Inhibition of Cross-Reactive Carbohydrate Determinant–Specific IgEs Reduce False Positive Reponses to Multiple Allergen Simultaneous Test–Immunoblot Assay: A Case Study

Cross-reactive carbohydrate determinants (CCDs) are simple carbohydrates linked to amino acid chains; they are found in pollens, vegetable foods, insect, and Hymenoptera venoms and are broadly cross-reactive with CCD-specific IgE antibodies. A man in his fifties was evaluated using a multiple allergen simultaneous test–immunoblot assay. On the PROTIA Allergy-Q 64 inhalant panel (ProteomeTech, Korea), reactions to 37 of 59 antigens were observed except mammalian antigens, and cross-reactivity owing to anti-CCD antibodies was suspected. After ProGlycAn CCD-blocker (ProGlycAn, Austria) treatment, the patient exhibited no response to CCD allergens, and the number of allergens showing positive reactions was reduced to 15. We further tested a total of 7 samples from patients who were suspected to have CCD-related cross-reactivity. For these 8 patients, the average number of positive reactions to allergens was reduced from 33 (range 24-36) to 8 (range 0-19) after CCD-blocker treatment. We concluded that CCD-blocker treatment in sample with anti-CCD antibodies can reduce the false positive reponse and provide more specific information about allergens.

A Multiplex Phytosterol Assay Utilizing Gas Chromatography-Mass Spectrometry for Diagnosis of Inherited Lipid Storage Disorders

No abstract available.

Report on the External Quality Assessment Scheme for Hemoglobin A1c in Korea (2016–2017)

As part of the clinical chemistry programs of the Korean Association of External Quality Assessment Service, we organized trials to conduct external quality assessment of hemoglobin A1c (HbA1c) in 2016 and 2017. We delivered three commutable whole blood specimens for each trial. The overall response rates were 96.4% in 2016 and 98.4% and 99.6% in the first and second trials in 2017, respectively. Two types of reports were generated: a common report including the mean, standard deviation, coefficient of variation, median, minimum value, and maximum value according to manufacturer and instrument, as well as a summary report of the results from each participating laboratory, including a histogram for each specimen, Levey-Jennings chart for serial percent deviations, and table showing the target value, upper and lower limits, percent deviation, and grade. From 2016, the acceptance criterion was changed from ±8.0% to ±6.7%. The overall acceptable rates for the survey were 91.5%, 91.4%, 94.9%, and 89.0% for the first trial in 2016 through the second trial in 2017, respectively. The requisite continual improvement in the quality of HbA1c testing can be achieved through participation in similar accuracy-based proficiency testing programs.

Evaluation of the Self-Testing Blood Glucose Monitoring System GlucoDr.S According to ISO 15197:2013 Guidelines

BACKGROUND: The performance of the self-monitoring of blood glucose in patients with diabetes should be properly evaluated to ensure strict glycemic control. This study evaluated the self-testing Blood Glucose Monitoring System GlucoDr.S™ (All Medicus Co., Ltd., Korea). METHODS: This study recruited 120 patients. Use of the glucometer was evaluated according to ISO 15197:2013 guidelines. The YSI 2300 STAT PLUS Glucose Analyzer (YSI Life Sciences, USA) was used as the reference device. RESULTS: The standard deviation and coefficients of variation ranges for measurement repeatability and intermediate measurement precision conducted with 10 meters and 3 reagent lots on the same day were 2.7–3.2 mg/dL (0.99. The influence effect of hematocrit and the 24 interference agents was not significant, except for xylose. A system accuracy test was conducted with 100 subjects taking duplicate measurements from each of the 3 reagent lots. When glucose levels were 95% of the samples were within ±15 mg/dL and within ±15% of the average measured values of the reference measurement, respectively. In Consensus Error grid analysis, all results were distributed in zone A and B. The results of the user performance evaluation using 115 lay persons were also included in the acceptance range. CONCLUSION: The GlucoDr.S™ showed acceptable performance according to the ISO 15197:2013 guidelines and could be a clinically useful self-testing glucometer.

Measurement of Succinylacetone Using HPLC-Tandem Mass Spectrometry and Establishment of a Cut-off Value

BACKGROUND: Newborn screening of tyrosinemia type 1 is important for identifying infants at risk for developing this disease before life-threatening symptoms occur. It is difficult to differentiate between tyrosinemia type 1 and transient neonatal tyrosinemia (TNT) by analyzing tyrosine alone. Thus, succinylacetone must be analyzed. In this study, we measured succinylacetone in dried blood spot (DBS) by HPLC-tandem mass spectrometry (HPLC-MS/MS) and established cut-off values. METHODS: We used the hydrazine derivatization method to measure succinylacetone in 127 DBSs showing normal results in the newborn screening test and 93 DBSs showing increased tyrosine levels. We established cut-off values using the 99.9th percentile value or median+5 standard deviation value. RESULTS: Succinylacetone levels determined by our method were well-correlated with the results recommended by the Centers for Disease Control and Prevention for proficiency testing (r=0.9968). The succinylacetone levels in normal newborn DBSs were significantly lower than those in DBSs with high tyrosine levels (P < 0.001). The cut-off values were calculated to be 1.3 µM from the results of 127 normal DBS samples and 2.2 µM from 220 DBSs, including in 93 newborns with TNT. CONCLUSIONS: Measurement of succinylacetone in DBSs by HPLC-MS/MS is useful in individuals with increased tyrosine concentrations and can be used for rapid differential diagnosis of tyrosinemia when an appropriate cut-off value is established.

Effects of Pyridoxal-5'-Phosphate on Aminotransferase Activity Assay

BACKGROUND: Pyridoxal-5'-phosphate (P5P), a coenzyme of the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) reactions, is required to measure aminotransferase levels (IFCC method). However, a modified IFCC method that uses a reagent devoid of P5P is commonly used in laboratories in Korea. To determine the differences between the two methods, we compared aminotransferase levels measured by using the IFCC method and modified IFCC method. METHODS: Serum levels of AST and ALT, with and without P5P, were measured in 2,318 patients. Based on the allowable limits of performance set by the Royal College of Pathologists of Australasia (RCPA), differences between the two methods were analyzed under various conditions. RESULTS: Higher AST and ALT values were obtained by the IFCC method compared to modified IFCC method, showing significant differences between the two methods (AST, 5.8±14.2 IU/L; ALT, 2.8±6.9 IU/L) (P<0.001). Values exceeding RCPA criteria were more frequently observed in emergency orders (AST, 65.8%; ALT, 14.4%) than in routine orders (AST, 3.2%; ALT, 9.6%), as well as in inpatient wards (AST, 70.4%; ALT, 18.5%) compared to outpatient clinics (AST, 56.6%; ALT, 10.0%). However, the differences between the two methods were not significant among the disease groups, except for the acute myocardial infarction group. CONCLUSIONS: The method using reagents without P5P underestimated aminotransferase activity. The effect of P5P was more significant in patients with acute myocardial infarction, considered as P5P-deficient. In conclusion, the IFCC method with P5P should be applied for measuring AST and ALT serum levels.

Performance Evaluation of a Point-of-care Test, ‘Samsung LABGEO PA CHF Test’, for the Amino-terminal Pro-brain Natriuretic Peptide

BACKGROUND: The amino-terminal pro-brain natriuretic peptide (NT-proBNP) is a useful biomarker for the diagnosis of acute congestive heart failure. A point-of-care test (POCT) could rapidly detect the presence of NT-proBNP during emergencies. We evaluated the analytical performance of the new Samsung LABGEO PA CHF Test (Samsung Electronics, Korea). METHODS: Based on the guidelines of the Clinical and Laboratory Standards Institute (CLSI), we compared the precision, linearity, and method with those of the E170 (Roche Diagnostics, Switzerland). Matrix comparison between the NT-proBNP values in whole blood and plasma was also performed, and the reference interval was determined using residual samples from healthy adults selected based on the evaluation criteria. RESULTS: The Samsung LABGEO PA CHF Test provided results in approximately 18 min. The coefficient of variation (CV) of within-laboratory precision was below 6.8%. A desirable linearity was observed in the range of 0–10,000 pg/mL, with R²=0.99. The correlation with E170 was also excellent (N=108, r=0.96). NT-proBNP values in the whole blood were correlated with those in the plasma (N=36, r=0.99). The reference interval for the circulating NT-proBNP concentration was determined in 118 plasma samples from healthy subjects (26-75 yr of age). The 97.5th percentile was found to be 58.3 pg/mL. CONCLUSIONS: The Samsung LABGEO PA CHF Test demonstrated a good analytical performance. It could be a powerful tool as a POCT for clinical practice, particularly during emergencies.

Annual Report on the External Quality Assessment Scheme for Routine Clinical Chemistry in Korea (2016)

In 2016, the clinical chemistry proficiency-testing program consisted of 21 programs, including the general chemistry program of the Korean Association of External Quality Assessment Service. The general chemistry program consisted of 28 test items and was conducted using two level control materials four times per year. Based on the information and results for each test item entered by each institution, statistical analysis data according to test method, instrument, and reagent were reported. The report comprised a general statistics report showing the characteristics of all participating institutions and a separate institutional report showing the evaluation data of individual institutions. The statistics included the number of participating institutions and the mean, standard deviation, coefficient of variation, median, minimum, and maximum values for each group. Each report was composed of a table, histogram, and Levey-Jennings chart showing the statistics for each test item. The results of each institution and the statistics for each classification are presented in the table showing the statistics, and a standard deviation index is presented together with a method classification and a classification by reagent companies. A total of 14 items, including albumin, were evaluated by more than 1,000 institutions. There was no significant difference in the distribution of the measurement methods compared with those used in the previous year. The coefficient of variation showed a tendency to increase as the concentration of the level control material decreased and as the number of participating institutions decreased for each test item. Most of them showed a coefficient of variation within 10%. These statistical data will be useful when interpreting the survey results from the institutions and selecting a test method.

Annual Report on the External Quality Assessment Scheme for Clinical Chemistry in Korea (2015)

The Clinical Chemistry Subcommittee of the Korean Association for External Quality Assessment Service conducted external quality assessments in 2015. This included general chemistry and blood gas measurements as part of a scheme of six trials, comprising three samples each. All control materials were included at the same time. The overall response rates were 94.4% for general chemistry and 92.2% for blood gas. The parameters tested included sodium, potassium, chloride, blood urea nitrogen, glucose, calcium, phosphorus, uric acid, creatinine, bilirubin, total protein, albumin, total cholesterol, triglyceride, aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase, lactate dehydrogenase, gamma glutamyl transferase, high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol for general chemistry, and pH, partial pressure of carbon dioxide (pCO₂), and partial pressure of oxygen (pO₂) for blood gas assessments. Two types of reports were generated: a method summary, including mean, standard deviation, and coefficient of variation for each test method; and a result summary of each participating laboratory, including mean, standard deviation, number of peer groups, and standard deviation index and variance index scores of each laboratory. The overall quality performance in 2015 was similar to that of previous years, and showed lower interlaboratory variation than that in 2014. The requisite continual improvement in clinical chemistry testing quality can be achieved through participation in similar proficiency testing programs.

Evaluation of Analytical Performance of the D-100 Hemoglobin Testing System for Hemoglobin A1c Assay

BACKGROUND: The hemoglobin A1c (HbA1c) level is widely used to diagnose and monitor glycaemic control in people with diabetes mellitus, and various methods are used for its determination. The D-100 hemoglobin testing system (Bio-Rad Laboratories, USA) is a fully automated, high-throughput glycohaemoglobin analyzer based on an ion-exchange high-performance liquid chromatographic method. Here, we evaluated the analytical performance of a newly developed HbA1c analyzer. METHODS: Precision, linearity, and comparison to the Variant II Turbo analyzer (Bio-Rad Laboratories, USA) were evaluated according to the Clinical Laboratory Standards Institute guidelines. Carryover, bias from the value assigned by the HbA1c Network Laboratory of Korea Centers for Disease Control and Prevention, and the vulnerability to interference by hemoglobin variants frequently found in Korea were also assessed. Statistical analyses were performed using Excel 2010 (Microsoft Co., USA) and MedCalc ver. 14.12.0 (MedCalc Software bvba, Belgium). RESULTS: The coefficients of variation for repeatability and within-device precision were less than 1.08% in National Glycohaemoglobin Standardization Program (NGSP) unit and less than 1.68% in international system of unit at all three levels. The calibration curve was linear, with R²=0.996 in the range of 4.6% to 15.4% in NGSP unit. The results highly correlated with those produced by Variant II Turbo (r=0.998). The 95% confidence interval for differences from the assigned values was -3.3% to 2.9%. No significant interferences of haemoglobin variants were observed except for Hemoglobin Yamagata. CONCLUSIONS: The D-100 hemoglobin testing system showed excellent precision, linearity, and good correlation with the Variant II Turbo analyzer and agreement with the assigned values. Therefore, its analytical performance is satisfactory for diabetes diagnosis and treatment monitoring.

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 Pearson's 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.

Evaluation of the Urinary Glucose Tetrasaccharide Assay Using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry for Diagnosis of Pompe Disease

We evaluated the urinary glucose tetrasaccharide (Glc4) assay using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The calibration curve was linear over a range of 5-500 micromol/L. Performance parameters such as intra- and inter-day imprecision CVs were 6.52-14.6% and 11.5-13.2%, respectively. The mean concentrations of urinary Glc4 in 27 normal controls and 3 pseudodeficiency patients were 1.5 and 12.1 mmol/mol creatinine, respectively. Urinary Glc4 concentration in a patient with Pompe disease was 171.3 mmol/mol creatinine, which decreased to 130.9 mmol/mol following enzyme replacement therapy. Based on our results, we suggest that the urinary Glc4 assay using UPLC-MS/MS can be a reliable diagnostic tool for identification of patients with Pompe disease.

Annual Report on the External Quality Assessment Scheme for Clinical Chemistry in Korea (2014)

The Clinical Chemistry subcommittee of The Korean Association of External Quality Assessment Service conducted external quality assessments in 2014. This included general chemistry and blood gas measurements as part of a scheme of six trials, comprising of three samples each. All control materials were sent at the same time. The overall response rates were 93.4% for general chemistry and 90.0% for blood gas. The parameters tested included sodium, potassium, chloride, blood urea nitrogen, glucose, calcium, phosphorus, uric acid, creatinine, bilirubin, total protein, albumin, total cholesterol, triglyceride, AST, ALT, alkaline phosphatase, lactate dehydrogenase, gamma glutamyl transferase, HDL cholesterol, and LDL cholesterol for general chemistry and pH, partial pressure of carbon dioxide, and partial pressure of oxygen for blood gas assessment. Two types of reports were generated, namely, a method summary report including mean, standard deviation, and coefficient of variation, for each test method, as well as a result summary report of each participating laboratory, including mean, standard deviation, number of peer groups, and standard deviation index and variance index scores of each laboratory. The overall quality performance in 2014 was similar to that of previous years and as compared to results from 2013, the inter-laboratory variation was lower. The requisite continual improvement in the quality of clinical chemistry testing can be achieved through participation in similar proficiency testing programs.

Six Years' Experience of Accuracy-Based Proficiency Testing for HbA1c in Korea

BACKGROUND: The proficiency testing (PT) program for HbA1c, performed by the Korean Association of External Quality Assessment Service (KAEQAS), first started in 2007. From 2007 to 2008, the results were assessed using means as the standard within a peer group (identical method group). However, the assessment method changed to accuracy-based PT in 2009. This study aimed to analyse the results of an external quality assessment of HbA1c from 2009 to 2014. METHODS: Based on the data obtained from the external quality assessment of HbA1c from 2009 to 2014, we analysed the number of participating institutions, response rate, 'unacceptable' result rate, bias from the target value, and CVs according to each instrument code. RESULTS: The number of participating institutions was only 180 in 2009. However, it increased over the next 5 years, and as of 2014, 345 institutions were enrolled. The response rates were 93.8% to 99.1%. Since 2009, the measurement method changed and most of the participating institutions now use the high-performance liquid chromatography (HPLC) method. As of 2014, the HPLC method showed small bias from the target value and inter-laboratory CVs (<3.5%), demonstrating satisfactory performance. Immunoassays and point-of-care testing (POCT) demonstrated relatively unsatisfactory performance, showing larger inter-laboratory CVs compared to those obtained with the HPLC method, with some of them exceeding the acceptance limit of +/-8% of the target value. CONCLUSIONS: As of 2014, relatively large-scale laboratories are participating in the accuracy-based PT for HbA1c. According to the accuracy-based PT for HbA1c, POCT showed the highest 'unacceptable' rate and imprecision. Therefore, small-scale laboratories mostly using POCT for HbA1c measurement should be encouraged to participate in the accuracy-based PT program for HbA1c, and the external quality assessment program undertaken by KAEQAS should be expanded.

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

No abstract available.

Biochemical and Genetic Analysis of Seven Korean Individuals With Suspected Metachromatic Leukodystrophy

Metachromatic leukodystrophy (MLD) is an autosomal recessive disease caused by a deficiency in arylsulfatase A (ARSA). However, decreased ARSA activity is also observed in pseudodeficiency (PD). To distinguish between MLD and PD, we performed gene mutation and sulfatide analyses by using dried blood spots (DBSs) from seven Korean individuals who underwent an analysis of ARSA activity. DNA was extracted from DBSs, and PCR-direct sequencing of ARSA was performed. The cDNA obtained was analyzed to confirm a novel mutation. Of the seven subjects, three were confirmed as having MLD, one was confirmed as having MLD-PD, one was confirmed as having PD, and the remaining two were obligate heterozygotes. We verified the novel pathogenic variant c.1107+1delG by performing familial and cDNA analyses. Sulfatide concentrations in DBSs were analyzed and were quantified by using ultra-performance liquid chromatography and tandem mass spectrometry, respectively. Total sulfatide concentration was inversely correlated with ARSA activity (Spearman's coefficient of rank correlation, P=0.929, P=0.0025). The results of this mutational and biochemical study on MLD will increase our understanding of the genetic characteristics of MLD in Koreans.

Annual Report on the External Quality Assessment Scheme in Clinical Chemistry in Korea (2013) / 임상검사와정도관리

The Clinical Chemistry subcommittee of the Korean Association for Quality Assurance for Clinical Laboratory conducted external quality assessments in 2013. This included general chemistry and blood gas measurements as part of a scheme of six trials, comprising of three samples each. All control materials were sent at the same time. The overall response rates were 90.8% for general chemistry and 88.0% for blood gas. The parameters tested included sodium, potassium, chloride, blood urea nitrogen, glucose, calcium, phosphorus, uric acid, creatinine, bilirubin, total protein, albumin, total cholesterol, triglyceride, AST, ALT, alkaline phosphatase, lactate dehydrogenase, and gamma glutamyl transferase for general chemistry and pH, pCO2, and pO2 for blood gas assessment. Two types of reports were generated, namely, method summary reports including mean, standard deviation, and coefficient of variation, according to the test method as well as result summary reports of each participating laboratory, including mean, standard deviation and number of peer groups, and standard deviation index and variance index score of each laboratory. The overall quality performance in 2013 was similar to that of previous years, and as compared to results from 2012, the inter-laboratory variation had decreased. The requisite continual improvement in quality of clinical chemistry testing can be achieved through participation in similar proficiency testing programs.

Annual Report on the External Quality Assessment Scheme in Clinical Chemistry in Korea (2013) / 임상검사와정도관리

The Clinical Chemistry subcommittee of the Korean Association for Quality Assurance for Clinical Laboratory conducted external quality assessments in 2013. This included general chemistry and blood gas measurements as part of a scheme of six trials, comprising of three samples each. All control materials were sent at the same time. The overall response rates were 90.8% for general chemistry and 88.0% for blood gas. The parameters tested included sodium, potassium, chloride, blood urea nitrogen, glucose, calcium, phosphorus, uric acid, creatinine, bilirubin, total protein, albumin, total cholesterol, triglyceride, AST, ALT, alkaline phosphatase, lactate dehydrogenase, and gamma glutamyl transferase for general chemistry and pH, pCO2, and pO2 for blood gas assessment. Two types of reports were generated, namely, method summary reports including mean, standard deviation, and coefficient of variation, according to the test method as well as result summary reports of each participating laboratory, including mean, standard deviation and number of peer groups, and standard deviation index and variance index score of each laboratory. The overall quality performance in 2013 was similar to that of previous years, and as compared to results from 2012, the inter-laboratory variation had decreased. The requisite continual improvement in quality of clinical chemistry testing can be achieved through participation in similar proficiency testing programs.

Evaluation of the T-KB-H and 3-HB Kits for the Measurement of Serum Ketone and beta-Hydroxybutyric Acid

BACKGROUND: Diabetes mellitus and alcohol consumption are the most common causes of ketoacidosis in adults. Recently, beta-hydroxybutyric acid (betaHBA) was reported to be a potential serum biomarker in the diagnosis and monitoring of ketoacidosis. We evaluated the performance of T-KB-H and 3-HB kits for the measurement of ketone bodies [acetoacetate (AcAc)+betaHBA] and betaHBA, respectively. METHODS: Quantitative enzymatic assays were performed using the T-KB-H and 3-HB kits (Nittobo Medical Co., Japan) and the Architect ci16200 Integrated System (Abbott Laboratories, USA). Simultaneously, the ketone body levels in these serum samples were determined by gas chromatography-mas spectrometry (GC-MS). We evaluated precision and linearity of these kits and correlation with GC-MS, and established reference intervals in children and adults. RESULTS: The coefficients of variation for the T-KB-H and 3-HB kits were less than 4.0% at analyte levels of 50, 100, and 400 micromol/L. Linearity was observed for AcAc and betaHBA over a 0-1,000 micromol/L range (R2<0.99). Results from the T-KB-H and 3-HB kits were in good agreement with those from the GC-MS analysis, with correlation coefficients of 0.94 for AcAc and 0.96 for betaHBA. Reference intervals determined for the T-KB-H kit were 9.8-270.1 micromol/L and 18.5-531.8 micromol/L in children and adults, respectively. For the 3-HB kit, the reference intervals were 6.4-234.0 micromol/L and 16.0-437.2 micromol/L in children and adults, respectively. CONCLUSIONS: The T-KB-H and 3-HB kits displayed good precision, clinically acceptable linearity, and reliable correlation with an established assay. This indicates that the kits can be used clinically for measuring serum ketone bodies.

Measurement of Serum Levels of 25-Hydroxyvitamin D3 and 25-Hydroxyvitamin D2 Using Diels-Alder Derivatization and Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry

BACKGROUND: Vitamin D has been recently shown to play important roles in the functioning of various systems. Most of the current analytical methods for measuring vitamin D levels are based on immunoassays. We simultaneously measured the levels of 25-hydroxyvitamin D3 [ 25(OH)D3 ] and 25-hydroxyvitamin D2 [ 25(OH)D2 ] in human serum by performing ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) after Diels-Alder derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) and evaluated the performance of our method. METHODS: After liquid-liquid extraction, samples were dried under N2 at 50degrees C for 1 hr followed by Diels-Alder derivatization with ethyl acetate containing 0.1 mg/mL PTAD. The samples were resuspended in 60 microL of methanol:10 mM ammonium formate solution (1:1, V/V). C18 UPLC column and positive ion multiple reaction monitoring transitions such as m/z 558.35-->298.1, 25(OH)D3; m/z 570.35-->298.1, 25(OH)D2; and m/z 564.35-->298.1, hexadeuterated-25(OH)D3 were used for UPLC-MS/MS. RESULTS: The within-run imprecision (CVs) for 25(OH)D3 and 25(OH)D2 were 3.5-4.0% and 3.8-4.2%, respectively, and the corresponding between-run CVs were 3.3-5.5% and 4.7-5.8%. The lower limit of quantification for 25(OH)D3 and 25(OH)D2 were 0.5 and 1.0 ng/mL, respectively. The curve for interassay calibration variability data obtained over concentrations of 0-120 ng/mL for 25(OH)D3 and 0-90 ng/mL for 25(OH)D2 was linear and reproducible [ 25(OH)D3, R2=0.993; 25(OH)D2, R2=0.998]. The total 25(OH)D levels in Koreans (average, 18.7 ng/mL) were lower than those in American Caucasians, and the percentage of people with total 25(OH)D levels under 10 ng/mL was 8.1%. CONCLUSIONS: Our method to measure 25(OH)D3 and 25(OH)D2 levels by performing UPLC-MS/MS after PTAD derivatization showed good performance as a sensitive and reproducible method for routine analysis of vitamin D status.