Development and validation of an ultraperformance liquid chromatography-tandem mass spectrometry method for quantitation of total 3,3',5-triiodo-L-thyronine and 3,3',5,5'-tetraiodo-L-thyronine in rodent serum.

Many environmental chemicals are known to disrupt thyroid function. Measurement of thyroid hormones in animal studies provides useful information to understand the effects of environmental chemicals on thyroid hormone metabolism. We report an efficient method, utilizing a protein precipitation followed by ultraperformance liquid chromatography-tandem mass spectrometry analysis, to quantitate total 3,3',5-triiodo-L-thyronine (triiodothyronine, T3) and total 3,3',5,5'-tetraiodo-L-thyronine (thyroxine, T4) in rodent serum. The use of synthetic serum for calibration standards eliminated the interferences from endogenous total T3 and T4 and allowed the experimental lower limits of quantitation (LOQ) to be set at the required concentration (T3, 20 ng/dL; T4, 0.5 µg/dL) to allow quantitation of endogenous concentrations. The method was linear (r>0.99; range 20.0-600 ng/dL T3, 0.500-15 µg/dL T4) with good assay recoveries (90.4-107%) for both analytes. Intra- and inter-day accuracy, estimated as percent relative error, were ≤ ±7.6% and intra- and inter-day precision, estimated as the relative standard deviation, were ≤ 5.3% for both analytes. The method may easily be adapted to a well-plate format thereby further improving the efficiency. Total T3 and T4 concentrations were stable in male and female rat and mouse serum when stored in the freezer (~ -70 °C) for up to 62 d with determined values within 92.8-111% of day 0 for both analytes. The method can be extended to quantitate total T3 and T4 concentrations in humans or other species with minimal optimization.

Development and Validation of an Analytical Method for Quantitation of Alpha-Pinene Oxide in Rodent Blood and Mammary Glands by GC-MS.

Alpha-pinene is a monoterpene found in the oil of coniferous trees and has a wide variety of applications. Alpha-pinene oxide (APO) is a potential reactive metabolite of alpha-pinene in rodents. The objective of this work is to validate a gas chromatography-mass spectrometry method to quantitate APO in rat and mouse blood and mammary glands in support of studies investigating the toxicity and toxicokinetic behavior of alpha-pinene. The method was validated in male Sprague Dawley rat blood over the concentration range of 5-250 ng/mL. Matrix standard curves were linear (r ≥ 0.99), and accuracy (percent relative error, %RE) was ≤±15% for standards at all levels. Intra- and interday precision (percent relative standard deviation, %RSD) and accuracy (%RE) were evaluated at three concentration levels (10, 50 and 200 ng/mL) and were ≤6.3% and ≤±5.4%, respectively. The limit of detection, determined from the SD of the limit of quantitation (5 ng/mL), was 1.06 ng/mL. Standards as high as 25,000 ng/mL could be accurately quantified after diluting to the validated range (%RE ≤ ±7.1%; %RSD ≤ 5.8%). APO was stable in rat blood for at least 70 days in frozen storage (-80°C). APO could accurately be quantified in male and female Hsd:Sprague Dawley® SD® rat and B6C3F1 mouse blood (mean %RE ≤ ±5.3%; %RSD ≤ 7.8%) and female B6C3F1 and Sprague Dawley rat mammary glands (mean %RE ≤ ±14.6%; %RSD ≤ 8.1%) using a primary matrix standard curve. These results demonstrate that the method is suitable for the analysis of APO in rodent blood and mammary glands generated from toxicokinetic and toxicology studies.

Development of an Analytical Method for Quantitation of 2,2'-Dimorpholinodiethyl Ether (DMDEE) in Rat Plasma, Amniotic Fluid and Fetal Homogenate by UPLC-MS-MS for Determination of Gestational and Lactational Transfer in Rats.

2,2'-Dimorpholinodiethyl ether (DMDEE) is a specialty amine catalyst used in the production of flexible foams, adhesives and coatings. The potential for occupational exposure to DMDEE is high, but toxicity data are very limited. The objective of this work was to develop a method to quantitate DMDEE in biological matrices to assess gestational and lactational transfer of DMDEE in rats following exposure of dams The method used protein precipitation, followed by removal of phospholipids and analysis of supernatant by ultra-performance liquid chromatography-tandem mass spectrometry. Rat fetuses were homogenized in water prior to protein precipitation and delipidation procedures. The method was evaluated in male Sprague Dawley rat plasma over the concentration range 5 to 1000 ng/mL. The method was linear (r ≥ 0.99), accurate (mean relative error (RE) ≤ ±11.9%) and precise (relative standard deviation (RSD) ≤ 2.7%). The mean absolute recovery was 106%. The limit of detection was 0.262 ng/mL. Standards as high as ∼100,000 ng/mL could be successfully diluted into the calibration range (mean %RE = -14.9; %RSD = 0.5). The method was evaluated in Sprague Dawley rat dam plasma, post-natal day 4 pup plasma, gestational day (GD) 18 amniotic fluid and fetal homogenate (mean %RE ≤ ±11.9; %RSD ≤ 2.3). Concentrations of DMDEE in rat dam plasma, amniotic fluid and fetal homogenate stored for at least 29 days and in pup plasma for at least 18 days at -80°C were within 87.7 to 99.5% of Day 0 concentrations, demonstrating that DMDEE is stable in these matrices. The method was used to quantitate DMDEE in rat plasma, amniotic fluid and fetus samples from a dose range finding toxicology study in which dams were dosed via gavage with DMDEE from GD 6 at doses of 0 (control), 62.5 and 250 mg/kg/day. DMDEE concentration increased with the dose in all matrices examined. The concentration in GD 18 fetuses was almost 2-fold higher than GD 18 dams demonstrating gestational transfer of DMDEE. However, the concentration in post-natal day 4 pup plasma was more than an order of magnitude lower than corresponding dam plasma suggesting less potential for transfer of DMDEE from dams to pups via lactation. There was no significant difference in concentration for male and female pup plasma.

Development and Validation of an Analytical Method for Quantitation of Monobutylphthalate, a Metabolite of Di-n-Butylphthalate, in Rat Plasma, Amniotic Fluid, Fetuses and Pups by UPLC-MS/MS.

Phthalates have been used for decades as softening agents in the production of plastics, but in recent years have been extensively investigated for their potential hazards to human health and the environment. Di-n-butyl phthalate (DBP), with widespread exposure occurring through a variety of consumer products such as cosmetics and pesticides, is a suspected carcinogen and an endocrine system disruptor in both humans and laboratory animals. Its predominant metabolite is the monoester, monobutyl phthalate (MBP), which can serve as a marker of exposure. To support toxicological studies of DBP in pregnant and lactating rats and their offspring, a novel ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for quantitation of MBP in rat plasma, amniotic fluid, fetuses and whole pup samples. Plasma samples were extracted using a simple protein precipitation with acetonitrile. Extraction and delipidation of pup homogenate was performed using acetonitrile and then submerging the vials in liquid nitrogen. Extracts were analyzed by UPLC-MS/MS in the negative ion mode. The method was successfully validated over the concentration ranges 25-5,000 ng/mL in female Sprague Dawley (SD) rat plasma and 50-5,000 ng/g in SD pup homogenate. Matrix calibration curves were linear (r ≥ 0.99), and the percent relative error (%RE) values were ≤ ±15% for standards at all levels. Absolute recoveries were > 92% in both matrices. The limits of detection (LODs) were 6.9 ng/mL in plasma and 9.4 ng/g in pup homogenate. Acceptable intra- and interday accuracy and precision were demonstrated by mean %RE ≤ ±7.5 and relative standard deviation (%RSD) ≤ 10.1%. Extract stability was demonstrated for ~6 days at various temperatures and freeze-thaw stability was demonstrated after 3 cycles over 3 days. Secondary matrix evaluation was performed for MBP in amniotic fluid and pooled fetus homogenate (mean %RE ≤ ±11.5 and %RSD ≤ 13.7). These data demonstrate that this simple method is suitable for determination of MBP in plasma, amniotic fluid, fetus and pup samples from toxicological studies of DBP.

Development and Validation of an Analytical Method for Quantitation of Alpha-pinene in Rodent Blood and Mammary Gland by Headspace GC-MS.

Alpha-pinene (AP), produced by pine trees and other plants, is the main component of turpentine and is used as a fragrance and flavor ingredient. Exposure occurs via use of personal care and household cleaning products and in the lumber industry. Despite widespread exposure, toxicity data for AP are limited. The objective of this work was to develop and validate a method to quantitate AP in rodent blood and mammary glands, in support of toxicokinetic and toxicology studies of AP. The method uses 100 µL of blood or ~100 mg of mammary gland with analysis by headspace gas chromatography-mass spectrometry. The samples are diluted with internal standard (2H3-AP, IS) and sealed in headspace vials; mammary glands are homogenized within the vial. The vials are equilibrated briefly at 60°C before a headspace sample is analyzed. The method was validated in Sprague Dawley rat blood over the range 5-500 ng/mL and mammary gland over the range 100-5000 ng/g. The method was linear (r ≥0.99), accurate (mean relative error (RE) ≤±13.4%) and precise (relative standard deviation (RSD) ≤7.1%) in both matrices. Recoveries incorporating IS were ≥88.7% at all concentrations in both tissues. Standards as high as 1500 ng/mL in blood and 20,000 ng/g in mammary gland could be analyzed using lower injection volume or extrapolating the calibration curve beyond the upper limit of quantitation (mean %RE ≤±18.7; %RSD ≤2.2). Loss of AP occurred during overnight autosampler storage as well as frozen storage in as few as 15 days, but incorporation of IS prior to storage corrected for the loss such that calculated concentrations were within 84.7-117% of day 0 concentrations following frozen storage up to ≥32 days in both matrices. Matrix evaluation was performed in Hsd:Sprague Dawley®SD® rat and B6C3F1 mouse blood and mammary glands (mean %RE ≤±9.2; %RSD ≤4.3). These data demonstrate that the method is suitable for determination of AP in rodent blood and mammary glands.

Development and Validation of an Analytical Method for Quantitation of Sulfolane in Rat and Mouse Plasma by GC-MS.

Sulfolane is an industrial solvent commonly used for extraction of aromatic hydrocarbons in the oil refining process, as well in the purification of natural gas. Its wide use and high solubility in water has led to contamination of groundwater. The objective of this work was to develop and validate an analytical method to quantitate sulfolane in rodent plasma in support of the National Toxicology Program toxicology and toxicokinetic studies of sulfolane. The method uses extraction of plasma with ethyl acetate and analysis by gas chromatography-mass spectrometry with electron ionization. The method was validated in male Sprague Dawley (SD) rat plasma over the concentration range of 20-100,000 ng/mL. The method was linear (r ≥ 0.99), accurate (mean relative error (RE) ≤ ±5.1%) and precise (relative standard deviation (RSD) ≤ 2.9%). The absolute recovery was ≥74%. The limit of detection was 0.516 ng/mL. Standards as high as ~2.5 mg/mL could be successfully diluted into the calibration range (mean %RE ≤ ±4.5; %RSD ≤ 4.6). Extracted samples were stable for at least 3 days at ambient and refrigerated temperatures, and freeze/thaw stability in matrix was demonstrated after three cycles over 3 days (calculated concentrations within 90.8-102% of Day 0 concentrations). Sulfolane was stable in frozen plasma for at least 75 days at -80°C (calculated concentrations within 93.0-98.1% of Day 0 concentrations). Matrix evaluation was performed for sulfolane in female SD rat plasma and male and female B6C3F1 mouse plasma (mean %RE ≤ ±4.9; %RSD ≤ 3.3). These data demonstrate that the method is suitable for determination of sulfolane in rodent plasma.

Determination of L-ephedrine, pseudoephedrine, and caffeine in rat plasma by liquid chromatography-tandem mass spectrometry.

A rapid and simple liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous determination of L-ephedrine, pseudoephedrine, and caffeine in male Fisher-344 rat plasma at nanogram-per-milliliter concentrations for use in support of toxicology studies. Only 25 µL of plasma is required, and extraction is performed using a simple, single-step protein precipitation. The method was validated over a range of 2.09 to 5460 ng/mL for L-ephedrine, 2.09 to 5050 ng/mL for pseudoephedrine and 2.03 to 5340 ng/mL for caffeine. A binary gradient elution at 0.3 mL/min was used with a Waters XBridge Phenyl (2.1 × 150 mm, 3.5 µm) column and a Waters XBridge Phenyl 2.1- × 10-mm guard column at ambient temperature. The mobile phase consisted of 10 mM ammonium acetate in water (pH 5.0) and methanol. Caffeine trimethyl-(13)C(3) was used as the internal standard. The method was evaluated for linearity, recovery, precision, accuracy, and stability, and it was successfully applied in toxicokinetic studies of ephedrine, administered alone, in combination with caffeine, and in the herbal source Ma Huang.

Application of chemoproteomics to drug discovery: identification of a clinical candidate targeting hsp90.

A chemoproteomics-based drug discovery strategy is presented that utilizes a highly parallel screening platform, encompassing more than 1000 targets, with a focused chemical library prior to target selection. This chemoproteomics-based process enables a data-driven selection of both the biological target and chemical hit after the screen is complete. The methodology has been exemplified for the purine binding proteome (proteins utilizing ATP, NAD, FAD). Screening of an 8000 member library yielded over 1500 unique protein-ligand interactions, which included novel hits for the oncology target Hsp90. The approach, which also provides broad target selectivity information, was used to drive the identification of a potent and orally active Hsp90 inhibitor, SNX-5422, which is currently in phase 1 clinical studies.