Hye-Ran Yoon

Quantitative analysis of acyl-lysophosphatidic acid in plasma using negative ionization tandem mass spectrometry.

Analysis of acyl-lysophosphatidic acids (LPAs) has clinical importance as a potential biomarker for ovarian and other gynecological cancers or obesity from the point of view of prevention. Here we report a simple sample preparation and analytical method with high sensitivity and specificity for the early detection of gynecological cancers to improve the overall outcome of this disease. We established a novel quantification method for acyl-LPAs in plasma by electrospray negative ionization tandem mass spectrometry (MS-MS) using multiple reaction monitoring mode without conventional TLC step. Protein-bound lipids, acyl-LPAs in plasma were extracted with methanol/chloroform (2:1) containing LPA C(14:0) as internal standard under acidic conditions. Following back-extraction with chloroform and water, the centrifuged lower phase was evaporated and reconstituted in methanol and then analyzed. Using ESI-MS-MS with negative ionization MRM mode, all the species of LPAs were completely separated from plasma matrix without severe interference. For MRM mode, Q1 ions selected were m/z 409, 433, 435, 437 and 457 which corresponds to molecular mass [M-H](-) of C(16:0), C(18:2), C(18:1), C(18:0) and C(20:4) LPA, respectively. Q2 ions selected for MRM was m/z 79, phosphoryl product. Using MS-MS with MRM mode, all the species of LPAs were completely separated from plasma matrix without severe interference. This method allowed simultaneous detection and quantification of different species of LPAs in plasma over a linear dynamic range of 0.01-25 micromol/l. The method detection limit was 0.3 pmol/ml with correlation coefficient of 0.9983 in most LPAs analyzed. When applied to plasma from normal and gynecological cancer patients, this new method differentiated two different groups by way of total LPA level.

Two Step Derivatization for the Analyses of Organic, Amino Acids and Glycines on Filter Paper Plasma by GC-MS/SIM

A rapid dried-filter paper plasma-spot analytical method was developed to quantify organic acids, amino acids, and glycines simultaneously in a two-step derivatization procedure with good sensitivity and specificity. The new method involves a two-step trimethylsilyl (TMS) - trif-luoroacyl (TFA) derivatization procedure using GC-MS/ selective ion monitoring (GC-MS/SIM). The dried-filter paper plasma was fortified with an internal standard (tropate) as well as a standard mixture of distilled water and methanol. Methyl orange was added to the residue as an indicator. N-methyl-N-(trimethylsilyl-trifluoroacetamide) and N-methyl-bis-trifluoroacetamide were then added and heated to 60°C for 10 and 15 min to produce the TMS and TFA derivatives, respectively. Using this method, the silylation of carboxylic functional groups was carried out, which was followed by the trifluoroacyl derivatization of the amino functional group. The derivatives were analyzed by GC-MS/SIM. A calibration cure showed a linear relationship for the target compounds between concentrations of 10-500 ng/mL The limit of detection and quantification on a plasma spot were 10-90 ng/mL (S/N=9) and 80-500 ng/ mL, respectively. The correlation coefficient ranged from 0.938 and 0.999. When applied to the samples from positive patients, the method clearly differentiated normal subjects from the patients with various metabolic disorders such as PKU, MSUD, OTC and a Propionic Aciduria. The new developed method might be useful for making a rapid, sensitive and simultaneous diagnosis of inherited organic and amino acid disorders. In addition, this method is expected to be an alternative method for screening newboms for metabolic disorders in laboratories where expen-sive MS/MS is unavailable.

PHEX gene mutations and genotype-phenotype analysis of Korean patients with hypophosphatemic rickets.

X-linked hypophosphatemic rickets (XLH) results from mutations in the PHEX gene. Mutational analysis of the PHEX gene in 15 unrelated Korean patients with hypophosphatemic rickets revealed eight mutations, including five novel mutations, in nine patients: two nonsense mutations, two missense mutations, one insertion, and three splicing acceptor/donor site mutations. Of these, c.64G>T, c.1699C>T, c.466_467 insAC, c.1174-1G>A, and c.1768+5G>A were novel mutations. To analyze the correlation between genotype and phenotype, phenotypes were compared between groups with and without a mutation, in terms of mutation location, mutation type, and sex. Skeletal disease tended to be more severe in the group with a mutation in the C-terminal half of the PHEX gene, but no genotype-phenotype correlation was detected in other comparisons. Further extensive studies of the PHEX gene mutations and analyses of the genotype-phenotype relationships are required to understand PHEX function and the pathogenesis of XLH.

A pulsed amperometric detection method of galactose 1-phosphate for galactosemia diagnosis

Galactose 1-phosphate uridyltransferase deficiency causes the accumulation of galactose and galactose 1-phosphate (Gal 1-P) in the blood. We describe a new pulsed amperometric detection method for determining Gal 1-P levels as a pathognomic marker for the diagnosis of galactosemia. The method uses high-performance anion-exchange chromatography with pulsed amperometric detection. In an anion-exchange column, the analytes were separated in 5 min by the eluent mixture of 40 mM NaOH and 40 mM Na(2)CO(3). The detection limit (signal to noise ratio of 3) to Gal 1-P was 30 microg/dL. The linear dynamic range was 3.0-50 mg/dL (r(2)=0.9999). The mean recoveries of Gal 1-P for intra- and interday assays were 97.55-103.78%. This method clearly separated the type I galactosemia patients from the normal group and is a practical procedure for the rapid diagnosis of galactosemia.

Simultaneous diagnostic method for phenylketonuria and galactosemia from dried blood spots using high-performance liquid chromatography-pulsed amperometric detection

We developed a simultaneous diagnostic method for phenylketonuria (PKU) and galactosemia through simultaneous determination of phenylalanine (Phe) and galactose (Gal) by high-performance liquid chromatography (HPLC) with pulsed amperometric detection (PAD). The intra- and inter-day precisions were <5.8%, with satisfactory mean recoveries (98.2-105%). For all PKU-positive samples, Phe levels were above the cut-off value (>30.0 mg/L), but Gal levels were nearly zero. For 77% of galactosemia-positive samples, Phe levels were above the cut-off value, but Gal levels were above the cut-off value (>80.0 mg/L) for all samples. Our HPLC-PAD method can reduce the false-positive rate of misdiagnosis for PKU and galactosemia.

Determination of phenylalanine in blood by high-performance anion-exchange chromatography―pulsed amperometric detection to diagnose phenylketonuria

We have developed a high-performance anion-exchange chromatography with pulsed amperometric detection method for the detection of phenylalanine (Phe) and diagnosis of phenylketonuria (PKU). Sample pretreatment steps were simplified without derivatization. The analyte was separated within 5 min. The detection limit (S/N=3) for Phe was 50 pg. Linear dynamic range was 1.23-14.43 mg/dL (r(2)=0.9999) for a dried blood spot. The mean recoveries of Phe for intra- and inter-day assays were found to be 96.87-104.16%. This method clearly differentiated PKU-positive groups from normal groups, and proved to be a practical procedure for rapid screening and follow-up monitoring of PKU.

Chromatographic diagnosis of maple syrup urine disease by measuring the l-alloisoleucine/l-phenylalanine ratio in dried blood spots

A high-performance ligand-exchange chromatography with ultraviolet detection method for confirmation diagnosis of maple syrup urine disease (MSUD) was developed that relies on the determination of branched-chain amino acids (BCAAs) and Phe levels in blood. The dynamic ranges for the BCAAs and Phe were 50-1000 μM (r(2)=0.9982-0.9996) and 74-873 μM (r(2)=0.9992) from a dried blood spot, and the BCAA detection limits (S/N=3) were 0.43-1.91 μM. The mean recoveries of BCAA for intra- and inter-day assays were 92.1-103.0%. The ranges of alloisoleucine (Allo-Ile)/Phe ratio were ND-0.04 and 1.5-2.4 for PKU and MSUD patient samples, respectively. The lowest ratio (1.5) of the MSUD samples was 37.5 times higher than the highest ratio (0.04) of the PKU samples. Therefore, the Allo-Ile/Phe ratio was very useful biomarker for confirmation diagnosis of MSUD.

Galactosemia Screening by Simultaneous Blood Spot Quantification of Galactose and Galactose 1-Phosphate

Galactosemia, caused by enzyme deficiencies of galactose metabolism, leads to the accumulation of galactose (Gal)1 and galactose 1-phosphate (Gal 1-P) in blood. The disease is classified into 3 types. The concentrations of Gal and Gal 1-P in galactose 1-phosphate uridyltransferase (GALT) or galactokinase (GALK) deficiency are quite different (1). Therefore, simultaneous monitoring of Gal and Gal 1-P in blood is useful to distinguish infants with GALT deficiency (type I) from those with GALK deficiency (type II). Of the various methods for determining Gal or Gal 1-P in blood spots, none are suitable for simultaneous and direct measurement because of the requirements of special reagents or multistep and time-consuming procedures (2)(3). We developed a novel method based on high-performance anion-exchange chromatography (HPAEC)–pulsed amperometric detection (PAD) that simultaneously measures Gal and Gal 1-P concentrations in patient blood spots. We prepared blood spot standards using healthy male adult blood and stock solutions of Gal and Gal 1-P. We collected blood spot specimens (70 controls and 12 patients) from Korean newborns within 3 days after birth. All of the samples were identified by quantifying Gal concentrations using the enzymatic colorimetric method (ECM) (3). Two 3.2 mm–diameter filter paper …

Methionine/galactose ratio on newborn blood spots useful for reduction of false positives for homocystinuria and galactosemia by high-performance anion-exchange chromatography with pulsed amperometric detection

BACKGROUND Methionine (Met) in blood and urine is a useful diagnostic marker for homocystinuria (HCU). However, galactosemia could be misdiagnosed as HCU when Met is used as the sole marker, since elevated excretion of Met presents in both galactosemia and HCU. Use of a more specific diagnostic marker in addition to Met is therefore necessary for reduction of false positive results for HCU as well as confirmative diagnosis of HCU. METHODS Chromatographic separation was performed using an anion-exchange column. The levels of Met and galactose (Gal) on blood were measured and Met/Gal ratios were calculated from blood spot samples from 300 normal volunteers, eight galactosemia patients, and three HCU patients. RESULTS The Met/Gal ratio ranged 0-4.95 for normal blood spots (n=300), 0-0.22 for galactosemia samples (n=8), and >1250 for HCU patient samples. CONCLUSIONS Separation, extraction, and deproteinization procedures were established for Met and Gal in blood spots. And Met/Gal ratio allowed HCU to clearly distinguish from galactosemia. As a way of second tier confirmative analysis, the ratio is the best way to reduce false positives. The assay is most appropriate to reduce false positives in labs that do not screen for galactosemia.

A Simple, Rapid and Reliable Method to Determine Imipramine and Desipramine in Mouse Serum Using Ultra-High-Performance Liquid Chromatography–Quadrupole-Time-of-Flight Mass Spectrometry

A rapid and sensitive ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometric (UHPLC-Q-TOF-MS) method was developed for quantification of imipramine, one of the most widely used tricyclic antidepressants, and desipramine, an active metabolite of imipramine, in mouse serum. The developed method included a simple protein precipitation with acetonitrile in 50 μL of serum and analyte separation on an Acquity UPLC BEH C18 column using a gradient elution of acetonitrile with 0.1% formic acid and 20 mM ammonium formate. As a result, the entire analysis time was <20 min including the sample preparation and the LC-MS analysis. The limit of quantification was 5.0 ng mL(-1) for both imipramine and desipramine, and calibration curves were linear over the concentration range of 5.0-1,000.0 and 5.0-250.0 ng mL(-1) for imipramine and desipramine, respectively. Intraday precisions at three levels were 2.2-3.6 and 1.7-4.2% for imipramine and desipramine, respectively, whereas interday precisions were 2.6-5.0 and 2.0-8.4% for imipramine and desipramine, respectively. Accuracy ranged between 93.6 and 106.6% for imipramine and 94.1 and 106.4% for desipramine. Absolute recovery was 96.0-97.6% for imipramine and 87.0-99.5% for desipramine. Finally, the described method was applied to mice administered with imipramine, demonstrating the suitability for quantification of imipramine and desipramine for therapeutic drug monitoring or bioequivalence studies.