LC–MS/MS Assays to Quantify Sulfatides and Lysosulfatide in Cerebrospinal Fluid of Metachromatic Leukodystrophy Patients

Figures & data

Figure 1 Structures of sulfatides & lysosulfatide.

Figure 2 Extracted, representative LC–MS/MS chromatograms from the cerebrospinal fluid sulfatides assays.

The 15 sulfatide species at the LLOQ (0.020 μg/ml) that comprised the assessment of total sulfatide levels from samples.

^†Internal standard (C18:0-D₃-sulfatide) at the spiking concentration (50 ng/ml).

cps: Counts per second.

Figure 3 Extracted, representative LC–MS/MS chromatograms from the cerebrospinal fluid lysosulfatide assays.

(A) Lysosulfatide at the LLOQ (0.02 ng/ml). (B) Internal standard (N-acetyl-sulfatide) at the spiking concentration (50 ng/ml).

cps: Counts per second.

Figure 4 Representative assay calibration curves.

(A) First run of cerebrospinal fluid sulfatides assay. (B) First run of cerebrospinal fluid lysosulfatide assay.

IS: Internal standard.

Figure 5 Box and whisker plots of cerebrospinal fluid sulfatide and lysosulfatide concentrations in children without metachromatic leukodystrophy and in children with metachromatic leukodystrophy.

(A) Sulfatides. (B) Lysosulfatide. Sulfatide concentrations were below the limit of quantification, defined as half of the LLOQ value (0.0103 μg/ml; panel A dashed line) after factoring in the volume change in final solution, in 13 of the 60 CSF samples from children without MLD, and lysosulfatide concentrations were below the limit of quantification (0.0103 ng/ml; panel B dashed line) in 59 of the 60 CSF samples from children without MLD, skewing the distribution of these box and whisker plots. For children with MLD, none of the CSF sulfatide concentrations were below the limit of quantification and one CSF lysosulfatide concentration was below the limit of quantification. The data points above the plot for children without MLD may be considered outliers; however, an outlier analysis was not performed. P-values are for two-sample t-tests for differences between mean values (denoted as crosses) between children without MLD and children with MLD.

^†Concentrations for children with MLD are baseline concentrations.

CSF: Cerebrospinal fluid; MLD: Metachromatic leukodystrophy.

Figure 6 Correlation of cerebrospinal fluid sulfatide and lysosulfatide concentrations with baseline Gross Motor Function Measure-88 scores in children with metachromatic leukodystrophy.

(A) Baseline CSF sulfatide levels. (B) Baseline CSF lysosulfatide levels. P-values are based on Spearman’s correlation tests.

CSF: Cerebrospinal fluid; GMFM-88: Gross Motor Function Measure-88.

Table 1 Mass transition and elution times for the 19 sulfatide species monitored by the cerebrospinal fluid sulfatides assay.

Reference	Sulfatide name	Included in sulfatide quantification	Transitions	Typical elution time (min)
778	C16:0	No	m/z 778.5→97.0	1.48
804	C18:1	Yes	m/z 804.5→97.0	1.66
806	C18:0	Yes	m/z 806.5→97.0	1.79
822	C18:0-OH	Yes	m/z 822.5→97.0	1.69
834	C20:0	Yes	m/z 834.6→97.0	2.04
850	C20:0-OH	No	m/z 850.6→97.0	1.94
860	C22:1	Yes	m/z 860.6→97.0	2.11
862	C22:0	Yes	m/z 862.6→97.0	2.34
876	C23:0 or C22:1-OH (d18:2–22:0-OH)	Yes	m/z 876.6→97.0	2.47
878	C22:0-OH	Yes	m/z 878.6→97.0	2.26
886	d18:2–C24:1	Yes	m/z 886.6→97.0	2.17
888	C24:1	Yes	m/z 888.6→97.0	2.37
890	C24:0	Yes	m/z 890.6→97.0	2.60
902	d18:2–24:1-OH (d18:1–C24:2-OH) or d18:1–25:1 (d18:2–25:0)	Yes	m/z 902.6→97.0	2.48
904	C24:1-OH	Yes	m/z 904.6→97.0	2.32
906	C24:0-OH	Yes	m/z 906.6→97.0	2.52
916	C26:1	Yes	m/z 916.6→97.0	2.64
932	C26:1-OH	No	m/z 932.6→97.0	2.48
934	C26:0-OH	No	m/z 934.6→97.0	2.70

Table 2 Intra- and interday accuracy (%bias) and precision (%CV) using the sulfatide and lysosulfatide quality controls in human cerebrospinal fluid.

Analyte	Nominal concentration	Endogenous concentration	Endogenous concentration in QC solution^†	Intrarun (n = 6 samples)			Inter-run (n = 3 runs [18 samples in total]^‡)
				Mean calculated concentration	%bias	%CV	Concentration	%bias	%CV
Sulfatides (concentrations in μg/ml)
LLOQ QC	0.02	0.0720	0.0684	0.0898	1.6	2.2	0.0864	5.2	3.9
Low QC	0.06	0.0720	0.0684	0.123	-3.9	5.7	0.121	-0.3	2.4
Mid QC	0.30	0.0720	0.0684	0.354	-3.8	4.2	0.346	-4.4	2.1
High QC	0.75	0.0720	0.0684	0.773	-5.5	3.6	0.758	-6.7	1.8
Lysosulfatide (concentrations in ng/ml)
LLOQ QC	0.02	0.00143	0.00136	0.0207	-3.3	12.7	0.0243	-3.7	14.3
Low QC	0.06	0.00143	0.00136	0.0550	-10.4	5.6	0.0625	-4.2	10.4
Mid QC	0.30	0.00143	0.00136	0.273	-9.3	1.7	0.293	-3.8	6.1
High QC	0.75	0.00143	0.00136	0.698	-7.1	4.5	0.734	-2.8	5.2

† Endogenous concentration taking into account the volume change after spiking in QC solution.

‡ There were 17 samples in total for the LLOQ QC for lysosulfatide owing to exclusion of an outlier.

LLOQ: Lower limit of quantitation; QC: Quality control.

Data sharing statement

The authors certify that this manuscript reports the secondary analysis of clinical trial data that have been shared with them, and that the use of this shared data is in accordance with the terms (if any) agreed upon their receipt. The source of this data is NCT01510028. Shire (a Takeda company) does not plan to share data supporting the results reported in this article as there is a reasonable likelihood that individual patients could be re-identified (due to the limited number of study participants/study sites).