Key words

1 Introduction

5-Methyltetrahydrofolate (5-MTHF) is the predominant form of folate in cerebrospinal fluid (CSF). Testing for 5-MTHF in CSF is useful to determine a deficiency of folate in the central nervous system. Low 5-MTHF levels are associated with inborn errors of metabolism affecting folate metabolism and in dietary deficiency of folate. Disorders associated with low folate include anemia, developmental delay, seizures, depression, dementia, cerebral folate deficiency , and Kearns-Sayre syndrome [1]. More recent reports indicate that there is reduced uptake of 5-MTHF across the blood–brain barrier due to the presence of auto-antibodies to the folate receptor at the choroid plexus. Patients with cerebral folate deficiency (CFD) are characterized by normal plasma folate in the presence of decreased concentration of 5-MTHF in CSF [2, 3]; patients with CFD have neurological complications. The following is a simple high performance liquid chromatography -tandem mass spectrometry (HPLC -MS/MS) method for determination of cerebrospinal fluid 5-MTHF as a clinical diagnostic test.

2 Materials

2.1 Samples

Human lumbar CSF—Specimen drawn any time during the day will be acceptable. No patient preparation is required. Optimal volume of 1.0 ml (minimum 0.25 mL) CSF should be collected in tube 2 (or 1, 4 or 5) of the collection kit provided by the testing laboratory or in a regular CSF collection tube. If the CSF is clear, the sample should be immediately frozen at the bedside on dry ice. If blood contaminated, the sample should be placed on wet ice, centrifuged within 5 min, and the clear CSF transferred to another vial and frozen on dry ice as soon as possible. CSF is stored at −80 °C until time of testing.

2.2 Solvents and Reagents

  1. 1.

    Magnesium chloride × 6H2O, ACS grade.

  2. 2.

    Sodium phosphate dibasic anhydrous, ACS grade.

  3. 3.

    Mobile Phase A (0.1 % formic acid in HPLC -grade water): In a hood add 1 mL of formic acid to a 1 L volumetric flask, bring to volume with water, and mix. Stable at room temperature, 18–24 °C, up to 3 months.

  4. 4.

    Mobile Phase B (0.1 % formic acid in methanol): In a hood, add 1 mL of formic acid to a 1 L volumetric flask, bring to volume with methanol, and mix. Stable at room temperature, 18–24 °C, up to 3 months.

  5. 5.

    10× Artificial CSF (aCSF): 1450 mM NaCl, 27 mM KCl, 10 mM MgCl2, 12 mM CaCl2, 20 mM Na2HPO4.

    1. (a)

      Weigh the following and combine in 100 mL volumetric flask containing 50 mL water: 8.474 g NaCl, 0.201 g KCl, 0.203 g MgCl2, 0.176 g CaCl2, 0.284 g Na2HPO4.

    2. (b)

      Bring to volume with water.

    3. (c)

      Add small magnetic stir bar and mix on magnetic stirrer until dissolved.

    4. (d)

      Adjust pH to 7.4 with 85 % phosphoric acid.

    5. (e)

      Store at 2–8 °C for up to 1 year.

  6. 6.

    1× aCSF: Add 1 mL 10× aCSF to a 10 mL volumetric flask and bring to volume with water. 1× aCSF is stable for up to 8 h at 2–8 °C and must be made fresh daily.

2.3 Internal Standards and Standards

  1. 1.

    Primary standard: 5-MTHF ((6S)-5-Methyl-5,6,7,8-tetrahydrofolic acid, calcium salt) (Schircks Laboratories).

  2. 2.

    Primary internal standard (I.S.): 5-MTHF-13C5 (Calcium-l-Mefolinate-13C5) (Merck Eprova).

  3. 3.

    5-MTHF Standard Stock Solution (1 mM): Add 49.8 mg 5-MTHF to 100 mL volumetric flask, bring to volume with water containing 1 mg/mL ascorbic acid. Wrap flask with foil and sonicate for 5 min. Store in 125 μL aliquots at −80 °C for up to 4 years (see Note 1 ).

  4. 4.

    5-MTHF-13C5 I.S. Stock Solution (1 mM): Add 5 mg 5-MTHF-13C5 to 10 mL volumetric flask, bring to volume with water containing 1 mg/mL ascorbic acid. Wrap flask with foil and sonicate for 5 min. Store in 125 μL aliquots at −80 °C for up to 4 years (see Note 1 ).

  5. 5.

    I.S. Working Solution (5-MTHF-13C5 prepared in water containing ascorbic acid and dithiothreitol): Add 2 μL 1 mM 5-MTHF-13C5 to 2 mL of water containing 40 mg ascorbic acid and 18 mg dithiothreitol in a 2 mL screw-top tube and mix by vortex. Working internal standard may be stored in the refrigerator at 0–10 °C for up to 8 h. Volume of internal standard may be increased to process the number of specimens within the assay.

2.4 Calibrators and Controls

  1. 1.

    Calibrators: 5-MTHF Working Standard Curve, dilute stock solution 1 mM 5-MTHF as follows:

    1. (a)

      Dilution A (100 μM): Add 100 μL of 1 mM 5-MTHF stock solution to 900 μL of 1× aCSF and mix well by vortex.

    2. (b)

      Dilution B (10 μM): Add 100 μL Dilution A to 900 μL of 1× aCSF and mix well by vortex.

    3. (c)

      Dilution C (1 μM): Add 100 μL Dilution B to 900 μL of 1× aCSF and mix well by vortex.

    4. (d)

      Working Standard Curve (25–400 nM): Add 400 μL of Dilution C to 600 μL of 1× aCSF and mix well by vortex. Perform four additional serial dilutions by adding 500 μL of previous standard to 500 μL of 1× aCSF. This will provide a calibration curve of (400, 200, 100, 50, 25 nM). Working standard curve may be stored in the refrigerator at 4 °C for up to 8 h (see Note 2 ).

  2. 2.

    Normal Control: (5-MTHF = 80–240 nM target value):

    1. (a)

      Prepare 10 mL pooled CSF.

    2. (b)

      Assay pooled CSF to quantitate the native concentration of 5-MTHF.

    3. (c)

      Spike or dilute pooled CSF with diluted stock standard or water to obtain a final concentration of 80–240 nM 5-MTHF. Store in 80 μL aliquots at −80 °C for up to 4 years (see Note 1 ).

  3. 3.

    Abnormal Control: (5-MTHF = 20–40 nM target value):

    1. (a)

      Prepare 10 mL pooled CSF.

    2. (b)

      Assay pooled CSF to quantitate the native concentration of 5-MTHF.

    3. (c)

      Spike or dilute pooled CSF with diluted stock standard or water to obtain a final concentration of 20–40 nM 5-MTHF. Store in 80 μL aliquots at −80 °C for up to 4 years (see Note 1 ).

2.5 Analytical Equipment and Supplies

  1. 1.

    Shimadzu Prominence liquid chromatograph system with ABSciex 4000QTRAP® with Analyst software version 1.6.2.

  2. 2.

    Analytical Column: Phenomenex Synergi-Hydro, 4 μm, 150 × 3 mm.

  3. 3.

    Guard Column: Phenomenex Security Guard, 5 μm, 4 × 3 mm.

  4. 4.

    1.5 mL microcentrifuge tubes.

3 Methods

3.1 Sample Preparation

  1. 1.

    To labeled 1.5 mL microcentrifugal units, pipette 50 μL sample (calibrators, controls, patient CSF).

  2. 2.

    Add 50 μL of 5-MTHF-13C5 I.S. Working Solution.

  3. 3.

    Cap and vortex mix tubes at maximum speed for 3 s.

  4. 4.

    Centrifuge for 10 min at 14,000 × g.

  5. 5.

    Transfer 90 μL of prepared sample into corresponding work list position in 96-well microtiter plate and cover with silicone cover.

  6. 6.

    Place completed 96-well microtiter plate onto refrigerated autosampler (4 °C).

  7. 7.

    Inject 10 μL of sample onto HPLC -ESI-MS/MS. Representative HPLC-ESI-MS/MS ion chromatograms for 5-MTHF and I.S. are shown in Figs. 1 and 2 (see Note 3 ).

    Fig. 1
    figure 1

    HPLC -ESI-MS/MS ion chromatogram of 5-MTHF 100 nM standard. [5-MTHF 1 (m/z 460.3 > 313.3), 5-MTHF 2 (m/z 460.3 > 194.5), and 5-MTHF-13C5 (m/z 465.3 > 313.3)]

    Full size image
    Fig. 2
    figure 2

    HPLC -ESI-MS/MS ion chromatogram of 5-MTHF abnormal QC (30 nM). [5-MTHF 1 (m/z 460.3 > 313.3), 5-MTHF 2 (m/z 460.3 > 194.5), and 5-MTHF-13C5 (m/z 465.3 > 313.3)]

    Full size image

3.2 Data Analysis

  1. 1.

    Instrumental operating parameters are given in Table 1.

    Table 1 HPLC -ESI-MS/MS operating conditions
    Full size table
  2. 2.

    Data are analyzed using Analyst software version 1.6.2 (AB Sciex).

  3. 3.

    Standard curves are generated based on linear regression of the analyte/I.S. peak-area ratio (y) versus analyte concentration (x) using the primary ions indicated in Table 2.

    Table 2 HPLC -ESI-MS/MS operating conditions
    Full size table
  4. 4.

    Acceptability of each run is confirmed if the calculated control concentrations fall within two standard deviations of the target values. Inter-day precision was evaluated by repeated analysis of bi-level QC material analyzed in duplicate over a period of 20 different days.

  5. 5.

    Liquid chromatography retention time window limits for 5-MTHF and 5-MTHF-13C5 are set at 3.7 (±0.2) min.

  6. 6.

    The assay has a lower limit of quantitation of 3 nM for 5-MTHF, with imprecision of <6 % over the entire range. See Note 4 for information regarding ion suppression studies. See Table 3 for age-specific reference range [4].

    Table 3 Age-specific reference range for CSF 5-MTHF
    Full size table

4 Notes

  1. 1.

    Individual sets of 5-MTHF Standard and Internal Standard Stock Solutions and controls can be pre-aliquoted and frozen until use in each analytical run. For each set pipette specified volume of stock standards/control solution into 1.5 mL microfuge tubes and freeze at −80 °C until use. Thaw completely before use. Stable for 4 years at −80 °C.

  2. 2.

    A new standard curve should be prepared with each analytical run to optimize method performance.

  3. 3.

    The controls are analyzed at the beginning of analysis, every five unknowns and at the end of the assay as analysis verification.

  4. 4.

    Ion suppression effects were evaluated by sample infusion method. No significant interferences or ion suppression was identified.