Development,validation and comparison of LC–MS/MS and RIA methods for quantification of vertebrates-like sex-steroids in prosobranch molluscs

The role of vertebrate-like sex-steroids (testosterone, T, progesterone, P, and 17β-estradiol, E2) in molluscs is still debated, but they could represent potential biomarkers of endocrine disruption. A radioimmunoassay (RIA) and a liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) methods have been developed and compared to measure their levels in a gastropod snail Potamopyrgus antipodarum. Both methods showed a good reproducibility despite the complex matrix and the very low levels of vertebrate-like sex-steroids. Only T and P were detected using the LC–MS/MS method, while the RIA method reached lower detection limits and enabled the detection of all three steroids. Results indicated that T and P were mainly present as unconjugated forms. Both methods were compared in the analysis of snails exposed to waste water treatment plant effluents and led to the same conclusions concerning the modulation of steroids levels. Moreover, they both were in agreement concerning T measurements. On the other hand, LC–MS/MS appeared to be more suitable when analyzing P levels due to a low sensitivity of the RIA method. As E2 was not measured using the LC–MS/MS method because of a higher detection limit compared to the other steroids, the results obtained with the RIA method should be interpreted with caution. LC–MS/MS remains the gold standard for sex-steroid determinations, however a relevant and alternative method based on RIA was developed, requiring fewer organisms. RIA seems a promising method as a screening tool for experimental use, allowing comparison of sex-steroid levels in the mudsnail both in laboratory and in field experiments.     

Validation and application of an LC–MS/MS method for quantitation of three fatty acid ethanolamides as biomarkers for fatty acid hydrolase inhibition in human plasma

Endogenous ethanolamides (fatty acid amides), including arachidonyl ethanolamide (anandamide, AEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA), are substrates of fatty acid amide hydrolase (FAAH). FAAH may play an important role for pain, anxiety/depression, and metabolic disorders. Ethanolamides are considered to be potential pharmacodynamic biomarkers to determine target engagement for FAAH inhibition by novel pharmaceutical agents. A highly selective, sensitive, and high-throughput liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous quantitation of AEA, OEA, and PEA in human plasma. The method employed D₄-AEA, D₄-OEA, and ¹³C₂-PEA as “surrogate analytes” to establish the concentration–mass response relationship, i.e. a regression equation. The concentrations of AEA, OEA, and PEA were calculated based on the regression equations derived from the surrogate analytes. This approach made it possible to prepare calibration standard and quality control (QC) samples in plasma devoid of interferences from the endogenous analytes. The analytical methodology required 150 μL of human plasma that was processed via liquid–liquid extraction (LLE) using a 96-well plate format. Chromatographic separation was achieved with a reversed-phase high performance liquid chromatography (HPLC) column using gradient elution, and the run time was 3 min. The method was fully validated and it demonstrated acceptable accuracy, precision, linearity, and specificity. The lower limit of quantitation (LLOQ) was 0.1/0.5/0.5 ng/mL for AEA/OEA/PEA, which was sensitive enough to capture the basal plasma levels in healthy subjects. Bench-top stability in plasma, freeze–thaw stability in plasma, frozen long-term stability in plasma, autosampler stability, and stock solution stability all met acceptance criteria (%Bias within ±12.0%). Characterization of stability in purchased/aged blood indicated that ethanolamides are subject to degradation mediated by intracellular membrane-bound FAAH, which has been shown to be inhibited by phenylmethylsulfonyl fluoride (PMSF). In the presence of PMSF, ethanolamide levels increased slightly over time, suggesting that blood cells release ethanolamides into plasma. Whole blood stability conducted in fresh blood immediately following collection revealed that there was significant elevation of ethanolamide concentrations (∼1.3–2.0-fold on ice and ∼1.5–3.0-fold at room temperature by 2 h), indicating that de novo synthesis and release from blood cells were the predominant factors affecting ethanolamide concentrations ex vivo. Accordingly, conditions that ensured rapid separation of plasma from blood cells and consistency in the blood harvesting procedures were established and implemented for clinical studies to minimize the ex vivo elevation of plasma ethanolamide concentrations. The variability (intra-subject and inter-subject) of plasma ethanolamide levels was evaluated in healthy subjects during a Phase 0 study (no drug administration) that simulated the design of single-ascending dose and multiple-ascending dose clinical trials in terms of sample collection time points, population, food, and activity. The data indicated there was relatively large inter- and intra-subject variation in plasma ethanolamide concentrations. In addition, apparent variations due to time of day and/or food effects were also revealed. Understanding the variability of ethanolamide levels in humans is very important for study design and data interpretation when changes in ethanolamide levels are used as target engagement biomarkers in clinical trials.     

A new modified Edman procedure for analysis of N-terminal valine adducts in hemoglobin by LC–MS/MS

A rapid and sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for simultaneous determination of adducts from acrylamide, glycidamide and ethylene oxide to N-terminal valines in hemoglobin (Hb) was developed. This new procedure is based on the same principles as the N-alkyl Edman procedure for analysis of adducts from electrophilic agents to N-terminal valines in Hb. The N-substituted valines can be detached, enriched and measured selectively as thiohydantoins by the use of an Edman reagent, in this case fluorescein isothiocyanate (FITC). This procedure is denoted as the “adduct FIRE procedure” as the FITC reagent is used for measurement of adducts (R) formed from electrophilic compounds with a modified Edman procedure. In this study, fluorescein thiohydantoin (FTH) analytes of N-substituted valines from acrylamide, glycidamide and ethylene oxide, as well as their corresponding hepta- and tri-deuterium-substituted analogues, were synthesized. These analytes (n = 8) were then characterized by LC–MS/MS (ESI, positive ion mode) and obtained product ions were interpreted. A considerable work with optimization of the FIRE procedure™, resulted in a procedure in which low background levels of the studied adducts could be measured from 250 μL lyzed whole blood samples (human non-smokers). The analytes were enriched and purified with solid phase extraction columns and analyzed by LC–MS/MS with LOQ down to 1 pmol adduct/g Hb. Compared to other procedures for determination of N-terminal Hb adducts, the introduction of FITC has led to a simplified procedure, where whole blood also can be used, giving new opportunities and reduced hand on time with increased sample throughput.     

A method for the analysis of six thyroid hormones in thyroid gland by liquid chromatography–tandem mass spectrometry

Perchlorate can competitively inhibit iodide uptake by the thyroid gland (TG) via the sodium/iodide symporter, consequently reducing the production of thyroid hormones (THs). Until recently, the effects of perchlorate on TH homeostasis are being examined through measurement of serum levels of TH, by immunoassay (IA)-based methods. IA methods are fast, but for TH analysis, they are compromised by the lack of adequate specificity. Therefore, selective and sensitive methods for the analysis of THs in TG are needed, for assessment of the effects of perchlorate on TH homeostasis. In this study, we developed a method for the analysis of six THs: l-thyroxine (T₄), 3,3′,5-triiodo-l-thyronine (T₃), 3,3′,5′-triiodo-l-thyronine (rT₃), 3,5-diiodo-l-thyronine (3,5-T₂), 3,3′-diiodo-l-thyronine (3,3′-T₂), and 3-iodo-l-thyronine (3-T₁) in TG, using liquid chromatography (LC)–tandem mass spectrometry (MS/MS). TGs used in this study were from rats that had been placed on either iodide-deficient diet or iodide-sufficient diet, and that had either been provided with perchlorate in drinking water (10 mg/kg/day) or control water. TGs were extracted by pronase digestion and then analyzed by LC–MS/MS. The instrumental calibration range for each TH ranged from 1 to 200 ng/ml and showed a high linearity (r > 0.99). The method quantification limits (LOQs) were determined to be 0.25 ng/mg TG for 3-T₁; 0.33 ng/mg TG for 3,3′- and 3,5-T₂; and 0.52 ng/mg TG for rT₃, T₃, and T₄. Rats were placed on an iodide-deficient or -sufficient diet for 2.5 months, and for the last 2 weeks of that period were provided either perchlorate (10 mg/kg/day) in drinking water or control water. Iodide deficiency and perchlorate administration both reduced TG stores of rT₃, T₃, and T₄. In iodide-deficient rats, perchlorate exacerbated the reduction in levels of THs in TG. With the advances in analytical methodology, the use of LC–MS/MS for measurement of hormone levels in TG will allow more comprehensive evaluations of the hypothalamic-pituitary–thyroid axis.     

Quantitative analysis of biomarkers,drugs and toxins in biological samples by immunoaffinity chromatography coupled to mass spectrometry or tandem mass spectrometry: A focused review of recent applications

Immunoaffinity chromatography (IAC), mass spectrometry and especially tandem mass spectrometry (MS/MS) represent the most efficient and reliable analytical techniques for specific isolation, unequivocal identification and accurate quantification of numerous natural and synthetic substances in biological samples. This review article focuses on the combined use of these outstanding methodologies in basic and clinical research and in life sciences for the quantitative analysis of low- and high-molecular mass biomarkers, drugs and toxins in urine, plasma or serum samples, in tissue and other biologicals systems published in the last decade. The analytes discussed in some detail include the biomarkers of oxidative stress 15(S)-8-iso-prostaglandin F_2α {15(S)-8-iso-PGF_2α} and 3-nitrotyrosine, the major urinary metabolite of the lipid mediators cysteinyl leukotrienes, i.e., the leukotriene E₄ (LTE₄), melatonin, and the major collagen type II neoepitope peptide in human urine.     

A sensitive liquid chromatography–electrospray tandem mass spectrometric method for lancemaside A and its metabolites in plasma and a pharmacokinetic study in mice

A high-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) method employing electrospray ionization (ESI) has been developed for simultaneous determination of lancemaside A (3-O-β-d-glucuronopyranosyl-3β, 16α-dihydroxyolean-12-en-28-oic acid 28-O-β-d-xylopyranosyl(1→3)-β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl ester) and its metabolites in mouse plasma. When lancemaside A (60 mg/kg) was orally administered to mice, echinocystic acid was detected in the blood. T_max and C_max of the echinocystic acid were 6.5 ± 1.9 h and 56.7 ± 29.1 ppb. Orally administered lancemaside A was metabolized to lancemaside X (3β, 16α-dihydroxyolean-12-en-28-oic acid 28-O-β-d-xylopyranosyl(1→3)-β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl ester) by intestinal microflora in mice, which was metabolized to echinocystic acid by intestinal microflora and/or intestinal tissues. Human intestinal microflora also metabolized lancemaside A to echinocystic acid via lancemaside X. These results suggest that the metabolism by intestinal microflora may play an important role in pharmacological effects of orally administered lancemaside A.     

A rubber transfer gasket to improve the throughput of liquid–liquid extraction in 96-well plates: Application to vitamin D testing

Background

The unmitigated rise in demand for the assessment of vitamin D status has taxed the ability of clinical mass spectrometry laboratories to preserve turn-around times. We aimed to improve the throughput of liquid–liquid extraction of plasma/serum for the assay of 25-hydroxy vitamin D.

Methods

We designed and fabricated a flexible rubber gasket that seals two 96-well plates together to quantitatively transfer the contents of one plate to another. Using the transfer gasket and a dry-ice acetone bath to freeze the aqueous infranatant, we developed a novel liquid–liquid extraction workflow in a 96-well plate format. We applied the technology to the mass spectrometric quantification of 25-hydroxy vitamin D.

Results

Cross-contamination between wells was ≤0.13%. The interassay imprecision over 132 days of clinical implementation was less than 10%. The method compared favorably to a standard liquid–liquid extraction in glass tubes (Deming slope = 1.018, S_x|y = 0.022). The accuracy of the assay was 102–105% as assessed with the recently released control materials from NIST.

Conclusions

The development of a plate-sealing gasket permits the liquid–liquid extraction of clinical specimens in a moderate-throughput workflow and the reliable assay of vitamin D status. In the future, the gasket may also prove useful in other sample preparation techniques for HPLC or mass spectrometry.     

Simultaneous determination of vitexin-4″-O-glucoside,vitexin-2″-O-rhamnoside,rutin and vitexin from hawthorn leaves flavonoids in rat plasma by UPLC–ESI-MS/MS

A sensitive and accurate ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC–ESI-MS/MS) method was developed and validated for the simultaneous determination of vitexin-4″-O-glucoside (VGL), vitexin-2″-O-rhamnoside (VRH), rutin (RUT) and vitexin (VIT) in rat plasma after intravenous administration of hawthorn leaves flavonoids (HLF). Following protein precipitation by methanol, the analytes were separated on an ACQUITY UPLC BEH C₁₈ column packed with 1.7 μm particles by gradient elution using a mobile phase composed of acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.20 mL/min. The analytes and diphenhydramine (internal standard, IS) were detected in the multiple reaction monitoring (MRM) mode by means of an electrospray ionization (ESI) interface (m/z 292.96 for vitexin-4″-O-glucoside, m/z 293.10 for vitexin-2″-O-rhamnoside, m/z 299.92 for rutin, m/z 310.94 for vitexin and m/z 166.96 for IS). The calibration curve was linear over the range 10–40,000 ng/mL for vitexin-4″-O-glucoside, 10–50,000 ng/mL for vitexin-2″-O-rhamnoside, 8–1000 ng/mL for rutin and 16–2000 ng/mL for vitexin. The intra- and inter-run precisions (relative standard deviation, RSD) of these analytes were all within 15% and the accuracy (the relative error, RE) ranged from −10% to 10%. The stability experiment indicated that the four analytes in rat plasma samples and plasma extracts under anticipated conditions were stable. The developed method was applied for the first time to pharmacokinetic studies of the four bioactive compounds of hawthorn leaves flavonoids following a single intravenous administration of 20 mg/kg in rats.     

An automated screening method for drugs and toxic compounds in human serum and urine using liquid chromatography–tandem mass spectrometry

A fully automated screening using liquid chromatography–mass spectrometric method applying data-dependent acquisition was developed to identify toxicologically relevant substances in serum and urine. A library including more than 405 spectra of about 365 compounds (main drugs and important metabolites) was established. An easy to use program was created to automate and accelerate library search. Drugs were identified based on their relative retention times, molecular ions and fragment ions. Limits of detection were tested with 100 of the 365 compounds the majority of these were lower than 100 μg/l (67%). The developed LC–MS–MS system seems to be a valuable alternative to other general unknown screening methods allowing fast and specific identification of drugs in serum and urine samples.     

Chromatographic separation and sensitive determination of teriflunomide,an active metabolite of leflunomide in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive, selective and high throughput liquid chromatography tandem mass spectrometry (LC–ESI-MS/MS) method has been developed for the determination of teriflunomide, an active metabolite of leflunomide in human plasma. Plasma samples were prepared by liquid–liquid extraction of teriflunomide and valsartan as internal standard (IS) in ethyl acetate from 200 μL human plasma. The chromatographic separation was achieved on an Inertsil ODS-3 C18 (50 mm × 4.6 mm, 3 μm) analytical column using isocratic mobile phase, consisting of 20 mM ammonium acetate–methanol (25:75, v/v), at a flow-rate of 0.8 mL/min. The precursor → product ion transition for teriflunomide (m/z 269.0 → 82.0) and IS (m/z 434.1 → 350.3) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and negative ion mode. The method was validated over a wide dynamic concentration range of 10.1–4001 ng/mL. Matrix effect was assessed by post-column infusion experiment and the mean process efficiency were 91.7% and 88.2% for teriflunomide and IS respectively. The method was rugged and rapid with a total run time of 2.0 min and is applied to a bioequivalence study of 20 mg leflunomide (test and reference) tablet formulation in 12 healthy Indian male subjects under fasting condition.