scholarly journals Pilot Study for the Standardization of Insulin Immunoassays with Isotope Dilution–Liquid Chromatography/Tandem Mass Spectrometry

2007 ◽  
Vol 53 (8) ◽  
pp. 1462-1469 ◽  
Author(s):  
Diego Rodríguez-Cabaleiro ◽  
Katleen Van Uytfanghe ◽  
Veronique Stove ◽  
Tom Fiers ◽  
Linda M Thienpont
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Pilot Study ◽  
Total Error ◽  
Method Comparison ◽  
Tandem Ms ◽  
Tandem Mass ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Lc Tandem Ms

Abstract Background: An international working group convened by the American Diabetes Association (ADA) called for a reference measurement procedure for use in a trueness-based standardization project of insulin immunoassays. In view of this demand, we conducted a pilot study to investigate the feasibility of such a standardization project with our isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LC/tandem MS) procedure. Methods: We evaluated the precision, accuracy, and limit of quantification (LoQ) of the ID-LC/tandem MS procedure by use of insulin-free serum supplemented with insulin to give 3 pools with concentrations of 0.0796, 0.769, and 5.56 μg/L. We conducted a pilot method comparison study with 4 immunoassays and 80 samples from fasting and glucose-stimulated patients. Results: The within-run and total imprecision (CV) ranged from 3.2% to 6.3% and from 4.9% to 12.1% (listing sequence from the high to the low pool). The recovery from supplemented insulin-free sera ranged from 101.8% to 104.1%, and the LoQ was 0.07 μg/L (12 pmol/L). Weighted Deming regression and correlation analysis of the method-comparison data showed considerable between-assay variation for the immunoassays but, with the exception of one assay, excellent correlation with ID-LC/tandem MS. Recalibration of the immunoassay results considerably reduced the between-assay variation. Moreover, after recalibration, 3 of the 4 assays fulfilled the total error specification of 32% proposed by the ADA Workgroup. Conclusions: Recalibration of insulin assays by regression equations established from method comparison with ID-LC/tandem MS can result in successful standardization and fulfillment of the total error criterion proposed by the ADA Workgroup.

scholarly journals State-of-the-Art of Serum Testosterone Measurement by Isotope Dilution–Liquid Chromatography– Tandem Mass Spectrometry

2008 ◽  
Vol 54 (8) ◽  
pp. 1290-1297 ◽  
Author(s):  
Linda M Thienpont ◽  
Katleen Van Uytfanghe ◽  
Stuart Blincko ◽  
Carol S Ramsay ◽  
Hui Xie ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Isotope Dilution ◽  
Total Error ◽  
Serum Testosterone ◽  
Tandem Mass ◽  
Chromatography Tandem Mass Spectrometry ◽  
Deming Regression ◽  
Liquid Chromatography Tandem Mass

Abstract Background: The recent interest of clinical laboratories in developing serum testosterone assays based on isotope dilution–liquid chromatography–tandem mass spectrometry (ID-LC-MS/MS) stems from the lack of accuracy of direct immunoassays. In this study, we assessed the accuracy and state of standardization (traceability) of 4 published ID-LC-MS/MS procedures in a method comparison with an ID–gas chromatography (GC)–MS reference measurement procedure listed in the database of the Joint Committee for Traceability in Laboratory Medicine. Methods: The study used 58 specimens from different patient categories. Each specimen was measured in triplicate (ID-LC-MS/MS) and quadruplicate (ID-GC-MS) in independent runs. Results: The testosterone concentrations by ID-GC-MS were 0.2–4.4 nmol/L (women), 0.2–2.0 nmol/L (hypogonadal man), and 10.1–31.3 nmol/L (normogonadal men). For ID-GC-MS, the CV was nearly constant, with a median of 1.0%; for ID-LC-MS/MS, it was concentration-dependent, with a median of up to 8%. Weighted Deming regression gave mean slopes, intercepts, and correlation coefficients of 0.90–1.11, −0.055–0.013 nmol/L, and 0.993–0.997, respectively. The % difference plot showed between 7% and 26% of the results outside a total error limit of 14%, with median deviations from ID-GC-MS between −9.6 and 0.4%. Conclusions: This study demonstrated fairly good accuracy and standardization of the tested ID-LC-MS/MS procedures. Performance differences between procedures were evident in some instances, due to improper calibration and between-run calibration control. This emphasizes the need for thorough validation, including traceability, of new ID-LC-MS/MS procedures.

scholarly journals Sample Multiplexing: Increased Throughput for Quantification of Total Testosterone in Serum by Liquid Chromatography-Tandem Mass Spectrometry

2020 ◽  
Vol 66 (9) ◽  
pp. 1181-1189 ◽  
Author(s):  
Julia D Colletti ◽  
Mildred M Redor-Goldman ◽  
Agustin E Pomperada ◽  
Amit K Ghoshal ◽  
William W Wu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Method Comparison ◽  
Total Testosterone ◽  
Tandem Mass ◽  
Limit Of Quantification ◽  
Chromatography Tandem Mass Spectrometry ◽  
Deming Regression ◽  
Liquid Chromatography Tandem Mass

Abstract BACKGROUND For high-volume assays, optimizing throughput reduces test cost and turn-around time. One approach for liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays is sample multiplexing, wherein the analyte of interest is derivatized in different specimens with reagents of different molecular weight (differential mass tagging). Specimens can then be combined and simultaneously analyzed within a single injection to improve throughput. Here we developed and validated a quantitative, sample-multiplexed LC-MS/MS assay for serum total testosterone (TT) based on this approach. METHODS For the sample-multiplexed assay, calibrators, controls, and patient specimens were first extracted separately. After mass tagging with either methoxyamine or hydroxylamine, they were combined and injected into the LC-MS/MS system. To evaluate assay performance, we determined limit of quantification (LOQ), linearity, recovery, and imprecision. A method-comparison study was also performed, comparing the new assay with the standard LC-MS/MS assay in 1574 patient specimens. RESULTS The method was linear from 2.5 to 2000 ng/dL, with accuracies from 93% to 104% for both derivatives. An LOQ of 1.0 ng/dL was achieved. Intra-assay and total CVs across 4 quality control concentrations were less than 10%. The assay demonstrated good agreement (Deming regression, 1.03x + 6.07) with the standard LC-MS/MS assay for the patient specimens tested (TT, 3 to 4862 ng/dL). CONCLUSION Sample multiplexing by differential mass tagging of TT increases LC-MS/MS throughput 2-fold without compromising analytical accuracy and sensitivity.

scholarly journals Rapid Liquid Chromatography-Tandem Mass Spectrometry Method for Routine Analysis of Cyclosporin A Over an Extended Concentration Range

2002 ◽  
Vol 48 (1) ◽  
pp. 69-76 ◽  
Author(s):  
Brian G Keevil ◽  
David P Tierney ◽  
Donald P Cooper ◽  
Michael R Morris
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Cyclosporin A ◽  
Pharmacokinetic Studies ◽  
Tandem Ms ◽  
Tandem Mass ◽  
Transplant Recipients ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

Abstract Background: Cyclosporin A (CsA) is commonly measured by immunoassay techniques that have a limited analytical range. The consequence of this is that low CsA concentrations that may be clinically significant are difficult to measure and that high concentrations require sample dilution, which introduces error and increases cost. More specific assays, such as HPLC, do not have the required turnaround times for busy transplant clinics. Methods: CsA was measured in whole blood from 180 cardiac and lung transplant recipients by a liquid chromatography-tandem mass spectrometry (MS) assay, and the results were compared with the Dade Behring Emit assay. Proteins were precipitated with acetonitrile containing ascomycin as internal standard. We used isocratic elution on a Supelco CN column (33 × 3.0 mm; 3- μm bead size) with a mobile phase of 65% aqueous acetonitrile containing ammonium acetate (2 mmol/L) and formic acid (1 g/L), at a flow rate of 0.5 mL/min, with a sample injection volume of 6 μL. We used positive-ion electrospray MS to monitor the ammonium adducts of the compounds of interest decomposing under controlled conditions to the most dominant fragments of the individual molecules. Calibration curves used linear least-squares regression with 1/x weighting. Results: Maximum sensitivity was obtained by monitoring fragmentation of the ammonium adducts m/z 1220→m/z 1203 for CsA and m/z 809→m/z 765 for ascomycin. Sample throughput, including preparation time, was 30 samples in 1.5 h with an injection-to-injection cycle time of 1.5 min. The calibration curve was linear to 5000 μg/L, with a detection limit of 0.03 μg/L and a limit of quantification of 1 μg/L. Regression analysis [tandem MS method (y) and Emit assay (x)] yielded a slope of 1.09 (± 0.03), an intercept of 6.2 (± 4.5) μg/L, and Sy|x = 27 μg/L. Conclusions: Tandem MS assay is a realistic alternative to immunoassay for the routine monitoring of CsA in transplant recipients. Its wide dynamic range has utility for pharmacokinetic studies of CsA.

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