madman
Super Moderator
Sample Multiplexing: Increased Throughput for Quantification of Total Testosterone in Serum by Liquid Chromatography-Tandem Mass Spectrometry
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 LCMS/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 the 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. A 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 þ 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.
Introduction
A goal of large clinical laboratories is to optimize throughput, especially for high-volume liquid chromatography-tandem mass spectrometry (LC-MS/ MS) assays such as serum total testosterone (TT). Reducing the turn-around time for patient testing is key but must be done in a manner that maintains the quality of the assay. Increasing the speed of analysis on LCMS/MS platforms can be accomplished by shortening and optimizing liquid chromatography (LC) methods through the appropriate selection of solvents, LC columns, and gradient conditions. Multiplexing LC systems that operate simultaneously in tandem with a single mass spectrometer can also increase throughput.
Once LC parameters have been optimized to maximize the speed of analysis, the options to further increase LC-MS/MS throughput become limited. One methodology, sample multiplexing, involves introducing more than one sample per injection into the LC-MS/MS instrument (1, 17-19). This approach uses reagents of differing mass (differential mass tagging) to derivatize separate specimens, which are then combined prior to injection. The individual specimens are identified based on their corresponding modified molecular weights (1, 17-19).
Methoxyamine (MOA) and hydroxylamine (HOA), popular Girard reagents used in both gas chromatography-tandem mass spectrometry and LC-MS/MS applications, enhance the ionization of ketosteroids (2–6, 16, 20). We chose MOA and HOA as derivatization reagents for TT because of the similarity in reaction conditions and unique, stable derivatives (Fig. 1) (2–6, 20). Both reagents react with the carbonyl group of testosterone to form an oxime derivative (2–6, 11, 16, 20). Our goal was to develop and validate a sample multiplexing assay using MOA and HOA to quantitate TT in serum via LC-MS/MS.
Discussion
Improved LC-MS/MS throughput by sample multiplexing helped offset the increase in complexity of the sample preparation process relative to the standard HTLC method. The ability to analyze 2 samples within a single injection resulted in a reduction in solvent usage (further achieved by decreasing both the loading and eluting flow rates) and a decrease in the number of LCMS/MS instruments required to maintain the desired turn-around time. This, in conjunction with the additional sample clean-up steps incorporated to help preserve LC-MS/MS cleanliness, yielded a potential for lower instrument maintenance costs. The use of an automated liquid handler for sample preparation minimized the required manual sample manipulation, resulting in low imprecision of technical replicates, consistent batch-to-batch results, and a decrease in the possibility of cross-contamination between patient specimens and reagents due to human error. Automation was also critical in minimizing the impact of a lengthier sample preparation process relative to the standard method (about 4.25 h versus 1.5 h to process 384 samples). Inaccurate analysis of the mixed patient specimens was further prevented by derivatizing testosterone with reagents that allowed for a rapid and stable reaction. Additionally, each derivative was identified with unique ion pairs, and the corresponding chromatographic peaks were adequately resolved.
With confidence in the reliability and accuracy of the new method, the standard methodology was replaced with the new, multiplexed assay as the routine clinical assay to quantitate TT in serum and is now currently operational within Quest Diagnostics.
In summary, our results support sample multiplexing by differential mass tagging as an acceptable alternative to the standard LC-MS/MS assay for quantifying TT in serum. The approach achieves the higher LCMS/MS throughput required for this high-volume assay without compromising accuracy or analytical sensitivity. Additionally, automation minimized the impact of the increased complexity of sample preparation relative to the standard method.
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 LCMS/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 the 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. A 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 þ 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.
Introduction
A goal of large clinical laboratories is to optimize throughput, especially for high-volume liquid chromatography-tandem mass spectrometry (LC-MS/ MS) assays such as serum total testosterone (TT). Reducing the turn-around time for patient testing is key but must be done in a manner that maintains the quality of the assay. Increasing the speed of analysis on LCMS/MS platforms can be accomplished by shortening and optimizing liquid chromatography (LC) methods through the appropriate selection of solvents, LC columns, and gradient conditions. Multiplexing LC systems that operate simultaneously in tandem with a single mass spectrometer can also increase throughput.
Once LC parameters have been optimized to maximize the speed of analysis, the options to further increase LC-MS/MS throughput become limited. One methodology, sample multiplexing, involves introducing more than one sample per injection into the LC-MS/MS instrument (1, 17-19). This approach uses reagents of differing mass (differential mass tagging) to derivatize separate specimens, which are then combined prior to injection. The individual specimens are identified based on their corresponding modified molecular weights (1, 17-19).
Methoxyamine (MOA) and hydroxylamine (HOA), popular Girard reagents used in both gas chromatography-tandem mass spectrometry and LC-MS/MS applications, enhance the ionization of ketosteroids (2–6, 16, 20). We chose MOA and HOA as derivatization reagents for TT because of the similarity in reaction conditions and unique, stable derivatives (Fig. 1) (2–6, 20). Both reagents react with the carbonyl group of testosterone to form an oxime derivative (2–6, 11, 16, 20). Our goal was to develop and validate a sample multiplexing assay using MOA and HOA to quantitate TT in serum via LC-MS/MS.
Discussion
Improved LC-MS/MS throughput by sample multiplexing helped offset the increase in complexity of the sample preparation process relative to the standard HTLC method. The ability to analyze 2 samples within a single injection resulted in a reduction in solvent usage (further achieved by decreasing both the loading and eluting flow rates) and a decrease in the number of LCMS/MS instruments required to maintain the desired turn-around time. This, in conjunction with the additional sample clean-up steps incorporated to help preserve LC-MS/MS cleanliness, yielded a potential for lower instrument maintenance costs. The use of an automated liquid handler for sample preparation minimized the required manual sample manipulation, resulting in low imprecision of technical replicates, consistent batch-to-batch results, and a decrease in the possibility of cross-contamination between patient specimens and reagents due to human error. Automation was also critical in minimizing the impact of a lengthier sample preparation process relative to the standard method (about 4.25 h versus 1.5 h to process 384 samples). Inaccurate analysis of the mixed patient specimens was further prevented by derivatizing testosterone with reagents that allowed for a rapid and stable reaction. Additionally, each derivative was identified with unique ion pairs, and the corresponding chromatographic peaks were adequately resolved.
With confidence in the reliability and accuracy of the new method, the standard methodology was replaced with the new, multiplexed assay as the routine clinical assay to quantitate TT in serum and is now currently operational within Quest Diagnostics.
In summary, our results support sample multiplexing by differential mass tagging as an acceptable alternative to the standard LC-MS/MS assay for quantifying TT in serum. The approach achieves the higher LCMS/MS throughput required for this high-volume assay without compromising accuracy or analytical sensitivity. Additionally, automation minimized the impact of the increased complexity of sample preparation relative to the standard method.
