Analysis of Dried Blood Spots by Paper Spray Ionization - MS
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
TISSUE(S): Blood
PROVIDER: ST000229 | MetabolomicsWorkbench |
REPOSITORIES: MetabolomicsWorkbench
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Project description:In this research, a UHPLC-MS/MS method was developed and validated for the determination of zonisamide in dried plasma spots (DPS) and dried blood spots (DBS). Detection of zonisamide and internal standard, 1-(2,3-dichlorphenyl)piperazine, was carried out in ESI+ mode by monitoring two MRM transitions per analyte. Total run time, less than 2.5 min, was achieved using Acquity UPLC BEH Amide (2.1 × 100 mm, 1.7 µm particle size) column with mobile phase comprising acetonitrile-water (85:15%, v/v) with 0.075% formic acid. The flow rate was 0.225 mL/min, the column temperature was 30 °C and the injection volume was 3 µL. Desolvation temperature, desolvation gas flow rate, ion source temperature and cone gas flow rate were set by the IntelliStart software tool in combination with tuning. All of the Guthrie cards were scanned, and DPS/DBS areas were determined by the image processing tool. The influence of hematocrit values (20-60%) on accuracy and precision was evaluated to determine the range within which method for DBSs is free from Hct or dependency is within acceptable limits. The validated method was applied to the determination of zonisamide levels in DPS and DBS samples obtained from patients confirming its suitability for clinical application. Finally, the distribution of zonisamide into the red blood cells was estimated by correlating its DPS and DBS levels.
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Project description:Lysosomal acid lipase deficiency (LAL-D) (OMIM: 278000) is a lysosomal storage disorder with two distinct disease phenotypes such as Wolman disease and cholesteryl ester storage disorder (CESD), characterized by an accumulation of endocytosed cholesterol in the body. Due to the presence of multiple lipases in DBS, previous studies measured LAL enzyme activity in the presence of Lalistat-2, an established LAL-specific inhibitor (Hamilton J et al Chim Clin Acta (2012) 413:1207-1210). Alternatively, a novel substrate specific for LAL has been reported very recently (Masi S. et al Clin Chem (2018) 64:690-696). In this study, we examined the LAL enzyme activity of a Japanese population with the LAL-specific substrate using liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based enzyme assay whether an affected individual can be identified among this population. To achieve this, we first performed assay validation using LC-MS/MS. Under our experimental setting, typically we obtained LAL enzyme activity for QC High (100% enzyme activity) as 261.9 ± 3.2 μmol/h/L (n = 5) and for QC Low as (5% enzyme activity) as 14.7 ± 0.5 μmol/h/L (n = 5). The percentage of coefficient of variation for interday assay for QC High was 9.6% (n = 4) and for QC Low was 7.9% (n = 4), respectively. Based on these results, we further examined the LAL enzyme activity of control Japanese population and that of affected individuals with Wolman disease and CESD. The averaged enzyme activity for control newborns, Wolman, and CESD was 123.9 ± 53.9 μmol/h/L (n = 131), 6.6 ± 0.9 μmol/h/L (n = 3), and 4.8 ± 0.3 μmol/h/L (n = 3), respectively. These results suggest that an LAL-D-affected individual can be readily identified by enzyme activity using LC-MS/MS-based technique.
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Project description:This study focused on the simultaneous detection of amphetamine, 3,4-methylenedioxy methamphetamine, morphine, benzoylecgonine, and 11-nor-9-carboxy-tetrahydrocannabinol in whole blood and dried blood spot (DBS). It is aimed to select a solvent mixture for liquid-liquid extraction technique employing liquid chromatography-tandem mass spectrometry (LC-MS-MS). The obtained DBS results were compared with the whole blood samples results. A simple, rapid, and reliable LC-MS-MS method was developed and validated for all analytes in whole blood and DBS. LC was performed on a Hypersil Gold C18 column with an initial gradient of 0.01% formic acid, 5 mM ammonium format buffer in water, and acetonitrile at 0.3 ml/min with 7.5 min runtime. A methanol:acetonitrile (40:60 v/v) mixture was selected for both matrices. Limit of quantitation (LOQ) values were 10-25 ng/mL; linear ranges were LOQ-500 ng/ml for all analytes; correlation coefficients were greater than 0.99, and all calibrator concentrations were within 20%. Analytical recovery in blood and DBS ranged from 84.9% to 113.2% of the expected concentration for both intra- and inter-day. Analytes were stable for 1, 10, and 30 days after three freeze/thaw cycles. It was determined that the variances of the results obtained with the two matrices in the comparison study were equal for each analyte, and the results were highly correlated (r = 0.9625). A sensitive, accurate, and reliable chromatographic method was developed to determine amphetamine, 3,4-methylenedioxy methamphetamine, morphine, benzoylecgonine, and cannabis, by performing the same preliminary steps with whole blood and dried blood spots. It was observed that the results obtained in these two matrices were compatible and interchangeable when statistically compared.
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