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 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 Assessment of androgen concentration in women: liquid chromatography–tandem mass spectrometry and extraction RIA show comparable results

2011 ◽  
Vol 165 (6) ◽  
pp. 925-933 ◽  
Author(s):  
Femi Janse ◽  
Martinus J C Eijkemans ◽  
Angelique J Goverde ◽  
Eef G W M Lentjes ◽  
Annemieke Hoek ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Polycystic Ovary ◽  
Intraclass Correlation ◽  
Total Testosterone ◽  
Tandem Mass ◽  
Good Precision ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

ObjectiveThe measurement of serum testosterone in women is challenging due to lack of trueness, precision, and sensitivity of various available testosterone assays. Accurate assessment of testosterone in women is crucial especially in conditions associated with alleged over- or under-production of testosterone, such as in polycystic ovary syndrome (PCOS) or primary ovarian insufficiency (POI). The aim of this study was to measure and compare androgen concentrations in women with PCOS, POI, and female controls and to evaluate the performance of extraction RIA and liquid chromatography–tandem mass spectrometry (LC–MS/MS) in these women.DesignCross-sectional study.MethodsCarefully phenotyped women with POI (n=208) or PCOS (n=200) and 45 healthy, regularly cyclic female controls were included. Method comparison analyses were performed for total testosterone, androstenedione (AD), and DHEA, as measured by LC–MS/MS and extraction RIA.ResultsAll androgen levels were significantly elevated in women with PCOS compared with POI patients (P<0.05) and controls (P<0.05). Women with POI presented with similar androgen concentrations as controls, except for AD. Compared with measurements by extraction RIA, testosterone, DHEA, and AD concentrations measured by LC–MS/MS were systematically lower. However, using extraction RIA and LC–MS/MS, testosterone, DHEA, and AD measurements were shown to have good agreement as assessed by Bland–Altman analysis and intraclass correlation coefficient: 0.95 (95% confidence interval 0.94–0.91), 0.83 (0.79–0.86), and 0.96 (0.95–0.97) respectively.ConclusionsLC–MS/MS, compared with a labor-intensive extraction RIA, shows good precision, sensitivity, and high accuracy for measuring female testosterone, DHEA, and AD concentrations under various clinical conditions. LC–MS/MS, therefore, represents a convenient and reliable assay for both clinical and research purposes, where androgen measurement in women is required.

scholarly journals A novel high-throughput assay for the measurement of salivary progesterone by liquid chromatography tandem mass spectrometry

2018 ◽  
Vol 56 (1) ◽  
pp. 64-71 ◽  
Author(s):  
Lina Schiffer ◽  
Joanne E Adaway ◽  
Elizabeth S Baranowski ◽  
Wiebke Arlt ◽  
Brian G Keevil
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Menstrual Cycle ◽  
Tandem Mass Spectrometry ◽  
High Throughput ◽  
Large Set ◽  
Tandem Mass ◽  
Limit Of Quantification ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

Background Liquid chromatography tandem mass spectrometry (LC-MS/MS) enables specific and sensitive quantification of steroids with a high throughput. Saliva sampling is advantageous for multisample profiling over longer periods of time, as it is non-invasive, cheap, can be carried out at home and does not require the attendance of clinical personnel. We developed a rapid LC-MS/MS for the measurement of salivary progesterone, frequently assessed as ovulation marker in patients desiring fertility. Methods Samples (300 μL) were prepared by supported liquid extraction using dichloromethane and were reconstituted in 40% methanol. Chromatography was performed using a C8 column with a water/methanol gradient containing 0.1% formic acid and 2 mmol/L ammonium acetate. Quantification was performed with a Waters TQ-S mass spectrometer. Results Total run time was 5.5 min. The lower limit of quantification was 20 pmol/L (1.2 fmol on column). Inter- and intra-assay comparison showed coefficients of variation and bias between measured and nominal concentrations of less than 11%. Mean recovery was 91%. Interference with a large set of natural and synthetic steroids was excluded. The assay was successfully applied to measure progesterone variation during the menstrual cycle ( n = 9) and diurnal variations during luteal phase ( n = 7) in regularly cycling women. Discussion We present a novel LC-MS/MS assay for the determination of salivary progesterone with high-throughput potential. The applicability of the assay for progesterone profiling during the menstrual cycle is demonstrated.

Enantioselective Plasma Pharmacokinetic Study of a Novel Anti- Sichistosomiasis Agent P96 in Rat by Liquid Chromatography-tandem Mass Spectrometry

2019 ◽  
Vol 15 (4) ◽  
pp. 379-387
Author(s):  
Ran Meng ◽  
Danlu Zhang ◽  
Jianbo Ji ◽  
Lingyun Hu ◽  
Dequn Sun ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Rat Plasma ◽  
Drug Candidate ◽  
Tandem Mass ◽  
Limit Of Quantification ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Positive Mode

Background: 2-Cyclopentanecarbonyl-1,2,3,6,7,11b-hexahydro-pyrazino[2,1- a]isoquinolin- 4-one (P96), was found to be a novel drug candidate with one chiral center to treat schistosomiasis caused by Schistosoma japonicum. </P><P> Objective: To study pharmacokinetic characteristics, a simple, rapid and sensitive liquid chromatography- tandem mass spectrometry (LC-MS/MS) method was developed and fully validated for the quantification analysis of P96 in rat plasma. Methods: Chromatographic separation was performed on a C18 column with gradient eluted mobile phase composed of acetonitrile and water at a flow rate of 0.5 mL/min. Detection was performed on a triple-quadrupole tandem mass spectrometer using positive mode electrospray ionization in the multiple reactions monitoring (MRM) mode. Results: Excellent linearity was observed in the range of 3-900 ng/mL with the lower limit of quantification of 3 ng/mL in rat plasma for P96. The intra- and inter-day precisions exhibited less than 6.6%. Mean recoveries ranged from 96.9% to 102.4%. This method was applied to investigate the enantioselective differences on the pharmacokinetics between (R,S)-P96 and its enantiomers in rats after oral administration. The enantioselective differences of (R)-P96, (S)-P96 and (R,S)-P96 were found and compared. Conclusion: The established method was found to be accurate, precise, and sensitive and can be applied to investigate the stereoselective differences on pharmacokinetics between rac-P96 and its enantiomers.

Quantification by Liquid Chromatography Tandem Mass Spectrometry of Mycophenolic Acid and Its Phenol and Acyl Glucuronide Metabolites

2006 ◽  
Vol 52 (10) ◽  
pp. 1962-1964 ◽  
Author(s):  
Gunnar Brandhorst ◽  
Frank Streit ◽  
Sandra Goetze ◽  
Michael Oellerich ◽  
Victor William Armstrong
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Mycophenolic Acid ◽  
Solid Phase ◽  
Tandem Mass ◽  
Limit Of Quantification ◽  
Acyl Glucuronide ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

Abstract Background: We developed and validated a rapid and reliable liquid chromatography–tandem mass spectrometry (LC-MS/MS) procedure for the quantification of mycophenolic acid (MPA) and its phenol glucuronide (MPAG) and acyl glucuronide (AcMPAG) metabolites. Methods: We performed protein precipitation on all samples (calibrators, quality controls, and patient samples) and then subjected them to online solid-phase extraction followed by reversed-phase liquid chromatography for 4.0 min. The carboxybutoxy ether of MPA (MPAC) was used as the internal calibrator. The separated compounds (MPA, MPAG, AcMPAG, and MPAC) were detected by electrospray ionization-coupled MS/MS. We compared LC-MS/MS results with results for the same samples obtained with a validated HPLC procedure with an ultraviolet detector. Results: Comparison with the validated HPLC-ultraviolet procedure demonstrated good agreement. The Passing–Bablok regression was y = 0.968x − 0.058 for MPA, y = 1.08x − 1.697 for MPAG, and y = 0.952x + 0.076 for AcMPAG. Assay imprecision showed a CV &lt;10% at 3 concentrations for each compound. The lower limit of quantification was 0.1 mg/L for MPA, 1.0 mg/L for MPAG, and 0.05 mg/L for AcMPAG. The mean analytical recovery was 90%–110%. The assay was linear from 0.1 to 50 mg/L for MPA (r = 0.9987), from 1 to 500 mg/L for MPAG (r = 0.9999), and from 0.05 to 10 mg/L for AcMPAG (r = 0.9988). Quantification of the compounds was not affected by in-source fragmentation or ion suppression. Conclusion: The LC-MS/MS assay described here is valid and reliable for the quantification of total MPA, MPAG, and AcMPAG in serum.

Survey of Deoxynivalenol and Aflatoxin B1 in Instant Noodles and Bread Consumed in Thailand by Using Liquid Chromatography–Tandem Mass Spectrometry

2016 ◽  
Vol 79 (7) ◽  
pp. 1269-1272 ◽  
Author(s):  
SASITHORN PRALATNET ◽  
SARANYA POAPOLATHEP ◽  
MARIO GIORGI ◽  
KANJANA IMSILP ◽  
SUSUMU KUMAGAI ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Aflatoxin B1 ◽  
Urban Areas ◽  
Tandem Mass ◽  
Limit Of Quantification ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Instant Noodles

ABSTRACT One hundred wheat product samples (50 instant noodle samples and 50 bread samples) were collected from supermarkets in Bangkok, Thailand. Deoxynivalenol (DON) and aflatoxin B1 (AFB1) contamination in these products was analyzed using a validated liquid chromatography–tandem mass spectrometry method. The limit of quantification values of DON and AFB1 in the instant noodles and bread were 2 and 1 ng g−1, respectively. The survey found that DON was quantifiable in 40% of collected samples, in 2% of noodles (0.089 μg g−1), and in 78% of breads (0.004 to 0.331 μg g−1). AFB1 was below the limit of quantification of the method in all of the tested samples. The results suggest that the risk of DON exposure via noodles and breads is very low in urban areas of Thailand. No risk can be attributable to AFB1 exposure in the same food matrices, but further studies with a larger sample size are needed to confirm these data.

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.

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