Key words

1 Introduction

Methotrexate (MTX) is a folic acid antagonist that is widely used as an immunosuppressant and chemotherapeutic agent. MTX exerts its cytotoxic effects by competitively inhibiting dihydrofolate reductase (DHFR), the enzyme responsible for converting folates to tetrahydrofolates; the folate carrier that functions in the transfer of carbon units. A normal dividing cell uses large amounts of reduced folates to maintain ongoing purine and thymidine synthesis and the demand is even greater for rapidly dividing malignant cells [1].

High-dose MTX is mainly used for the treatment of leukemia and osteosarcoma. Intermediate and lower dose MTX regimens are used to treat malignant gestational trophoblastic disease, breast and bladder cancer, ALL, and acute promyelocytic leukemia [1, 2]. In addition to its antiproliferative activity, MTX also has anti-inflammatory and immunomodulating properties and is a first-line treatment for a growing number of autoimmune rheumatologic, dermatologic, and gasteroenterologic conditions [1, 2]. After high-dose administration of MTX serum levels must be monitored to determine when to administer leucovorin, a folic acid analog that bypasses the enzyme inhibition caused by MTX and reverses its toxicity [3]. Patients in renal failure who are given high-dose MTX are sometimes given carboxypeptidase-G2 (CPDG2) (CPDG2) to reverse the effects of MTX [3–5]. CPDG2 is an enzyme that converts MTX into glutamate and 4-amino-4-deoxy-N10-methylpteroic acid (DAMPA) that are much less toxic and readily excreted. DAMPA cross-reacts considerably in immunoassays rendering them unsuitable for monitoring patients who have been given CPDG2 therapy [6].

MTX assays using mass spectrometry have been described previously [7–9]. However the objective of this study was to develop an MTX assay performed by LC-MS with the following characteristics: analytically sensitive with a clinically useful dynamic range; good specificity with no interference from metabolites or other compounds; suitable analytical transferability for a high volume clinical laboratory, and the accurate measurement of 7-OH MTX and DAMPA to support clinical trials utilizing CPDG2 and related compounds. The following chapter describes a rapid and simple turbulent flow method implementing positive heated electrospray ionization for the accurate and precise determination of MTX, 7-OH MTX, and DAMPA concentrations in serum.

2 Materials

2.1 Samples

Serum samples are required. All samples should be processed and analyzed within 4 h of collection or refrigerated for analysis up to 24 h after collection or frozen for analysis up to 6 months.

2.2 Solvents and Reagents

  1. 1.

    Human drug-free pooled normal serum (UTAK Laboratories).

  2. 2.

    Mobile Phase A (10 mM ammonium formate/0.1 % formic acid in water): Remove 8.4 mL of water from a 4 L bottle. Add 2.8 mL of ammonium hydroxide, cap, and invert ten times. Add 5.6 mL of formic acid and degas for 5 min by sonication. The mobile phase is stable at room temperature, 18–24 °C, up to 1 month.

  3. 3.

    Mobile phase B (10 mM ammonium formate/0.1 % formic acid in methanol): Remove 8.4 mL of methanol from a 4 L bottle. Add 2.8 mL of ammonium hydroxide, cap, and invert ten times. Add 5.6 mL of formic acid and degas for 5 min by sonication. The mobile phase is stable at room temperature, 18–24 °C, up to 1 month.

  4. 4.

    Mobile phase C (acetonitrile/2-propanol/acetone, 6:3:1): In a 1000 mL graduated cylinder, add 600 mL acetonitrile, 300 mL of 2-propanol, and 100 mL of acetone into a 2 L HPLC solvent bottle. Degas the solution for 5 min by sonication. The mobile phase is stable at room temperature, 18–24 °C, up to 1 month.

  5. 5.

    Autosampler aqueous wash (water/acetic acid/acetonitrile, 8.8:1:0.2): In a 500 mL graduated cylinder, add 440 mL of water, 50 mL of acetic acid, and 10 mL of acetonitrile and transfer into an HPLC wash bottle. Degas the solution for 5 min by sonication. The wash solution is stable at room temperature, 18–24 °C, up to 1 month.

  6. 6.

    Autosampler organic wash (acetonitrile/2-propanol/acetone, 6:3:1): In a 1000 mL graduated cylinder, add 600 mL acetonitrile, 300 mL of 2-propanol, and 100 mL of acetone and transfer into a 2 L HPLC solvent bottle. Degas the solution for 5 min by sonication. The mobile phase is stable at room temperature, 18–24 °C, up to 1 month.

  7. 7.

    Extraction solution (30 ng/mL MTX-D3 in methanol with 0.1 % formic acid): Add approximately 150 mL of methanol and 200 μL formic acid into a 200 mL volumetric flask. Add 60 μL of MTX-D3 stock (100 μg/mL) to the same volumetric flask. Bring to volume with methanol and mix well. The extraction solution is stable for up to 6 months when stored at −20 °C.

2.3 Internal Standards and Standards

  1. 1.

    Primary standards: MTX (Sigma-Aldrich), 7-OH MTX (Santa Cruz Biotechnology Inc.), and DAMPA (Schircks Laboratories).

  2. 2.

    Primary internal standard (I.S.): MTX-D3 (Cerilliant) 100 μg/mL in 0.1 N sodium hydroxide.

  3. 3.

    Standard stock solutions:

    1. (a)

      Methotrexate MTX (1 mg/mL): Using an analytical balance weigh 25 mg and place in a 25 mL volumetric flask. Bring to volume with methanol containing 0.1 N sodium hydroxide and mix well. Then prepare a 100 μg/mL stock solution from the previous 1 mg/mL stock solution: Add 900 μL of methanol to a 2 mL amber vial and add 100 μL of MTX stock (1 mg/mL) and mix well. Both stock solutions are stable up to 6 months when stored at −20 °C.

    2. (b)

      7-Hydroxymethotrexate, 7-OH MTX (1 mg/mL): Using an analytical balance weigh 25 mg and place in a 25 mL volumetric flask. Bring to volume with methanol containing 0.1 N sodium hydroxide and mix well. Then prepare a 100 μg/mL stock solution from the previous: Add 900 μL of methanol to a 2 mL amber vial and add 100 μL of 7-OH MTX stock (1 mg/mL) and mix well. Both stock solutions are stable for up to 6 months when stored at −20 °C.

    3. (c)

      DAMPA (1 mg/mL): Using an analytical balance weigh 25 mg and place in a 25 mL volumetric flask. Bring to volume with methanol containing 0.1 N sodium hydroxide and mix well. Then prepare a 100 μg/mL stock solution from the previous: Add 900 μL of methanol to a 2 mL amber vial and add 100 μL of DAMPA stock (1 mg/mL) and mix well. Both stock solutions are stable for up to 6 months when stored at −20 °C.

    4. (d)

      Combined standard (MTX, 7-OH MTX, and DAMPA): To obtain a combined standard containing, 4.54 μg/mL, 9.40 μg/mL, and 3.25 μg/mL respectively, add 1656 μL of drug-free serum to a 2 mL amber glass vial. Then add 91, 188, and 65 μL of MTX, 7-OH MTX, and DAMPA from each respective 100 μg/mL stock solution and mix well. Stock solution is stable for up to 6 months when stored at −20 °C.

2.4 Calibrators and Controls

  1. 1.

    Calibrators: Prepare calibrators 1–8 by making serial dilutions of the combined standard according to Table 1. For each dilution step add the appropriate amount of previous solution as shown in the table to a 10.0 mL volumetric flask and fill with drug-free human serum. Vortex mix the volumetric flask after each dilution step (see Note 1 ).

    Table 1 Calibrator preparation
    Full size table
  2. 2.

    Controls: MTX controls were purchased from UTAK laboratories at the following concentrations: 0.023, 0.034, 0.227, and 0.341 μg/mL. Currently there are no commercially available controls for 7-OH MTX and DAMPA. To prepare 7-OH MTX and DAMPA controls follow Table 2 for the procedure. For each control add the appropriate amount of 100 μg/mL stock solution into a 10 mL volumetric flask then bring to volume with drug-free serum. These two sets of controls are made on separate days and from separate lots of material than the calibrators (see Note 1 ).

    Table 2 7-OH MTX and DAMPA control preparation
    Full size table
  3. 3.

    Check the new lot of standards by verifying five unknown patient samples concentrations with the current lot of calibrators. The agreement between the two calculated concentrations must be within 10 %.

  4. 4.

    Establish a range for the new lot of controls by collecting data points over 20 consecutive runs and establish the mean and standard deviation.

2.5 Analytical Equipment and Supplies

  1. 1.

    Thermo Scientific Transcend TLX-2 with Agilent 1200 pumps coupled to a TSQ Quantum Ultra triple quadrupole mass spectrometer running Aria software 1.6.3 (Thermo Scientific).

  2. 2.

    TurboFlow column (TFC): Cyclone-P 50 × 0.5 mm, 60 μm particle size, 60 Å pore size (Thermo Scientific). Analytical column: Hypersil Gold C8 2.1 × 50 mm, 5 μm particle size (Thermo Scientific).

  3. 3.

    Column heater (Thermo Scientific).

  4. 4.

    Eppendorf 1.5 mL microcentrifuge tubes and National Scientific 2 mL amber glass vials with inserts and pre-slit caps or equivalent.

2.6 Instrument Operating Conditions

  1. 1.

    Turbulent flow liquid chromatography (TFLC) : Chromatographic separations were performed using a Thermo Scientific Transcend TLX-2 which was comprised of a PAL autosampler (CTC Analytics), a low-pressure mixing quaternary pump (loading pump), a high-pressure binary pump (eluting pump), and a six-valve switching module with six-port valves (Fig. 1). The system was controlled via Aria software, version 1.6.2. The TurboFlow column used was a Cyclone-P 50 × 0.5 mm, 60 μm particle size, and a 60 Å pore size. The analytical HPLC column was a Hypersil Gold C8 2.1 × 50 mm, 5 μm particle size. The temperature of the analytical column was maintained at 70 °C using a column heater. The analytes were loaded on the TurboFlow column in 100 % mobile phase A and transferred to the HPLC column with 80 % mobile phase B using a 200 μL transfer loop. The loading and eluting mobile phase composition for the HPLC column was identical to that of the TurboFlow column. The integration parameters for all four analytes were similar with a baseline window of 20, area noise factor of 5, peak noise factor of 10, and an integration window of 15 s. The retention times of the TFLC-ESI-MS/MS ion chromatograms of the analytes can be seen in Fig. 2.

    Fig. 1
    figure 1

    Transcend TLX-2 valve configuration

    Full size image
    Fig. 2
    figure 2

    TFLC-ESI-MS/MS ion chromatograms of MTX, 7-OH MTX, DAMPA, and MTX-D3 (I.S.) product ions

    Full size image
  2. 2.

    Tandem mass spectrometry : Mass spectrometric detection was performed using a Thermo Scientific TSQ Quantum Ultra triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source operating in a positive ion mode. MS/MS conditions are depicted in Table 4. Nitrogen (99.995 % purity) was used as the desolvation gas, and ultra-pure argon (99.999 % purity) was used as the collision gas. The mass transitions from the protonated molecular ion [M+H]+ to the most abundant product ions were used as the quantifying ions for each analyte (Table 5). The SRM acquisition method was run in unit resolution (0.7) in both Q1 and Q3 with a scan width and scan rate of 0.050 m/z and 0.100 s, respectively.

3 Methods

3.1 Stepwise Procedure

  1. 1.

    Run a system suitability to confirm the system performance (see Note 2 ).

  2. 2.

    To a 1.5 mL microcentrifuge tubes pipette 100 μL serum (calibrators, controls, or patient specimen) (see Note 3 ).

  3. 3.

    Add 200 μL extraction solution.

  4. 4.

    Cap and vortex each sample vigorously.

  5. 5.

    Centrifuge for 10 min at 13,000 × g.

  6. 6.

    Transfer 200 μL of the supernatant into glass inserts and place in 2 mL amber glass vials.

  7. 7.

    Place all samples in the autosampler and inject 50 μL of the sample into the TFLC-ESI-MS/MS. Ion chromatograms for all analytes are shown in Fig. 2.

3.2 Analysis

  1. 1.

    Instrumental operating parameters are shown in Tables 3 and 4.

    Table 3 TurboFlow and HPLC operating parameters
    Full size table
    Table 4 MS/MS tune settings
    Full size table
  2. 2.

    The data are analyzed using LCquan 2.6 software (Thermo Scientific).

  3. 3.

    Standard curves are based on linear regression analysis for MTX, 7-OH MTX, and DAMPA. Weighted linear regression models with weights inversely proportional to the X values were used. The analysis compared I.S. peak area to sample peak area (y-axis) versus analyte concentration (x-axis) using the quantifying ions indicated in Table 5.

    Table 5 Analyte precursor and product ions
    Full size table
  4. 4.

    Acceptability of each run is confirmed if the calculated control concentrations fall within two standard deviations of the target mean values. Target values are established as the mean of 20 separate runs. If any control is greater than three standard deviations from the mean the run cannot proceed and troubleshooting procedure must commence.

  5. 5.

    Typical coefficients of correlation of the standard curve are >0.995 (see Note 4 ).

4 Notes

  1. 1.

    Individual sets of calibrators and controls can be aliquoted and stored at −80 °C for 1 year. For each calibrator or control, aliquot 125 μL into a glass insert and place in a 2 mL amber glass vial and cap. Thaw completely before use.

  2. 2.

    A system suitability should be performed each day the method is run. The suitability includes running a test mix with all analytes to ensure proper retention time, integration, and sensitivity.

  3. 3.

    A new standard curve should be generated with each analytical run to ensure method performance.

  4. 4.

    The MTX, 7-OH MTX, and DAMPA assays were linear from 0 to 0.454 μg/mL, 0.007 to 0.940 μg/mL, and 0.003 to 0.325 μg/mL, respectively. Dilutions of 10-, 100-, and 1000-fold were validated for all analytes. Within-day and between-day precisions at concentrations spanning the analytical measurement ranges were less than 10 % for all three analytes.