Development and Validation of Liquid Chromatography-Tandem Mass Spectrometry Method for Pharmacokinetic Evaluation of 7ß-(3-Ethyl-cis-crotonoyloxy)-1α-(2-methylbutyryloxy)-3,14-dehydro-Z-notonipetranon in Rats.

Recently, potent neuroprotective and anti-diabetic effects of 7ß-(3-Ethyl-cis-crotonoyloxy)-1α-(2-methylbutyryloxy)-3,14-dehydro-Z-notonipetranone (ECN), a sesquiterpenoid isolated from Tussilago farfara Linnaeus, have been elucidated. To facilitate further pre-clinical evaluation in rats, an analytical method for the determination of ECN in rat plasma was developed and optimized by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasma samples were pretreated by the protein precipitation method with an acetonitrile solution of losartan (LST) as the internal standard. Chromatographic separation was performed using a an Octadecyl-silica (ODS) column (2.6 µm, 100 x 4.6 mm) in the isocratic mode. The mobile phase, comprising 10 mM ammonium formate in water pH 5.75) and acetonitrile (11:89, v/v), was eluted at a flow rate of 0.4 mL/min. Mass spectrometric detection was performed in the multiple reaction monitoring mode with positive electrospray ionization, and the mass transitions of ECN and LST were m/z 431.3 to 97.3 and m/z 423.1 to 207.2, respectively. The calibration curves of spiked plasma samples were linear in the 10.0-10,000 ng/mL range (r2 > 0.996). The lower limit of quantification (LLOQ) was determined as 10.0 ng/mL. Validation was conducted in the LLOQ, and three quality control (QC) sample levels (10.0, 25.0, 3750, and 7500 ng/mL) were studied. Among them, the relative standard deviation for the within- and between-run precisions was under 9.90%, and the relative error of the accuracies was within the -8.13% to 0.42% range. The validated method was successfully employed to investigate the pharmacokinetic properties of ECN in rats, which revealed the linear pharmacokinetic behavior of ECN for the first time.

Bioavailability and Pharmacokinetics of Anisatin in Mouse Blood by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Anisatin is a neurotoxic sesquiterpene dilactone wildly found in plants of the family Illiciaceae. Due to morphological similarities among Illiciaceae fruits, fatal poisonings are frequent. OBJECTIVE: This study is aimed at developing a rapid, simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to determine anisatin's bioavailability in mouse blood and the method's application to pharmacokinetics. METHODS: Blood samples were preprocessed by protein precipitation using acetonitrile. Salicin (internal standard, IS) and anisatin were gradient-eluted by a mobile phase of methanol and water (0.1% formic acid) in a UPLC BEH C18 column. This step involved using an electrospray ionization source of anisatin at a mass-to-charge ratio (m/z) of 327.1 → 127.0 and IS at m/z 285.1 → 122.9 in the negative ion mode with multiple reaction monitoring. RESULTS: The calibration curve ranged from 1 to 2000 ng/ml (r > 0.995), with the method's accuracy ranging from 86.3% to 106.9%. Intraday and interday precision were lower than 14%, and the matrix effect was between 93.9% and 103.3%. The recovery rate was higher than 67.2%. CONCLUSIONS: The developed UPLC-MS/MS method was successfully used for a pharmacokinetic study of oral (1 mg/kg) and intravenous (0.5 mg/kg) administration of anisatin to mice-the absolute bioavailability of anisatin in the mouse blood was 22.6%.

Validation and application of a novel UHPLC-MS/MS method for the measurement of furanodienone in rat plasma.

A sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was established to analyze furanodienone in rat plasma. In the process of chromatographic separation, selected reaction monitoring transitions for furanodienone and patchouli alcohol (internal standard, IS) were m/z 231.1 → 83.2 and m/z 205.1 → 95.1, respectively. Great linearity of furanodienone in plasma samples was found in the corresponding concentration range (r > 0.995). Intra- and inter-day precisions (RSD, %) were <11.3% in plasma, and the accuracy (RE, %) was within ±10.7%. This method was used to the furanodienone study on rat pharmacokinetics after a single oral dose of 10 mg/kg of furanodiene. The results indicated that the maximum observed plasma concentration was 52.4 ± 19.1 ng/ml at 1.2 ± 0.7 h with an elimination half-life of 2.2 ± 0.7 h. The obtained data indicated that furanodienone could be moderately distributed and eliminated.

The profiling and identification of the absorbed constituents and metabolites of Naoshuantong capsule in mice biofluids and brain by ultra- fast liquid chromatography coupled with quadrupole-time-of-flight tandem mass spectrometry.

Naoshuantong capsule (NSTC) is an oral traditional Chinese medicine formula used widely in the clinic for ischemic stroke. The absorbed ingredients and metabolites of NSTC have never been reported before. In this study, a method incorporating rapid resolution liquid chromatography with quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was used to identify absorbed ingredients and metabolites after oral administration of NSTC. A total of 15 constituents were detected and identified as prototypes of NSTC. 109 metabolites related to catechin, gallic acid, paeoniflorin, chlorogenic acid, protocatechuate, typhaneoside, ß-elemene, calycosin were identified in serum, urine and brain. 19 metabolites of typhaneoside, 3 metabolites of ß-elemene, 12 metabolites of calycosin were reported for the first time. This is the first time to explore the absorption and metabolism of NSTC. The work will provide helpful information for further research of the mechanism and application of NSTC.

Fate of ptaquiloside-A bracken fern toxin-In cattle.

Ptaquiloside is a natural toxin present in bracken ferns (Pteridium sp.). Cattle ingesting bracken may develop bladder tumours and excrete genotoxins in meat and milk. However, the fate of ptaquiloside in cattle and the link between ptaquiloside and cattle carcinogenesis is unresolved. Here, we present the toxicokinetic profile of ptaquiloside in plasma and urine after intravenous administration of ptaquiloside and after oral administration of bracken. Administered intravenously ptaquiloside, revealed a volume of distribution of 1.3 L kg-1 with a mean residence-time of 4 hours. A large fraction of ptaquiloside was converted to non-toxic pterosin B in the blood stream. Both ptaquiloside and pterosin B were excreted in urine (up to 41% of the dose). Oral administration of ptaquiloside via bracken extract or dried ferns did not result in observations of ptaquiloside in body fluids, indicating deglycosolidation in the rumen. Pterosin B was detected in both plasma and urine after oral administration. Hence, transport of carcinogenic ptaquiloside metabolites over the rumen membrane is indicated. Pterosin B recovered from urine counted for 7% of the dose given intravenously. Heifers exposed to bracken for 7 days (2 mg ptaquiloside kg-1) developed preneoplastic lesions in the urinary bladder most likely caused by genotoxic ptaquiloside metabolites.

Stereo-Selective Pharmacokinetics of Ilimaquinone Epimers Extracted from a Marine Sponge in Rats.

An ilimquinone (IQ) mixture isolated from Hippiospongia metachromia, consisting of IQ and epi-ilimaquinone (epi-IQ), exerts anti-HIV, anti-microbial, anti-inflammatory, and anti-cancer effects. An HPLC-MS/MS method was developed for simultaneous determination of the two epimers in rat plasma, separating them using a biphenyl column. Ascorbic acid is added during the sample preparation to ensure the stability of both isomers. The plasma concentrations of the isomers were monitored following intravenous and oral administration of the IQ mixture in rats as well as the individual epimers that were separately orally administered. Compare to IQ, epi-IQ was much more stable in rat plasma, likely due to its configurations of decalin. Both substances decayed in more than bi-exponential pattern, with an elimination rate constant of 1.2 h-1 for IQ and 1.7 h-1 for epi-IQ. The epi-IQ was distributed more widely than IQ by about two-fold. Consequently, the clearance of epi-IQ was greater than that of IQ by about three-fold. The oral absolute bioavailability for IQ was 38%, and, that for epi-IQ, was 13%. Although the systemic exposure of IQ was greater than that of epi-IQ by ~8.7-fold, the clearance of each isomer was similar when administered either orally or intravenously, when normalized for bioavailability. The stereo-specific behavior of the isomers appears to originate from differences in both their tissue distribution and gastrointestinal permeability.

[Determination of coriatin and corianin in plasma and urine using ultra-performance liquid chromatography-triple quadrupole mass spectrometry].

An ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS) method has been developed for the determination of coriatin and corianin in plasma and urine, which are the biomarkers of poisoning caused by Coriaria sinica Maxim. Plasma and urine samples were extracted and purified using a solid supported liquid/liquid extraction method. Chromatographic separation was performed on a Cortecs C18 column (100 mm×2.1 mm, 1.6 µm) using a gradient elution of methanol and water. Coriatin and corianin were detected using negative electrospray ionization tandem mass spectrometry in multiple reaction monitoring (MRM) mode and quantified via a matrix working standard curve internal standard method; florfenicol was used as the internal standard. The assay was linear in the calibration range of 0.03-5.0 µg/L for coriatin and 0.3-50 µg/L for corianin in plasma, and 0.1-10 µg/L and 1-100 µg/L for coriatin and corianin in urine, respectively. The average recoveries were 86.2%-110% for coriatin and corianin in plasma and urine with relative standard deviations of 5.1%-14.6% (n=6). The limits of detection (S/N=3) for coriatin and corianin were 0.01 µg/L and 0.1 µg/L in plasma, and 0.03 µg/L and 0.3 µg/L in urine, respectively. The method is simple, sensitive and accurate for the determination of coriatin and corianin in plasma and urine for toxicological purposes.

HPLC-MS/MS analysis of ilimaquinone and its application in a pharmacokinetic study in rats.

Ilimaquinone, a metabolite isolated from the marine sponge Hippiospongia metachromia, has antimicrobial, cytotoxic, anti-HIV, anti-inflammatory, and anti-cancer activities. A new quantitative analytical method for determination of ilimaquinone in rat plasma using HPLC-MS/MS was developed and validated. Ascorbic acid was added to ensure the stability of ilimaquinone in plasma. After protein precipitation using acetonitrile plus diclofenac as an internal standard, the analytes were chromatographed on a biphenyl column with a mobile phase of methanol and water (8:2, v/v, including 0.1% formic acid). This method was successfully applied in a pharmacokinetic study of ilimaquinone after oral administration in rats.

Resveratrol and Its Human Metabolites-Effects on Metabolic Health and Obesity.

Resveratrol is one of the most widely studied polyphenols and it has been assigned a plethora of metabolic effects with potential health benefits. Given its low bioavailability and extensive metabolism, clinical studies using resveratrol have not always replicated in vitro observations. In this review, we discuss human metabolism and biotransformation of resveratrol, and reported molecular mechanisms of action, within the context of metabolic health and obesity. Resveratrol has been described as mimicking caloric restriction, leading to improved exercise performance and insulin sensitivity (increasing energy expenditure), as well as having a body fat-lowering effect by inhibiting adipogenesis, and increasing lipid mobilization in adipose tissue. These multi-organ effects place resveratrol as an anti-obesity bioactive of potential therapeutic use.

Determination of Penicillium griseofulvum-oriented pyripyropene A, a selective inhibitor of acyl-coenzyme A:cholesterol acyltransferase 2, in mouse plasma using liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic studies.

In this study, we developed a method for the determination of Penicillium griseofulvum-oriented pyripyropene A (PPPA), a selective inhibitor of acyl-coenzyme A:cholesterol acyltransferase 2, in mouse and human plasma and validated it using liquid chromatography-tandem mass spectrometry. Pyripyropene A (PPPA) and an internal standard, carbamazepine, were separated using a Xterra MS C18 column with a mixture of acetonitrile and 0.1% formic acid as the mobile phase. The ion transitions monitored in positive-ion mode [M + H]+ of multiple-reaction monitoring (MRM) were m/z 148.0 from m/z 584.0 for PPPA and m/z 194.0 from m/z 237.0 for the internal standard. The detector response was specific and linear for PPPA at concentrations within the range from 1 to 5,000 ng/mL. The intra-/inter-day precision and accuracy of the method was acceptable by the criteria for assay validation. The matrix effects of PPPA ranged from 97.6 to 104.2% and from 93.3 to 105.3% in post-preparative mouse and human plasma samples, respectively. PPPA was also stable under various processing and/or handling conditions. Finally, PPPA concentrations in the mouse plasma samples could be measured after intravenous, intraperitoneal, or oral administration of PPPA, suggesting that the assay is useful for pharmacokinetic studies on mice and applicable to human studies.

Pharmacokinetics and tissue distribution study of bisabolangelone from Angelicae Pubescentis Radix in rat using LC-MS/MS.

A sensitive and accurate LC-MS/MS method was established for quantifying bisabolangelone in rat plasma and tissues. Bisabolangelone was isolated and purified from Angelicae Pubescentis Radix. The pharmacokinetic and tissue distribution of bisabolangelone after administration to rat was performed by LC-MS/MS. Separation was carried out on a C8 (4.6 × 100 mm, 1.8 µm) column. The MS/MS transitions of bisabolangelone and tussilagone (internal standard) were set at m/z 249.1 → 109.1 and m/z 391.4 → 217.4, respectively. The lower limit of quantification in plasma and other tissues ranged from 1 to 4 ng/mL. The biosamples were prepared using protein precipitation method with acetonitrile. The recovery was >92%. The results showed that values of maximum concentrations and area under the curve depended linearly on the studied doses (2.5, 5 and 7.5 mg/kg body weight). The other ingredients in Angelicae Pubescentis Radix extract possibly reduce the absorption of bisabolangelone in rat. Tissue distribution revealed that bisabolangelone was widely distributed in vivo. The highest and lowest concentrations of bisabolangelone were found in the stomach and in the brain, respectively. It was concluded that the newly established HPLC-MS/MS method was suitable to describe the pharmacokinetic characteristics of bisabolangelone in rat after administration.

UPLC-MS/MS of Atractylenolide I, Atractylenolide II, Atractylenolide III, and Atractyloside A in Rat Plasma after Oral Administration of Raw and Wheat Bran-Processed Atractylodis Rhizoma.

Atractylodis Rhizoma is the dried rhizome of Atractylodes lancea (Thunb.) DC. or Atractylodes chinensis (DC.) Koidz and is often processed by stir-frying with wheat bran to reduce its dryness and increase its spleen tonifying activity. However, the mechanism by which the processing has this effect remains unknown. To explain the mechanism based on the pharmacokinetics of the active compounds, a rapid, sensitive ultra-performance liquid chromatography-tandem mass spectrometry method was developed to analyze atractylenolides I, II, and III, and atractyloside A simultaneously in rat plasma after oral administration of raw and processed Atractylodis Rhizoma. Acetaminophen was used as the internal standard and the plasma samples were pretreated with methanol. Positive ionization mode coupled with multiple reaction monitoring mode was used to analyze the four compounds. The method validation revealed that all the calibration curves displayed good linear regression over the concentration ranges of 3.2⁻350, 4⁻500, 4⁻500, and 3.44⁻430 ng/mL for atractylenolides I, II, and III, and atractyloside A, respectively. The relative standard deviations of the intra- and inter-day precisions of the four compounds were less than 6% with accuracies (relative error) below 2.38%, and the extraction recoveries were more than 71.90 ± 4.97%. The main pharmacokinetic parameters of the four compounds were estimated with Drug and Statistics 3.0 and the integral pharmacokinetics were determined based on an area under the curve weighting method. The results showed that the integral maximum plasma concentration and area under the curve increased after oral administration of processed Atractylodis Rhizoma.

Simultaneous Quantitation of Isoprenoid Pyrophosphates in Plasma and Cancer Cells Using LC-MS/MS.

Isoprenoids (IsoP) are an important class of molecules involved in many different cellular processes including cholesterol synthesis. We have developed a sensitive and specific LC-MS/MS method for the quantitation of three key IsoPs in bio-matrices, geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP). LC-MS/MS analysis was performed using a Nexera UPLC System connected to a LCMS-8060 (Shimadzu Scientific Instruments, Columbia, MD) with a dual ion source. The electrospray ionization source was operated in the negative MRM mode. The chromatographic separation and detection of analytes was achieved on a reversed phase ACCQ-TAG Ultra C18 (1.7 µm, 100 mm × 2.1 mm I.D.) column. The mobile phase consisted of (1) a 10 mM ammonium carbonate with 0.1% ammonium hydroxide in water, and (2) a 0.1% ammonium hydroxide in acetonitrile/methanol (75/25). The flow rate was set to 0.25 mL/min in a gradient condition. The limit of quantification was 0.04 ng/mL for all analytes with a correlation coefficient (r2) of 0.998 or better and a total run time of 12 min. The inter- and intra-day accuracy (85⁻115%) precision (<15%), and recovery (40⁻90%) values met the acceptance criteria. The validated method was successfully applied to quantitate basal concentrations of GPP, FPP and GGPP in human plasma and in cultured cancer cell lines. Our LC-MS/MS method may be used for IsoP quantification in different bio-fluids and to further investigate the role of these compounds in various physiological processes.

Pharmacokinetic study comparing pure desoxo-narchinol A and nardosinonediol with extracts from Nardostachys jatamansi.

Nardostachyos Radix et Rhizoma (NR) is a valuable medicinal herb widely used in Korea, India, and China for the treatment of many diseases. Desoxo-narchinol A (DA) and nardosinonediol (ND) are the two main bioactive compounds belonging to the sesquiterpene group. Desoxo-narchinol A possesses anti-inflammatory activity while ND exhibits anti-depressant and cardioprotective activities. A pharmacokinetic study is important to decide whether the isolated compounds or the NR extract have better pharmacological activity. Hence, we developed an analytical method for studying the pharmacokinetics of DA and ND after oral administration of the pure compounds and herbal extract. An optimized liquid chromatography-mass spectrometry method (LC-MS/MS) with solid-phase extraction (SPE) for sample preparation was developed. A ZORBAX Extend C18 column (2.1 × 50 mm, 3.5 µm) was used under gradient elution with acetonitrile and 0.1% formic acid in water as the mobile phase. Validation experiments assessing accuracy, precision, and stability were satisfactory; the lower limit of quantification was 5 ng/mL. For the pharmacokinetic study, three groups of rats were administrated pure DA, pure ND, or NR extract orally. Concentrations of DA and ND in their plasma were determined by the developed method. Pharmacokinetic parameters, including the time to achieve maximum plasma concentration (Tmax) and the area under the plasma concentration curve from time zero to infinity (AUC0-∞), were compared for the herbal extract and pure compounds. The Tmax of the pure compound and the NR extract for DA was 7.50 and 8.33 min, respectively, compared to 5.00 and 5.83 min for the pure compound and the NR extract for ND, respectively. The AUC0-∞ of the pure compound and the NR extract for DA was 156.34 and 133.90 µg min/mL, respectively, and that for the NR extract for ND was 6.42 and 4.15 µg min/mL, respectively. LC-MS/MS was used to determine DA and ND in rat plasma. The pharmacokinetic profile of each pure compound and those in the extract were characterized and compared.

High norepinephrinergic orthostatic hypotension in early Parkinson's disease.

INTRODUCTION: Plasma norepinephrine concentration reflects lesions causing OH. We investigate whether patients with high norepinephrinergic orthostatic hypotension (OH) whose supine plasma norepinephrine concentration (NEsupine) is above the mean value in all patients with Parkinson's disease (PD) have central sympathetic denervation. METHODS: We analyzed data from 110 non-demented patients with early de novo PD who underwent cardiovascular examinations. We divided the patients into three groups according to the presence or absence of orthostatic hypotension and NEsupine: patients without OH, patients with OH+high NEsupine, and patients with OH+low NEsupine. RESULTS: The mean NEsupine in all patients was 251.6 pg/ml. Twelve patients (10.9%) had OH+high NEsupine (≥251.6 pg/ml), and 45 patients (40.9%) had OH+low NEsupine (<251.6 pg/ml). OH was more pronounced in patients with OH+high NEsupine than in those with OH+low NEsupine (p = 0.024). Vasopressin release and percent increase of NE after orthostatic stress were well preserved in patients with OH+low NEsupine, but not in patients with OH+high NEsupine. Cognition was lower in patients with OH+high NEsupine than in patients with OH+low NEsupine (p = 0.019) and was associated with vasopressin release during orthostatic stress on multiple regression analysis. The degree of cardiac sympathetic denervation did not differ between two groups with OH. CONCLUSIONS: Patient with PD and high norepinephrinergic OH are a subset of patients who have early cognitive decline and impaired vasopressin release. Vasopressin release after orthostatic stress was closely related to global cognition in PD.

Determination and pharmacokinetic study of guaiol in rat plasma by gas chromatography-mass spectrometry with selected ion monitoring.

Guaiol has been used for thousands of years as a traditional Uygur medicine and is the primary active component found in Ferula ferulaeoides (Steud.) Korov (F. ferulaeoides). In our present study, a rapid, selective, and sensitive method of monitoring selected ions was established based on gas chromatography-mass spectrometry. This method was optimized for the quantification and pharmacokinetic analysis of guaiol in rat plasma following oral administration of chloroform extract from Ferula ferulaeoides. Plasma was extracted using liquid-liquid extraction with ethyl acetate and was analyzed on a HP-5MS column (30 m × 250 µm × 0.25 µm) with a mass selective detector. Detection was carried out under selected ion monitoring mode, and three selected ion monitoring ions (m/z 59.1, 107.1, and 161.1 for guaiol) were used for the quantitative determination of that under investigation. The assay demonstrated excellent linearity in the range of 1-200 ng/mL (r = 0.9993, n = 8) in the case of guaiol measured in rat plasma. The limit of detection and the limit of quantification for guaiol in rat plasma were found to be 0.25 ng/mL and 1 ng/mL, respectively. Intra-day and inter-day precisions were expressed as the relative standard deviation for the method and were in the range of 97.49%-106.16% and 97.04%-105.91%, respectively. Extraction efficiencies were all determined to be >90%, and recoveries were ranged from 91.25% to 96.24%. This method has been successfully applied for the pharmacokinetic evaluation of chloroform extract isolated from F. ferulaeoides following a single oral administration dose (157.5 mg/kg) in rats. The guaiol pharmacokinetic study demonstrated that the half-life of guaiol was 9.18 ±â€¯3.75 h, the mean residence time was 9.07 ±â€¯3.86 h, the maximum guaiol concentration in the plasma was 28.63 ±â€¯6.82 ng/mL, and the maximum time guaiol was in the plasma was 0.50 h.

A Sensitive LC-MS-MS Method for Quantification of 1,6-O,O-Diacetylbritannilactone in Rat Plasma and its Application in a Pharmacokinetic Study.

1,6-O,O-Diacetylbritannilactone is a natural sesquiterpene lactone isolated from Inula britannica that has displayed cytotoxic effects against several human cancer cell lines. In this study, a selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed for determination of 1,6-O,O-diacetylbritannilactone in rat plasma. Chromatographic separation was achieved on an Agilent C18 column (4.6 mm × 75 mm, 3.5 µm) with methanol and water (80:20, v/v) as the mobile phase. An ESI source was applied and operated in positive ion mode; selected-reaction monitoring was used for quantification using target fragment ions m/z 373.2→312.9 for 1,6-O,O-diacetylbritannilactone and m/z 331.2→144.1 for the IS. Calibration plots were linear in the range of 1.5-1350 ng/mL for 1,6-O,O-diacetylbritannilactone in rat plasma. Intra- and inter-day precisions were <8.5%, and the accuracy ranged from -2.7 to 12.8%. The LC-MS-MS method was successfully applied in a pharmacokinetic study of 1,6-O,O-diacetylbritannilactone in rats.

ß-Caryophyllene protects against alcoholic steatohepatitis by attenuating inflammation and metabolic dysregulation in mice.

BACKGROUND AND AIMS: ß-Caryophyllene (BCP) is a plant-derived FDA approved food additive with anti-inflammatory properties. Some of its beneficial effects in vivo are reported to involve activation of cannabinoid CB2 receptors that are predominantly expressed in immune cells. Here, we evaluated the translational potential of BCP using a well-established model of chronic and binge alcohol-induced liver injury. METHODS: In this study, we investigated the effects of BCP on liver injury induced by chronic plus binge alcohol feeding in mice in vivo by using biochemical assays, real-time PCR and histology analyses. Serum and hepatic BCP levels were also determined by GC/MS. RESULTS: Chronic treatment with BCP alleviated the chronic and binge alcohol-induced liver injury and inflammation by attenuating the pro-inflammatory phenotypic `M1` switch of Kupffer cells and by decreasing the expression of vascular adhesion molecules intercellular adhesion molecule 1, E-Selectin and P-Selectin, as well as the neutrophil infiltration. It also beneficially influenced hepatic metabolic dysregulation (steatosis, protein hyperacetylation and PPAR-α signalling). These protective effects of BCP against alcohol-induced liver injury were attenuated in CB2 receptor knockout mice, indicating that the beneficial effects of this natural product in liver injury involve activation of these receptors. Following acute or chronic administration, BCP was detectable both in the serum and liver tissue homogenates but not in the brain. CONCLUSIONS: Given the safety of BCP in humans, this food additive has a high translational potential in treating or preventing hepatic injury associated with oxidative stress, inflammation and steatosis. LINKED ARTICLES: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

Bioanalysis of farnesyl pyrophosphate in human plasma by high-performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry and hybrid quadrupole Orbitrap high-resolution mass spectrometry.

The isoprenoids farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are pivotal intermediates for cholesterol homeostasis and cell signaling in the mevalonate pathway. We developed a sensitive and selective high-performance liquid chromatography tandem triple quadrupole mass spectrometry (LC-QQQ-MS) method for FPP in human plasma without the need for a derivatization process. We optimized the sample preparation procedure to extract FPP and 13C5-FPP (as internal standard) from sample fluids using methanol. Phosphate-buffered saline was used as the surrogate matrix for the preparation of calibration curves and quality control samples. Using an XBridge C18 column (3.5 µm, 2.1 × 100-mm ID) with gradient elution composed of 10 mmol/L ammonium carbonate/ammonium hydroxide (1000:5, v/v) and acetonitrile/ammonium hydroxide (1000:5, v/v) provided the sharp peaks of FPP and 13C5-FPP in human plasma. The calibration curve ranged from 0.2 to 20 ng/mL in human plasma with acceptable intra-day and inter-day precision and accuracy. The sensitivity of this bioanalytical method was sufficient for clinical analysis. The endogenous FPP plasma concentrations in 40 human healthy volunteers ascertained by LC-QQQ-MS and high-performance liquid chromatography tandem hybrid quadrupole Orbitrap high-resolution mass spectrometry (LC-Q-Orbi-MS) were comparable. Furthermore, the endogenous GGPP in human plasma was selectively detected for the first time by LC-Q-Orbi-MS. In conclusion, a sensitive bioanalytical method for FPP in human plasma by means of LC-QQQ-MS and LC-Q-Orbi-MS was developed in this study. Taking into account the versatility of LC-Q-Orbi-MS, the simultaneous detection of FPP and GGPP may be feasible in clinical practice.

High resolution mass spectrometry based method applicable for a wide range of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors in blood serum including intermediates and products of the cholesterol biosynthetic pathway.

Statins belong to the major class of hypolipidemic drugs. They act as competitive inhibitors of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in the cholesterol biosynthetic pathway. This inhibition not only leads to the depletion of cholesterol and its fatty acid esters, but also to the depletion of the intermediates of this metabolic pathway (mainly pyrophosphates), which can play an important role in tumor proliferation. The aim of the current study was to establish a versatile multi-analyte method capable of quantitative determination of various currently-used statins, together with free cholesterol (FC), cholesterol esters (CEs), and some key intermediates of the mevalonate pathway occurring in human serum. Various methods of sample preparation were examined in order to minimize the content of potentially interfering serum proteins, and simultaneously to assure acceptable recovery of the target analytes. Following protein precipitation with 2-propanol, separation of the sample components using ultra-high performance liquid chromatography coupled with tandem high resolution mass spectrometry (U-HPLC-HRMS/MS) was performed, employing a hyphenated quadrupole Orbitrap mass analyzer. The potential of the developed method was validated on human serum samples from patients treated with statins. This versatile method possesses wide applicability, in both clinical and experimental medicine.