A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay to profile 20 plasma steroids in endocrine disorders

2020 ◽  
Vol 58 (9) ◽  
pp. 1477-1487 ◽  
Author(s):  
Zhenxin Wang ◽  
Hao Wang ◽  
Yingfei Peng ◽  
Fangjun Chen ◽  
Lin Zhao ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Polycystic Ovary ◽  
Endocrine Disorders ◽  
Tandem Mass ◽  
Limit Of Quantification ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Plasma Steroids

AbstractBackgroundLiquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assays are employed in more and more clinical laboratories to quantify steroids. The steroid quantification by LC-MS/MS shows great value in screening or diagnosing endocrine disorders; however, the number of functional steroids included in the LC-MS/MS methods is still limited.MethodsHere, we describe the performance and validation of a 20-steroid plasma panel by LC-MS/MS. The panel included progestogens (including mineralocorticoids and glucocorticoids), androgens and estrogens biosynthesized in steroid metabolic pathways. The LC-MS/MS method was validated according to guidance documents, and subsequently employed to profile steroid changes in endocrine disorders.ResultsUsing LC-MS/MS, 20 steroids were separated and quantified in 8 min. Coefficients of variation (CVs) of the 20 analytes at the lower limit of quantification (LLoQ) were all less than 15% (ranging from 1.84% to 14.96%). The linearity of the assay was demonstrated by all the R2 values greater than 0.995. Individual plasma steroids changed significantly in patients with subclinical Cushing’s syndrome (SCS) and polycystic ovary syndrome (PCOS) – 17-hydroxypregnenolone (17-OH-PR), testosterone (T) and dihydrotestosterone (DHT) were significantly decreased in SCS patients, while in PCOS patients, pregnenolone, corticosterone (CORT), androstenedione (A4) and T were significantly increased and DHT was decreased.ConclusionsThe LC-MS/MS method we developed for the quantification of 20 plasma steroids is clinical practicable. The steroid profiling data using this assay indicate its screening value for endocrine disorders. To further explore the value of the assay, more investigations are however needed.

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.

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.

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 Candidate Reference Measurement Procedures for Serum 25-Hydroxyvitamin D3 and 25-Hydroxyvitamin D2 by Using Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry

2011 ◽  
Vol 57 (3) ◽  
pp. 441-448 ◽  
Author(s):  
Hedwig CM Stepman ◽  
An Vanderroost ◽  
Katleen Van Uytfanghe ◽  
Linda M Thienpont
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Isotope Dilution ◽  
Tandem Mass ◽  
Limit Of Quantification ◽  
Validation Data ◽  
Reference Measurement ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

BACKGROUND 25-hydroxyvitamin D [25(OH)D] assays are characterized by poor between-assay comparability. This result emphasizes the need for reference measurement procedures (RMPs) to establish calibration traceability and assist in method validation. We aimed at developing candidate RMPs on the basis of isotope- dilution liquid chromatography–tandem mass spectrometry (ID-LC-MS/MS) for separate quantification of serum 25(OH)D2 and 25(OH)D3. METHODS Hexa-deuterated 25(OH)D3/D2 was added to serum. This mixture was extracted with n-hexane and fractionated on Sephadex LH-20 before 2-dimensional LC-MS/MS. In the first dimension, both procedures used a C4 column; however, in the second dimension, the 25(OH)D2 procedure used a C18 and the 25(OH)D3 procedure used a Zorbax SB-CN column. Calibration was traceable to the NIST Standard Reference Material (SRM) 2972. Validation comprised assessment of interference and limit of quantification/detection. Imprecision and trueness were validated by analysis of the SRM 972 against specifications (CV &lt;5% and bias &lt;1.7%). The expanded uncertainty for quadruplicate measurements was estimated. RESULTS Testing of potentially interfering substances was negative. Interference by 3-epi-25(OH)D3 was resolved by sufficient chromatographic resolution. The limits of quantification/detection were 1.1 nmol/L and 0.09 pmol/L for 25(OH)D3 and 1.2 nmol/L and 0.05 pmol/L for 25(OH)D2. Mean total CVs and differences from the SRM 972 target (± 1-sided 95% CI) were 2.1% and 1.1% ± 1.5% [25(OH)D3] and 3% and 1.3% ± 0.6% [25(OH)D2], respectively. The respective expanded uncertainties were 3.4% and 3.9%. CONCLUSIONS From the validation data, we conclude that we achieved our objective of 2 state-of-the-art candidate RMPs for serum 25(OH)D3 and 25(OH)D2.

scholarly journals Simultaneous Measurement of Serum Testosterone and Dihydrotestosterone by Liquid Chromatography–Tandem Mass Spectrometry

2008 ◽  
Vol 54 (11) ◽  
pp. 1855-1863 ◽  
Author(s):  
Steve Shiraishi ◽  
Paul W N Lee ◽  
Andrew Leung ◽  
Victor H H Goh ◽  
Ronald S Swerdloff ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Premenopausal Women ◽  
Serum Testosterone ◽  
Reference Intervals ◽  
Tandem Mass ◽  
Limit Of Quantification ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

Abstract Background: Recent reports have described inherent problems with androgen immunoassays compared with mass spectrometry analyses. Methods: We developed a method for measuring serum testosterone (T) and 5α-dihydrotestosterone (DHT) simultaneously via liquid–liquid extraction followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) with positive-mode electrospray ionization. Results: The DHT and T calibrators showed a linear response from 0.069 nmol/L to 34.4 nmol/L and 69.3 nmol/L, respectively. T interference in the DHT assay and vice versa were negligible. Within- and between-run imprecision values were &lt;5% for both analytes. Percent recoveries of T and DHT spiked into samples at concentrations spanning the calibration curve were 100%–113% and 98%–107%, respectively. The lower limit of quantification was 0.069 nmol/L for both steroids. Serum T concentrations measured by LC-MS/MS were different from those obtained by RIA, especially at lower T concentrations. Serum DHT concentrations measured by LC-MS/MS were markedly lower than those generated by RIA because of the nonselectivity of the RIA without chromatography. The reference intervals (mean ± 2 SDs) determined for T and DHT were 9.2–33.7 nmol/L and 0.47–2.65 nmol/L, respectively, for 113 healthy adult men and 0.33–2.02 nmol/L and 0.09–0.91 nmol/L, respectively, for 133 healthy premenopausal women. Conclusions: We have developed and validated a selective and precise method for simultaneous measurements of serum T and DHT that can be adopted for routine measurements of these androgens in health and disease in men and women.

scholarly journals Comparison of Sample Preparation and Determination of 60 Veterinary Drug Residues in Flatfish Using Liquid Chromatography-Tandem Mass Spectrometry

Molecules ◽  
2020 ◽  
Vol 25 (5) ◽  
pp. 1206
Author(s):  
Joohye Kim ◽  
Hyunjin Park ◽  
Hui-Seung Kang ◽  
Byung-Hoon Cho ◽  
Jae-Ho Oh
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Sample Preparation ◽  
Tandem Mass Spectrometry ◽  
Tandem Mass ◽  
Limit Of Quantification ◽  
Chromatography Tandem Mass Spectrometry ◽  
Fish Samples ◽  
Liquid Chromatography Tandem Mass ◽  
Fishery Products

This study was performed to optimize the analytical method for multi-residues of 60 compounds in flatfish samples. Three sample preparation methods were tested to identify the optimal recovery conditions for target analytes. As a result, 10 mL of water/acetonitrile (1:4, v/v) was used to extract analytes from fish samples. For purification, C18 and 10 mL of acetonitrile saturated hexane were used to treat the samples. After evaporation and reconstitution, the fish samples were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry. The proposed method was validated according to the CODEX guidelines (CAC/GL-71). Our results showed the recoveries of 73.2%–115% and coefficients of variation of 1.6%–22.1%. The limit of quantification was 0.0005–0.005 mg/kg in the fishery products. In analysis of real samples, no samples exceeded the limit of quantification. This analytical method can be used for multi-residue screening and confirmation of the residues of veterinary drugs in fishery products.

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