Sun-Hee Jun

Multiplex enzyme assay for galactosemia using ultraperformance liquid chromatography-tandem mass spectrometry.

BACKGROUND Galactosemia is one of the most important inherited disorders detected by newborn screening tests. Abnormal results in screening tests should be confirmed by enzyme activity assays, but existing methods are time and labor intensive. We developed a novel multiplex enzyme assay for galactosemia using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). METHODS [(13)C6]-galactose, [(13)C2]-galactose-1-phosphate, and UDP-glucose were used as substrates for 3 galactose-metabolizing enzymes. The end products from the combined reaction mixtures, [(13)C6]-galactose-1-phosphate, UDP-[(13)C2]-galactose, and UDP-galactose, were simultaneously measured using UPLC-MS/MS. Linearity, imprecision, ion suppression, and the effects of substrate were evaluated to determine assay performance. Enzyme activities from 35 healthy individuals, 8 patients with enzyme deficiency, and 18 mutant cells were analyzed. RESULTS Substrates, products, and internal standards from the mixture of 3 enzyme reactions were clearly separated by using UPLC-MS/MS, with an injection cycle time of 10 min. Ion suppression was 0.1%-2.5%, the interassay imprecision of UPLC-MS/MS was 3.3%-10.6% CV, and the linearity of each system was good (R(2) = 0.994-0.999). Patient samples and mutated cells showed consistently low enzyme activities compared with those of normal individuals and wild-type cells. CONCLUSIONS This method allows for a high-throughput and reproducible multiplex enzyme assay for galactosemia in erythrocytes.

Ultra‐performance liquid chromatography/tandem mass spectrometry for determination of sulfatides in dried blood spots from patients with metachromatic leukodystrophy

RATIONALE Metachromatic leukodystrophy (MLD) is a genetic autosomal recessive disease caused by a deficiency in arylsulfatase A. Accumulated sulfatides can be detected in the urine and detection of sulfatiduria is a useful test for diagnosis and monitoring. To our knowledge, no studies have explored the accumulation of sulfatides in dried blood spots (DBSs). We developed an ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method for measuring sulfatides in DBSs from patients with MLD. METHODS DBSs were eluted with internal standard. After mixing and centrifugation, the organic layer was transferred to a 96-well microplate and dried, then resuspended in methanol/propanol solution. Samples were analyzed on an UPLC system. Total running time was 4 min. Quantification was achieved by multiple reaction monitoring using a tandem mass spectrometer. We evaluated the precision, linearity, and ion suppression of the method and analyzed sulfatide concentrations in DBS specimens from MLD patients (n = 9), pseudodeficiency (PD) patient (n = 1), obligate heterozygotes (OH) (n = 2) and normal controls (n = 124). RESULTS In negative-ion mode, sulfatides species subjected to collision-induced dissociation readily fragment to produce an intense ion at m/z 96.8 (HSO4(-)). The precisions of low and high concentration controls ranged from 5.4 to 19.9%. The sulfatides produced linear responses. Molecular species of sulfatides were barely detected in DBSs from normal individuals and the PD-OH group [mean (range), 0.07 (<0.05-0.34) and 0.13 (<0.05-0.22) µg/mL, respectively]. In contrast, the DBSs from MLD patients showed a marked increase in several molecular species of sulfatide [mean (range), 2.02 (1.18-3.89) µg/mL]. CONCLUSIONS Simultaneous detection for sulfatides using UPLC/MS/MS can be successfully applied to DBS analysis. This method provides a fast and effective screening and monitoring tool for the diagnosis and treatment of MLD.

Performance evaluation of SD A1cCare as a HbA1c analyzer for point-of-care testing.

OBJECTIVES Some studies have shown that a rapid feedback of HbA1c results is helpful for controlling plasma glucose levels. Point-of-care (POC) instruments that are fast, portable, and easy to use are suitable for rapid determination of HbA1c levels. Here, we evaluated the analytical performance of a newly developed POC HbA1c analyzer, SD A1cCare (SD Biosensor, Inc.). DESIGN AND METHODS The precision, linearity, and correlation with the Variant II Turbo instrument (Bio-Rad Laboratories, Inc.) were evaluated according to CLSI guidelines for SD A1cCare. All tests were performed according to the manufacturer instructions, and statistical analyses, including linear regression and Passing-Bablok regression, were performed. Bias from the IFCC reference targets was also evaluated with 12 duplicate specimens (n=24 in total). RESULTS The coefficients of variation based on EP9-A2 protocol were 2.6% in SI unit and 1.8% in NGSP unit. The calibration curve was linear, with R(2)=0.9911 in the range of 23.5 to 125.1 mmol/mol in SI units (4.3% to 13.6% in NGSP units). The results of the SD A1cCare correlated with those of the Variant II Turbo (r=0.986). Deviations from IFCC targets at 30, 60, and 90 mmol/mol IFCC levels were -1.95, -1.85, and -1.74 mmol/mol, respectively. CONCLUSIONS The SD A1cCare analyzer showed excellent precision, linearity, correlation with the Variant II Turbo analyzer, and accuracy with IFCC targets. Therefore, it may be suitable for HbA1c assays in the POC setting and in small laboratories.

Multiplex Assay of Second-Line Anti-Tuberculosis Drugs in Dried Blood Spots Using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry

As dried blood spots (DBSs) have various advantages over conventional venous blood sampling, some assays for detection of one or two anti-tuberculosis (TB) drugs in DBSs have been developed. However, there are no assays currently available for the simultaneous measurement of three or more anti-TB drugs in DBSs. In this study, we developed and evaluated a multiplex method for detecting nine anti-TB drugs including streptomycin, kanamycin, clarithromycin, cycloserine, moxifloxacin, levofloxacin, para-aminosalicylic acid, prothionamide, and linezolid in DBSs by using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Seventy-nine patient samples of DBS were analyzed on the UPLC-MS/MS system. All drug concentrations were determined within 4 min, and assay performance was evaluated. All drugs were clearly separated without ion suppression. Within-run and between-run precisions were 1.7-13.0% and 5.7-17.0%, respectively, at concentrations representing low and high levels for the nine drugs. Lower limits of detection and quantification were 0.06-0.6 and 0.5-5.0 µg/mL, respectively. Linearity was acceptable at five level concentrations for each drug. Correlations between drug concentrations in plasma and DBSs by using Passing-Bablock regression and Pearsons rho (ρ, 0.798-0.989) were acceptable. In conclusion, we developed a multiplex assay to measure nine second-line anti-TB drugs in DBSs successfully. This assay provided convenient and rapid drug quantification and could have applications in drug monitoring during treatment.

Direct assay of iduronate-2-sulfatase for Hunter disease using UPLC-tandem mass spectrometry and fluorogenic substrate

OBJECTIVE We devised iduronate-2-sulfatase (IDS) enzyme activity assays by combining fluorometric substrate and LC-MS/MS based detection. DESIGN AND METHODS 4-Methylumbelliferyl α-L-idopyranosiduronic acid 2-sulfate (IDS-S) was used as a substrate for IDS. Its enzymatic product, 4-methylumbelliferyl α-L-idopyranosiduronic acid (IDS-P) and internal standard, 4-methylumbelliferyl α-L-idopyranoside (IDS-IS), were directly measured by UPLC-MS/MS. We determined the precision of our enzyme assay and the effects of sample amounts and incubation time based on the results. Dried blood spots (DBSs) of 110 normal newborns and three patients with Hunter disease were analyzed. RESULTS IDS-IS, IDS-P and IDS-S were fully separated using UPLC without any ion suppressions. The intra- and inter-assay precisions were 8.5-10.5% and 11.9-15.3%, respectively. The amount of product obtained was proportional to the number of DBSs and increased linearly with the incubation period from 0 to 15 h. The enzyme activities in DBSs from three patients with MPS II were markedly lower than those in the DBSs of 110 normal newborns. CONCLUSION To the best of our knowledge, this is the first report describing the use of LC-MS/MS for the diagnosis of Hunter disease with a commercially available substrate. Our method would be a rapid and effective screening tool for the diagnosis of Hunter disease with further study.

Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Measurement of Leukocyte Arylsulfatase A Activity Using a Natural Substrate

Biochemical diagnosis of metachromatic leukodystrophy (MLD) is usually performed by measuring arylsulfatase A (ARSA; EC3.1.6.8) activity with artificial substrates (p-nitrocatechol sulfate or 4-methylumbelliferyl sulfate) in leukocytes or cultured skin fibroblasts [1, 2]. Unfortunately, these artificial substrates are also substrates for several other enzymes, including arylsulfatase B [3, 4]. Thus, quantitation of residual activity could be inaccurate, especially in the context of MLD variants and ARSA pseudodeficiency. In this study, we evaluated the feasibility of ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS) in a leukocyte ARSA assay using a natural sulfatide substrate, and we compared this approach with a traditional spectrophotometric assay using a synthetic substrate. The substrate and internal standard (IS) were N-octadecanoyl-sulfatide and N-octadecanoyl-D35-psychosine (Matreya LLC, Pleasant Gap, PA, USA), respectively. We used C18 β-D-glucosyl ceramide (Avanti Polar Lipids Inc., Alabaster, AL, USA) instead of C18 β-D-galactosyl ceramide as the standard because of commercial availability. The enzyme reaction cocktail contained 2.08 g/L sodium taurodeoxycholate, 33 mmol/L MnCl2, 0.08 mol/L sodium acetate buffer (pH 4.5), and 6.20 µmol/L substrate. Seventy microliters of reaction cocktail and 30 µL of sonicated leukocyte solution were incubated at 37℃ for 1 hr and were quenched with 100 µL of ethyl acetate-methanol solution. Ten microliters of 1 µmol/L IS solution and 300 µL of both ethyl acetate and water were added to the reaction vial and centrifuged at 15,700 g for 10 min. Two hundred microliters of the top organic layer was dried under N2 gas and resuspended in 70 µL of methanol. And 7 µL was injected into a Waters ACQUITY UPLC system (Waters, Milford, MA, USA). The mobile phase was a mixture that was 97% methanol containing 3% of 0.05 mol/L ammonium formate solution in water at a flow rate of 0.5 mL/min. A Quattro Premier XE MS/MS (Waters) was operated by using the following settings: cone voltage, 25, 30, and 25 V; collision energy, 35, 40, and 35 V; and multiple reaction monitoring transition in positive ion mode, m/z 808.5→264.3, m/z 763.7→265.3, and m/z 728.5→264.3 for the substrate, IS, and product, respectively. The amount of product was calculated from the calibration curves constructed with five concentrations of C18 s-D-glucosyl ceramide (0-687 nmol/L), and the enzyme activities were expressed in nmol/min/mg protein. Substrates, products, and IS were fully separated by using UPLC with an HSS T3 1.8-µm column (2.1×50 mm) with a 3 min chromatographic separation time (Fig. 1). The amount of product obtained was proportional to the volume of leukocytes used in the assay (10, 20, 30, 40, and 50 µL of leukocyte extract) and increased linearly with the incubation period (0, 0.5, 1, 2, and 3 hr). We chose a 30 µL leukocyte volume and a 1 hr incubation time for all subsequent enzymatic assays. The within- and between-run imprecision (CVs), as determined by 10 replicated analyses and 5 consecutive runs using a normal control sample, were 7.7% and 14.5%, respectively. Fig. 1 Representative ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) multiple reaction monitoring (MRM) chromatograms from the mixture of S, IS and standard (A) and the mixture of ARSA enzyme reactions in the leukocyte samples ... To validate the capability of our system to detect MLD patients, three MLD patients, one pseudodeficiency, one obligate heterozygote and 38 normal adults were examined. All patients exhibited reduced enzyme activity using synthetic substrates, confirmed by mutation analysis. As expected, the leukocytes of MLD patients exhibited consistently lower enzyme activities than those of the obligate heterozygote, pseudodeficiency, and normal adults without overlapping values (Fig. 2A). The provisional cut-off value was estimated as 1.23 mmol/min/mg protein. Passing-Bablok regression analysis revealed that the UPLC-MS/MS method and the traditional colorimetric assay [1] compared favorably (r=0.8019) (Fig. 2B). Fig. 2 Comparison of arylsufatase A activities measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in leukocytes samples from three patients with MLD (all 2-yr-old females) and one patient with pseudodeficiency (PD, a 47-yr-old ... Recently, reports on multiplex enzyme assay screening of dried blood spots (DBS) for lysosomal storage disorders have engendered interest in the use of MS/MS for newborn screening [5, 6]. Although assays using natural sulfatide substrates are more complicated because of the poor water solubility of sulfatides [7], utilization of natural substrates is increasingly required becausethe ARSA assay may soon be incorporated into newborn screening programs. In this regard, mass spectrometry is accepted as a valuable tool for the analysis of lipids and lipid-metabolizing enzymes using a natural substrate system. In this study, we investigated the feasibility of UPLC-MS/MS for use in a leukocyte ARSA assay using a natural sulfatide substrate. To the best of our knowledge, this is the first study to report the feasibility of UPLC-MS/MS for diagnosing MLD. The assay performance for our devised method in terms of precision and correlation with a traditional spectrophotometric method was within the generally acceptable standard and could be adopted for newborn screening of DBSs for MLD. More experiments would be needed to develop the assay for routine application.

Application of the isoniazid assay in dried blood spots using the ultra-performance liquid chromatography-tandem mass spectrometry

OBJECTIVES Therapeutic drug monitoring (TDM) of anti-tuberculosis (TB) drugs is important for proper treatment of TB. Dried blood spots (DBSs) are widely used for TDM because of their several advantages. Rifampicin and pyrazinamide assays with DBSs have already been developed. However, isoniazid (INH) assay for capillary DBSs have not been reported because of INH instability. We developed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for measuring INH concentrations in venous and capillary DBSs. METHODS Each DBS was analyzed on an UPLC system. INH and internal standard (IS) concentrations were determined by multiple-reaction monitoring in positive ion mode. Analytical performances, including precision, linearity, and comparison of different types of specimens were determined. Further, the stability of INH in venous DBSs was tested. RESULTS INH and IS were clearly separated in the UPLC-MS/MS system without matrix effect. Within-run precision and between-day precision were 2.68-8.02% and 2.54-5.45%, respectively. INH concentrations in venous DBS showed proportional bias compared with those in plasma (Slope: 0.8704) with good correlation. INH concentration in capillary DBS was slightly but not significantly higher than that in venous DBS. CONCLUSIONS The findings of our study show that the analytical performance of this novel method for capillary and venous DBSs was clinically acceptable for the TDM of INH.