Sensitive LC MS-MS Research Articles

A simultaneous, high-throughput and sensitive method for analysing 13 neonicotinoids and metabolites in urine using a liquid chromatography–tandem mass spectrometry

A simultaneous, high-throughput and sensitive method for analysing nine neonicotinoid pesticides (NEOs) and four metabolites (NEOms) in urine using a liquid chromatography-tandem mass spectrometry (LC–MSMS) was developed. The method detection limit (MDL) and lowest concentration minimum reporting limit (LCMRL) of the nine NEOs were 0.0013–0.048 ng/ml and 0.0050–0.17 ng/ml, respectively. The MDL and LCMRL of the four NEOms were 0.0052–0.52 ng/ml and 0.011–1.6 ng/ml, respectively. Intermediate precision for the nine NEOs and four NEOms was 7.5–12.5% and 7.4–10.9%, respectively. Accuracy for the nine NEOs and four NEOms was 3.83–5.60% and 3.01–29.2%, respectively. The developed method was applied to analyse urine samples collected from participants of a large-scale birth cohort study, namely, the Japan Environment and Children's Study (JECS).•The NEO and NEOm concentrations in 100 µl urine samples were analysed using a highly sensitive LC–MSMS.•Automated solid phase extraction in a 96-well plate was utilised to achieve high-throughput analysis.•Intermediate precision and accuracy were less than 12.5% and 94.8–99.1%, respectively.

Clinical impact of using sensitive LC-MSMS based testosterone analysis for prostate cancer patients; A requirement for testosterone analysis in castrated men?

The present study aims to examine the distribution, sources and potential risks of toxic metals in the northern Bay of Bengal, Bangladesh. We found Cu, Pb, Zn and Hg exhibited similar spatial distribution pattern. Influenced by the Ganges-Brahmaputra River and the Karnafuli River, there were higher concentrations of these metals associated with the finer sediment and higher TOC in the northeastern portion of the study area. Moreover, coal transportation was assumed to account for the distinctive spatial distribution of As with higher concentration down the Port of Chittagong in the eastern boundary. Chemical-screening level assessment demonstrated the majority of the metals exceeded the threshold effect values, indicating certain possibility of adverse effect. The concentrations of Ni were higher than the possible hazardous values, suggesting high possibility of harmful consequences. The uncontaminated sediments mainly distributed in northwestern and the central portions affected by the delta erosion and marine transported sediments.

A bad salad seasoning: When aconite confused with Couscouil

Description of a fatal case of aconite poisoning after ingestion of leaves of Aconitum napellus , documented by the dosages of 3 aconite alkaloids on various matrices in a deceased patient. After accidental ingestion of aconite plant as a Couscouil salad, a 78-years-old man with non-insulin-dependent diabetes mellitus, and his wife felt buccal paraesthesia and numbness. Quickly, these symptoms become widespread. The man finally admitted in reanimation unit where he developed cardiovascular decompensation on acute arrhythmia and deceased. To confirm the aconite toxidrome, several samples were collected from the wife and the deceased patient. Large screenings of drugs and toxics were performed on blood samples. An aconite specific and sensitive LCMSMS (3200 QTrap, ABSciex) method was developed to determine aconite alkaloids aconitine (A), hypaconitine (H) and mesaconitine (M). Clinical and autopsy samples were analysed after precipitation with methanol [1] , [2] . Bile and urine were hydrolysed before extraction. The limit of quantitation was 1 ng/mL for A, H and M. The toxicological findings were only amiodarone, lidocaine (intensive care) and metformin in non-toxic level in the fatal case. Aconitine was the only alkaloid found in all samples, with traces only for the wife. Measured concentrations in autopsy samples are presented in Table 1 . The concentration of A measured in peripheral blood was higher than those reported in other aconitine-related deaths [2] , even if the digestive absorption might have not been finished. The heart/peripheral concentrations ratio is about 3, which is in accordance to the heart accumulation and the cardiotoxicity of the compound. The concentrations of A in spleen, lung and heart were similar (about 4-fold the peripheral blood). In contrast, diffusion in skeletal muscle was close to blood distribution (34.2 ng/mL). Both diffusion in brain and vitreous humor represented half of blood. Unchanged and conjugated A forms seemed to be low compared to the blood concentrations and the other published cases despite liver and kidney distributions, suggesting a potential renal impairment during resuscitation cares. As another French intoxication case, H and M were not found, may be because of a lack of these compounds in the French species of Aconite. Blood A concentration above 30 ng/mL seems to be lethal even with appropriate cares. In France, toxicological investigation should focus only on A in peripheral blood, and if not available postmortem distribution must be taken into account. Vitreous humor could have an interest.

Abstract P4-10-14: Impact of route of administration of estradiol (oral vs. transdermal) on genotoxic estrogens concentrations in girls with ovarian failure due to Turner syndrome: Potential implications for breast cancer prevention

Abstract Objective: The established link between estrogen and breast cancer occurs via both estrogen receptor (ER)-mediated and non ER-mediated mechanisms. The term genotoxic estrogens describes mutagenic metabolites, including estrogen catechols and quinones, which have been linked to breast carcinogenesis in post-menopausal women. Recent data showed that childhood obesity is associated with significantly higher levels of genotoxic estrogens in the blood compared with lean children, raising the possibility of a potential pathogenic roles of these metabolites in breast cancer starting even prior to the onset of puberty (Mauras et al: J Clin Endocrinol Metab. 100:2322, 2015). A finding of higher levels of genotoxic estrogens associated with oral estradiol may have breast cancer prevention implications.We hence aimed to assess if the route of administration of 17β estradiol (E2) affects the accumulation of genotoxic estrogen metabolites in a model of ovarian failure in young girls with Turner Syndrome. Methods: Stored plasma were used from 40 adolescents with Turner's who participated in a previous 12 months randomized controlled trial of the metabolic impact of E2 orally (2mg/d) vs. transdermally (100μg/d). The doses of oral and transdermal E2 were determined to result in similar plasma levels of unconjugated E2. Previously we had reported that despite the similar plasma levels of unconjugated E2, the oral E2 administration route was associated with higher levels of biologically active estrogen activity than the transdermal route (Torres-Santiago L et al:J Clin Endocrinol Metab. 98:2716, 2013). In this study, we measured 12 estrogen metabolites (conjugated and unconjugated) using a highly sensitive LCMSMS assay. Results from 48 normally menstruating adolescents were used for comparison. Results: After treatment, least square mean (SE) total (conjugated plus unconjugated) E2 and estrone (E1) concentrations were higher in the oral vs. transdermal group (p<0·0001), as were catechol-estrogens 4-OH-E2 (149 vs. 28 (49) pmol/L), 2-OH-E2 (300 vs 76 (52)), 4-OH-E1 (450 vs 105 (113)), 2-OH-E1 (304 vs 740 (684)) and 16α-OH-E1 (3007 vs 157 (534)) (<0·001 between groups). Levels were much closer to controls in the transdermal group. Conclusions: Common feminizing doses of oral estradiol for 12 months result in greater accumulation of unphysiologic, genotoxic estrogens than transdermal estradiol, expanding concerns about oral estrogens' first hepatic passage contributing to the accumulation of these metabolites. These metabolites have the potential for inducing breast cancer in post-menopausal women. These results suggest the potential benefit of preferential use of transdermal versus oral estrogens as replacement and possibly contraceptive options, in the prevention of breast cancer. Further studies to assess long-term risks of these metabolites in women taking different forms of estrogen replacement are needed. Citation Format: Colon-Otero G, Torres-Santiago L, Santen R, Mericq V, Ross J, Damaso L, Hossain J, Wang Q, Mesaros C, Blair IA, Mauras N. Impact of route of administration of estradiol (oral vs. transdermal) on genotoxic estrogens concentrations in girls with ovarian failure due to Turner syndrome: Potential implications for breast cancer prevention [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-10-14.

High-sensitivity determination of estrogens in fish plasma using chemical derivatization upstream UHPLC–MSMS

This article describes the development and validation of a sensitive LC–MSMS method for determination of estrogen in fish plasma. Dansyl chloride derivatization of the phenol functional group in estrogen was used to enhance the response to atmospheric pressure ionization leading to improve the sensitivity. Individual 13C internal standards were selected after comparison with deuterated standards. Liquid-liquid extraction (ethyl acetate or methyl tert-butyl ether) and protein precipitation (acetonitrile, methanol or acetone) were compared for the extraction and clean-up of estrogens from fish plasma. Ethyl acetate was selected as the best alternative with recovery ranging from 61 to 96% and matrix effect ranging from 88 to 106%. Limits of quantification ranged from 0.5 to 1pg/mL showing a gain in sensitivity of 10,000 times over electrospray ionization of underivatized estrogens. Accuracy and precision were validated over three consecutive days and the method was applied to measure estrogen in sea lamprey (Petromyzon marinus) and lake trout (Salvelinus namaycush) plasma. Estrone and estriol were detected in fish below 1ng/mL in plasma, justifying the need of a highly sensitive LC–MSMS quantification method.

Development and Validation of a Sensitive LC-MS-MS Method for the Determination of Adefovir in Human Serum and Urine: Application to a Clinical Pharmacokinetic Study.

A rapid and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of adefovir (PMEA,9-(2-phosphonylmethoxyethyl) adenine) concentration in human serum and urine. The analysis was performed on a negative ionization electrospray mass spectrometer via multiple reaction monitoring. The monitored transitions were set at m/z 272.0 → 134.0 and m/z 276.0 → 149.8 for PMEA and internal standard, respectively. After protein precipitation, samples were separated by high-performance liquid chromatography on a reversed-phase Dikma Diamonsil C18 (250 × 4.6 mm; 5 µm) column with a mobile phase of 0.1 mM ammonium formate buffer-methanol. The calibration curves were linear over the serum concentration range 0.5-1,000 ng/mL and urine concentration range 2.0-1,000 ng/mL. The intra- and interday precision values of PMEA in both serum and urine were lower than 18.16% for low quality control and 13.70% for medium and high quality control. The accuracy, recovery, matrix factor and stability were also within the acceptable limits. The developed method was successfully applied to the pharmacokinetic study of following oral administration of single dose of pradefovir mesylate (10, 30, 60, 90 and 120 mg) and adefovir dipivoxil (10 mg) to healthy Chinese volunteers.

Highly Sensitive LC-MS-MS Method for the Determination of Tacrine in Rat Plasma: Application to Pharmacokinetic Studies in Rats.

A rapid and highly sensitive assay method has been developed and validated for the estimation of tacrine in rat plasma using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode. The assay procedure involves a simple liquid-liquid extraction of tacrine and phenacetin (internal standard, IS) from rat plasma using ethyl acetate. Chromatographic separation was achieved with 0.2% formic acid : acetonitrile (30 : 70, v/v) at a flow rate of 0.50 mL/min on an Atlantis dC18 column with a total run time of 3.0 min. The MS-MS ion transitions monitored were 199.10 → 171.20 for tacrine and 180.10 → 110.10 for IS. Method validation was performed as per United States Food and Drug Administration (US FDA) guidelines and the results met the acceptance criteria. The lower limit of quantification achieved was 0.008 ng/mL and linearity was observed from 0.008 to 53.4 ng/mL. The intra- and inter-day precision was in the range of 2.76-12.5 and 5.15-12.8%, respectively. This novel method has been applied to a pharmacokinetic study in rats.

Exposure pathways of anticoagulant rodenticides to nontarget wildlife

Second-generation anticoagulant rodenticides are widely reported to contaminate and poison nontarget wildlife, primarily predatory birds and mammals. Exposure pathways, however, have not been well defined. Here, we examined potential movement of rodenticides from deployment of bait to exposure of small mammals and other biota. At two adjacent working farms, we placed baits containing either brodifacoum or bromadiolone. We monitored movement of those compounds to the surrounding environment by collecting small mammals, birds, and invertebrates. Similar collections were made at a third agricultural setting without active bait deployment, but located among intensive livestock production and regular rodenticide use by farmers. Livers and whole invertebrate samples were analyzed for rodenticides using a sensitive LC-MSMS method. Norway rats (Rattus norvegicus) from both baited and non-baited farms had residues of brodifacoum or bromadiolone, implicating rats as an important exposure pathway to wildlife. Among 35 analyzed nontarget small mammals, a single vole had high hepatic residues (18.6 μ/g), providing some indication of a small mammal pathway. One song sparrow (Melospiza melodia) sample from a baited farm contained 0.073 μg/g of brodifacoum in liver, while 0.39 μg/g of diphacinone was measured in a pool of carrion beetles (Dermestes spp.) from the non-baited farm area, implicating avian and invertebrate components in exposure pathways. Regurgitated pellets of barn owl (Tyto alba) selected randomly from baited farms contained no detectable rodenticide residues, while 90% of owl pellets collected from a variety of farms, and selected for the presence of rat fur, contained detectable anticoagulant residues. We recorded behavior of a captive sample of a representative songbird, the house sparrow (Passer domesticus); they readily entered bait stations and fed on (unloaded) bait.

Development and Validation of a Sensitive LC–MS-MS Assay for the Quantification of Betulonic Acid in Rat Plasma

A rapid, sensitive and reliable liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of betulonic acid in rat plasma. The plasma samples were prepared by protein precipitation. Chromatographic separation was achieved on an Agela MG-C18 analytical column (50 × 2.1 mm, 5 µm) using methanol-water-formic acid (80:20:0.1, v/v/v) as the mobile phase with a constant flow rate of 0.6 mL/min. Mass detection was conducted by electrospray ionization in positive ion multiple reaction monitoring mode. The calibration curves were linear over a concentration range of 3.00-3,000 ng/mL for the analyte. The intra-day and inter-day accuracy and precision were within the acceptable limits of ±15% at all concentrations. The method was successfully applied for the estimation of betulonic acid in rat plasma from a preclinical pharmacokinetics study.

Determination of the Phosphorylated Metabolites of Gemcitabine and of Difluorodeoxyuridine by LCMSMS

Gemcitabine is an established chemotherapy agent in several solid tumors. Its mechanism of action has been theoretically established and this is supported with strong experimental evidence. However, certain aspects of the resistance mechanism for this agent remain elusive. We present a method of analysis using tandem liquid chromatography and mass spectrometry that provides a broader, yet more focused view of the action of gemcitabine and its primary metabolite, difluorodeoxyuridine in relation to the (deoxy) nucleoside and (deoxy) nucleotide pools in tumor cell lines. Alcoholic cytosole extracts were incubated with alkaline phosphatase reducing the nucleotide pools to their respective nucleosides. Determination of the nucleoside content by a sensitive LCMSMS method before and after incubation enables the calculation of the total amount of phosphorylation of each (deoxy) nucleoside in the cell. Incubation with clinically relevant levels of gemcitabine (dFdC) or difluorodeoxyuridine (dFdU) for 24 hours enabled the determination of the changes in the (deoxy) nucleotide pools in relation to chemotherapeutic and toxicological effects. Confirmation of the presence of dFdC phosphorylation is presented as well as direct evidence of dFdU phosphorylation after both dFdC and dFdU treatment. Differences in the nucleotide pools are presented after dFdC and dFdU incubation, indicating that dFdU might have more chemotherapeutic properties than previously believed.

Simultaneous estimation of 16α-hydroxycleroda-3,13(14) Z-dien-15,16-olide from Polyalthia longifolia and its metabolite in hamster plasma: application to pharmacokinetic study

A selective and sensitive LC-MS-MS method was developed and validated for simultaneous estimation and pharmacokinetic studies of 16α-hydroxycleroda-3,13(14) Z-dien-15,16-olide (K-09) obtained from Polyalthia longifolia and its metabolite (K-9T), a novel antidyslipidemic agent. Sample clean-up involved liquid-liquid extraction of both the analytes and internal standard (rosuvastatin) from 200 μL of hamster plasma. The analytes were chromatographically separated on a Symmetry-Shield C₁₈ (5 µm, 4.6 × 150 mm) column, using acetonitrile-0.1% aqueous formic acid (92:08, v/v) as the mobile phase. Detection was performed using negative ion electrospray ionization in multiple reaction monitoring mode. The MS/MS response was linear over the concentration range 1.56-200 ng/mL, with a correlation coefficient (r²) of 0.998 or better. The within- and between-batch precisions (relative standard deviation, %RSD) and the accuracy (percentage bias) were within acceptable limits as per FDA guidelines. The validated method was successfully applied to reveal the pharmacokinetic parameters of K-09 and metabolite after oral administration. This method will therefore be highly useful for future studies of K-09 and metabolite K-9T pharmacokinetics in preclinical and clinical studies.

Simultaneous Determination of Berberine and Palmatine in Rabbit Plasma by LC-MS–MS and Its Application in Pharmacokinetic Study After Oral Administration of Coptidis and Coptidis–Gardeniae Couple Extract

A valid and sensitive LC-MS–MS method is adopted for pharmacokinetics study of berberine and palmatine in rabbit plasma. After mixing with internal standard tetrahydroberberine, plasma samples were pretreated with 1.5 mL acetonitrile. Chromatographic separation was on a C18 column using a mixture of water (containing 10 mmol L−1 ammonium acetate, pH 3.5) and acetonitrile (50∶50, v/v) as mobile phase. The detection was performed by selected ion monitoring mode via electrospray ionization source operating in the positive ionization mode. The method was linear over the concentration range of 2.0–200.0 ng mL−1 for berberine and 1.0–100.0 ng mL−1 for palmatine. The lowest limits of quantitation (LLOQ) were 2.0 ng mL−1 for berberine and 1.0 ng mL−1 for palmatine. The intra- and inter-day precision values were less than 14.3% and the deviations were within ±11.0%. The fully validated LC-MS–MS method has been successfully applied to a pharmacokinetic study of berberine, palmatine in rabbit plasma after oral administration of Coptidis and coptidis–gardeniae couple extract. The results indicated that the plasma profiles of the two compounds in rabbit confirmed to one-compartment open model and the combinational utilization with Gardeniae could increase the bioavailability of berberine and palmatine, the two major active components of Coptidis.