Implication of Phospholipids in Signiﬁcant
Matrix Effects in Negative Ion ESI-MS/MS
Tianyi Zhang, Min Meng,
KC Van Horne, and
Patrick K. Bennett Matrix effects are a major concern for quantitative bioanalytical mass spectrometry. Endogenous
Tandem Labs, phospholipids from blood-derived and tissue matrices have previously been shown to be the primary
Salt Lake City, Utah source of matrix effects and ionization suppression when using positive ion electrospray ionization
(ESI).1-5 This work expands the previous work to provide information on the primary sources of matrix
effects and ionization suppression when using negative ion ESI.
Many separate factors can interfere with proper quantitation in bioanalytical LC/MS/MS. Among the
most signiﬁcant of these are ionization suppression/enhancement and matrix effects. Ionization
suppression/enhancement is a reduction/increase of detected signal that occurs when one or more
species are ionized concurrently. Species that may fall into this category include eluent modiﬁers and
analytical system contaminants (for example, salts) and both endogenous and exogenous species.
Matrix effects are cross-sample differences (i.e., suppression or enhancement) in detected signal that
may result from varying sample composition within a particular sample set and a given analytical
method. Matrix effects can cause a number of analytical problems, including erroneous quantitative
results if stable label internal standard(s) are not used as well as resulting in the inaccurate
determination of a method LLOQ.
PPE extract: 200 µL of human plasma + 500 µL MeOH. Followed by centrifugation,
evaporation, and reconstitution.
Lipid extract: 200 µL of human plasma + 300 µL 10 mM NH4 OAc pH 4.6+1.5 mL
CHCl3 + 0.5 mL MeOH. Followed by vortex-mixing, centrifugation,
evaporation, and reconstitution.
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Category Figures 1-6 Figures 7-8
Column: Phenomenex 5 µm Luna C18(2) Luna Phenyl-Hexyl 50x2 mm, 5 µm
column, 2mm x 30mm
Flow rate: 400 µL/minute 800 µL/minute
Mobile phase A: 0.2% Formic acid in water 10 mM ammonium acetate, pH unadjusted
Mobile phase B: 0.2% Formic acid in acetonitrile 90/10 MeCN/A
Linear gradient: 0’(20%B)-10’(90%B)-15’(90%B) 0’(30%)B-24’(100%B); 24.1’, 30%B
Mass spectrometer: MDS Sciex API 3000 (HSID)
Ionization source: TurboIonSpray
Ionization mode: Negative ionization and positive ionization modes
HPLC: Shimadzu 10 ADvP gradient system
Autosampler: Perkin-Elmer PE200
Sample: - Human plasma protein precipitation extract (PPE)
- Human plasma lipid extract (Lipid)
Injection volume: 20 µL
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Method Results Figures
1. Q1 Full Scan (1a) and Four major peaks were detected at 4.9-5.9 min. They eluted at organic Figure 1
Spectrum (1b) of Human Plasma composition of 40-60%, which is the typical elution composition for most
PPE Extract Under Positive Ion pharmaceutical analytes. The Q1 spectrum of the peak at 5.11 min showed
Mode. two major ion clusters: m/z 496, the monomer of lysophophostidylcholine,
and m/z 992, the dimer of lysophophostidylcholine.
2. Q1 Full Scan (2a) and The same sample from Figure 1 was analyzed back-to-back under negative Figure 2
Spectrum (2b) of Human Plasma ion mode. Three major peaks were detected at 4.9-5.9 min. Their elution
PPE Extract Under Negative Ion times correlate to the peaks shown in Figure 1. The Q1 spectrum at 5.11
Mode. min showed two major ion clusters at m/z 540, presumably the monomer of
the formate adduct of lysophophostidylcholine, and m/z 1036, the formate
adduct of the dimer of lysophophostidylcholine.
3. Q1 Full Scan (3a) and Similar to Figure 1, four major peaks were detected at 4.9-5.9 min. In Figure 3
Spectrum (3b) of Human Plasma addition, the Q1 spectrum of the peak at 5.06 min showed the same
Lipid Extract Under Positive Ion phospholipid ions at m/z 496 and 992 as in Figure 1.
4. Q1 Full Scan (4a) and The same sample from Figure 3 was analyzed back-to-back under negative Figure 4
Spectrum (4b) of Human Plasma ion mode. Similar to Figure 2, three major peaks were detected in Q1 full
Lipid Extract Under Negative Ion scan. The Q1 spectrum at 5.13 min showed the same ion clusters at m/z
Mode. 540 and 1036 that were identiﬁed as phospholipids in Figure 2.
5. Q1 SIM Chromatograms of the Three major Q1 Extracted Ion Chromatograms at m/z 520, 496, and 524 are Figure 5
Main Peaks from Figure 1 Positive shown in Figure 1. These correlate to lysophophostidylcholines 18:2, 16:0,
Q1 Full Scan. and 18:0, respectively.
6. Q1 SIM Chromatograms of Three major Q1 Extracted Ion Chromatograms at m/z 564, 540, and 568 are Figure 6
the Main Peaks from Figure 2 shown in Figure 2. Based on the retention time and the masses, these are
Negative Q1 Full Scan. the negative ion adducts of the same lysophophostidylcholines in Figure 5
(m/z 564 = 520, 540 = 496, and 568 = 524).
7. Ionization Enhancement as a Drug infusion was conducted to examine the effect of phospholipids from Figure 7
Result of Lipids Under Negative human plasma extract injection. The observed MRM drug peaks
Ion Mode. (Figure 7) correlate to the lipid peaks from plasma (Figure 8), indicating
signal enhancement as a result of lipids under negative ion mode.
8. Lipid Peaks and Their m/z From Lipid peaks from human plasma extract and their m/z were obtained from Figure 8
Human Plasma Extract Under Q1 scan under negative ion mode.
Negative Ion Mode.
9. Product Ions from the Infusion Product ion spectra were obtained using the fraction-collected samples. Table 1
Experiment Collected at 5.0-5.5 The subsequent product ion scans showed that there were several common
Minutes. product ions, i.e., m/z 134, 224, 255, 277, 345, and 506. The products
m/z 255, 277, and 281 are typically from the fatty acid fragment of
phospholipids and lysophospholipids.
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Table 1. Product Ions From Infusion Experiment (collected from 5-5.5 min)
Precursor Product Ion Product Ion Product Ion Product Ion Product Ion Product Ion Product Ion Product Ion Product Ion
Ions 1 2 3 4 5 6 7 8 9
345.1 134.2 277.1 345
450.4 224 255.1
540.5 224.3 255.1 480.4
564.5 279 504.2
566.6 223.7 281.2 506.4
577.6 277.2 429.3
645.2 277.1 345 480.1
772.5 277.2 480.1
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Endogenous phospholipids are found in plasma at signiﬁcant concentrations, appear at variable levels
organism–to-organism, and are typically not completely removed during extractions. Phospholipids
contains two distinguishing moieties: a) a polar head group substituent, which includes an ionizable
organic phosphate moiety and b) one or two long chain fatty acid ester groups, which impart
considerable hydrophobicity to the molecule.
Most phospholipids are ionized efﬁciently under positive ion mode due to the presence of a quaternary
nitrogen atom. However, phospholipids also generate negative ions through a demethlyation product.
This can result from the formation of a cluster with a corresponding counter ion such as acetate or
chloride. The cluster ions can be activated to remove a methyl group as well as the positive charge
from the choline residue.6
The negative ionization of phospholipids is less pronounced compared to positive ion mode. However,
because of the ionic nature, the abundance, and the hydrophobicity of phospholipids in biological
matrix, it makes them logical candidates to inﬂuence ionization in negative ion mode electrospray MS
1. In contrast to the ionization suppression observed previously in positive ion mode, signal
enhancement in negative ion mode has been observed correlating to the lipid peaks. The
correlation of the elution peaks under both ionization modes suggested that they are from the
same endogenous materials, i.e., phospholipids.
2. The mid-elution peaks in negative ion mode were further investigated. The product ion spectra
yielded several common fatty acid fragments.
3. The precursor ions were decoded by fragment, adduct, or dimerization. Several common
phospholipid ions merged, which suggested that ionization occurred under rather complicated
Bennett PK and Van Horne KC. Identiﬁcation of the Major Endogenous and Persistent Compounds in
Plasma, Serum, and Tissue That Cause Matrix Effects With Electrospray LC/MS Techniques. Presented
at the American Association of Pharmaceutical Scientists Conference, Salt Lake City, UT, October 2003.
Bennett PK and Van Horne KC. Preventing Matrix Effects By Using New Sorbents to Remove
Phospholipids From Biological Samples. Presented at the American Association of Pharmaceutical
Scientists Conference, Salt Lake City, UT, October 2003.
Bennett PK and Liang H. Overcoming Matrix Effects Resulting From Biological Phospholipids Through
Selective Extractions in Quantitative LC/MS/MS. Presented at the 52nd ASMS Conference on Mass
Spectrometry, Nashville, TN, May 2004.
Van Horne KC, Meng M, Marquardt R, and Bennett PK. Investigation of Analyte Recoveries From a
New Sorbent Designed to Remove Phospholipids and Reduce Associated Matrix Effects. Presented at
the 52nd ASMS Conference on Mass Spectrometry, Nashville, TN, May 2004.
Meng M and Bennett PK. Source for Imprecision Resulting From Ionization Suppression From Strongly
Retained Phospholipids and Dioctyl Phthalate. Presented at the 52nd ASMS Conference on Mass
Spectrometry, Nashville, TN, May 2004.
Murphy RC. Mass Spectrometry of Phospholipids: Tables of Molecular and Product Ions. Denver:
Illuminati Press. 2002.
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