Implication of Phospholipids in Significant Matrix Effects in

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					                       Implication of Phospholipids in Significant
                       Matrix Effects in Negative Ion ESI-MS/MS


Tianyi Zhang, Min Meng,
                           Purpose
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.



                           Introduction
                           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.



                           Methods

                           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.




                                                                                                               Tandem Labs      1
                  Samples

                  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



                  HPLC Conditions

                  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




2   Tandem Labs
Method                              Results                                                                       Figures
                                                                                                                  Tables



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.
Mode.


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.


                                                                                                             Tandem Labs     3
Table 1

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

    277.2    134.1

    345.1    134.2                                 277.1                    345

    450.4                 224         255.1

    540.5                224.3        255.1                                                     480.4

    555.6

    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




Figure 1




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Figure 2




Figure 3




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Figure 4




Figure 5




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Figure 6




Figure 7




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Figure 8




8   Tandem Labs
Discussion
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
sources.

Conclusion
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
      conditions.

    References
1
 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.
2
 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.
3
 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.
4
 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.
5
 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.
6
 Murphy RC. Mass Spectrometry of Phospholipids: Tables of Molecular and Product Ions. Denver:
Illuminati Press. 2002.




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