Data in support for the measurement of heparan sulfate and dermatan sulfate by LC–MS/MS analysis

This article provides supplementary data for the paper “LC–MS/MS method for simultaneous quantification of heparan sulfate and dermatan sulfate in urine by butanolysis derivatization” (Forni et al., 2018). Several parameters were tested to optimize sample preparation by butanolysis in order to carry out simultaneous quantifications of HS and DS by tandem mass spectrometry. Here we describe step-by-step instructions to perform HS and DS analysis in urine samples using external calibration curves of standards of known concentration. Sample are quantified by interpolation from the calibration curve and reported in µg/mL. Then, HS and DS are normalized to creatinine concentration and reported as mg/g uCr.


a b s t r a c t
This article provides supplementary data for the paper "LC-MS/ MS method for simultaneous quantification of heparan sulfate and dermatan sulfate in urine by butanolysis derivatization" (Forni et al., 2018). Several parameters were tested to optimize sample preparation by butanolysis in order to carry out simultaneous quantifications of HS and DS by tandem mass spectrometry.
Here we describe step-by-step instructions to perform HS and DS analysis in urine samples using external calibration curves of standards of known concentration. Sample are quantified by interpolation from the calibration curve and reported in mg/mL. Then, HS and DS are normalized to creatinine concentration and reported as mg/g uCr. .

Value of the data
The described method allows the simultaneous quantitation of dermatan sulfate (DS) and heparan sulfate (HS) in urine by LC-MS/MS, so as to facilitate differential diagnoses in MPS and targeted patient follow up.
The protocol for preparing samples involves the chemical cleavage of glycosaminoglycans (GAGs) in a butanolysis reaction. The high yield product permits a reproducible quantitation of HS and DS even if a small amount of sample is used.
The method could become a useful and reliable test in clinical laboratories where mass spectrometry is commonly used in several areas of diagnostics.
Given that the method can be applied to different type of matrices including dried blood spots, it could be modified and adapted for second tier testing of positive samples in newborn screening programmes for lysosomal storage disorders.

Data
The reported dataset includes five figures. Figs. 1-4 provide experimental data for the optimization of time-temperature and reagent volume parameters for butanolysis reaction of HS and DS. Fig. 5 provides short-term stability data relating to storage conditions. The details on the operating procedure for the quantitative analysis of HS and DS in human urine are given below.

Experimental design, materials and methods
2.1. Operating procedure for sample preparation 1. Keep the urine sample at room temperature until completely thawed. Gently invert the tube to ensure that the urine specimen is homogeneous. 2. Filter $ 1-2 mL of urine with 0.22-mm syringe filters (Merck KGaA, Darmstadt, Germany) and transfer to 1.5 mL tube. 3. Analyze all urine samples for creatinine in order to measure an equal concentration of urine for each patient: if the initial creatinine of the sample (i-uCr) is 4100 mg/mL: a) dilute the filtered urine in deionized water until a final creatinine concentration (f-uCr) of 100 m g/mL is reached:   10. Combine DS samples into the correspondingly labeled tube containing HS for each patient (final volume 1 mL) 11. Vortex for 15 s.
Samples can be kept for two days at 4-8°C.

Calibration standard solutions
12. Prepare two stock solutions for DS and HS at a concentration of 3 and 1 g/L in water, respectively. 13. Prepare a calibration standard stock solution HS7 at 50 mg/L by diluting 1:20 the stock solution at 1 g/L in water. Prepare calibration standard stock solutions from HS6 to HS1 by a serial dilution 1:2 from HS7 calibration standard stock solutions in water. 14. Prepare a calibration standard stock solution DS7 at 200 mg/L by diluting 1:15 the stock solution at 3 g/L in water. Prepare DS6 to DS1 calibration standard stock solutions by a serial dilution 1: 2 from DS7 in water. 15. The calibration standard stocks solutions are as follows:  18. Transfer 5 mL of each calibration standard to a labeled tube and proceed to point 4.
Normalize the results value for urinary creatinine and express it as mg/g of uCr.

Optimization of sample preparation
In order to determine the optimal reaction conditions for HS derivatization, a set of experimental runs was conducted to assess the effects of temperature, time and derivatization reagent volume on yield.
Taking into account the results published by Trim [2], we tested temperatures around 100°C. Three sets of spiked samples with fixed HS concentration were incubated at 100, 90 and 80°C and analyzed every 30 min. Each experiment was performed in six replicates to estimate the variability of results.
The boxplot in Fig. 1 shows the median, interquartile range, and outliers for each dataset. A further dataset was collected to determine the effect of incubation time on the HS peak area counts over a period of 6 h at 90°C (Fig. 2).
To establish the optimum reaction parameters for DS butanolysis the same experimental procedure used for HS analysis was conducted.
In order to check if different volumes of derivatization reagent corrupt sensitivity, a set of samples for each analyte was incubated with increased amounts of 3 N HCl in n-Butanol (Fig. 4) QC samples with low and high concentrations were maintained at four different temperatures for 1 month and analyzed at regular time intervals (Fig. 5).