, where y is the MTX/SIL-IS peaks area ratio, and x is the nominal MTX concentration.\u003c/p\u003e\n\u003cp\u003eAccuracy and precision were evaluated in nine repetitions for LLOQ, LQC, MQC\u003csub\u003e1\u003c/sub\u003e, MQC\u003csub\u003e2\u003c/sub\u003e and HQC during intra-run (intra-day) and between-run (between-day) experiments (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The acceptance criteria for all quality control levels were fulfilled according to ICH M10 guidelines [9].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab3\" style=\"width: 534px;\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eAccuracy and precision experiment results [n\u0026thinsp;=\u0026thinsp;9].\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth style=\"width: 74px;\" align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth style=\"width: 84px;\" align=\"left\"\u003e\n\u003cp\u003eLLOQ\u003c/p\u003e\n\u003cp\u003e0.001 \u0026micro;mol/L\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"width: 84px;\" align=\"left\"\u003e\n\u003cp\u003eLQC\u003c/p\u003e\n\u003cp\u003e0.035 \u0026micro;mol/L\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"width: 80px;\" align=\"left\"\u003e\n\u003cp\u003eMQC\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003e0.25 \u0026micro;mol/L\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"width: 80px;\" align=\"left\"\u003e\n\u003cp\u003eMQC\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003e2.50 \u0026micro;mol/L\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"width: 87.926px;\" align=\"left\"\u003e\n\u003cp\u003eHQC\u003c/p\u003e\n\u003cp\u003e20.00 \u0026micro;mol/L\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth style=\"width: 499.926px;\" colspan=\"6\" align=\"left\"\u003e\n\u003cp\u003eIntra-run (intra-day) accuracy and precision\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 74px;\" align=\"left\"\u003e\n\u003cp\u003eC\u003csub\u003eMTX\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003e[\u0026micro;mol/L]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 84px;\" align=\"left\"\u003e\n\u003cp\u003e0.011 \u0026plusmn; 0.005\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 84px;\" align=\"left\"\u003e\n\u003cp\u003e0.033 \u0026plusmn; 0.004\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 80px;\" align=\"left\"\u003e\n\u003cp\u003e0.246 \u0026plusmn; 0.028\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 80px;\" align=\"left\"\u003e\n\u003cp\u003e2.474 \u0026plusmn; 0.171\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.926px;\" align=\"left\"\u003e\n\u003cp\u003e20.198 \u0026plusmn; 0.673\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 74px;\" align=\"left\"\u003e\n\u003cp\u003eAccuracy [%]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 84px;\" align=\"left\"\u003e\n\u003cp\u003e104.67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 84px;\" align=\"left\"\u003e\n\u003cp\u003e94.61\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 80px;\" align=\"left\"\u003e\n\u003cp\u003e98.31\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 80px;\" align=\"left\"\u003e\n\u003cp\u003e99.98\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.926px;\" align=\"left\"\u003e\n\u003cp\u003e100.99\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 74px;\" align=\"left\"\u003e\n\u003cp\u003ePrecision [%]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 84px;\" align=\"left\"\u003e\n\u003cp\u003e12.38\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 84px;\" align=\"left\"\u003e\n\u003cp\u003e11.23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 80px;\" align=\"left\"\u003e\n\u003cp\u003e9.83\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 80px;\" align=\"left\"\u003e\n\u003cp\u003e6.92\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.926px;\" align=\"left\"\u003e\n\u003cp\u003e3.33\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 499.926px;\" colspan=\"6\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eBetween-run (inter-day) accuracy and precision\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 74px;\" align=\"left\"\u003e\n\u003cp\u003eC\u003csub\u003eMTX\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003e[\u0026micro;mol/L]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 84px;\" align=\"left\"\u003e\n\u003cp\u003e0.011 \u0026plusmn; 0.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 84px;\" align=\"left\"\u003e\n\u003cp\u003e0.035 \u0026plusmn; 0.003\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 80px;\" align=\"left\"\u003e\n\u003cp\u003e0.255 \u0026plusmn; 0.022\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 80px;\" align=\"left\"\u003e\n\u003cp\u003e2.334 \u0026plusmn; 0.120\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.926px;\" align=\"left\"\u003e\n\u003cp\u003e20.064 \u0026plusmn; 0.475\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 74px;\" align=\"left\"\u003e\n\u003cp\u003eAccuracy [%]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 84px;\" align=\"left\"\u003e\n\u003cp\u003e102.34\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 84px;\" align=\"left\"\u003e\n\u003cp\u003e99.05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 80px;\" align=\"left\"\u003e\n\u003cp\u003e102.18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 80px;\" align=\"left\"\u003e\n\u003cp\u003e93.37\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.926px;\" align=\"left\"\u003e\n\u003cp\u003e100.24\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 74px;\" align=\"left\"\u003e\n\u003cp\u003ePrecision [%]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 84px;\" align=\"left\"\u003e\n\u003cp\u003e9.97\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 84px;\" align=\"left\"\u003e\n\u003cp\u003e9.81\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 80px;\" align=\"left\"\u003e\n\u003cp\u003e8.54\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 80px;\" align=\"left\"\u003e\n\u003cp\u003e5.14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.926px;\" align=\"left\"\u003e\n\u003cp\u003e2.30\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eHQC \u0026ndash; higher quality control; LLOQ \u0026ndash; lower limit of quantification; LQC \u0026ndash; lower quality control; MQC \u0026ndash; medium quality control; MTX \u0026ndash; methotrexate.\u003c/p\u003e\n\u003cp\u003eCarry-over was assessed experimentally. For this purpose, a sequence was applied in which an HQC sample was injected directly after a blank sample without the analyte. According to EMA guidelines, the acceptance criteria for MTX are less than 20% and less than 5% for IS. The carry-over effect obtained for 10 samples was negligible and met the acceptance criteria for MTX (0.6912\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2491) and IS (0.02682\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01933).\u003c/p\u003e\n\u003cp\u003eDilution integrity parameter was verified by preparing a sample to which a working solution of the analyte at a concentration of 50 \u0026micro;mol/L was added. The sample was then diluted in ratios of 1:10 and 1:5 using a blank matrix to achieve the target concentrations (5 and 10 \u0026micro;mol/L). Five samples were prepared for each dilution level, and it was verified whether the obtained concentrations aligned with the calibration curve. According to the guidelines, accuracy should be within \u0026plusmn;\u0026thinsp;15%, and precision should not exceed 15%. For dilution 1:10, the mean concentration with SD was 4.6712\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2387 (mean accuracy and precision: 93.42% and 5.11%, respectively). In case of dilution 1:5, the mean concentration with SD was 10.0018\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6051 (mean accuracy and precision: 100.02% and 6.05%, respectively) [9].\u003c/p\u003e\n\u003cp\u003eMatrix effect (ME), process efficiency (PE), and absolute recovery (AR) were evaluated according to the well-known approaches of Taylor et al. and Matuszewski et al. for LQC and HQC samples [10,11]. The results for six independent repetitions were calculated for analyte and analyte to IS ratio for evaluation the compensation of matrix effect by using SIL-IS:\u003c/p\u003e\n\u003c/div\u003e\n\u003cul\u003e\n\u003cli\u003e\n\u003cp\u003eLQC: -14.20% \u0026plusmn; 3.01% (ME), 72.56% \u0026plusmn; 2.98% (PE) and 68.15% \u0026plusmn; 4.15% (AR);\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eLQC/IS ratio: -1.02% \u0026plusmn; 0.29 (ME), 98.06% \u0026plusmn; 3.06% (PE) and 97.99% \u0026plusmn; 5.18% (AR);\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eHQC: -11.48% \u0026plusmn; 4.09% (ME), 71.24% \u0026plusmn; 6.26% (PE) and 63.81% \u0026plusmn; 7.16% (AR);\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eHQC/IS ratio: -0.95% \u0026plusmn; 0.30 (ME), 99.15% \u0026plusmn; 5.93% (PE) and 98.37% \u0026plusmn; 6.18% (AR);\u003c/p\u003e\n\u003c/li\u003e\n\u003c/ul\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eThe serum samples spiked with LQC/HQC MTX levels remained stable during a minimum of five freeze-thaw cycles; however, samples stored for extended periods must be protected from light exposure. Additionally, the stability of extracts in the autosampler was evaluated at 4\u0026deg;C after 24 hours, 2, 5, and 7 days of storage. The results for LQC ranged from 96.20\u0026ndash;101.45%, while those for HQC ranged from 92.47\u0026ndash;100.67%. Serum samples spiked with LQC/HQC levels remained stable during storage in amber test tubes for 6 months at -20\u0026deg;C, with 98.99% and 100.66% of the mean nominal MTX value, respectively (n\u0026thinsp;=\u0026thinsp;3). Exposure to sunlight appeared to reduce sample stability, as indicated by three-day bench-top stability tests. The experiment was performed in triplicate, with mean results for light-exposed and light-protected samples as follows: 78.25% / 84.23% versus 79.87% / 90.45% for LQC and HQC, respectively.\u003c/p\u003e\n\u003cp\u003eThe ISR experiment was conducted on all clinical samples included in the study. All determined MTX concentrations met the criteria (see Section \u003cspan class=\"InternalRef\"\u003e2.6\u003c/span\u003e), with a mean difference of -2.15% \u0026plusmn; 0.49% [9].\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3. Selected results of EMIT and EIA methods calibration, control and stability evaluation\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eThe systems have been calibrated and controlled (using QCs) at the beginning of each daily batch run. Mean interday/intraday precisions (calculated as CV%; n\u0026thinsp;=\u0026thinsp;6) were 5.46%/8.34% and 4.63%/9.16% for EMIT and EIA, respectively. Mean calibration parameters (n\u0026thinsp;=\u0026thinsp;6) were:\u003c/p\u003e\n\u003c/div\u003e\n\u003cul\u003e\n\u003cli\u003e\n\u003cp\u003eEMIT: y\u0026thinsp;=\u0026thinsp;1.009x-0.004 (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.998),\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eEIA: y\u0026thinsp;=\u0026thinsp;1.013x\u0026thinsp;+\u0026thinsp;0.011 (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.998),\u003c/p\u003e\n\u003c/li\u003e\n\u003c/ul\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003efor observed versus expected MTX levels.\u003c/p\u003e\n\u003cp\u003eThe carry-over effect, observed after aspiration of a blank sample following the highest commercial quality control, was not detected. The LOD and LLOQ values are shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Manufacturers indicated dilution integrity, set at 1:6 and 1:10 (or multiples), for EMIT and EIA, respectively. Onboard stability of reagents was assessed as 7 weeks for EMIT and 5 weeks for EIA.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4. Results of MTX Quantification in Clinical Serum Samples\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eIn the clinical part of the presented study, 53 clinical samples from patients receiving MTX intravenously under the HDMTX protocol were analyzed using three methods: LC-MS/MS (reference method), EMIT, and EIA. The results of the assays are presented in the plot (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) and in the Supplementary File. Mean results with ranges (min/max) were as follows: 1.404 (0.024\u0026ndash;13.800) for LC-MS/MS, 1.322 (0.026\u0026ndash;16.091) for EIA, and 1.291 (0.040\u0026ndash;9.140) for EMIT.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4. Cross- and Clinical Validation\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eDuring cross- and clinical validation, the three sets of paired results for MTX determination in clinical samples were evaluated, namely: MTX\u003csub\u003eEMIT\u003c/sub\u003e \u003cem\u003eversus\u003c/em\u003e MTX\u003csub\u003eLC-MS/MS\u003c/sub\u003e, MTX\u003csub\u003eEMIA\u003c/sub\u003e \u003cem\u003eversus\u003c/em\u003e MTX\u003csub\u003eLC-MS/MS,\u003c/sub\u003e and MTX\u003csub\u003eEMIT\u003c/sub\u003e \u003cem\u003eversus\u003c/em\u003e MTX\u003csub\u003eEIA\u003c/sub\u003e. The results of regression studies (Passing-Bablok test), bias estimation (Bland-Altman plots) and other correlation studies are comprehensively presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e and in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab4\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eResults of cross- and clinical validation for determined results of MTX determination using EMIT, EIA and LC-MS/MS methods [n\u0026thinsp;=\u0026thinsp;53 triplicated results]. Acceptance criteria based on literature [12\u0026ndash;14].\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eStatistical tool\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eAnalyzed relationship\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMTX\u003csub\u003eLC-MS/MS\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eversus\u003c/em\u003e MTX\u003csub\u003eEMIT\u003c/sub\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMTX\u003csub\u003eLC-MS/MS\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eversus\u003c/em\u003e MTX\u003csub\u003eEIA\u003c/sub\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMTX\u003csub\u003eEMIT\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eversus\u003c/em\u003e MTX\u003csub\u003eEIA\u003c/sub\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePassing-Bablok\u003c/p\u003e\n\u003cp\u003eequation\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMTX\u003csub\u003eLC-MS/MS\u003c/sub\u003e=\u003c/p\u003e\n\u003cp\u003e1.0726(MTX\u003csub\u003eEMIT\u003c/sub\u003e)-0.0084\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMTX\u003csub\u003eLC-MS/MS\u003c/sub\u003e=\u003c/p\u003e\n\u003cp\u003e0.9914(MTX\u003csub\u003eEIA\u003c/sub\u003e)-0.0096\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMTX\u003csub\u003eEMIT\u003c/sub\u003e=\u003c/p\u003e\n\u003cp\u003e0.9299(MTX\u003csub\u003eEIA\u003c/sub\u003e)\u0026thinsp;+\u0026thinsp;0.0041\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSlope\u003c/p\u003e\n\u003cp\u003e(95% CI)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.0726\u003c/p\u003e\n\u003cp\u003e(0.9722 to 1.1789)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.9914\u003c/p\u003e\n\u003cp\u003e(0.9108 to 1.1724)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.9299\u003c/p\u003e\n\u003cp\u003e(0.8729 to 1.0069)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIntercept\u003c/p\u003e\n\u003cp\u003e(95% CI)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-0.0084\u003c/p\u003e\n\u003cp\u003e(-0.0230 to 0.0093)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-0.0096\u003c/p\u003e\n\u003cp\u003e(-0.0292 to 0.0106)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0041\u003c/p\u003e\n\u003cp\u003e(-0.0150 to 0.0161)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBland-Altman\u003c/p\u003e\n\u003cp\u003ebias [%]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.7328\u003c/p\u003e\n\u003cp\u003e(-1.8404 to 17.3061)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-2.4510\u003c/p\u003e\n\u003cp\u003e(-14.1862 to 9.2842)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-9.9995\u003c/p\u003e\n\u003cp\u003e(-18.3460 to -1.6529)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e% of samples in LoA\u003c/p\u003e\n\u003cp\u003e(bias\u0026thinsp;\u0026lt;\u0026thinsp;20%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e66.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e71.70\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e67.92\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e% of samples in LoC\u003c/p\u003e\n\u003cp\u003e(bias\u0026thinsp;\u0026lt;\u0026thinsp;15%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e54.72\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e59.40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e56.60\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRandom differences (RSD)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.4301\u003c/p\u003e\n\u003cp\u003e(-0.8430 to 0.8430)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.5364\u003c/p\u003e\n\u003cp\u003e(-1.0514 to 1.0514)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.8214\u003c/p\u003e\n\u003cp\u003e(-1.6100 to 1.6100)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSpearman Correlation coefficient (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.9300\u003c/p\u003e\n\u003cp\u003e(0.8820 to 0.9590)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.9110\u003c/p\u003e\n\u003cp\u003e(0.8490 to 0.9480)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.9530\u003c/p\u003e\n\u003cp\u003e(0.9190 to 0.9730)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eLoA \u0026ndash; analytical limit of bias agreement (\u0026lt;\u0026thinsp;20%), LoC \u0026ndash; clinical limit of bias agreement (\u0026lt;\u0026thinsp;15%), EMIT - Enzyme-Multiplied Immunoassay, EIA- Homogeneous Enzyme Immunoassay, LC-MS/MS - Liquid Chromatography \u0026ndash; Tandem Mass Spectrometry.\u003c/p\u003e\n\u003cp\u003eHowever, the Passing-Bablok regression analysis confirmed the interchangeability between paired results (slope differences\u0026thinsp;\u0026lt;\u0026thinsp;10%, and 0/1 values within intercept/slope 95% CI) [12\u0026ndash;14]. The clinical validation, based on clinical limits of agreement, did not confirm equivalence between the analyzed results (less than 67% of paired samples showed a bias\u0026thinsp;\u0026lt;\u0026thinsp;15%). The analytical limit of bias agreement (mean bias\u0026thinsp;\u0026lt;\u0026thinsp;20% for over 67% of paired results) was acceptable in almost all cases of the evaluated relationships (slightly lower in MTX\u003csub\u003eLC-MS/MS\u003c/sub\u003e compared to MTX\u003csub\u003eEMIT\u003c/sub\u003e). Strong positive correlations were identified in all analyzed scenarios.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThis study evaluated three methods\u0026mdash;EMIT, EIA, and LC-MS/MS\u0026mdash;for measuring methotrexate (MTX) levels in plasma from pediatric patients receiving high-dose methotrexate (HDMTX). Results indicated that LC-MS/MS was the most accurate and reliable technique, whereas immunoassays offered practical advantages for routine therapeutic drug monitoring. The LC-MS/MS method complied with the validation criteria outlined in the ICH M10 guidelines, confirming its suitability for clinical use.\u003c/p\u003e\u003cp\u003eA limited number of LC-MS/MS studies have been published on the determination of MTX in plasma or serum [15\u0026ndash;19]. Most required 100 \u0026micro;L or more of biological matrix for MTX measurement, while our method used only 40 \u0026micro;L. Reducing the sample volume is especially important in pediatric populations. Protein precipitation with an organic solvent, using MTX-d\u003csub\u003e3\u003c/sub\u003e as an internal standard, proved effective for serum sample preparation, ensuring sufficient sensitivity and addressing matrix effects and other interferences. Following guidelines, it is essential to use a stable isotope labelled internal standard (SIL-IS) whenever possible. In literature, both unrelated internal standards (URIS) and SIL-IS (\u003csup\u003e13\u003c/sup\u003eC,D\u003csub\u003e3\u003c/sub\u003e-MTX and MTX-D\u003csub\u003e3\u003c/sub\u003e) have been successfully applied. For instance, Tripathy et al. demonstrated the successful use of aminoacetophenone for MTX determination via LC-MS/MS, yielding satisfactory validation results and adequate compensation for matrix effects. Our investigation demonstrated strong correlations; however, it also revealed that immunoassays frequently failed to stay within the \u0026lt;\u0026thinsp;15% bias threshold essential for interchangeable use with LC-MS/MS, especially in pediatric oncology. Interestingly, in some triplicated results, the EIA method produced higher results compared to LC-MS/MS, whereas EMIT results for MTX determination were lower than those of LC-MS/MS. The ARK test for MTX showed less cross-interference with 7-OH-MTX than the Syva\u003csup\u003e\u0026reg;\u003c/sup\u003e test and is characterized by a lower limit of quantification (10 times more sensitive than the Syva\u003csup\u003e\u0026reg;\u003c/sup\u003e test). These differences may also contribute to the inaccuracies observed in the immunochemical applications presented in this study. It should be noted that during leucovorin/glucarpidase rescue treatment after MTX infusion, the actual levels of its metabolites in serum may increase. Conversely, higher metabolite levels may lead to an overestimation of results in immunoassays due to potential cross-reactivity. It appears that relying solely on immunoassay results, particularly in complex clinical situations, may unnecessarily prolong leucovorin/glucarpidase rescue therapy, thereby delaying chemotherapy and increasing healthcare costs [18\u0026ndash;26].\u003c/p\u003e\u003cp\u003eThe results of the presented study confirmed previous findings regarding LC-MS/MS as the gold standard, due to its superior specificity and sensitivity, especially in complex clinical situations where metabolites may interfere with immunoassay results. For example, Bouqui\u0026eacute; et al. demonstrated an excellent correlation between the LC-MS/MS method and the FPIA method (Abbott TDx\u0026trade;). Still, they concluded that none of the immunoassays could be used after glucarpidase administration. In such cases, LC-MS/MS appears to be more suitable because of its good selectivity, especially for MTX and its metabolites [23, 24].\u003c/p\u003e\u003cp\u003eCross-reactivities are not characteristic only of DAMPA and 7-OH-MTX. It should be noted that MTX polyglutamates, mainly present intracellularly in RBC, can cause a high overestimation of results. Particular attention should be paid to hemolysis of the sample, as some MTXPGs are usually present in the blood plasma fraction [1, 25].\u003c/p\u003e\u003cp\u003eThe study with high application potential in pediatric oncology, published recently by Opitz et al., described volumetric absorptive microsampling (VAMS) as an alternative to daily TDM service of MTX [26]. The application of VAMS strategies is not possible with immunoassays due to the low volume of capillary dried blood collected by the device (10, 20, and 30 \u0026micro;L). Therefore, LC-MS/MS remains the method of choice for this type of sample.\u003c/p\u003e\u003cp\u003eIn the same study, the authors concluded that the mean difference between the tested ARK\u0026trade; immunoassay and LC-MS/MS was approximately \u0026minus;\u0026thinsp;27% (with SD of 41%) [26]. It was indicated that the ARK test significantly overestimates MTX concentration compared to other immunoassays and LC-MS/MS methods. These overestimations are more pronounced at lower MTX concentrations, as observed in the presented study. Consequently, Opitz et al. proposed revised dose regimens for MTX based on the assay type, whether immunoassay (IA) or LC-MS/MS [26]. For example, after 54 hours of infusion, the estimated MTX level in serum should be \u0026le;\u0026thinsp;0,25 \u0026micro;mol/l when measured using IA (ARK\u0026trade; test) and \u0026lt;\u0026thinsp;0.12 \u0026micro;mol/l when measured using LC-MS/MS [26]. Our study also confirms the need to adjust MTX thresholds depending on the assay type for safe leucovorin dosing.\u003c/p\u003e\u003cp\u003eThe choice of analytical method for determining methotrexate depends not only on economic factors but also on the specific clinical scenario, such as implications for metabolite excretion, the patient\u0026rsquo;s clinical condition, or unusual drug distribution that may lead to toxicity. Examples of clinical situations, along with the proposed analytical technique, are presented in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe examples of high-dose methotrexate protocol and proposed analytical approaches\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClinical specific situation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProposed method\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJustification\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eType of oncology disorder (typical dose):\u003c/p\u003e\u003cp\u003e\u0026bull; ALL (1\u0026ndash;5 g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003cp\u003e\u0026bull; osteosarcoma (8\u0026ndash;12 g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003cp\u003e\u0026bull; PCNSL (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\ge\\:\\)\u003c/span\u003e\u003c/span\u003e 3 g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003cp\u003e\u0026bull; NHL (3\u0026ndash;8 g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003cp\u003e\u0026bull; choriocarcinoma (0.5\u0026ndash;1.5 g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEMIT/EIA\u003c/p\u003e\u003cp\u003eEMIT/EIA\u003c/p\u003e\u003cp\u003eEMIT/EIA\u003c/p\u003e\u003cp\u003eEMIT/EIA\u003c/p\u003e\u003cp\u003eLC-MS/MS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eThe immunoassays enabled a rapid turnaround time for administering an appropriate dose of leucovorin rescue therapy in the high-dose MTX protocol. However, this type of therapy with MTX may produce high levels of metabolites, which can interfere within 24\u0026ndash;48 hours after drug administration. Cross-reactivity is acceptable during the initial stage of renal clearance of MTX/metabolites (\u0026lt;\u0026thinsp;24 hours). In some clinical situations\u0026mdash;such as delayed elimination, abnormal drug distribution, or renal impairment\u0026mdash;the interference caused by metabolites may be observed; in these cases, LC-MS/MS is the preferred method.\u003c/p\u003e\u003cp\u003eIn lower doses (e.g., choriocarcinoma), the sensitivity of the immunoassay may be insufficient. If MTX is administered directly to the CNS (with CSF as the preferred matrix for MTX measurement), LC-MS/MS is also recommended.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMTX concentration level:\u003c/p\u003e\u003cp\u003e\u0026bull; 0.2\u0026ndash;1.0 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003eM\u003c/p\u003e\u003cp\u003e\u0026bull; \u0026lt;\u0026thinsp;0.2 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEMIT/EIA\u003c/p\u003e\u003cp\u003eLC-MS/MS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eImmunoanalyses are suitable for detecting higher concentrations but often overestimate results due to the presence of metabolites. Accurate measurement of low concentrations, especially those occurring more than 48 hours after MTX administration, is essential for patient safety and ongoing therapy with leucovorin/glucarpidase. Therefore, the higher costs are justified in clinical practice.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of analytes simultaneously determined:\u003c/p\u003e\u003cp\u003e\u0026bull; MTX only\u003c/p\u003e\u003cp\u003e\u0026bull; MTX with other anticancer drugs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEMIT/EIA\u003c/p\u003e\u003cp\u003eLC-MS/MS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSelectivity is a key advantage of LC-MS/MS. When multiple analytes need to be determined simultaneously, this technique remains the preferred method over immunochemical techniques.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eType of laboratory:\u003c/p\u003e\u003cp\u003e\u0026bull; in small hospital\u003c/p\u003e\u003cp\u003e\u0026bull; in large oncology centre\u003c/p\u003e\u003cp\u003e\u0026bull; reference laboratory\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEMIT/EIA\u003c/p\u003e\u003cp\u003eLC-MS/MS or IAs\u003c/p\u003e\u003cp\u003eLC-MS/MS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eImmunoassays are cost-effective, quick, and entirely adequate for daily practice. Additionally, their low unit cost and ease of integration with a biochemistry analyzer make them appealing for both small and larger TDM laboratories. Conversely, LC-MS/MS enables the simultaneous measurement of multiple drugs, thereby enhancing cost efficiency, particularly when testing large batches of samples.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003eALL \u0026ndash; acute lymphoblastic leukaemia; CSF \u0026ndash; cerebrospinal fluid; CNS \u0026ndash; central nervous system; EMIT \u0026ndash; enzyme-multiplied immunoassay technique; EIA \u0026ndash; enzyme immunoassay; IA \u0026ndash; immunoassay; LC-MS/MS \u0026ndash; liquid chromatography\u0026ndash;tandem mass spectrometry; NHL \u0026ndash; Non-Hodgkin lymphoma; PCNSL \u0026ndash; primary central nervous system lymphoma\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn daily laboratory practice, the calibration process and regular external proficiency testing are essential for ensuring reliable and high-quality assay results. In the presented study, international proficiency testing was employed to evaluate the tested method (Reference Institute for Bioanalytics, Bonn, Germany).\u003c/p\u003e\u003cp\u003eThe limited number of patient samples may restrict the ability to draw broad conclusions about the clinical equivalence of the tested methods for MTX determination (LC-MS/MS, EMIT and EIA). However, the sample size was adequate for meaningful cross-correlation and clinical analyses. Future research should include a larger number of samples from multiple TDM laboratories.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThis study successfully developed and validated a fast, sensitive LC-MS/MS method for measuring methotrexate (MTX) levels in plasma from oncology patients receiving high-dose therapy. It complied with rigorous international standards (ICH M10, EMA, IATDMCT) and showed excellent analytical performance. A comparative analysis indicated that while immunochemical assays (EMIT and EIA) provide acceptable accuracy for routine monitoring, they can cross-react with MTX metabolites, such as DAMPA, potentially leading to overestimated MTX levels and unnecessary extension of leucovorin rescue therapy. Although immunoassays are practical due to their ease, lower cost, and rapid results, LC-MS/MS remains the definitive method for MTX quantification in critical cases requiring maximum specificity and accuracy. In summary, LC-MS/MS should be the preferred method for monitoring MTX in high-dose treatments, especially when precise measurement is crucial for patient safety and effective therapy. Nonetheless, immunoassays remain an accessible and cost-effective alternative when LC-MS/MS is not available, underscoring their ongoing importance in clinical routine TDM services for MTX.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eConceptualization, A.C. and A.K.; methodology, A.C. and A.K.; software, A.C, M.P. and A.S.; validation, A.M., A.C. and A.K.; formal analysis, A.M., A.C. and M.S.; investigation, A.K.; resources, A.C.; data curation, A.K.; writing—original draft preparation, A.C., A.M., M.P., A.S. and M.S.; writing—review and editing, A.K.; visualization, A.K.; supervision, A.K.; project administration, A.C. and A.K.; funding acquisition, A.C., A.M. and A.K. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The research (especially LC-MS/MS tests) was partially funded by the Medical University of Warsaw, grant number 3/F/MG/N/24.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eThis study was conducted in accordance with the principles of the Declaration of Helsinki. The presented study presented results of routine diagnostic testing for methotrexate in patients treated at the Children’s Memorial Institute in Warsaw. Approval reference number: 2377/2023.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eInformed consent was obtained from all subjects involved in the study as routine diagnostic process of MTX measurements in the Children’s Memorial Health Institute in Warsaw.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement: The d\u003c/strong\u003eata will be made available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e The authors thank Altium Poland Sp. z.o.o for the possibility of testing the VITROS 5600 system for MTX measurements based on the Altium and CMHI agreement: 2377/2023. The part of the presented study is Aleksandra Mikulska’s master’s thesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKocur A, Mikulska A, Moczulski M, Pawiński T. Therapeutic Drug Monitoring of Low Methotrexate Doses for Drug Exposure and Adherence Assessment\u0026mdash;Pre-Analytical Variables, Bioanalytical Issues, and Current Clinical Applications. Int J Mol Sci. 2024;25:13430.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLev\u0026ecirc;que D, Becker G, Toussaint E, Fornecker L-M, Paillard C. Clinical Pharmacokinetics of Methotrexate in Oncology. Int J Pharmacokinet. 2017;2:137\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMikulska A, Kocur A. 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Anal Methods. 2014;6:178\u0026ndash;86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/C3AY40815A\u003c/span\u003e\u003cspan address=\"10.1039/C3AY40815A\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBouqui\u0026eacute; R, Gr\u0026eacute;goire M, Hernando H, Azoulay C, Dailly E, Monteil-Gani\u0026egrave;re C, Pineau A, Deslandes G, Jolliet P. Evaluation of a Methotrexate Chemiluminescent Microparticle Immunoassay: Comparison to Fluorescence Polarization Immunoassay and Liquid Chromatography-Tandem Mass Spectrometry. Am J Clin Pathol. 2016;146:119\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHayashi H, Fujimaki C, Tsuboi S, Matsuyama T, Daimon T, Itoh K. Application of Fluorescence Polarization Immunoassay for Determination of MethotrexatePolyglutamates in Rheumatoid Arthritis Patients. Tohoku J Exp Med. 2008;215:95\u0026ndash;101.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOpitz P, Fobker M, Fabian J, Hempel G. Development and Validation of a Bioanalytical Method for the Quantification of Methotrexate from Serum and Capillary Blood Using Volumetric Absorptive Microsampling (VAMS) and OnLine Solid Phase Extraction LCMS. J Chromatogr A. 2024;1715:464610.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"pharmacological-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prep","sideBox":"Learn more about [Pharmacological Reports](https://link.springer.com/journal/43440)","snPcode":"43440","submissionUrl":"https://submission.springernature.com/new-submission/43440/3","title":"Pharmacological Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"methotrexate, LC-MS/MS, EMIT, EIA, therapeutic drug monitoring, pediatric oncology","lastPublishedDoi":"10.21203/rs.3.rs-7529927/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7529927/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMethotrexate (MTX) is a widely used chemotherapeutic agent in pediatric oncology, where high-dose protocols (HDMTX; \u0026gt;500 mg/m\u0026sup2;) are standard for treating hematological and central nervous system malignancies. Due to its narrow therapeutic index and potential for severe toxicity, therapeutic drug monitoring (TDM) of plasma MTX concentrations is essential to guide leucovorin rescue therapy and prevent adverse effects. The presented study aimed to compare the analytical performance of two immunoassays\u0026mdash;enzyme-multiplied immunoassay technique (EMIT) and enzyme immunoassay (EIA)\u0026mdash;against a newly developed and validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The LC-MS/MS assay demonstrated excellent linearity, sensitivity (LLOQ\u0026thinsp;=\u0026thinsp;0.01 \u0026micro;mol/L), and precision, meeting ICH M10 regulatory guidelines. Clinical samples from pediatric patients receiving HDMTX were analyzed using all three methods. Results showed strong correlations (r\u0026thinsp;\u0026gt;\u0026thinsp;0.93) between methods; however, immunoassays exhibited biases related to cross-reactivity with MTX metabolites such as DAMPA (2, 4-diamino-N(10)-methylpteroic acid) and 7-OH-MTX, which may lead to overestimation of MTX levels and unnecessary prolongation of leucovorin rescue. While immunoassays remain practical for routine monitoring due to their accessibility and speed, LC-MS/MS provides superior accuracy and should be the method of choice in critical clinical situations. These findings underscore the importance of selecting the appropriate assay in optimizing HDMTX therapy and ensuring patient safety.\u003c/p\u003e","manuscriptTitle":"Novel LC-MS/MS Method for Measuring Methotrexate in High-Dose Therapy: A Comparative Study with Commercial EMIT and EIA Immunoassays","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 11:09:13","doi":"10.21203/rs.3.rs-7529927/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-17T06:23:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-22T09:05:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-22T08:26:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"307313882539511532579020839105526551449","date":"2025-09-15T09:05:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"339573140065960133101748338784556561444","date":"2025-09-12T09:53:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"144251601416828519927041473464035451640","date":"2025-09-12T08:33:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"232650321210810722363344260182993479859","date":"2025-09-12T07:56:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-10T07:45:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-05T15:13:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-05T03:01:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Pharmacological Reports","date":"2025-09-03T19:33:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"pharmacological-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prep","sideBox":"Learn more about [Pharmacological Reports](https://link.springer.com/journal/43440)","snPcode":"43440","submissionUrl":"https://submission.springernature.com/new-submission/43440/3","title":"Pharmacological Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c37732d1-359c-41b3-a4c5-43785ae3f1c1","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T16:10:33+00:00","versionOfRecord":{"articleIdentity":"rs-7529927","link":"https://doi.org/10.1007/s43440-025-00807-5","journal":{"identity":"pharmacological-reports","isVorOnly":false,"title":"Pharmacological Reports"},"publishedOn":"2025-11-19 15:59:10","publishedOnDateReadable":"November 19th, 2025"},"versionCreatedAt":"2025-09-17 11:09:13","video":"","vorDoi":"10.1007/s43440-025-00807-5","vorDoiUrl":"https://doi.org/10.1007/s43440-025-00807-5","workflowStages":[]},"version":"v1","identity":"rs-7529927","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7529927","identity":"rs-7529927","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}