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Title: Determination of Folates in Human Plasma Using Hydrophilic Interaction ChromatographyTandem Mass Spe


1
Determination of Folates in Human Plasma Using
Hydrophilic Interaction ChromatographyTandem
Mass Spectrometry
Spiros D. Garbis, Alida Melse-Bonnstra, Clive E.
West, and Richard B. van Breeman Department of
Medicinal Chemistry and Pharmacognosy, College of
Pharmacy, University of Illinois at Chicago,
Department of Human Nutrition and Epidemiology,
Wageningen University, The Netherlands, and
Department of Gastroenterology, University
Medical Centre, Nijmegen, The Netherlands
Barrett Little March 31, 2006
2
What are Folates?
  • The term folates refers to folic acid (a) and its
    derivatives that are found in the human body
  • The derivatives of folic acid found in human
    plasma are tetrahydrofolate (b),
  • 5-methyltetrahydrofolate (c), and
    5-formyltetrahydrofolate (d)

c.
a.
b.
d.
3
Why Folates Are Important
  • Folic acid is a B vitamin important in cellular
    function
  • Folates are needed for the synthesis of adenine
    and thymine, two of the four nucleic acids that
    make up DNA.
  • A deficiency in folate is linked to many health
    disorders
  • Alzheimers Disease
  • Depression
  • Cardiovascular Disease
  • Cancer
  • Neural Tube Defects (Spina Bifida) in Unborn
    Children
  • Anemia
  • The body is unable to make or store folic acid so
    we must obtain folate from the food we eat
  • Quantitative analysis of folates in the human
    body can help prevent, diagnose, and treat many
    of these diseases

4
Shortcomings of the Classic Methods to Determine
Total Folate Content
  • Microbiological assay using the bacteria
    Lactobacillus casei
  • Based on the quantitative relationship between
    folate content and growth of Lactobacillus casei
  • Limited by a lack of information on the
    individual folate forms, multiple interferences
    from the sample matrix, and significant growth
    response differences to various folate forms
  • Various High-Performance Liquid Chromatography
    (HPLC) methods with UV, fluorescence, or
    electrochemical detection
  • HPLC methods include reversed-phase, affinity,
    ion exchange, and ion pair chromatography
  • All methods lack specificity and sensitivity and
    require complex sample preparation
  • Gas Chromatography/mass spectrometry (GC/MS)
  • Has substantial amount of selectivity
  • Requires elaborate sample preparation including
    chemical derivatization that results in new
    sources of experimental error and an increase in
    the possibility of folate degradation
  • HPLC-mass spectrometry
  • Selectivity too low for detection of specific
    folates in human plasma
  • Heat pretreatment may contribute to folate
    degradation

5
A New Method
  • Goal The development of a more selective, fast,
    robust, and more sensitive method to analyze
    folic acid and its derivatives in human plasma
  • An HPLC-Tandem Mass Spectrometric method
    (LC-MS-MS) was chosen for evaluation
  • Ideally, the method could simultaneously measure
    levels of folic acid, tetrahydrofolate,
    5-methyltetrahydrofolate, and 5-formyl-tetrathyd
    rofolate in human plasma

6
What is HPLC?
  • HPLC is an analytical process utilizing special
    instruments designed to separate, quantify, and
    analyze components of a chemical mixture
  • HPLC has two phases
  • A stationary phase which refers to the solid
    support contained within the HPLC column
  • The column is usually packed with silica
    particles that have a bonded phase attached to
    them designed to non-covalently interact with the
    analyte molecules
  • The mobile phase refers to solvent continuously
    being pumped through the stationary phase
  • The sample solution is injected into the mobile
    phase and flows with the mobile phase through the
    stationary phase
  • The components of the solution will migrate
    through the column according to the non-covalent
    interactions of the components with the
    stationary phase
  • The mobile phase can be altered to in order to
    manipulate the interactions of the components of
    the solution with the stationary phase

7
Reversed Phase vs. Normal Phase HPLC
  • Reversed Phase HPLC
  • Non-polar stationary phase and polar mobile phase
  • Results in the elution of hydrophilic compounds
    in the sample more quickly than hydrophobic
    compounds
  • Stationary phase is commonly composed of
    hydrophobic alkyl chains bonded to silica
    particles with lengths of C4, C8, and C18
  • Normal Phase HPLC
  • Polar stationary phase and a less polar mobile
    phase
  • Results in the elution of hydrophobic compounds
    in the sample more quickly than hydrophilic
    compounds
  • Stationary phase is commonly composed of polar
    materials such as cyano, amino, and diol

8
A Variation of Normal Phase HPLC
  • Hydrophilic Interaction Chromatography (HILIC) is
    a variation of normal phase chromatography where
    solvents that are miscible water are used
  • Stationary phase is polar and mobile phase is
    highly organic with a small amount of water (75
    acetonitrile/25 water)
  • Retention of highly polar analyte molecules
  • Advantages
  • Enhanced sensitivity in mass spectrometry
  • Shortens sample preparation procedure

9
Electrospray Ionization (ESI)
  • The authors chose a LC-MS-MS system that utilized
    electrospray ionization tandem mass spectrometry
  • Electrospray ionization tandem mass spectrometry
    was preferred because it is highly compatible
    with HPLC
  • Electrospray is a method of generating a very
    fine liquid aerosol through electrostatic
    charging
  • How it works
  • The analyte molecules are introduced to the ESI
    source in solution as the eluent flow from the
    HPLC system
  • The analyte solution flow passes through the
    electrospray needle that has a large voltage
    applied to it
  • The voltage forces the spraying of charged
    droplets of analyte solution from the needle with
    a surface charge of the same polarity to the
    charge on the needle
  • The charged droplets are repelled by the needle
    and travel towards the counter electrode
  • As the droplets travel the space between the
    needle and the counter electrode solvent
    evaporation occurs and the droplets become
    increasingly smaller
  • This pushes the like-charged analyte molecules
    closer together until they reach a point called
    the Rayleigh limit where the surface tension can
    no longer sustain the charge
  • A Coulombic explosion occurs and rips the
    droplets apart
  • This process occurs repeatedly until the analyte
    enters the gas phase as an ion
  • The method is a soft method of ionization because
    little or no fragmentation occurs
  • The analyte ions then enter the first mass
    spectrometer
  • Voltage can be adjusted to produce negative or
    positive charged analyte ions

10
ESI Schematic
11
ESI Schematic
12
Tandem Mass Spectrometry
  • After electrospray ionization, the analyte ions
    enter the first of two mass spectrometers
  • In the first MS the m/z ratio is measured for the
    analyte ions that have not been fragmented
  • Before being detected by the second mass
    spectrometer, the analyte ions are subjected to
    collision induced dissociation
  • The precursor ions are bombarded by a high energy
    gas (Argon) that causes the ions to fragment
  • The fragmented ions are then detected and
    measured by the second mass spectrometer

13
The Experiment
  • Seven different HPLC columns and eleven mobile
    phases were evaluated for the separation of the
    folate species spiked into human plasma under
    conditions compatible with electrospray
    ionization mass spectrometry (Table 1)
  • Stationary Phases
  • HPLC reversed phase systems (1-4)
  • C18 on porous silica
  • C18 on non-porous silica
  • C18 on copolymer support
  • HPLC normal phase systems (5-7)
  • Cyanopropyl silica
  • Aminopropyl silica
  • HILIC polyhydroxyethyl aspartamide
  • Mobile Phases
  • Each reversed phase system utilized two different
    mobile phases
  • A 9010 mixture of 20-25 mM ammonium
    acetate/acetonitrile at a pH of 7 for negative
    ion electrospray (favors deprotonation of folate
    species)
  • A 9010 mixture of 20-25 mM ammonium
    formate/acetonitrile at a pH of 3.5 for positive
    ion electrospray (favors protonation of folate
    species)
  • Normal phase systems 5 and 6
  • A 6040 mixture of methanol/acetonitrile
  • Normal phase system 7 (HILIC)
  • An isocratic 7525 mixture of acetonitrile/water
    with 5 mM ammonium acetate at a pH of 6.9

14
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15
Results
  • Reversed Phase HPLC
  • pH of 7 (negative ion electrospray)
  • Retention times were all less than 3.5 minutes
    which are inadequate for separation
  • Coeluting plasma components suppressed ionization
  • pH of 3.5 (positive ion electrospray)
  • Retention times increased to between 5 and 14
    minutes
  • Coeluting plasma components interfered with
    analyte detection
  • Conclusions on reversed phase methods
  • Although acidification resulted in separation of
    the folates, the positive ion electrospray mass
    spectra were complicated by the appearance of
    sodium and potassium adducts of the analyte (from
    plasma components)
  • Preliminary extraction of the plasma sample was
    necessary prior to LC-MS analysis to eliminate
    interference from the plasma components
  • In general, the folates were too polar for
    adequate retention and separation on reversed
    phase columns

16
Results Why Negative Ion Electrospray is
Preferred to Positive Ion Electrospray
  • Figure 1. Comparison of (A) positive ion and (B)
    negative ion electrospray mass spectra of folic
    acid. A variable mixture of cationized species
    including protonated molecules, sodium adducts,
    and potassium adducts were detected in positive
    ion mode, whereas only abundant deprotonated
    molecules were detected in negative ion mode.
  • 2Na (m/z 22)
  • 2K (m/z 38)
  • Only negative ion electrospray ionization was
    utilized further

17
Results (contd)
  • Normal Phase HPLC
  • Cyanopropyl silica column
  • Retention time was gt80 minutes
  • Broad and tailing peaks were detected BAD
  • Preliminary extraction of the plasma sample was
    also necessary prior to LC-MS analysis in order
    to eliminate interference from the plasma
    components
  • Aminopropyl silica column
  • No sample ions were detected
  • HILIC polyhydroxyethyl aspartamide
  • Resulted in baseline resolution of the four
    folate species

18
MS-MS Product Ion Spectra
Folic Acid
5-methyltetrahydrofolate
m/z 458.1 missing
5-formyltetrahydrofolate
m/z 444.1 missing
Tetrahydrofolate
m/z 472.1 missing
  • .Figure 2. (A) Negative ion electrospray tandem
    mass spectrum with CID of the deprotonated
    molecule of folic acid (precursor ion of m/z
    440.1). (B) Negative ion electrospray product ion
    mass spectrum with CID of deprotonated
    tetrahydrofolate (precursor ion of m/z 444.1).
    Note that the deprotonated molecule of m/z 444.1
    fragmented so extensively that only product ions
    were detected. (C) Negative ion electrospray
    tandem mass spectrum with CID of deprotonated
    5-methyltetrahydrofolate (precursor ion of m/z
    458.1). Under the conditions of this CID
    analysis, the deprotonated molecule of m/z 458.1
    fragmented so extensively that no intact
    precursor ions were detected. (D) Negative ion
    electrospray tandem mass spectrum with CID of the
    deprotonated molecule of 5-formyltetrahydrofolate
    (precursor ion of m/z 472.1). Note that the
    deprotonated molecule of m/z 472.1 completely
    dissociated during CID.

19
LC-MS-MS Results
  • Based on the MS-MS results, the characteristic
    transitions for each folate species were selected
    to be measured for LC-MS-MS quantitative analysis
    as follows
  • Folic acid- m/z 440?311 (elimination of a
    glutamyl group from the deprotonated molecule)
  • Tetrahydrofolate- m/z 444?176 (transition to
    glutamate product ion from the deprotonated
    molecule)
  • 5-methyltetrahydrofolate- m/z 458?329
    (elimination of a glutamyl group from the
    deprotonated molecule)
  • 5-formyltetrahydrofolate- m/z 472?315
    (elimination of glutamyl group and CO from the
    deprotonated molecule)
  • The results are shown for the LC-MS-MS analysis
    in Figure 3.

Figure 3. LC-MS-MS analysis of folate species
spiked into human plasma. HILIC was used with
negative ion electrospray mass spectrometric
detection.
20
An Analysis on Unspiked Human Plasma Sample
  • Standard curves for each of the folate species
    were obtained
  • An unspiked sample of human plasma was
    quantitatively analyzed for its
    5-methyl-tetrahydrofolate composition by the
    LC-MS-MS method already described
  • Based on the standard curve, the level of
    5-methyltetrahydrofolate in the unspiked sample
    was determined to be 6.7 µg/L

Figure 4. LC-MS-MS quantitative analysis of
5-methyltetrahydrofolic acid in unspiked human
plasma. Based on the standard curve the level of
folic acid in this sample was determined to be
6.7 µg/L
21
Conclusions
  • HILIC proved successful in the separation of all
    four folate species
  • The use of a predominately organic mobile phase
    was very compatible with negative ion
    electrospray mass spectrometric detection
  • Since the hydrophobic species in the plasma were
    eluted at the solvent front and were directed to
    waste, HILIC provided on-column sample cleanup
    and eliminated the need for extraction of the
    plasma samples prior to LC-MS-MS analysis
  • Degradation of the folate species was minimized
    by simplifying and increasing the speed of sample
    preparation
  • Recoveries of the folate species from the plasma
    samples exceeded 97
  • The measurement of endogenous levels of
    5-methyltetrahydrofolate had never been reported
    using mass spectrometry-based methods previous to
    this paper

22
A Special Thanks to
  • Dr. Stevenson (Steve-O)
  • Dr. Miller (Bert)
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