An improved method for glycosaminoglycan analysis by LC-MS/MS of urine samples collected on filter paper.
Author(s): Auray-Blais C, Lavoie P, Zhang H, Gagnon R, Clarke JT, Maranda B, Young SP, An Y, Millington DS
Publication: Clin Chim Acta, 2012, Vol. 413, Page 771-8
PubMed ID: 22285314 PubMed Review Paper? No
Purpose of Paper
This paper investigated the stability of dermatan sulfate (DS) and heparan sulfate (HS) in dried urine specimens during storage and freeze-thaw cycling and investigated the effect of mucopolysaccharidoses (MPS) diagnosis and treatment on DS and HS levels.
Conclusion of Paper
Only minor changes (less than 20%) in the concentrations of DS or HS were observed when dried urine specimens were stored for 6 weeks at room temperature, 4˚C, -20˚C, or -80˚C and the authors report no effect of up to three freeze-thaw cycles. Dried urine from patients with MPS had higher levels of DS and HS than urine from healthy individuals and, as expected, DS and HS values declined after enzyme replacement therapy (ERT) treatment.
Studies
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Study Purpose
This study investigated the stability of DS and HS in dried urine specimens during storage and freeze-thaw cycling and investigated the effect of MPS diagnosis and treatment on DS and HS levels. Urine from 21 patients with MPS and 52 healthy patients (27 male, 25 female) was collected on Whatman paper and dried overnight at room temperature. Urine was extracted by placing 5 cm punches of the dried urine specimens in ammonium hydroxide and shaking for 10 min. Extracted urine was evaporated under nitrogen and analyzed by LC-MS. To assess intra-day variability, five urine specimens were obtained on a single day from two MPS patients. To assess inter-day variation, four urine specimens were collected from two MPS patients on different days. Stability during storage and freeze-thaw cycling was determined using urine specimens spiked with 18.75 and 68.75 µg/mL DS and HS.
Summary of Findings:
Use of dried urine allowed for recovery of 87-95% of the spiked in DS and 99-103% of the spiked in HS. When stored for six weeks at room temperature, 4˚C, -20˚C, or -80˚C, levels of DS changed by less than 18% and levels of HS changed by less than 20%. The authors report no effect of up to three freeze-thaw cycles on HS and DS in dried specimens. DS and HS were no quantifiable in urine from healthy individuals, but were higher in patients with MPS. After ERT treatment, DS and HS values declined in a single MPSI patient and a single MPSII patient. The inter-day variation in DS was 11.1-15.8% and inter-day variation in HS was 6.7-7.4%. The intra-day variation in DS was 6.4-7.7% and the intra-day variation in HS was 2.7-6.7%.
Biospecimens
Preservative Types
- Other Preservative
- Frozen
Diagnoses:
- Normal
- Other diagnoses
Platform:
Analyte Technology Platform Carbohydrate LC-MS or LC-MS/MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Diagnosis/ patient condition Healthy
MPSI
MPSII
Preaquisition Other drugs None
ERT
Storage Storage temperature Room temperature
4˚C
-20˚C
-80˚C
Storage Freeze/thaw cycling 0 cycles
1 cycle
2 cycles
3 cycles
Biospecimen Aliquots and Components Biospecimen components 68.75 µg/mL DS
68.75 µg/mL HS
18.75 µg/mL DS
18.75 µg/mL HS
Biospecimen Acquisition Time of biospecimen collection Collection on 4 different days
Collection at 5 times in one day
Storage Storage duration 0 weeks
2 weeks
6 weeks