Accepted Manuscript

A validated HPLC-MS/MS method for estimating the

concentration of the ganglioside, GD2, in human plasma or serum

C.M. Busch, A.V. Desai, G.S. Moorthy, E. Fox, F.M. Balis

PII: S1570-0232(18)30934-6
DOI: doi:10.1016/j.jchromb.2018.10.010
Reference: CHROMB 21404
To appear in: Journal of Chromatography B
Received date: 13 June 2018
Revised date: 21 September 2018
Accepted date: 11 October 2018

Please cite this article as: C.M. Busch, A.V. Desai, G.S. Moorthy, E. Fox, F.M. Balis , A
validated HPLC-MS/MS method for estimating the concentration of the ganglioside, GD2,
in human plasma or serum. Chromb (2018), doi:10.1016/j.jchromb.2018.10.010

This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.
 ACCEPTED MANUSCRIPT
A Validated HPLC-MS/MS Method for Estimating the Concentration of the
Ganglioside, GD2, in Human Plasma or Serum

C. M. Busch1, A. V. Desai2, G. S. Moorthy1, E. Fox1, F. M. Balis1 ; 1 The Children’s Hospital of Philadelphia,

Philadelphia, PA, 2 University of Chicago Medicine and Biological Sciences, Chicago, IL.

Abstract:

T
IP
GD2 is a ganglioside found in the plasma membrane of the neural crest-derived cancer, neuroblastoma. GD2 is
shed into the circulation of patients with neuroblastoma and could serve as a tumor biomarker to monitor

CR
tumor burden or response to treatment. We developed and validated a high-performance liquid
chromatography tandem mass spectrometry (HPLC-MS/MS) method to quantify the D18:1-18:0 (C18) and the
D18:1-20:0 (C20) lipoforms of GD2 in human plasma and serum. Human brain derived GD2 containing a mixture

US
of C18 and C20 was used as the analytical standard. Samples were extracted with methanol containing
dueterated-GM1 (internal standard), and analytes were separated on a Phenomenex Kinetex C18 column eluted
with a gradient mobile phase composed of ammonium acetate buffer, methanol and isopropanol. An AB Sciex
AN
4500 QTRAP mass spectrometer in negative ion mode was used to quantify the doubly charged GD2 C18 and
C20 lipoform precursor ions (m/z 836.8 and m/z 850.8) that both yield a product ion of m/z 290.0. The
calibration curves were linear from 4 - 1000 ng/mL and 6 - 1500 ng/mL for GD2 C18 and C20 lipoforms
M

respectively. Inter-day and intra-day accuracy were within the acceptable validation range in plasma and
serum.
ED

Keywords: Ganglioside GD2, Neuroblastoma Biomarker, HPLC-MS/MS

PT
CE
AC
 ACCEPTED MANUSCRIPT
1. Introduction

Gangliosides are sialic acid-containing glycosphingolipids that are found primarily in the nervous system,
where they can account for up to 10-12% of the plasma membrane lipids [1]. Ceramide is the lipid portion that
resides in the membrane, and the glycan domain projects extracellularly [1]. The fatty acid chain length in the
ceramide moiety is variable, resulting in multiple lipoforms of each ganglioside [9].

GD2, which has a 5 member, branched oligosaccharide chain consisting of Glc-Gal1-(NeuAc)2,-GalNAc1

(Figure 1), is expressed on the surface of neuroectodermal-derived cancers, including the childhood cancers
neuroblastoma (NBL) and retinoblastoma (RBL), and melanoma [2]. GD2 is the target antigen of the therapeutic
monoclonal antibody, dinutuximab, which is approved by the U.S. Food and Drug Administration (FDA) for the

T
treatment of neuroblastoma.

IP
GD2 has been detected on >90% of NBL specimens by immunohistochemistry, [3,4] and G D2 is shed from
tumors and has been detected in the plasma or serum of patients with NBL (27/34 patients by thin layer

CR
chromatography [TLC]) and RBL (9/10 patients by quantitative immunostaining) [5-8]. Higher circulating GD2
concentrations (up to 2,160 nM) in patients with NBL were associated with higher tumor stage and more rapid
tumor progression and poorer survival [6,8]. Therefore, GD2 has the potential to serve as a circulating tumor

US
biomarker for NBL.
The TLC assay previously used to measure circulating GD2 concentrations is not a practical assay method for
AN
rapidly measuring GD2 concentrations in large clinical trials or the clinical laboratory setting. If GD2 proves to be
a useful tumor biomarker, our development and validation of a tandem mass spectrometry method for the
simultaneous measurement of the clinically relevant lipoforms of GD2 in human plasma and serum will be
M

clinically relevant.
ED

2. Materials and methods

2.1. Materials
PT

Purified Human brain-derived GD2 was purchased from EMD Millipore Corp. (Billerica, MA) and contains two
CE

dominant lipoforms of GD2 - D18: 1-18:0 (C18) and D18: 1-20:0 (C20). LC-MS grade Methanol, Isopropanol, and
Water were purchased from Honeywell Burdick & Jackson (Muskegon, MI). Internal standard of N-omega-
CD3-Octadecanoyl monosialoganglioside GM1 was purchased from Matreya Inc. (State College, PA).
AC

Ammonium Acetate was purchased from Sigma (St. Louis, MO). Pooled disease-free human plasma and serum
was obtained from The Children's Hospital of Philadelphia’s department of pathology & laboratory medicine
clinical laboratory.

2.2. Stock and Working Solutions

Analytical stock solutions (1 mg/mL of the free analyte) were prepared in 2:1 Chloroform: Methanol for both
GD2 and GM1-d3. Working solutions of 50,000, 5,000, 500, and 50 ng/mL total GD2 were prepared in 80%
methanol. Working solution of the internal standard GM1-d3 was prepared in methanol at a concentration of
10,000 ng/mL. All solutions were stored at -20 oC before use and were stable for at least 6 months.
 ACCEPTED MANUSCRIPT
2.3. Sample Extraction

Liquid-Liquid extraction sometimes combined with solid-phase extraction is commonly used to extract
gangliosides from various matrices [11-15]. Huang et al [16] described a simple extraction procedure utilizing
an eight-fold addition of methanol to plasma yielding near 100% extraction recovery. We selected this method
because of its simplicity and speed. Thirty L of blank plasma or serum samples, standards, and QCs were
pipetted into 1.5 mL polypropylene tubes. Eight times volume (240 L) of 100% methanol containing 100
ng/mL GM1-d3 internal standard was then added. Tubes were vortexed for 5 minutes, then centrifuged at
14000 x g for 20 min at 4°C. Supernatants were transferred to autosampler vials with glass micro-inserts for
LC-MS/MS analysis.

T
2.4. HPLC-MS/MS and Data Processing

IP
Extracted samples were separated utilizing a Shimadzu (Columbia, MD) Nexera XR system with two LC-20AD
delivery pumps, a DGU-20A5 vacuum degasser, a SIL-20AC autosampler, and a CBM-20A system controller.

CR
Ten l of each sample was injected onto a Phenomenex Kinetex C18 2.6 mm 100 Å, 2 x 50 mm column kept in a
column oven at 50°C. GD2 C18 and C20 lipoforms were separated with mobile phases (A) 2% methanol in 10
mM ammonium acetate in water and (B) methanol: isopropanol (90:10). Separation was optimized at a flow

US
rate of 0.5 mL/min. Eluent flow was diverted to waste during an isocratic hold of 30 sec with 50% B, and a
linear gradient was started from 50% to 95% B over 3.5 min. At 4 min, LC eluent flow was introduced into a
SCIEX 4500 QTRAP mass spectrometer (Framingham, MA) set for negative mode MRM to quantify the doubly
AN
charged GD2 C18 and C20 lipoform precursor ions (m/z 836.8 and m/z 850.8), as well as the singly charged
internal standard GM1-d3 (m/z 1547.8). All yield a product ion of m/z 290.0. Samples were analyzed for a total
M

of 4 min. At 6 min, the gradient was returned to 50% B. The column was equilibrated at this condition for 4
min until the next injection. Nitrogen was used for the collision gas, the curtain gas, and the carrier gas. Ion
spray voltage was set to -4500 Volts and capillary temperature was set to 500 oC. Analyst software version
ED

1.6.2 (Sciex) was used to process data.

PT

2.5. Assay Method Validation

The U.S. Food and Drug Administration (FDA) guidelines for bioanalytical method validation for industry [10]
CE

was followed to validate our assay method.

2.5.1 Precision, accuracy, and stability

AC

The intra-and inter- day accuracy and precision of this method were evaluated in plasma and serum. Accuracy
should not deviate greater than 20% at the LLOQ level and greater than 15% at all other levels. Precision was
determined by calculating the percent coefficient of variation (%CV) for each level. The %CV should not exceed
15%, except in LLOQ samples which should not exceed 20%. Stability studies of analytes in matrix at room
temperature were performed on replicate sets of both low and high level QC samples (n=3 for each). Analytes
in matrix frozen at -80 °C for one month, and frozen/thawed after three cycles were also measured (n=3 for
each level). Post-extraction analyte stability at room temperature for 12 hours in autosampler was measured.
2.5.2 Recovery and matrix effect
Recovery in plasma and serum was tested at three different concentrations (n=6 for each) of G D2 by comparing
the area ratios of GD2 spiked into extracted blank matrix (post-spike) versus GD2 extracted from spiked matrix
 ACCEPTED MANUSCRIPT
(pre-spike). Recovery was calculated as follows: % Recovery = (Average peak area ratio of pre-spike / Average
peak area ratio of post-spike) X 100.

To determine matrix effect in plasma and serum, the area ratio of analyte to internal standard at three levels
(Low, medium, and high) (n=6 for each) of GD2 spiked into extracted blank matrix (post-spike) versus the same
levels of GD2 in an aqueous solution were compared. Matrix effect was calculated as follows: Matrix effect =
(Average peak area ratio of post-spike / Average peak area ratio of aqueous mixture) X 100. A value of 100%
indicates no matrix effect, less than 100% indicates suppression and over 100% indicates enhancement.

3. Results and discussion

T
The commercially available analytical standard used for this assay was extracted from human brain and was a

IP
mixture of 2 predominant lipoforms of GD2 with fatty acid chain lengths of 18 and 20 carbons. Pure standards
of individual lipoforms are not currently available. Our chromatography conditions separate the lipoforms with

CR
retention times of 5.75 minutes for the C18 lipoform and 5.88 minutes for the C20 lipoform (Figure 2). In the
human brain extract, based on peak area ratios, 60% of the GD2 is the C20 lipoform and 40% of the GD2 is the
C18 lipoform. This 60:40 ratio of C20 to C18 lipoforms was used to generate separate standard curves for the 2

US
lipoforms. For example, 100 ng/mL of GD2 standard had 60 ng/mL of C20 and 40 ng/mL of C18. We
acknowledge that this assumption of identical ionization efficiencies of two isoforms can compromise the
accuracy of quantifying the absolute concentrations of GD2 lipoforms, but the relative concentration compared
AN
to baseline or across patients is more useful for tumor biomarkers. If GD2 proves to be a useful clinical or
research biomarker for NBL, synthesis or extraction of the individual lipoforms may become commercially
M

viable. With enzymatic treatment and purification, it is possible that pure standards for each lipoform can be
realized [23].
ED

3.1. HPLC-MS/MS analysis

PT

LC conditions were optimal in our assay with a C-18 column and eluting with 90% methanol: 10% isopropanol
and an aqueous phase of 2% methanol with 10mM ammonium acetate. Distinct separation of the two GD2
lipoforms as well as the internal standard was achieved within a retention time of 7 minutes. Previous studies
CE

achieved separation of various gangliosides with mobile phases of acetonitrile and aqueous phosphate or
ammonium acetate buffer with amino columns for separation [17-20], as well as with C-18 columns and
mobile phases of methanol-isopropanol with aqueous ammonium acetate buffer [16].
AC

Due to the relatively high molecular mass of GD2, the doubly charged precursor ions and their product ions
were selected for quantitation in this assay. Peak intensities were greatest utilizing negative ionization mode.
Precursor ions of m/z 836.8 and m/z 850.8 were used for the C18 and C20 lipoforms of GD2 respectively, and
m/z 1547.8 was used for GM1-d3 internal standard. All yielded the best product fragmentation ion of m/z 290.0
(Figure 3). LOD (limit of detection) was 2 ng/mL for C18 and 3 ng/mL for C20, and LLOQ (Lowest limit of
quantitation) was 4 ng/mL for C18 and 6 ng/mL for C20 in this assay.

3.2. Standard Addition Calibration Curve

Standards for the calibration curves were prepared in the range of 10 - 2500 ng/mL total GD2. Working
solutions were used to prepare 10X standard samples in 80% methanol. The 10X standards were then diluted
 ACCEPTED MANUSCRIPT
into human pooled plasma or serum for the final total GD2 concentrations of 10, 50, 100, 250, 500, 1000, and
2500 ng/mL used in the calibration curve. QC samples were prepared at 40, 80, 400, 800, and 1600 ng/mL
total GD2 using the same method. In order to rule out any matrix effect from the pooled human plasma or
serum, standard addition internal calibration was implemented, with GM1-d3 addition to the extraction
solution. Calibration curves for both lipoforms were found to be linear, with R 2 > 0.99 (Figure 4). Sample
plasma or serum concentrations of both lipoforms of GD2 were calculated using the corresponding regression
equation with Analyst software (SCIEX). High linearity, accuracy and precision of the calibration was achieved
in this assay by using the standard addition calibration method.

3.3. Precision, accuracy, and stability

T
Precision of this assay for both plasma and serum at three different concentrations was between 3% to 11%

IP
CV. Internal standard precision had a CV of less than 5% in both plasma and serum. Inter-day and intra-day
accuracy were within the acceptable validation range in plasma (Table 1) and serum. Stability studies

CR
performed on replicate sets of both low and high level QC samples (n=3 for each) showed that GD2 in human
plasma prior to extraction was stable at room temperature for at least 18 hours. Both lipoforms had a CV
between 2.4% to 8% and %Dev between -6% to +1% from 0 to 18 hours. Pre-extraction analytes in both low

US
and high level QC samples were found to be stable in plasma stored at -80 °C for at least 1 month. Stability of
pre-extracted GD2 in plasma was determined across five different concentrations between 10ng/ml and
2500ng/ml total GD2 (n=3) after three freeze thaw cycles (C18 CV range 1.2% to 10.7% with %Dev -5% and
AN
+1%, and C20 CV range 0.6% to 11.2% with %Dev -7% and +9%). Stability of post-extraction GD2 in plasma and
serum stored in the autosampler at room temperature for 12 hours was confirmed to simulate a typical HPLC-
MS/MS run. Low, medium, and high level QC samples were stable in plasma (%Dev -4% to +5% for C18, and
M

%Dev +1% to +7% for C20) and serum (%Dev -4% to +2% for C18, and %Dev -3% to +4% for C20).

3.4. Recovery and matrix effect

ED

Plasma recovery across three levels for the C18 lipoform ranged between 93% to 115% and in serum between
PT

92% to 96%. Recovery for the C20 lipoform was between 102% to 113% in plasma and between 90% to 108%
in serum. It has been reported that normal human plasma contains GD2 at levels between 0 – 9.4 ng/mL [21,
22]. The pool of control human plasma that we used during this study, contained less than 8 ng/mL total GD2.
CE

As previously reported, suppression of GD2 MS/MS signal due to matrix components was observed [16]. In this
current method, matrix had little impact on the calculation of GD2 concentration when internal standards and
AC

comparable matrix were used as evidenced by the high linearity, accuracy, and precision of the calibration
curve. There was little matrix effect noted in the C18 lipoform of GD2 in either plasma or serum (average across
levels of 102% in plasma and 110% in serum), but a decrease of area ratio across levels averaged 40% for
plasma and 48% for serum for the C20 lipoform was observed. The decrease was consistent across matrix and
was not concentration dependent. There was little matrix effect on the internal standard observed (101% in
plasma and 84% in serum) and the internal standard was not detected in normal control plasma or serum.

3.5. GD2 Lipoforms in Human Plasma/Serum

Unlike the human brain extract used for the standards, concentrations of the C18 lipoform were higher than
that of the C20 lipoform of GD2 observed in human plasma or serum samples from normal controls and
 ACCEPTED MANUSCRIPT
children with NBL. The ratio of C18 to C20 was highly variable, and in some cases the C20 lipoform was not
detected. GD3 was observed in normal control human plasma and serum at concentrations higher than that of
GD2 (data not shown).

3.6. Future Studies

Using this assay, GD2 will be quantified in stored serum samples from children with NBL taken at the time of
initial diagnosis and prior to treatment, from normal controls, and from children with other types of cancer.
GD2 will also be prospectively monitored over the course of treatment in children with NBL enrolled on the
upcoming Children’s Oncology Group high-risk NBL clinical trial.

T
Acknowledgements

IP
The authors thank Jessica Sima and Praveen Srivastava for technical assistance and guidance.

CR
Funding

US
This work was supported by Children’s Oncology Group Solid Malignancies Integrated Translational Science
Center (COGSM-ITSC) [U10CA180884-01] and Alex’s Lemonade Stand Foundation Center of Excellence Grant
[ALSF COE].
AN
4. References
M

[1] M. Gantner, G. Schwarzmann, K. Sandhoff, T. Kolter, Partial synthesis of ganglioside and lysoganglioside
lipoforms as internal standards for MS quantification, J. Lipid Res. 55 (2014) 2692–2704.
ED

doi:10.1194/jlr.D054734.
[2] M. Suzuki, N.-K. V Cheung, Disialoganglioside GD2 as a therapeutic target for human diseases, Expert Opin.
Ther. Targets. 19 (2015) 349–362. doi:10.1517/14728222.2014.986459.
PT

[3] Z. Wu, E. Schwartz, R. Seeger, S. Ladisch, Expression of GD2 Ganglioside by Untreated Primary Human
Neuroblastomas Expression of GD2Ganglioside by Untreated Primary Human Neuroblastomas1, 46 (1986)
CE

440–443.
[4] V.I. Poon, M. Roth, S. Piperdi, D. Geller, J. Gill, E.R. Rudzinski, D.S. Hawkins, R. Gorlick, Ganglioside GD2
AC

expression is maintained upon recurrence in patients with osteosarcoma, Clin. Sarcoma Res. 5 (2015) 4.
doi:10.1186/s13569-014-0020-9.
[5] S. Ladisch, Z. Wu, Detection of a Tumor-Associated Ganglioside in Plasma of Patients with Neuroblastoma,
Lancet. 325 (1985) 136–138.
[6] L. Valentino, T. Moss, E. Olson, H.J. Wang, R. Elashoff, S. Ladisch, Shed tumor gangliosides and progression
of human neuroblastoma, Blood. 75 (1990) 1564–7. http://www.ncbi.nlm.nih.gov/pubmed/2317562.
[7] J. Portoukalian, M.J. David, P. Gain, M. Richard, Shedding of GD2 ganglioside in patients with
retinoblastoma, Int J Cancer. 53 (1993) 948–951. doi:10.3390/md9040478.
[8] S. Ladisch, Z. Wu, S. Feig, L. Ulsh, E. Schwartz, G. Floutsis, F. Wiley, C. Lenarsky, R. Seecer, Shedding of GD2
Ganglioside by Human Neuroblastoma, Int. J. Cancer. 39 (1987) 73–76.
 ACCEPTED MANUSCRIPT
[9] S.B. Levery, Glycosphingolipid structural analysis and glycosphingolipidomics, Methods Enzymol. 405
(2006) 300–369. doi:10.1016/S0076-6879(05)05012-3.
[10] U.S. Food and Drug Administration, Guidance for Industry: Bioanalytical
Method Validation, U.S. Department of Health and Human Services, FDA, Center for Drug Evaluation and
Research, Rockville, MD, 2001.
[11] L.K. Sørensen, A liquid chromatography/tandem mass spectrometric approach for the determination of
gangliosides GD3 and GM3 in bovine milk and infant formulae, Rapid Commun. Mass Spectrom. 20 (2006)
3625–3633. doi:10.1002/rcm.
[12] L. Svennerholm, P. Fredman, A procedure for the quantitative isolation of brain gangliosides, Biochim.
Biophys. Acta (BBA)/Lipids Lipid Metab. 617 (1980) 97–109. doi:10.1016/0005-2760(80)90227-1.

T
[13] B. Fong, C. Norris, E. Lowe, P. McJarrow, Liquid chromatography-high-resolution mass spectrometry for

IP
quantitative analysis of gangliosides, Lipids. 44 (2009) 867–874. doi:10.1007/s11745-009-3327-1.

CR
[14] K. Ikeda, T. Shimizu, R. Taguchi, Targeted analysis of ganglioside and sulfatide molecular species by LC/ESI-
MS/MS with theoretically expanded multiple reaction monitoring, J. Lipid Res. 49 (2008) 2678–2689.
doi:10.1194/jlr.D800038-JLR200.

US
[15] A.D. Garcia, J.L. Chavez, Y. Mechref, Rapid and sensitive LC-ESI-MS of gangliosides, J. Chromatogr. B Anal.
Technol. Biomed. Life Sci. 947–948 (2014) 1–7. doi:10.1016/j.jchromb.2013.11.025.
[16] Q. Huang, X. Zhou, D. Liu, B. Xin, K. Cechner, H. Wang, A. Zhou, A new liquid chromatography/tandem
AN
mass spectrometry method for quantification of gangliosides in human plasma, Anal. Biochem. 455 (2014) 26–
34. doi:10.1016/j.ab.2014.03.014.
M

[17] D.J. Harvey, Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates, Mass
Spectrom. Rev. 18 (1999) 349–450. doi:10.1002/(SICI)1098-2787(1999)18:6<349::AID-MAS1>3.0.CO;2-H.
ED

[18] S. Ando, H. Waki, K. Kon, New solvent system for high-performance thin-layer chromatography and high-
performance liquid chromatography of gangliosides, J. Chromatogr. A. 405 (1987) 125–134.
doi:https://doi.org/10.1016/S0021-9673 (01)81754-4.
PT

[19] G. Gazzotti, S. Sonnino, R. Ghidoni, Normal-phase high-performance liquid chromatographic separation of

non-derivatized ganglioside mixtures, J. Chromatogr. A. 348 (1985) 371–378. doi:10.1016/S0021-
CE

9673(01)92475-6.
[20] S. Kirsch, J. Müthing, J. Peter-Katalinić, L. Bindila, On-line nano-HPLC/ESI QTOF MS monitoring of α2-3 and
α2-6 sialylation in granulocyte glycosphingolipidome, Biol. Chem. 390 (2009) 657–672.
AC

doi:10.1515/BC.2009.066.
[21] B. Sela, D. Herlyn, D. Iliopoulous, D. Guerry, H. Koprowski, Levels of Disialogangliosides in Sera of
Melanoma Patients Monitored by Sensitive Thin-Layer Chromatography and Immunostaining, J. Natl. Cancer
Institutes. 81 (1989) 1489–1492.
[22] G. Schulz, D.A. Cheresh, N.M. Varki, G. Schulz, D.A. Cheresh, N.M. Varki, A. Yu, L.K. Staffileno, R.A.
Reisfeld, Detection of Ganglioside G D2 in Tumor Tissues and Sera of Neuroblastoma Patients Detection of
Ganglioside GD2in Tumor Tissues and Sera of Neuroblastoma, 44 (1984) 5914–5920.
[23] F.-T. Huang, Y.-B. Han, Y. Feng, G.-Y. Yang, A facile method for controlling the reaction equilibrium of
sphingolipid ceramide N-deacylase for lyso-glycosphingolipid production, J. Lipid Res. 56 (2015) 1836–1842.
doi:10.1194/jlr.D061176.
 ACCEPTED MANUSCRIPT

C18 Intraday (n = 3) C18 Interday (n = 6 days)

Nominal Nominal
CV Accuracy CV Accuracy
Concentration Mean ± SD Concentration Mean ± SD
(%) (%) (%) (%)
(ng/ml) (ng/ml)
4 3.96 ± 0.6 15.1 99 4 3.9 ± 0.08 2.1 97
20 20.64 ± 1.3 6.3 103 20 20.9 ± 1.1 5.2 105
40 40.6 ± 3.3 8.2 102 40 41.5 ± 1.9 4.8 104
100 103.9 ± 8.0 7.8 104 100 103.1 ± 4.9 4.7 103

T
200 202.3 ± 9.92 4.9 101 200 192 ± 4.69 2.4 96

IP
400 392.04 ± 14.0 3.6 98 400 381.7 ± 18.8 4.9 96
1000 930.8 ± 52.9 5.7 93 1000 984.0 ± 30.45 3.1 98

CR
C20 Intraday (n = 3) C20 Interday (n = 6 days)

US
Nominal Nominal
CV Accuracy CV Accuracy
Concentration Mean ± SD Concentration Mean ± SD
(%) (%) (%) (%)
(ng/ml) (ng/ml)
AN
6 5.9 ± 0.5 9.1 99 6 6.1 ± 0.4 6.1 102
30 32.2 ± 0.1 0.1 107 30 32.5 ± 0.8 2.3 108
M

60 57.4 ± 3.5 6.0 96 60 58.3 ± 4.2 7.2 97

150 160.4 ± 11.7 7.3 107 150 153.8 ±10.0 6.5 102
ED

300 295.7 ± 7.3 2.5 99 300 295.0 ± 14.8 5.0 98

600 584.8 ± 15.7 2.7 97 600 569.2 ± 22.8 4.0 95
1500 1426.4 ± 86.5 6.1 95 1500 1450.0 ± 71.9 5.0 97
PT

Table 1. Intra- and inter-day precision and accuracy in plasma

CE
AC
 ACCEPTED MANUSCRIPT
Fig. 1 Structure of ganglioside GD2 (C18 lipoform)
Fig. 2 XIC of standard containing 40ng/mL C18 GD2, 60ng/mL C20 GD2, and 100 ng/mL GM1-d3 (A) and XIC of
Neuroblastoma patient sample (B).
Fig. 3 Product Ion (MS/MS) spectra for (A) C18 GD2, (B) C20 GD2, and (C) GM1-d3
Fig. 4 Calibration Curve of Both Lipoforms of GD2

T
IP
CR
US
AN
M
ED
PT
CE
AC
Figure 1
Figure 2
Figure 3
Figure 4