Title: Determination of Folates in Human Plasma Using Hydrophilic Interaction ChromatographyTandem Mass Spe
1Determination 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
2What 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.
3Why 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
4Shortcomings 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 -
5A 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
6What 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
7Reversed 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
8A 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
9Electrospray 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
10ESI Schematic
11ESI Schematic
12Tandem 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
13The 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(No Transcript)
15Results
- 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
16Results 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
17Results (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
18MS-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.
19LC-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.
20An 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
21Conclusions
- 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
22A Special Thanks to