Kinetics of 8-2 fluorotelomer alcohol and its metabolites, and liver glutathione status following daily oral dosing for 45 days in male and female rats

Fluorotelomer alcohols (FTOHs) are raw materials used in the manufacture of polymeric and surfactant products. Based on previous findings from single oral dosing in rats with radiolabeled 8-2 FTOH, glutathione (GSH) depletion and/or the presence of perfluorinated/polyfluorinated acids and aldehyde metabolites was hypothesized to account for the hepatocellular lesions observed in male rats from a 90-day subchronic oral dosing study. Further, the reported nephropathy in female rats from the subchronic experiment was hypothesized to have been initiated by a thiol metabolite produced by degradation of GSH conjugates. In the current investigation, the kinetics of 8-2 FTOH and its metabolites along with liver GSH status were evaluated in the rat following daily oral dosing with 8-2 FTOH for 45 days at 5 and 125 mg/kg/day. Liver GSH stores 1-2 h after dosing were unaffected, suggesting that GSH depletion is not likely a relevant mode of action in the liver. The tissue metabolite data indicate that the liver toxicity mode of action is likely associated with elevated levels of perfluoroalkyl acids found in males, since other polyfluorinated metabolites and 8-2 FTOH were present in livers from female rats at comparable or higher levels. Detection of the N-acetyl cysteine conjugate of the unsaturated parent telomer alcohol in urine from female rats and not male rats provides some evidence to support the mechanistic basis for the observed kidney effects. Further, the increasing levels of perfluorooctanoic acid (PFOA) in plasma from female rats over the 45-day dosing phase, while unexpected, may reflect an increased net absorption of 8-2 FTOH, slow elimination of intermediates in the metabolic pathway between 8-2 FTOH and PFOA, or altered kidney clearance. The results of this study have enhanced our understanding of 8-2 FTOH kinetics and metabolism and potential modes of action in the rat, which will guide the design of future studies for FTOHs and our need to define the mechanistic basis for the observed effects.     

Semi-automated solid-phase extraction procedure for the high-performance liquid chromatographic determination of alinastine in biological fluids

A solid-phase extraction (SPE) method for sample clean-up followed by a reversed-phase HPLC procedure for the assay of alinastina (pINN) in biological fluids is reported. The effects of the sample pH, composition of the washing and elution solvents and the nature of the SPE cartridge on recovery were evaluated. The selectivity of SPE was examined using spiked rat urine and plasma samples and the CH and PH cartridges gave rise to the cleanest extracts. The recoveries obtained in spiked rat urine and plasma samples were 91.2±2.7 and 99.9±2.8%, respectively. The proposed SPE method coupled off-line with a reserved-phase HPLC system with fluorimetric detection was applied to the quantitation of alinastine in real rat urine samples. The analytical method was also applied and validated for the determination of alinastine in dog plasma. The recovery from spiked dog plasma samples using the PH cartridge was around 65%. The within-day and between-day precisions were 7 and 12%, respectively. The detection and quantitation limits in dog plasma were 0.024 and 0.078 μg/ml, respectively.     

Distribution of the UV filter 3-benzylidene camphor in rat following topical application

A straightforward analytical method for determination of 3-benzylidene camphor (3-BC) in rat adipose tissue, brain, liver, muscle, plasma and testis following topical application was developed and validated. Three exposure levels (60, 180 and 540 mg kg(-1) day(-1)) were tested for 65 days in male Sprague-Dawley rats (24 days postnatal). Sample preparation involving homogenization and n-heptane or methanol extraction of the tissue was applied before injection into the LC-ESI-MS-MS system. The response was linear from 2 to 100 microg l(-1) for the qualifier and the quantifier MRM transitions (R(2) (quantifier) > 0.994). Detection limit of the method corresponded to 0.005 microg g(-1) tissue and 12.5 microg l(-1) plasma, respectively. Recovery was determined for all tissues (adipose tissue: 40%; all other tissues: 80-100%) at three individual levels. 3-(4-Methyl benzylidene camphor) (4-MBC) was used throughout the study as internal standard. 3-Benzylidene camphor was detected in all tissues at all exposure levels at concentrations between 0.05 microg g(-1) (liver) and 36 microg g(-1) (adipose tissue) and in plasma at 16-89 microg l(-1). The method allowed for the quantification of 3-benzylidene camphor in all tested tissues following topical application. Furthermore, it was shown that 3-benzylidene camphor can be found in various tissues in the rat following topical application. These findings may suggest that following use of 3-benzylidene camphor containing sunscreen, similar disposition and distribution may occur in humans.     

Quantification of ceramide species in biological samples by liquid chromatography electrospray ionization tandem mass spectrometry

We present an optimized and validated liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the simultaneous measurement of concentrations of different ceramide species in biological samples. The method of analysis of tissue samples is based on Bligh and Dyer extraction, reverse-phase high-performance liquid chromatography separation, and multiple reaction monitoring of ceramides. Preparation of plasma samples also requires isolation of sphingolipids by silica gel column chromatography prior to LC-ESI-MS/MS analysis. The limits of quantification were in a range of 0.01-0.50 ng/ml for distinct ceramides. The method was reliable for inter- and intraassay precision, accuracy, and linearity. Recoveries of ceramide subspecies from human plasma, rat liver, and muscle tissue were 78 to 91%, 70 to 99%, and 71 to 95%, respectively. The separation and quantification of several endogenous long-chain and very-long-chain ceramides using two nonphysiological odd chain ceramide (C17 and C25) internal standards was achieved within a single 21-min chromatographic run. The technique was applied to quantify distinct ceramide species in different rat tissues (muscle, liver, and heart) and in human plasma. Using this analytical technique, we demonstrated that a clinical exercise training intervention reduces the levels of ceramides in plasma of obese adults. This technique could be extended for quantification of other ceramides and sphingolipids with no significant modification.     

Ultra sensitive quantitation of endogenous oxytocin in rat and human plasma using a two-dimensional liquid chromatography-tandem mass spectrometry assay

Oxytocin (OT) is a neuropeptide with an extremely low endogenous level (low pg/ml) in human plasma. It is very challenging to develop a highly sensitive assay to measure endogenous OT, including radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Electrospray ionization (ESI) liquid chromatography–tandem mass spectrometry (LC–MS/MS) can provide high-throughput and selective methods for quantification of peptides in biological samples. A novel and highly sensitive two-dimensional LC–MS/MS (2D-LC–MS/MS) assay combining solid-phase extraction (SPE) has been developed and validated for the determination of endogenous OT in both human and rat plasma. The lower limit of quantification (LLOQ) was 1.00 pg/ml for human and 50.0 pg/ml for rat. Human plasma diluted with water (1:6, v/v) was successfully optimized as a surrogate matrix for human to prepare standard curves without endogenous interference. The extraction efficiency and absolute recovery were above 65.8% using the HLB SPE procedure, and matrix effects were lower than 12%. The method was validated in the range of 1.00–250 pg/ml for human plasma and 50.0–10,000 pg/ml for rat plasma with precision less than 12.7% and accuracy less than 7%.     

A rugged and accurate liquid chromatography-tandem mass spectrometry method for quantitative determination of BMS-790052 in plasma

To support toxicokinetic assessments, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of BMS-790052 in rat, dog, monkey, rabbit and mouse K(2)EDTA plasma. The drug was isolated from buffered samples using ISOLUTE C8 96-well solid phase extraction (SPE) plates. Chromatographic separation was achieved on a Waters Atlantis dC18 analytical column (2.1 mm × 50 mm, 5 μm) with detection accomplished using an API 4000 tandem mass spectrometer in positive ion electrospray and multiple reaction monitoring (MRM) mode. The standard curves, which ranged from 5.00 to 2000 ng/mL for BMS-790052, were fitted to a 1/x(2) weighted linear regression model. The intra-assay precision (%CV) and inter-assay precision (%CV) were within 8.5%, and the assay accuracy (%Dev) was within ±7.1 for rat, dog, monkey, rabbit and mouse K(2)EDTA plasma. This accurate, precise, and selective SPE/LC-MS/MS method has been successfully applied to analyze several thousands of non-clinical study samples.     

Rapid and simple one-step membrane extraction for the determination of 8-hydroxy-2'-deoxyguanosine in human plasma by a combination of on-line solid phase extraction and LC-MS/MS

A quantitative analytical method using automated on-line solid phase extraction (SPE) and liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) for the determination of 8-OHdG (8-hydroxy-2'-deoxyguanosine) in human plasma was developed and validated. A one-step membrane extraction method for the plasma sample preparation and a C18 SPE column with simple extraction and purification were used for the on-line extraction. A C18 column was employed for LC separation and ESI-MS/MS was utilized for detection. (15)N(5)-8-OHdG ((15)N(5)-8-hydroxy-2'-deoxyguanosine) was used as an internal standard for quantitative determination. The extraction, clean-up and analysis procedures were controlled by a fully automated six-port switch valve as one strategy to reduce the matrix effect and simultaneously improve detection sensitivity. Identification and quantification were based on the following transitions: m/z 284→168 for 8-OHdG and m/z 289→173 for (15)N(5)-8-OHdG. Satisfactory recovery was obtained, and the recovery ranged from 95.1 to 106.1% at trace levels in human plasma and urine, with a CV lower than 5.4%. Values for intraday and interday precision were between 2.3 and 6.8% for plasma and between 2.7 and 4.5% for urine, respectively. Values for the method accuracy of intraday and interday assays ranged from 93.0 and 100.5% for plasma and 110.2 and 119.4% for urine, respectively. The limits of detection (LOD) and LOQ were 0.008 ng/mL and 0.02 ng/mL, respectively.The applicability of this newly developed method was demonstrated by analysis of human plasma samples for an evaluation of the future risk of oxidative stress status in human exposure to nanoparticles and other diseases.     

Development and validation of a method for the confirmation of halofuginone in chicken liver and eggs using electrospray tandem mass spectrometry

A method is described for the quantitative confirmation of halofuginone (HFG) residues in chicken liver and eggs. This method is based on LC coupled to positive ion electrospray MS-MS of the tissue extracts, prepared by trypsin digestion of the tissues followed by liquid-liquid extraction and final clean-up using Solid Phase Extraction (SPE). The [M+H](+) ion at m/z 416 is monitored along with four transitions at m/z 398, 138, 120 and 100. The method has been validated according to the draft EU criteria for the analysis of veterinary drug residues at 15, 30 and 45 microg kg(-1) in liver and 5, 15 and 50 microg kg(-1) in eggs. The new analytical limits, CCalpha and CCbeta were calculated for liver and were 35.4 and 43.6 microg kg(-1), respectively.     

Rapid methods to determine procyanidins, anthocyanins, theobromine and caffeine in rat tissues by liquid chromatography-tandem mass spectrometry

Rapid, selective and sensitive methods were developed and validated to determine procyanidins, anthocyanins and alkaloids in different biological tissues, such as liver, brain, the aorta vein and adipose tissue. For this purpose, standards of procyanidins (catechin, epicatechin, and dimer B(2)), anthocyanins (cyanidin-3-glucoside and malvidin-3-glucoside) and alkaloids (theobromine, caffeine and theophylline) were used. The methods included the extraction of homogenized tissues by off-line liquid-solid extraction, and then solid-phase extraction to analyze alkaloids, or microelution solid-phase extraction plate for the analysis of procyanidins and anthocyanins. The eluted extracts were then analyzed by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry, using a triple quadrupole as the analyzer. The optimum extraction solution was water/methanol/phosphoric acid 4% (94/4.5/1.5, v/v/v). The extraction recoveries were higher than 81% for all the studied compounds in all the tissues, except the anthocyanins, which were between 50 and 65% in the liver and brain. In order to show the applicability of the developed methods, different rat tissues were analyzed to determine the procyanidins, anthocyanins and alkaloids and their generated metabolites. The rats had previously consumed 1g of a grape pomace extract (to analyze procyanidins and anthocyanins) or a cocoa extract (to analyze alkaloids) per kilogram of body weight. Different tissues were extracted 4h after administration of the respective extracts. The analysis of the metabolites revealed a hepatic metabolism of procyanidins. The liver was the tissue which produced a greater accumulation of these metabolites.     

Determination of tissue contributions to the circulating lipid pool in cold exposure via systematic assessment of lipid profiles

Plasma lipid levels are altered in chronic conditions such as type 2 diabetes and cardiovascular disease as well as during acute stresses such as fasting and cold exposure. Advances in MS-based lipidomics have uncovered a complex plasma lipidome of more than 500 lipids that serve functional roles, including as energy substrates and signaling molecules. This plasma lipid pool is maintained through regulation of tissue production, secretion, and uptake. A major challenge in understanding the lipidome complexity is establishing the tissues of origin and uptake for various plasma lipids, which is valuable for determining lipid functions. Using cold exposure as an acute stress, we performed global lipidomics on plasma and in nine tissues that may contribute to the circulating lipid pool. We found that numerous species of plasma acylcarnitines (ACars) and ceramides (Cers) were significantly altered upon cold exposure. Through computational assessment, we identified the liver and brown adipose tissue as major contributors and consumers of circulating ACars, in agreement with our previous work. We further identified the kidney and intestine as novel contributors to the circulating ACar pool and validated these findings with gene expression analysis. Regression analysis also identified that the brown adipose tissue and kidney are interactors with the plasma Cer pool. Taken together, these studies provide an adaptable computational tool to assess tissue contribution to the plasma lipid pool. Our findings have further implications in understanding the function of plasma ACars and Cers, which are elevated in metabolic diseases.     

Simultaneous quantification of beclomethasone dipropionate and its metabolite, beclomethasone 17-monopropionate in rat and human plasma and different rat tissues by liquid chromatography-positive electrospray ionization tandem mass spectrometry

A sensitive, rapid and selective liquid chromatography-positive electrospray ionization tandem mass spectrometry (LC-(ESI+)-MS-MS) method has been developed and validated for the simultaneous quantification of beclomethasone dipropionate (BDP) and its active metabolite, beclomethasone 17-monopropionate (17-BMP) in rat plasma and different tissues using fluticasone propionate (FP) as the internal standard. The method was validated over a linear range from 0.05 to 5 ng/ml for both analytes. A solid-phase extraction procedure was used for plasma samples and a liquid-liquid extraction procedure for tissues samples (lung, liver and kidney). The between-day and within-day coefficients of variation for all compounds were 相似文献   

Molecularly imprinted polymers for the determination of a pharmaceutical development compound in plasma using 96-well MISPE technology

The use of molecularly imprinted polymers (MIPs) as sorbents for the solid phase extraction (SPE) of a pharmaceutical compound in development, prior to quantitative analysis was investigated. Three MIPs were synthesised using a structural analogue as the template molecule. Each polymer was prepared with different monomers and porogens. The MIPs were then tested for their performance both in organic and aqueous environments, the final aim being to load plasma directly onto the polymers. At an early development stage, there is a limited amount of compound available. Due to this limitation, reducing the amount of template required for imprinting was investigated. A MIP capable of extracting the analyte directly from plasma was produced. The specificity of the polymer allowed the method to be validated at a lower sensitivity than a more conventional SPE assay. For the first time, MIPs were packed into 96-well blocks enabling high throughput analysis. The analytical method was fully validated for imprecision and inaccuracy down to 4 ng/ml in plasma.     

Simultaneous determination of nitrendipine and hydrochlorothiazide in spontaneously hypertensive rat plasma using HPLC with on-line solid-phase extraction

A HPLC method with on-line solid phase extraction (SPE) and DAD detection was developed for the simultaneous determination of nitrendipine and hydrochlorothiazide in spontaneously hypertensive rat (SHR) plasma. Plasma samples (100 μL) were injected directly onto a CAPCELL MF C(8) SPE column. High-abundance proteins and most matrixes in plasma were removed by on-line SPE technology, while nitrendipine and hydrochlorothiazide trapped on the SPE column were effectively separated on a C(18) analytical column. The column temperature was maintained at 20°C. The optimal detection wavelength was 237 nm for NTDP and 271 nm for HCTZ. The total analytical run time was 34 min. The proposed method was linear over the range 5-500 ng mL(-1) for nitrendipine and 10-1000 ng mL(-1) for hydrochlorothiazide. The lower limit of detection (LLOD) was 0.5 and 0.6 ng mL(-1) for nitrendipine and hydrochlorothiazide, respectively. The sensitivity and precision of the method were within acceptable limits during validation period. The method was successfully used to investigate the pharmacokinetic characteristics of nitrendipine and hydrochlorothiazide in spontaneously hypertensive rats.     

Immunochemical studies with soluble and mitochondrial pyruvate carboxylase activities from rat tissues

1. Pyruvate carboxylase (EC 6.4.1.1), purified from rat liver mitochondria to a specific activity of 14 units/mg, was used for the preparation of antibodies in rabbits. 2. Tissue distribution studies showed that pyruvate carboxylase was present in all rat tissues that were tested, with considerable activities both in gluconeogenic tissues such as liver and kidney and in tissues with high rates of lipogenesis such as white adipose tissue, brown adipose tissue, adrenal gland and lactating mammary gland. 3. Immunochemical titration experiments with the specific antibodies showed no differences between the inactivation of pyruvate carboxylase from mitochondrial or soluble fractions of liver, kidney, mammary gland, brown adipose tissue or white adipose tissue. 4. The antibodies were relatively less effective in reactions against pyruvate carboxylase from sheep liver than against the enzyme from rat tissues. 5. Pyruvate carboxylase antibodies did not inactivate either propionyl-CoA carboxylase or acetyl-CoA carboxylase from rat liver. 6. It is concluded that pyruvate carboxylase in lipogenic tissues is similar antigenically to the enzyme in gluconeogenic tissues and that the soluble activities of pyruvate carboxylase detected in many rat tissues do not represent discrete enzymes but are the result of mitochondrial damage during tissue homogenization.     

Effect of aflatoxin B1 on lipids of rat tissues.

The effect of aflatoxin B1 on lipids of liver, kidney, adipose and plasma of rats was studied. A single dose administration (6 mg/kg body weight) increased liver and kidney weights and their total lipids within 24 h. Increase in liver lipids was confined mainly to phospholipid and cholesterol, whereas triglycerides showed a significant decrease. Adipose tissue triglycerides were, however, increased. Plasma showed decreases in triglycerides, free fatty acids and cholesterol. Incorporation studies with palmitate-1-14C revealed increased incorporation in adipose tissue lipids and decreased incorporation in liver and plasma lipids, thereby indicating an increased synthesis of lipids in adipose. Their mobilization to plasma was, however, inhibited, hence the low levels of triglyceride in liver. But the adrenals showed hypo-activity upon aflatoxin B1 administration.     

Sensitive determination of aspirin and its metabolites in plasma by LC-UV using on-line solid-phase extraction with methylcellulose-immobilized anion-exchange restricted access media

We describe a sensitive determination of aspirin (ASA) and its three metabolites (salicylic acid [SA], 2,3-dihydroxybenzoic acid [2,3-DHBA], and 2,5-dihydroxybenzoic acid [gentisic acid (GA)]) in rat plasma. Analysis was carried out by on-line solid-phase extraction (SPE) using a methylcellulose-immobilized-strong anion-exchanger (MC-SAX), followed by liquid chromatography (LC) coupled with UV detection. The lower limits of quantitation for ASA and SA were 60 ng/mL in 100 microL of plasma, respectively. This method was validated with respect to intra- and inter-day precision, accuracy, and linearity up to concentrations of 20,000 ng/mL for ASA, SA, 2,3-DHBA and gentisic acid, respectively. The method was successfully applied to an analysis of the pharmacokinetics of ASA and SA in rats.     

Quantitative determination of salidroside in rat plasma by on-line solid-phase extraction integrated with high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

A method for the quantification of salidroside, a major biologically active compound in Rhodiola, in rat plasma by on-line SPE LC/MS/MS in negative electrospray mode was developed and validated. A column-switching instrument and two HPLC pumping systems were employed, and salicin was used as the internal standard. A Waters Oasis HLB extraction column and an Agilent TC-C(18) analytical column in a column-switching set-up with gradient elution were utilized. The MS/MS ion transitions monitored were m/z 299.0/119.0 and 285.1/122.9 for salidroside and salicin, respectively. The standard curves were linear within a range of 50-5000 ng/mL using weighted linear regression analysis (1/x). The intra- and inter-day coefficients of variance ranged from 1% to 9%. The recovery was above 90%. The freeze/thaw and long-term stability were validated. This method was subsequently applied to a pharmacokinetic study of salidroside in rats.     

Intertissue differences in the hysteretic behaviour of carnitine palmitoyltransferase in the presence of malonyl-CoA.

In the presence of malonyl-CoA, the overt form of carnitine palmitoyltransferase (CPT1) in mitochondria from rat liver, kidney cortex, heart, skeletal muscle and brown adipose tissue shows non-linear time courses, suggesting hysteretic behaviour. The pattern of this hysteresis is similar in heart, skeletal muscle and brown adipose tissue, but the hysteretic behaviour of the enzyme in these three tissues differs markedly from that seen in liver and kidney.     

Gas chromatography-tandem mass spectrometry assay for the quantification of four benzodiazepines and citalopram in eleven postmortem rabbit fluids and tissues, with application to animal and human samples

Pharmacokinetic studies and postmortem toxicological investigations require a validated analytical technique to quantify drugs on a large number of matrices. Three-step liquid/liquid extraction with online derivatization (silylation) ahead of analysis by gas chromatography-tandem mass spectrometry was developed and validated on rabbit specimens in order to quantify citalopram and 4 benzodiazepines (diazepam, nordazepam, oxazepam and temazepam) in 11 biological matrices (blood, urine, bile, vitreous humor, liver, kidney, skeletal muscle, brain, adipose tissue, bone marrow (BM) and lung). Since the 11 biological matrices came from the same animal species, full validation was performed on 1 matrix, bone marrow (considered the most complex), while the other 10 underwent partial validation. Due to non-negligible matrix effects, calibration curves were performed on each matrix. Within-day and between-day precision (less than 12.0% and 12.6%, respectively) and accuracy (from 88.9% to 106.4%) were acceptable on BM at both low and high concentrations. Assessment on the other matrices confirmed accuracy and within-day precision (less than 12%, and generally between 85.1% and 114.5%, respectively). The lower limit of quantification of the method was 1ng/g for nordazepam, 5ng/g for citalopram and 10ng/g for oxazepam, diazepam and temazepam. The combination of 3-step extraction and MS/MS detection provided good selectivity in all matrices, including the most lipid-rich. Application to real-case samples showed that the method was sensitive enough to describe distribution patterns in an animal experiment, and specific enough to detect molecules in highly putrefied samples from human postmortem cases.     

Validation of a UHPLC-FLD analytical method for the simultaneous quantification of aflatoxin B1 and ochratoxin a in rat plasma, liver and kidney

A rapid and simple method for the simultaneous quantification of AFB1 and OTA in rat plasma, liver and kidney by UHPLC-FLD has been successfully validated according to the following criteria: selectivity, stability, linearity, precision, accuracy, recovery, robustness and limits of quantification and detection. The extraction method, calibration curves and chromatographic conditions are common for the three matrices. Plasma and homogenized tissue samples (100 μL) were extracted with acetonitrile:formic acid mixture (99:1) (300 μL). Chromatographic separation was performed with a mixture of water and acetonitrile:methanol (50:50), both acidified with 0.5% of formic acid using a gradient profile. The method avoids the use of immunoaffinity columns and allows reduction of sample and solvent volumes as well as toxic wastes. The detection is based on a photochemical reaction which enhances the AFB1 response without affecting the OTA signal before reaching the fluorescent detector. The mycotoxin recovery for each matrix was very efficient, between 93% and 96% for AFB1 and between 94% and 96% for OTA. For both mycotoxins the LOQs were 2μg/L in plasma and 8μg/kg in liver and kidney. The method has successfully been applied to rat samples after a single oral administration of a mixture of AFB1 and OTA and it could be a useful tool in toxicokinetic and toxicological studies.