scholarly journals Increasing Liquid Chromatography–Tandem Mass Spectrometry Throughput by Mass Tagging: A Sample-Multiplexed High-Throughput Assay for 25-Hydroxyvitamin D2 and D3

2011 ◽  
Vol 57 (3) ◽  
pp. 431-440 ◽  
Author(s):  
Brian C Netzel ◽  
Kendall W Cradic ◽  
Eric T Bro ◽  
Adam B Girtman ◽  
Richard C Cyr ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Tandem Mass ◽  
Limits Of Detection ◽  
Sample Throughput ◽  
Chromatography Tandem Mass Spectrometry ◽  
Deming Regression ◽  
Liquid Chromatography Tandem Mass ◽  
Detection And Quantification

BACKGROUND The limits of chromatographic speed and mechanical frontend capabilities have been reached for many high-volume liquid chromatography–tandem mass spectrometry (LC-MS/MS) tests, curtailing the maximal achievable sample throughput. To overcome these boundaries, we developed and validated a derivatization-based sample-multiplex LC-MS/MS assay for detection of 25-hydroxyvitamins D2 and D3 [25(OH)D2 and 25(OH)D3], which increased sample throughput 5-fold. METHODS After separate derivatization with 1 of 5 different triazoline-diones (TADs), 5 calibrators, controls, or patient specimens were combined and injected together into an LC-MS/MS. On the basis of mass differences between TADs, the MS/MS quantified analyte and stable isotope internal standards for 25(OH)D2 and 25(OH)D3 for each respective multiplexed sample within the injection. Limits of detection and quantification, spiked recovery, linearity, imprecision, and patient results were determined and compared against our standard LC-MS/MS assay. RESULTS TAD multiplexing increased throughput on an LC-quadruplexed LC-MS/MS system from 60 samples/h to 300 samples/h. Limits of detection and quantification were 4.9 nmol/L [2 μg/L, 25(OH)D2], 2.2 nmol/L [0.9 μg/L, 25(OH)D3], and 10 nmol/L [4 μg/L, 25(OH)D2], 5 nmol/L [2 μg/L, 25(OH)D3], respectively. The assay was linear to 250 nmol/L (100 μg/L). Interassay CVs across the reportable range were 3.7%–15.2%. Spiked recoveries were 94%–119%. The method comparison with the standard LC-MS/MS method showed slopes of 0.96 and 0.97 (Deming regression) for 25(OH)D2 (n = 1733) and 25(OH)D3 (n = 7614) (R2=0.96 and 0.97), respectively. CONCLUSIONS Multiplexing samples by differential mass tagging in LC-MS/MS measurement of 25(OH)D2 and 25(OH)D3 allows for reliable quantification, with throughput increased over standard methods by the multiplexing factor.

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 Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry Candidate Reference Method for Total Testosterone in Human Serum

2013 ◽  
Vol 59 (2) ◽  
pp. 372-380 ◽  
Author(s):  
Julianne Cook Botelho ◽  
Christopher Shacklady ◽  
Hans C Cooper ◽  
Susan S-C Tai ◽  
Katleen Van Uytfanghe ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Isotope Dilution ◽  
Matrix Effects ◽  
Reference Method ◽  
Tandem Mass ◽  
Chromatography Tandem Mass Spectrometry ◽  
Deming Regression ◽  
Liquid Chromatography Tandem Mass

BACKGROUND We developed and evaluated a candidate reference measurement procedure (RMP) to standardize testosterone measurements, provide highly accurate and precise value assignments for the CDC Hormone Standardization Program, and ensure accurate and comparable results across testing systems and laboratories. METHODS After 2 liquid/liquid extractions of serum with a combination of ethyl acetate and hexane, we quantified testosterone by isotope-dilution liquid chromatography–tandem mass spectrometry with electrospray ionization in the positive ion mode monitoring 289→97 m/z (testosterone) and 292→112 m/z (3C13 testosterone). We used calibrator bracketing and gravimetric measurements to give higher specificity and accuracy to serum value assignments. The candidate RMP was evaluated for accuracy by use of NIST-certified reference material SRM971 and validated by split-sample comparison to established RMPs. We evaluated intraassay and interassay imprecision, measurement uncertainty, potential interferences, and matrix effects. RESULTS A weighted Deming regression comparison of the candidate RMP to established RMPs showed agreement with no statistical difference (slope 0.99, 95% CI 0.98–1.00, intercept 0.54, 95% CI −1.24 to 2.32) and a bias of ≤0.3% for NIST SRM971. The candidate RMP gave maximum intraassay, interassay, and total percent CVs of 1.5%, 1.4%, and 1.7% across the concentrations of testosterone typically found in healthy men and women. We tested structural analogs of testosterone and 125 serum samples and found no interferences with the measurement. CONCLUSIONS This RMP for testosterone can serve as a higher-order standard for measurement traceability and can be used to provide an accuracy base to which routine methods can be compared in the CDC Hormone Standardization Program.

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 Liquid Chromatography–Tandem Mass Spectrometry Assay for Simultaneous Measurement of Estradiol and Estrone in Human Plasma

2004 ◽  
Vol 50 (2) ◽  
pp. 373-384 ◽  
Author(s):  
Robert E Nelson ◽  
Stefan K Grebe ◽  
Dennis J O’Kane ◽  
Ravinder J Singh
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Simultaneous Measurement ◽  
Multiple Reaction Monitoring ◽  
Cross Reactivity ◽  
Tandem Mass ◽  
Limits Of Detection ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

Abstract Background: Estradiol (E2) and estrone (E1) measurements form an integral part of the assessment of female reproductive function and have expanding roles in other fields. However, many E1 and E2 immunoassays have limited functional sensitivity, suffer from cross-reactivity, and display poor intermethod agreement. To overcome these problems, we developed a sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay for the simultaneous measurement of E1 and E2. Methods: After dansyl chloride derivatization, samples were separated by fast gradient chromatography and injected into a tandem mass spectrometer after formation of positive ions with atmospheric pressure chemical ionization. The limits of detection and quantification, recovery, linearity, precision, and reference intervals were determined, and performance was compared with several immunoassays. Results: Total run time per sample was 5 min. The multiple-reaction monitoring ion pairs were m/z 506/171 for 3-dansyl-estradiol and m/z 504/171 for 3-dansyl-estrone. The limits of detection for E1 and E2 were 12.9 pmol/L (3.5 ng/L) and 10.3 pmol/L (2.8 ng/L), respectively. Interassay imprecision (CV) was 4–20% (n = 20). The limits of quantification (functional sensitivities) for E1 and E2 were 44.1 pmol/L (11.9 ng/L) and 23.2 pmol/L (6.3 ng/L), respectively. The assay was linear to >2200 pmol/L (∼600 ng/L) for each analyte. Recoveries were 93–108% for E1 and 100–110% for E2. No cross-reactivity was observed. Method comparison with several immunoassays revealed that the latter were inaccurate and prone to interferences at low E1 and E2 concentrations. Conclusions: LC-MS/MS allows rapid, simultaneous, sensitive, and accurate quantification of E1 and E2 in human serum.

scholarly journals Quantitative Insulin Analysis Using Liquid Chromatography–Tandem Mass Spectrometry in a High-Throughput Clinical Laboratory

2013 ◽  
Vol 59 (9) ◽  
pp. 1349-1356 ◽  
Author(s):  
Zhaohui Chen ◽  
Michael P Caulfield ◽  
Michael J McPhaul ◽  
Richard E Reitz ◽  
Steven W Taylor ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Human Insulin ◽  
High Throughput ◽  
Tandem Mass ◽  
Chromatography Tandem Mass Spectrometry ◽  
Deming Regression ◽  
Limit Of Quantitation ◽  
Liquid Chromatography Tandem Mass

BACKGROUND Circulating insulin concentrations reflect the amount of endogenous insulin produced by the pancreas and can be monitored to check for insulin resistance. Insulin is commonly measured using immunochemiluminometric assays (ICMA). Unfortunately, differing crossreactivities of the various ICMA antibodies have led to variability in assay results. In contrast, liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based approaches can provide a highly specific alternative to immunoassays. METHODS Insulin was extracted from patient serum and reduced to liberate the insulin B chain. Subsequent resolution of the peptide was achieved by LC coupled to triple-quadrupole MS. Selected-reaction monitoring of B-chain transitions was used for quantification. Recombinant human insulin was used as a calibrator and was compared against the National Institute for Biological Standards and Control (NIBSC) reference standard. Bovine insulin and a stable isotopic-labeled (13C/15N) human insulin B chain were used and compared as internal standards. RESULTS The LC-MS/MS assay described herein has been validated according to CLIA guidelines with a limit of detection of 1.8 μIU/mL (10.8 pmol/L) and a limit of quantitation of 3 μIU/mL (18.0 pmol/L). A correlation between the LC-MS/MS assay and a US Food and Drug Administration-approved ICMA was completed for patient samples and the resulting Deming regression revealed good agreement. A reference interval for the assay was established. CONCLUSIONS A simple, high-throughput, quantitative LC-MS/MS insulin assay traceable to the NIBSC standard has been successfully developed and validated.

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