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Diethylnitrosamine (DEN) was applied to create the primary liver cancer (PLC) animal model. In the study, the normal group, model group, cyclophosphamide (CTX) group, Cortex Juglandis Mandshuricae (CJM) extract group, myricetin group and myricitrin group were divided. LC-MS/MS technology was applied to determine the metabolites of liver tissue samples from different locations (nodular and non-nodular parts of liver tissue) in each group of rats. Through metabolomics research, the connection and difference of anti-PLC induced by the CJM extract, myricetin and myricitrin was analyzed. The surface of the liver tissues of rats in the model group was rough, dimly colored, inelastic, on which there were scattered gray white cancer nodules and blood stasis points. The number of cancer nodules was significantly reduced, and the degree of cell malignancy was low, but there were some inflammatory cell infiltrations, necrosis area and karyokinesis in the CJM extract group, myricetin group, myricitrin group and CTX group. The result of metabolic research indicated that 45 potential biomarkers of the PLC were found, as gamma-aminoisobutyrate, taurochenodeoxycholate, xanthurenic acid, etc. There were 22 differential metabolites in the CTX group, 16 differential metabolites in the CJM extract group, 14 differential metabolites in the myricetin group, 14 differential metabolites in the myricitrin group. [Display omitted] The LS-MS/MS method was established to study the metabolomics of the PLC. The CJM extract, myricetin, myricitrin have certain effects on the liver cancer. The urea cycle, ammonia recycling, etc. play an important role in treating PLC. The 45 potential biomarkers of the PLC were found, as gamma-aminoisobutyrate, etc. There were differential metabolites in different groups. [ABSTRACT FROM AUTHOR]

Enasidenib, an oral product for treating Acute Myeloid Leukemia, has been approved by FDA in Aug, 2017. In this study, we set up an ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for measuring Enasidenib and imatinib (internal standard, IS), simultaneously. Enasidenib and imatinib were separated on an ACQUITY UPLC BEH C 18 Column (2.1 mm × 50 mm, 1.7 μm, 132 Å). Mass detection was carried out by electrospray ionization in the position mode, and the multiple reaction monitoring transitions were m/z  474.23 → 456.17 and m/z  494.30 → 394.20 for Enasidenib and imatinib, respectively. Linearity (2 − 500 ng·mL −1 , R 2  > 0.999), precision and accuracy (RE < ± 15%), extraction recovery (≥ 96.69%), matrix effect (≥ 96.47%) and stability (RE < ± 10%) were validated which demonstrated the robustness of our method. This rapid, efficient and reliable UPLC-MS/MS method shows specificity and repeatability of Enasidenib in rat plasma and can be used in further pharmacokinetic studies. [ABSTRACT FROM AUTHOR]

Arecoline is the primary active and toxic constituent of areca nut. Arecaidine and arecoline N -oxide are two major active metabolites of arecoline. In this work, an accurate and simple high performance liquid chromatography tandem mass spectrometry method for simultaneous quantification of arecoline, arecaidine and arecoline N -oxide in rat plasma was developed and fully validated to study their pharmacokinetic behaviors in rats. After extracted from rat plasma by protein precipitation with methanol and then concentrated, the analytes were chromatographic separated on a Sepax Sapphire C 18 analytical column. The mobile phase consisted of methanol and 2 mM ammonium acetate buffer solution containing 0.2% ( v/v ) formic acid (8:92, v/v ) under isocratic elution. The analytes were detected by multiple reaction monitoring (MRM) with an electrospray ionization source in the positive ion mode. The transitions of m / z 156.2 → 53.2,  m / z 142.2 → 44.2 and m / z 172.2 → 60.2 were selected for arecoline, arecaidine and arecoline N -oxide, respectively. The method was linear over the concentration range of 0.5–100 ng/mL for arecoline, 5–5000 ng/mL for arecaidine and arecoline N -oxide with no carry-over effect. The accuracies and intra- and inter-batch precisions were all within the acceptance limits. No matrix effect and potential interconversion between the analytes and other metabolites were observed in this method. The validated method was further employed to a preclinical pharmacokinetic study of arecoline, arecaidine and arecoline N -oxide after oral treatment with 20 mg/kg arecoline to rats. [ABSTRACT FROM AUTHOR]

LNA-i-miR-221, a 13-mer oligonucleotide, is a new miR-221 inhibitor that could be used as a novel drug for multiple myeloma. Herein, an ion-pair reversed phase liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the quantification of LNA-i-miR-221 in rat plasma. Plasma samples were prepared with an initial phenol/chloroform/isoamyl alcohol liquid–liquid extraction followed by a solid phase extraction. Chromatographic separation was performed with a gradient system on a HALO C18 column using hexafluoro-2-propanol/triethylamine buffer and methanol as mobile phase at a flow rate of 0.4 mL/min. Under these conditions LNA-i-miR-221 and the analogue internal standard are co-eluted at 1.2 min. The detection was carried out in multiple reaction monitoring (MRM) mode using a negative electrospray ionization (ESI) interface. The assay showed a good linearity within the calibration range 10–10000 ng/mL. The precision, accuracy, and recovery values were found to be <15% (<20% at LLOQ), 100 ± 15%, and 97.6–103.7%, respectively. This method was successfully applied to measure the concentrations of LNA-i-miR-221 in plasma samples following the intravenous administration during a 4-week toxicity study in rats. [ABSTRACT FROM AUTHOR]

Mesupron, the first-in-class inhibitor of urokinase-type plasminogen activator (uPA) is known to regulate cell proliferation and migration, and is under investigation for the treatment of metastatic breast cancer. In this study, a quantification method was developed for the determination of mesupron in rat plasma using liquid chromatography with a tandem mass spectrometry (LC–MS/MS). After protein precipitation with acetonitrile including itraconazole (internal standard, IS), the analytes were chromatographed on a reversed phased column with a mobile phase of acetonitrile and water (7:3, v/v, including 0.1% formic acid). The ion transitions of the precursor to the product ion were principally protonated ion [M+H] + at m / z 630.4 → 398.3 for mesupron and 705.2 → 392.1 for the IS. The accuracy and precision of the assay were in accordance with FDA regulations for the validation of bioanalytical methods This method was successfully applied to a pharmacokinetic study of mesupron after intravenous administration in rats. [ABSTRACT FROM AUTHOR]

The investigation of the possible conversion of cannabidiol (CBD) into Δ 9 -tetrahydrocannabinol (THC) in vivo after oral administration of CBD is reported herein since recent publications suggested a rapid conversion in simulated gastric fluid. To this end, single high dose of CBD (50 mg/kg) was administered orally to rats and their blood was collected after 3 and 6 h. A highly sensitive and selective LC–MS/MS method was developed and fully validated in compliance with the Scientific Working Group of Forensic Toxicology (SWGTOX) standard practices for method validation in forensic toxicology. This method also involved the optimization of cannabinoids and their metabolites extraction in order to remove co-eluting phospholipids and increase the sensitivity of the MS detection. Neither THC nor its metabolites were detected in rat whole blood after 3 or 6 h from CBD administration. After oral administration, the amount of CBD dissolved in olive oil was higher than that absorbed from an ethanolic solution. This could be explained by the protection of lipid excipients towards CBD from acidic gastric juice. [ABSTRACT FROM AUTHOR]

A simple and rapid liquid chromatography–tandem mass spectrometry method was firstly developed for simultaneous quantification of neomangiferin, mangiferin, regaloside A, regaloside I, timosaponin BII, anemarsaponin E and timosaponin AIII in rat plasma after oral administration of Baihe Zhimu decoction, which plays an important role for the treatment of depression. The plasma samples were pretreated by a one-step direct protein precipitation with methanol. Separation of the seven components and scutellarin (IS) from endogenous components with high selectivity and sensitivity (LLOQ, 0.1–1.0 ng/mL) was achieved within 10 min using Poroshell 120 EC-C 18 column (150 mm × 3.0 mm, 2.7 μm). A gradient mobile phase consisting of acetonitrile and water (containing 5 mM ammonium acetate) was applied at a flow rate of 0.4 mL/min. Detection and measurement were performed on an AB Sciex QTRAP ® 5500 mass spectrometer in multiple reactions monitoring mode. The intra- and inter-day precisions were all within 15% and the accuracies were in the range of −10.4% to 14.5%. The recovery ranged from 90.8 to 113.8%. The validated method was successfully applied to pharmacokinetic study of the seven components in normal and chronic unpredicted mild stress-induced depression model rats. [ABSTRACT FROM AUTHOR]

An accurate and reliable UPLC–MS/MS method is reported for the quantification of endogenous Prostaglandin E2 (PGE 2 ) in rat colonic mucosa and polyps. This method adopted the “surrogate analyte plus authentic bio-matrix” approach, using two different stable isotopic labeled analogs — PGE 2 -d9 as the surrogate analyte and PGE 2 -d4 as the internal standard. A quantitative standard curve was constructed with the surrogate analyte in colonic mucosa homogenate, and the method was successfully validated with the authentic bio-matrix. Concentrations of endogenous PGE 2 in both normal and inflammatory tissue homogenates were back-calculated based on the regression equation. Because of no endogenous interference on the surrogate analyte determination, the specificity was particularly good. By using authentic bio-matrix for validation, the matrix effect and exaction recovery are identically same for the quantitative standard curve and actual samples – this notably increased the assay accuracy. The method is easy, fast, robust and reliable for colon PGE 2 determination. This “surrogate analyte” approach was applied to measure the Pirc (an Apc -mutant rat kindred that models human FAP) mucosa and polyps PGE 2 , one of the strong biomarkers of colorectal cancer. A similar concept could be applied to endogenous biomarkers in other tissues. [ABSTRACT FROM AUTHOR]

A sensitive, specific and rapid LC-ESI–MS/MS method has been developed and validated for the quantification of tideglusib in mice plasma using warfarin as an internal standard (I.S.) as per regulatory guidelines. Sample preparation was accomplished through liquid-liquid extraction process. Chromatographic separation was performed on Atlantis dC 18 column using mobile phase A (acetonitrile) and B (5 mM ammonium acetate in water) in a flow-gradient mode. Elution of tideglusib and the I.S. occurred at ∼2.06 and 1.29 min, respectively. The total chromatographic run time was 3.2 min. A linear response function was established in the concentration range of 20.2–1008 ng/mL. The intra- and inter-day accuracy and precision were in the range of 4.61–12.6 and 6.04–11.8%, respectively. This novel method has been applied to a pharmacokinetic study in mice. [ABSTRACT FROM AUTHOR]

Transcriptional coactivator with PDZ-binding motif (TAZ) is considered an attractive target for osteoporosis, obesity, and muscle regeneration. TM-53, a promising TAZ modulator, was recently introduced, and here, we developed a rapid, precise, and reliable analytical method for TM-53 and characterized its pharmacokinetic properties in rat plasma. The hybrid triple quadrupole/linear ion trap coupled to liquid chromatography method was developed and validated to quantify TM-53. Additionally, TM-53 concentrations in plasma were analyzed, and its pharmacokinetic parameters were calculated by non-compartmental analysis. Multiple reaction monitoring at m / z 569.4 → 207.1 showed the most sensitive signals for TM-53, and the linear scope of the standard curve was between 1.5 ng/mL and 500 ng/mL. The intra- and inter-day precisions of the quality control samples were <15%, and their accuracies were ranged from 86.2% to 111.0%. Furthermore, the matrix effects, extraction recoveries, and process efficiencies of this analytical method for evaluating TM-53 in rat plasma were 99.1%, 99.9%, and 99.1% respectively. In short- and long-term stability studies, TM-53 showed good stability under frozen conditions, but TM-53 hydrolysis in the plasma matrix was observed following storage at room temperature. This analytical method was successfully applied for pharmacokinetic analysis of TM-53 in rat plasma and demonstrated excellent sensitivity, selectivity, precision, and accuracy. These data indicated that this method can be applied for further preclinical studies of TM-53. [ABSTRACT FROM AUTHOR]

A robust high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS) assay was developed and qualified for the measurement of cyclic nucleotides (cNTs) in rat brain tissue. Stable isotopically labeled 3′,5′-cyclic adenosine- 13 C 5 monophosphate ( 13 C 5 -cAMP) and 3′,5′-cyclic guanosine- 13 C, 15 N 2 monophosphate ( 13 C 15 N 2 -cGMP) were used as surrogate analytes to measure endogenous 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP). Pre-weighed frozen rat brain samples were rapidly homogenized in 0.4 M perchloric acid at a ratio of 1:4 (w/v). Following internal standard addition and dilution, the resulting extracts were analyzed using negative ion mode electrospray ionization LC–MS/MS. The calibration curves for both analytes ranged from 5 to 2000 ng/g and showed excellent linearity (r 2 > 0.996). Relative surrogate analyte-to-analyte LC–MS/MS responses were determined to correct concentrations derived from the surrogate curves. The intra-run precision (CV%) for 13 C 5 -cAMP and 13 C 15 N 2 -cGMP was below 6.6% and 7.4%, respectively, while the inter-run precision (CV%) was 8.5% and 5.8%, respectively. The intra-run accuracy (Dev%) for 13 C 5 -cAMP and 13 C 15 N 2 -cGMP was <11.9% and 10.3%, respectively, and the inter-run Dev% was <6.8% and 5.5%, respectively. Qualification experiments demonstrated high analyte recoveries, minimal matrix effects and low autosampler carryover. Acceptable frozen storage, freeze/thaw, benchtop, processed sample and autosampler stability were shown in brain sample homogenates as well as post-processed samples. The method was found to be suitable for the analysis of rat brain tissue cAMP and cGMP levels in preclinical biomarker development studies. [ABSTRACT FROM AUTHOR]

QX-OH, a new synthetic local anesthetic, produced concentration-dependent, reversible, and long-acting local anesthesia in animal models, with moderate local toxicity. As part of preclinical research for drug development, we developed and validated a method for the determination of QX-OH in the plasma, muscle, and sciatic nerve using liquid chromatography–mass spectrometry. After a simple protein precipitation procedure, analysis was performed on an Extend C18 column (100 mm × 3 mm, 3.5 μm) by isocratic elution with 0.05% formic acid/acetonitrile (78:22, v/v) at a flow rate of 0.3 mL/min. A multiple-reaction monitoring mode at the transitions of m / z 279.1 → 102.1 for QX-OH and m / z 275.2 → 126.1 for an internal standard (ropivacaine hydrochloride) was used for the quantification, with a positive electrospray ionization interface. The approach was validated as per the U.S. Food and Drug Administration guidelines and successfully used in a pharmacokinetic study of QX-OH after a sciatic nerve block with 0.2 mL of 35 mM QX-OH. The results demonstrated that the new local anesthetic, QX-OH, had a high concentration in tissue, low systemic exposure, and long duration in the sciatic nerve. [ABSTRACT FROM AUTHOR]

The aim of the present investigation was to better understand the pharmacokinetic profile of bilberry (Vaccinium Myrtillus) anthocyanins and the role of glucose transporters (sGLT1 and GLUT2) on their absorption. In particular, the absorption of 15 different anthocyanins contained in a standardized bilberry extract (Mirtoselect ® ) was measured in rats by a validated LC-ESI–MS/MS approach. The plasma concentration peak (C max ) of 11.1 ng/mL was reached after 30 min and fasting condition significantly increased the bioavailability of anthocyanins by more than 7 fold in respect to fed rats. Glucose co-administration did not interfere with the overall anthocyanin uptake. Bioavailability of each anthocyanin was then estimated by comparing the relative content in plasma vs extract. The 15 anthocyanins behaved differently in term of bioavailability and both the aglycone and the sugar moiety were found to affect the absorption. For instance, arabinoside moiety was detrimental while cyanidin enhanced bioavailability. Computational studies permitted to rationalize such results, highlighting the role of glucose transporters (sGLT1 and GLUT2) in anthocyanins absorption. In particular a significant correlation was found for the 15 anthocyanins between sGLT1 and GLUT2 recognition and absorption. [ABSTRACT FROM AUTHOR]

In this study, various neurosteroids in brain and plasma were simultaneously determined using liquid chromatography-tandem mass spectrometry and their profile changes in a stress-induced rats were investigated. The investigated neurosteroids are as follows: progesterone (P4), 5α-dihydroprogesterone (5α-DHP), 5β-dihydroprogesterone, estrone, androstenedione (AE), cortisol, cortisone, corticosterone (CORT), dehydroepiandrosterone (DHEA), pregnanolone (3α,5β-THP), allopregnanolone (ALLO), 11-deoxycorticosterone (DOC), 11-deoxycortisol, pregnenolone (PREG), and 5α/5β-tetrahydrodeoxycorticosterone (5α/5β-THDOC). Brain and plasma samples were processed using solid-phase extraction with methanol and acetic acid (99:1), and derivatized with a hydroxylamine reagent. Separation was achieved within 13 min at a flow rate of 0.4 mL/min with a C18 column (3.0 × 50 mm, 2.7 μm). The triple quadrupole mass spectrometer was operated in the positive electrospray ionization mode. Using this method, the neurosteroid level variation was quantitated and investigated in the brain and plasma upon immobilization stress in rats. As a result, AE, CORT, DOC, P4, 5α-DHP, 5α/5β-THDOC, DHEA, 3α,5β-THP, ALLO, and PREG levels were significantly altered in both the brain and plasma samples when stress was induced. These findings demonstrated that stress leads to the alteration of the GABAergic neurosteroid profile. The present results will be helpful for furthering an understanding of the role of neurosteroids in stressed conditions. [ABSTRACT FROM AUTHOR]

A sensitive and robust LC–MS/MS method has been developed and validated to determine the concentrations of tacrolimus and its major metabolite 13-O-desmethyl tacrolimus (13-ODMT) in kidney tissue from rats who received tacrolimus intra-peritoneally at doses of 0.5 mg/kg and 2 mg/kg. The samples were prepared by a liquid-liquid extraction procedure using ethyl ether as the extraction solvent and ascomycin as the internal standard. Chromatographic separation was achieved using Phenomenex Kinetex column (2.6 μm C18 100 Å, 100 × 2.1 mm, Phenomenex, Torrance CA) and a gradient mobile phase of water and methanol-acetonitrile (50:50, v/v) both containing 0.1% formic acid. The limit of quantification was 0.25 ng/ml and the calibration curves covered a concentration range from 0.25 to 50 ng/ml. Intra-and inter-assay precision and accuracy for both tacrolimus and 13-ODMT were all within FDA guidelines for bioanalysis. Extraction efficiency for tacrolimus ranged from 67.00 to 74.90% and from 66.70 to 78.40% for 13-ODMT. Several challenges interfering with the performance of the method such as phospholipid build-up have also been addressed. Kidney tissue samples from six rats receiving either 0.5 or 2 mg/kg dose were analyzed and resulted in a median concentration of 11.54 and 0.72 ng/ml for tacrolimus and 13-ODMT, respectively, for the lower dose level, and a median concentration of 8.89 ng/ml and 1.50 ng/ml for tacrolimus and 13-ODMT, respectively, at the higher dose level. [ABSTRACT FROM AUTHOR]

Clematidis Radix et Rhizoma is a traditional Chinese medicine widely used for treating arthritic disease. Clematis triterpenoid saponins (TS) and clematichinenoside AR (C-AR) have been considered to be responsible for its antiarthritic effects. However, the underling mechanism is still unclear because of their low bioavailability. To address of this issue, metabolomics tools were performed to determine metabolic variations associated with rheumatoid arthritis (RA) and responses to Clematis TS, C-AR and positive drug (Triptolide, TP) treatments. This metabolomics investigation of RA was conducted in collagen-induced arthritis (CIA) rats. Liquid chromatography/mass spectrometry and multivariate statistical tools were used to identify the alteration of serum and urine metabolites associated with RA and responses to drug treatment. As a result, 45 potential metabolites associated with RA were identified. After treatment, a total of 24 biomarkers were regulated to normal like levels. Among these, PC(18:0/20:4), 9,11-octadecadienoic acid, arachidonic acid, 1-methyladenosine, valine, hippuric acid and pantothenic acid etc, were reversed in Clematis TS and C-AR groups. Tetrahydrocortisol was regulated to normal levels in Clematis TS and TP groups, while 3,7,12-trihydroxycholan-24-oic acid was regulated in C-AR and TP groups. Biomarkers like citric acid, p -cresol glucuronide, creatinine, cortolone were reversed in TP group. [ABSTRACT FROM AUTHOR]

A sensitive and rapid ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC–ESI–MS/MS) method was developed for the simultaneous analysis of anemoside B4, phellodendrine, berberine, palmatine, obakunone, esculin, esculetin, toosendanin (IS 1 of anemoside B4), tetrahydropalmatine (IS 2 of phellodendrine, berberine, palmatine and obakunone) and scopoletin (IS 3 of esculin and esculetin) and to compare the pharmacokinetics of these active ingredients in normal and ulcerative colitis rats. After methanol deproteinization, solvents were evaporated at 40 °C under a gentle stream of nitrogen. Chromatography was performed using a C18 column with a gradient elution of 0.1% aqueous formic acid and acetonitrile at 0.4 ml/min. Detection and measurement were performed on a 4000 QTRAP UPLC–MS/MS system from AB Sciex in the multiple reaction monitoring (MRM) mode. Phellodendrine, berberine, palmatine, obakunone, esculin, esculetin, tetrahydropalmatine (IS 2 ) and scopoletin (IS 3 ) were monitored under positive ionization conditions. Anemoside B4, and toosendanin (IS 1 ) were monitored under negative ionization conditions. The optimized mass transition ion-pairs ( m / z ) were 1221.1/750.7 for anemoside B4, 343.2/193.2 for phellodendrine, 337.1/321.0 for berberine, 353.0/336.9 for palmatine, 455.1/161.1 for obakunone, 341.2/179.2 for esculin, 179.1/123.0 for esculetin, 573.4/531.4 for toosendanin (IS 1 ), 356.2/192.2 for tetrahydropalmatine (IS 2 ) and 193.0/133.1 for scopoletin (IS 3 ). [ABSTRACT FROM AUTHOR]

A simple, sensitive and selective ultra-fast liquid chromatography-tandem mass spectrometry (UFLC–MS/MS) method was established for simultaneous determination and pharmacokinetic study of rosmarinic acid (RA), salvianolic acid D (Sal D), lithospermic acid (LA) and salvianolic acid B (Sal B) in rat plasma after intravenous administration of salvianolic acid for injection (SAFI). Three doses of administration, containing 14, 28 and 56 mg/kg, were investigated in this study. Plasma samples were pretreated using protein precipitation (PP) with pre-cooled acetonitrile. Chromatographic separation was achieved on a CORTECS™ UPLC C18 column (1.6 μm, 2.1 × 100 mm) with a mobile phase composed of 0.1% formic acid aqueous (V/V) and 0.1% formic acid acetonitrile (V/V). Analytes were detected using electrospray ionization (ESI) source in negative ionization mode and quantified in multiple reaction monitoring (MRM) mode. The validated method is stable and reliable. No significant difference of half lives ( t 1/2 ) of four analytes at three doses was observed. Area under the curve (AUC 0-∞ ) and peak concentration (C max ) of the four analytes demonstrated a linear increase in across the doses with the linear correlation r of each analyte at three doses were greater than 0.95. It indicated that the pharmacokinetic behavior of SAFI is positively related to dose at the range of 14–56 mg/kg. [ABSTRACT FROM AUTHOR]

A sensitive, selective and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of propofol (PRO) in rat serum and hair has been developed. 3-Bromomethyl-propyphenazone was used as derivatization reagent forming propofol-methyl-propyphenazone compound. The derivatization reaction was optimized and validated for maximum MS sensitivity. The MS instrumental sensitivity reached to 10 attogram. The serum samples were extracted by using Chromabond C 8 columns, while hair samples extracted with methanol. The tendency of volatility of PRO was minimized by adding triethylamine to the extract before the use of nitrogen gas for evaporation of solvent. The limit of quantitation (LLOQ) was 0.01 pg/mL and the assay was linear to 10000 pg/mL. The intra-and inter-day precision (RSD%) ranged from 0.33 to 3.44% while the accuracy (E r %, relative error) were −6.4 to 1.1%. The ionization suppression, due to reagent, was minimized by reacting the excess reagent with methanol, and eluting to waste before MS ionization source (2–4.5 min). The method was successfully applied for detection and determination of PRO in rat serum and hair after 7–28 days from administration of only one dose of propofol (10 mg/kg). [ABSTRACT FROM AUTHOR]

To investigate the pharmacokinetic (PK) interaction between telmisartan (Tel) and pitavastatin (Pit), a rapid and sensitive ultra performance liquid chromatography-tandem mass spectrometric assay method had been successfully established and fully validated for the simultaneous quantification of Tel and Pit in rat plasma. A simple protein precipitation procedure was adopted for the sample preparation with satisfactory extraction recovery for both analytes and the internal standard. The samples were chromatographed on an Inertsil ODS-3 C18 column (100 mm × 2.1 mm, 2 μm) using a mixture of acetonitrile and 10 mM ammonium acetate containing 0.1% formic acid (60: 40, v/v) as the mobile phase at a flow rate of 0.4 mL/min. The calibration curves obtained were linear (r > 0.99) over the concentration range of 2–200 ng/mL for Tel and 1–100 ng/mL for Pit, respectively. The validated method was successfully applied to the PK study and the data did not reveal any evidence for the potential drug–drug interaction (DDI) between Tel and Pit. This information would provide the evidence for clinical rational use of Tel and Pit, and this study might be applied in therapeutic drug monitoring in patients receiving Tel/Pit combinations or single drug. [ABSTRACT FROM AUTHOR]

Capilliposide B, a novel oleanane triterpenoid saponin isolated from Lysimachia capillipes Hemsl, showed significant anti-tumor activities in recent studies. To characterize the excretion of Capilliposide B, a reliable liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous determination of Capilliposide B and its active metabolite, Capilliposide A in rat urine and feces. Sample preparation using a solid-phase extraction procedure was optimized by acidification of samples at various degrees, providing extensive sample clean-up with a high extraction recovery. In addition, rat urinary samples were pretreated with CHAPS, an anti-adsorptive agent, for overcoming nonspecific analytes adsorption during sample storage and process. The method validation was conducted over the curve range of 10.0–5000 ng/ml for both analytes. The intra- and inter-day precision and accuracy of the QC samples showed ≤11.0% RSD and −10.4–12.8% relative error. The method was successfully applied to an excretion study of Capilliposide B following intravenous administration. [ABSTRACT FROM AUTHOR]

An amide derivative of gallic acid (GA), 3,4,5-trihydroxy-N-(8-hydroxyquinolin-2-yl)benzamide) (SG-HQ2) was recently synthesized, and its inhibitory actions were previously shown on histamine release and pro-inflammatory cytokine expression. In this study, a simultaneous quantification method was developed for the determination of SG-HQ2 and its possible metabolite, GA, in rat plasma using liquid chromatography with a tandem mass spectrometry (LC–MS/MS). After simple protein precipitation with acetonitrile including diclofenac (internal standard, IS), the analytes were chromatographed on a reversed phased column with a mobile phase of acetonitrile and water (60:40, v/v, including 0.1% formic acid). The ion transitions of the precursor to the product ion were principally protonated ion [M+H] + at m / z 313.2 → 160.6 for SG-HQ2, and deprotonated ions [M−H] − at m / z 168.7 → 124.9 for GA and 296.0 → 251.6 for the IS. The accuracy and precision of the assay were in accordance with FDA regulations for the validation of bioanalytical methods. This method was successfully applied to a pharmacokinetic study of SG-HQ2 after intravenous administration in rats. [ABSTRACT FROM AUTHOR]

An UPLC-MS/MS method for the quantification of indirubin in lithium heparinized rat plasma was developed and validated according to current international guidelines. Indirubin was extracted from rat plasma by using Waters Ostro™ pass-through sample preparation plates. The method was validated with a LLOQ of 5.00 ng/mL and an ULOQ of 500 ng/mL. The calibration curve was fitted by least-square quadratic regression, and a weighting factor of 1/X was applied. Recoveries of indirubin and I.S. were consistent and ≥75.5%. Stability studies demonstrated that indirubin was stable in lithium heparinized rat plasma for at least 3 freeze/thaw cycles, for 3 h at RT, for 96 h in the autosampler at 10 °C, and for 84 days when stored below −65 °C. Preliminary pharmacokinetic (PK) data were obtained from Sprague Dawley rats after intravenous administration of indirubin (2 mg/kg b.w.) and blood sampling up to 12 h after injection. PK parameters were determined by non-compartmental analysis. Indirubin had a half-life (t 1/2 ) of 35 min, and a relatively high clearance (CL) of 2.71 L/h/kg. [ABSTRACT FROM AUTHOR]

Ziyuglycoside I is one of the major active ingredients in Sanguisorba officinalis , a popular medicinal plant in China. In the present study, the metabolites of ziyuglycoside I in rat liver microsome and intestinal flora were identified and structurally characterized, and the metabolic rules were summed based on the LC–Q-TOF/MS system. Then, the metabolites in rat excreta samples were rapidly screened and identified according to the in vitro metabolic rules. Finally, ziyuglycoside I was incubated with fresh liver/lung/kidney/stomach homogenates to further explore the source of the metabolites and reveal the possible metabolic organs involved. Four metabolites in liver microsome were identified as M0-Glu, M0-CH 2 OH, M0-Glu + CH 3 , M0-Glu-Ara + CH 3 . In intestinal flora incubation system, 6 degradation products including M0-Glu-Ara + O, M0-Ara, M0-Glu-COOH, M0-Glu, M0-Glu-Ara + O and M0-Ara + H 2 O were tentatively identified by interpretation of their accurate MS 1 and MS 2 data. Fifteen metabolites in rat urine and feces were identified, and most of the metabolites were attributed to the transformation in liver microsome and intestinal flora. Specifically, more than a dozen of new metabolites were identified in rat fresh tissues, and ziyuglycoside II was confirmed as the major metabolite in rats. [ABSTRACT FROM AUTHOR]

A rapid, sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method has been developed for the simultaneous determination and pharmacokinetic investigation of Tetramethylpyrazine (TMP) and Ferulic acid (FA) in rat striatum. The method was validated over the concentration range of 1.15–505 ng/mL for TMP and 3.23–101 ng/mL for FA, with a lower limit of quantitation (LLOQ) of 1.15 ng/mL and 3.23 ng/mL, respectively. This method can be successfully applied in pharmacokinetic studies of TMP and FA in striatum of awake and anesthetic rats. The cerebral blood flow velocity (CBF) during middle cerebral artery occlusion (MCAO) was monitored by Laser speckle contrast imaging, to observe whether the compatibility of TMP and FA could improve CBF against cerebral ischemia/reperfusion (I/R) injury. Infarct volume was examined to evaluate severity of ischemic brain injury. The pharmacokinetic study indicated that T 1/2, C max, MRT and AUC 0-inf were changed after combined administration of TMP and FA, when compared with either drug alone both in awake and anesthetic groups. The pharmacodynamics results showed that co-administration of drugs could enhance the CBF during middle cerebral artery occlusion and reduced the infarct volume. Taken together, the compatibility treatment of TMP and FA might be a promising therapeutic strategy for ischemic stroke. Further study is required to optimize the compatibility proportion. [ABSTRACT FROM AUTHOR]

4-Methoxydiphenylmethane (4-MDM), a selective augmenter of Leukotriene A 4 Hydrolase (LTA 4 H), is a new anti-inflammatory compound for potential treatment of chronic obstructive pulmonary disease (COPD). Currently, there is no liquid chromatography tandem mass spectrometric (LC–MS/MS) method for the quantification of 4-MDM. A major barrier for developing the LC–MS/MS method is the inability of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) to ionize 4-MDM due to its hydrophobicity and lack of any functional group for ionization. With the advent of atmospheric pressure photoionization (APPI) technique, many hydrophobic compounds have been demonstrated to ionize by charge transfer reactions. In this study, a highly sensitive ultrapressure liquid chromatography tandem mass spectrometry assay using atmospheric pressure photoionization (UPLC–APPI-MS/MS) for the quantifications of 4-MDM in rat plasma has been developed and validated. 4-MDM was extracted from the plasma by solid phase extraction (SPE) and separated chromatographically using a reverse phase C 8 column. The photoionization (PI) was achieved by introducing anisole as a dopant to promote the reaction of charge transfer. The assay with a linear range of 5 (LLOQ)–400 ng mL −1 met the regulatory requirements for accuracy, precision and stability. The validated assay was employed to quantify the plasma concentrations of 4-MDM after an oral dosing in Sprague Dawley (SD) rats. [ABSTRACT FROM AUTHOR]

Rhodamine B is an illegal and potentially carcinogenic food dye. The aim of this study was to develop a convenient, rapid, and sensitive UHPLC-MS/MS method for pharmacokinetic studies in rats. Rat plasma samples were deproteinized with acetonitrile and separated by UHPLC on a reverse-phase C18e column (100 mm × 2.1 mm, 2 μm) using a mobile phase consisting of methanol—5 mM ammonium acetate (90:10, v/v). Detection was performed using a triple quadrupole tandem mass spectrometer in the selected reaction monitoring mode at [M] + ion m / z 443.39 → 399.28 for rhodamine B and [M+H] + ion m / z 253.17 → 238.02 for 5-methoxyflavone as the internal standard. This method was specific and produced linear results over a concentration range of 0.5–100 ng/mL, with a lower limit of quantitation of 0.5 ng/mL. All validation parameters, including the inter-day, intra-day, matrix effect, recovery, and stability in rat plasma, were acceptable according to the biological method validation guidelines developed by the FDA (2001). This method was successfully applied to a pharmacokinetic study in rats; oral administration of 1 mg/kg of rhodamine B yielded a time to maximum concentration ( T max ) of 1.3 ± 0.4 h and an elimination half-life of 8.8 ± 1.4 h, with a clearance of 229.7 ± 19.4 mL/h/kg. These pharmacokinetic results provide a constructive contribution to our understanding of the absorption mechanism of rhodamine B and support additional food safety evaluations. [ABSTRACT FROM AUTHOR]

A simple, sensitive and accurate ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of AKI603 in rat plasma has been firstly developed and validated. After a simple liquid–liquid extraction (LLE) with ethyl acetate, the analytes were separated on C18 column (2.1 × 100 mm, 1.9 μm, Thermo) by gradient elution with mobile phase of water (A) (containing 5 mM ammonium acetate and 0.1% formic acid) and methanol (B) with a flow rate of 0.3 mL min −1 and then analyzed by mass spectrometry in the positive multiple reactions monitoring (MRM) mode. The mass transitions monitored were m / z 410.0 → 352.9, m / z 457.1 → 367.9 for AKI603 and internal standard (Ly-7z), respectively. The developed method was validated for specificity, linearity and lower limit of quantification, intra- and inter-day precision and accuracy, extraction recovery, matrix effect and stability whose values satisfied the acceptable limits. The calibration curves for AKI603 was linear in concentration ranges of 0.025–5000 ng mL −1 . The method has been successfully used to the bioavailability study of AKI603 administered to rats intravenously (2.5 mg/kg) or orally (25 mg/kg). The oral bioavailability of AKI603 in rats was calculated as 28.7 ± 9.7%. [ABSTRACT FROM AUTHOR]

In this study, a sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed to determine mangiferin and neomangiferin in rat plasma simultaneously. Chromatographic separation was carried out on an Acquity UPLC BEH C18 column and mass spectrometric analysis was performed using a Xevo TQD triple quadruple mass spectrometer coupled with an electrospray ionization (ESI) source. The MRM transitions of m / z 423.2 → 303.1 and m / z 585.0 → 273.1 were used to quantify for mangiferin and neomangiferin, respectively. The linearity of this method was found to be within the concentration range of 5–2000 ng/mL for mangiferin, and 2–1000 ng/mL for neomangiferin in rat plasma, respectively. Only 3.0 min was needed for an analytical run. This assay was used to support a preclinical study to investigate the pharmacokinetics of mangiferin and neomangiferin in rats. [ABSTRACT FROM AUTHOR]

A sensitive UPLC–MS/MS method was developed and validated for the determination of brusatol in rat plasma. Chromatographic separation was carried out on a C 18 column using methanol and 10 mM ammonium acetate containing 0.1% (v/v) formic acid (55:45, v/v). The lower limit of quantification (LLOQ) was 1.0 ng/mL for brusatol in plasma. The intra- and inter-day precision for the analyte ranged from 3.2% to 9.2% and 1.3% to 7.8%, and the accuracy was between 97.3% and 108.5%. The method was successfully applied in a pharmacokinetic study of brusatol following intravenous injection (0.5, 1.0, and 2.0 mg/kg) of brusatol. [ABSTRACT FROM AUTHOR]

A rapid, sensitive, and accurate bioanalytical method was established for the quantitation and pharmacokinetic investigation of loratadine (LTD) in rat plasma by liquid chromatography–electrospray ionization mass spectrometry (LC-ESI-MS/MS) using loratadine-d5 as internal standard (ISTD). The analyte and ISTD were extracted by solid-phase extraction and chromatographic separation was achieved on Gemini NX- Reverse Phase C18 (50 × 4.6 mm;5 μ) using mobile phase mixture of 5 mM ammonium formate buffer in water (pH 3.5 ± 0.1 with formic acid), and acetonitrile (20:80 v/v), at a flow rate of 0.400 mL/min with injection volume of 10 μL. LTD and ISTD were detected at m / z 383.3 → 337.4 and 388.4 → 337.3 with retention time of 2.62 and 2.59 min, respectively. High sensitivity (1.0 ng/mL) was achieved using small volume of rat plasma (20 μL) and the method was validated over a linearity range of 1.05–405.41 ng/mL with high correlation coefficient (r = 0.9998). The extraction method displayed a mean process efficiency of 63.25 and 65.47% for LTD and ISTD, respectively. The validated method when successfully applied for quantification of LTD in rat plasma revealed enhanced bioavailability of orally administered LTD-loaded self-nanoemulsifying drug delivery system (SNEDDS) (C max , 466.65 ± 18.94 ng/mL and AUC 0-t 633.00 ± 12.44 ng-h/mL) over LTD-suspension (C max , 104.75 ± 2.87 ng/mL and AUC 0-t 287.00 ± 9.11 ng-h/mL). The in vivo-in silico prediction by the GastroPlus™ software showed good prediction accuracy for LTD-SNEDDS (fold error < 2). The Loo-Reigelman method (2-compartment) presented best model-fitting indicating adequate in vitro–in vivo correlations. Conclusively, the developed sensitive analytical method displayed enhanced systemic availability of LTD-SNEDDS, and the in vivo in silico approach revealed sufficiently good GI simulation. [ABSTRACT FROM AUTHOR]

A rapid and sensitive ultra fast liquid chromatography tandem mass spectrometry method (UFLC–MS/MS) was developed and validated for the simultaneous determination of six Saikosaponins (SSs) (SSa, SSb 1 , SSb 2 , SSd, SSc, SSf) of Bupleurum Dropping Pills (BDP) in rat plasma using chloramphenicol as the internal standard (IS). The SSs were separated using an ACQUITY UPLC ® BEH C 18 column (50 mm × 2.1 mm, 1.7 μm) and detection of these compounds were done by using a Qtrap 5500 mass spectrometer coupled with negative electrospray ionization (ESI) source under the multiple reaction monitoring (MRM) mode. According to regulatory guidelines, the established method was fully validated and results were showed within acceptable limits. The lower limit of quantifications (LLOQs) of all analytes were 0.2 ng/mL. The validated method was successfully applied into a pharmacokinetic study of orally administered BDP in rats. [ABSTRACT FROM AUTHOR]

A sensitive, simple and rapid analytical method based on a liquid chromatography–tandem mass spetrometry (LC–MS/MS) has been established and validated for the determination of stachyose in rat plasma. Plasma samples were prepared by protein precipitation with acetonitrile. Separation of stachyose and nystose (internal standard, IS) was achieved using acetonitrile-water (55:45, v/v) as the mobile phase at a flow rate of 1 ml/min for 6 min on an Asahipak NH 2 P-50 4E column with an Asahipak NH2P-50G 4A guard column. Detection and quantification were conducted by LC–MS/MS method in the negative ion mode using multiple reaction monitoring (MRM) transitions at m / z [M–H] − 665.4 → 383.1 for stachyose and 665.5 → 485.0 for IS, respectively. The method was linear over the concentration ranges of 100–30000 ng/ml with a lower limit of quantification (LLOQ) of 100 ng/ml. The intra- and inter- day precision were all within 8.7% and the accuracy ranged from 97.2–108.4% and 98.3–102.4%, respectively. Stability studies indicated that stachyose was stable under short-term, long-term and three freeze-thaw storage conditions. The method was successfully applied to a pharmacokinetic study involving pulmonary administration of micronized Rehmannia glutinosa oligosaccharides (RGOS) to rats. [ABSTRACT FROM AUTHOR]

Lurbinectedin (PM01183) is a new synthetic tetrahydroisoquinoline alkaloid that binds to selected sequences in the minor groove of DNA, inducing PM01183-DNA adducts that stall replication, DNA repair and transcription and gives rise to double-strand breaks and finally, caspase-dependent apoptotic cell death. PM01183 has demonstrated clinical antitumor activity in platinum resistant/refractory ovarian cancer patients. A rapid and sensitive liquid chromatography/tandem mass spectrometry assay was developed and validated to quantify PM01183 in plasma from nonclinical species. The bioanalysis consisted of a supported liquid extraction, followed by a gradient phase chromatography and, detection by positive ion electrospray tandem mass spectrometry. The calibration range for PM01183 was established using PM01183 standards from 0.1 to 100 ng/mL in blank plasma. The multiple reaction monitoring, based on the transition m / z 767.3 → 273.0, was specific for PM01183, and that based on the transition m / z 771.4 → 277.0 was specific for the internal standard (deuterated PM01183). No endogenous material interfered with the analysis of PM01183 and the internal standard from blank plasma. The limit of detection (LOD) of the assay was calculated as 0.025 ng/mL. The correlation coefficients for the calibration curves ranged from 0.9937 to 0.9987. The mean inter-day accuracies for all calibration standards ranged from 92 to 108% (≤8% bias), and the mean inter-day precision for calibration standards was always less than 12%. The mean intra and inter-day assay accuracy for all quality control replicates remained between 91 and 109%. The mean intra and inter-day assay precision was less than 10% for all QC levels. The method was validated to demonstrate the specificity, recovery, limit of quantification, accuracy and precision of measurements. The assay has been used to support preclinical pharmacokinetic and toxicokinetic studies of PM01183 in nonclinical species. The main PK parameters in dogs (3 male and 3 female, respectively) were calculated as follows: maximum concentration ( C max , 12.9 ± 0.6 and 10.2 ± 3.0 ng/mL) and the area under the plasma concentration-time curve (AUC, 24.9 ± 0.7 and 22.6 ± 6.1 ng h/mL). The results showed that plasma samples could be monitored for PM01183 for long enough to accurately estimate pharmacokinetics information. [ABSTRACT FROM AUTHOR]

Pinostilbene (3-methoxyresveratrol or trans -3,4′-dihydroxy-5-methoxystilbene) is a naturally occurring monomethylether analogue of resveratrol ( trans -3,5,4′-trihydroxystilbene) that exhibits various pharmacological activities. To further examine its medicinal potential, a sensitive LC–MS/MS method was developed and validated for the determination of pinostilbene in rat plasma. Heavy Isotope labelled resveratrol was used as an internal standard. The ESI was operated in its negative ion mode while pinostilbene and resveratrol were measured by multiple reaction monitoring (MRM) using precursor-to-product ion transitions of m / z 241 → 181 and m / z 233 → 191, respectively. This LC–MS/MS method had excellent selectivity, sensitivity (LLOQ = 1 ng/ml), accuracy (both intra- and interday analytical recovery within 100 ± 15%) and precision (both intra- and interday RSD < 15%). The matrix effect was insignificant. The pharmacokinetics of pinostilbene was subsequently profiled in Sprague–Dawley rats. Upon intravenous administration (5 or 10 mg/kg), pinostilbene displayed rapid clearance (Cl = 129 ± 42 or 107 ± 31 ml/min/kg) and extremely short mean transit time (MTT = 6.24 ± 0.41 or 8.52 ± 1.38 min). After oral dosing (50 mg/kg), the bioavailability of pinostilbene was limited but highly erratic ( F = 1.87 ± 2.67%). Pharmacokinetic comparison among pinostilbene, resveratrol and some resveratrol analogues suggested that stilbenes with meta-hydroxyl group(s) may be associated with metabolic instability and subsequently suffer from rapid clearance and low oral bioavailability. The information obtained from this study will facilitate further exploration on pinostilbene as well as other resveratrol analogues. [ABSTRACT FROM AUTHOR]

A simple and rapid quantitative UPLC–MS/MS method for simultaneous determination of picfeltarraenins IA and IB in rat plasma was developed and validated in accordance with the US FDA Bioanalytical Guidance (2001). Analytes were extracted from rat plasma by using methanol and separated on Agilent ZORBAX SB-C 18 (50 mm × 2.1 mm, 1.8 μm) column by using a mobile phase composed of methanol and water (70:30, v/v). Eluents were monitored by ESI tandem mass spectrometry detection with SRM mode using ion transitions m / z 785.4 → 639.5, m / z 815.5 → 669.5, and m / z 763.5 → 455.3 for picfeltarraenin IA, picfeltarraenin IB, and internal standard, respectively. The method was validated over the linear range of 11.5–1150 ng/mL and 13.0–1300 ng/mL. The developed analytical method was applied to support a pharmacokinetic study on simultaneous estimation of picfeltarraenins IA and IB in rats. [ABSTRACT FROM AUTHOR]

3- O -Acetyloleanolic acid (OAA) is a triterpenoid compound, and exerts an apoptosis in cancer cell lines, an inhibition of both atopic and allergic contact dermatitis in murine model, and a suppression of inflammatory bone loss in mice. OAA can be converted into oleanolic acid (OA) by hydrolysis in vivo , and OA exhibits several pharmacological effects as well. A liquid chromatographic method using tandem mass spectrometry (MS/MS) was developed for the simultaneous determination of OAA and OA in rat plasma. After liquid–liquid extraction with ethylacetate, both substances were chromatographed on a reversed phase column with a mobile phase of 0.1% formic acid aqueous solution and acetonitrile (1:9, v/v). The accuracy and precision of the assay were in accordance with FDA regulations for the validation of bioanalytical methods. This analytical method was successfully applied to monitor plasma concentrations of both substances over time following an intravenous administration of OAA in rats. [ABSTRACT FROM AUTHOR]

To establish a rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method for the determination of concentration of nintedanib and its metabolite BIBF 1202 in rat plasma. The nintedanib and its metabolite and the internal standard (diazepam) were separated on an Acquity UPLC BEH C18 chromatography column (2.1 mm × 50 mm, 1.7 μm) using gradient elution with a mobile phase of acetonitrile and 0.1% formic acid in water at a flow rate of 0.30 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m / z 540.3 → 113.1 for nintedanib, m / z 526.3 → 113.0 for BIBF 1202 and m / z 285.3 → 193.1 for diazepam (IS) using a positive electrospray ionization interface. The method was validated for 1.0–200 ng/mL for nintedanib and 0.5–100 ng/mL for BIBF 1202 using 100 μL of plasma sample. Total time for each chromatograph was 3.0 min. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels exhibited relative standard deviations (RSD) <10.8% and the accuracy values ranged from −11.9% to 10.4%. The method was successfully applied to a pharmacokinetic study of nintedanib and BIBF 1202 in rats after oral administration of nintedanib. [ABSTRACT FROM AUTHOR]

A specific, sensitive and rapid liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed and validated for simultaneous quantification of doxorubicin and curcumin in rat plasma after intravenous administration. The analytes of doxorubicin and curcumin were extracted with methanol precipitation using glibenclamide as internal standard (IS). The chromatographic separation was performed on a C 18 column with acetonitrile and 0.1% formic acid water as mobile phase and with gradient elution at a flow rate of 0.2 mL/min. Calibration curves were linear over the ranges of 2–8000 ng/mL for doxorubicin and 5–2000 ng/mL for curcumin ( r > 0.99). The lower limit of quantification (LLOQ) was 2 ng/mL for DOX and 5 ng/mL for Cur. Finally, this developed method was successfully applied in the pharmacokinetic study of doxorubicin and curcumin in rats and evaluated the effects of curcumin on the absorption of doxorubicin after intravenous administration. [ABSTRACT FROM AUTHOR]

A bioanalytical method for the quantification of rosiglitazone on rat dried blood spots (DBS) and rat urine using liquid chromatography, electrospray ionization coupled with tandem mass spectrometry (LC–ESI-MS/MS) was developed and validated. The chromatographic separation was achieved on a Nova-Pak C 18 Column (150 mm × 4.6 mm i.d., 4 μm), using 30 mM ammonium acetate (pH 4.0 adjusted with acetic acid) and acetonitrile (75:25, v/v) as a mobile phase at ambient temperature. LC–MS detection was performed with selected ion monitoring using target ions at m / z 358 and m / z 356 for rosiglitazone and pioglitazone respectively. The calibration curve showed a good linearity in the concentration range of 0.05–100 ng/mL. The effect of hematocrit, blood volume and punch location for DBS samples was studied. The mean recoveries of rosiglitazone from DBS and urine were 93.30% and 92.49% respectively. The intra and inter-day precisions of RSD were less than 4.82% in DBS as well as urine. The limit of detections and quantifications were 0.015 and 0.052 ng/mL in DBS and 0.023 and 0.075 ng/mL in urine samples respectively. The method was validated as per FDA guidelines and successfully applied to a pharmacokinetic study of rosiglitazone in rats. [ABSTRACT FROM AUTHOR]

Zhi-Zi-Da-Huang decoction (ZZDHD), consisting of Gardenia jasminoides Ellis, Rheum palmatum L., Citrus aurantium L. and Sojae Semen Praeparatum, is a widely used traditional Chinese medicine preparation for the treatment of acute or chronic hepatic diseases. In the present study, a sensitive and selective ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC–Q-TOF-MS) method was developed to separate and identify the absorbed components and metabolites in rat plasma after oral administration of ZZDHD. The plasma samples were pretreated by protein precipitation and separated on a Shim-pack XR-ODS C 18 column (75 mm × 3.0 mm, 2.2 μm) using a gradient elution program. Mass spectrometric detection was performed on an Agilent 6520 Q-TOF mass spectrometer equipped with electrospray ionization (ESI) source in positive and negative ion modes. By comparing the retention time, high resolution mass data of blank plasma and dosed plasma, a total of 43 constituents, including 21 prototype compounds and 22 metabolites were identified or tentatively characterized. Results indicated that glucuronidation and sulfation were the main metabolic pathways of iridoid glycosides and anthraquinones, glucuronidation was the main metabolic pathways of flavanone-related compounds. It is concluded the developed UHPLC–Q-TOF-MS method with high sensitivity and resolution is suitable for identifying and characterizing the absorbed components and metabolites of ZZDHD, and the results will provide essential data for further studying the relationship between the chemical components and pharmacological activity of ZZDHD. [ABSTRACT FROM AUTHOR]

Hordenine is an active compound found in several foods, herbs and beer. In this work, a sensitive and selective UPLC–MS/MS method for determination of hordenine in rat plasma was developed. After addition of caulophylline as internal standard (IS), protein precipitation by acetonitrile–methanol (9:1, v/v) was used as sample preparation. Chromatographic separation was achieved on a UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 μm) with acetonitrile (containing 10 mM ammonium formate) and water (containing 0.1% formic acid and 10 mM ammonium formate) as mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring (MRM) mode was used for quantification using target fragment ions m / z 166.1 → 121.0 for hordenine and m / z 205.1 → 58.0 for IS. Calibration plots were linear over the range of 2–2000 ng/mL for hordenine in rat plasma. Mean recoveries of hordenine in rat plasma were in the range of 80.4–87.3%. RSD of intra-day and inter-day precision were both <8%. The accuracy of the method ranged from 97.0% to 107.7%. The method was successfully applied to pharmacokinetic study of hordenine after oral and intravenous administration. [ABSTRACT FROM AUTHOR]

Notoginsenoside Fc (NGFc) is a protopanaxadiol-type (PPD-type) saponin from Panax notoginseng , which has perfect anti-platelet aggregatory effect. However, its pharmacokinetics and metabolism in vivo remain unknown. In this study, a simple and sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC–MS/MS) method was first developed for the determination of NGFc in rat plasma. After methanol-mediated protein precipitation, separation was achieved on a C 18 column with MS detection operated in negative SRM mode at m / z 604.56 → m / z 783.90 and m / z 799.93 → m / z 637.64 for NGFc and IS, respectively. The assay was linear over the concentration range ( r > 0.995) with the LLOQ of 0.002 μg/ml. The intra- and inter-day precisions (R.S.D.) were 2.45–12.36% and 3.67–14.22%, respectively; whereas accuracy ranged from (R.R.) 93.90% to 99.41%. The extraction recovery, stability, and matrix effect were within the acceptable limits. The validated LC–MS/MS method was successfully applied to the pre-clinical pharmacokinetic studies of NGFc in rat. After oral and intravenous administration, NGFc showed dose-independent pharmacokinetic behaviors with a t 1/2 of >22 h and its oral bioavailability was 0.10–0.14%. In addition, a total of 10 metabolites were detected and structurally characterized by UPLC–Q/TOF-MS technique, which suggested that deglycosylation was the major metabolic pathway for NGFc in rats. [ABSTRACT FROM AUTHOR]

Pseudolaric acid B (PAB), which is the main biologically active diterpene acid of Pseudolarix kaempferi , has presented anti-fungal, anti-tumor, anti-fertility, and anti-tubulin activities. In this study, a sensitive and selective liquid chromatography tandem mass spectrometry (LC–MS/MS) method with multiple-reaction monitoring mode was employed for quantification of PAB in rat plasma. The calibration curve was linear over the range of 0.86–288 ng/mL with correlation coefficient ( r ) greater than 0.995 for PAB, and the lower limit of quantification was 0.86 ng/mL in rat plasma. The accuracy of PAB was between −9.1% and 7.0% relative error, and precision ranged from 1.2 to 10.6% relative standard deviation. This method was successfully applied to the pharmacokinetic studies of PAB in Sprague-Dawley rats. After single intravenous administration of 2.0, 4.0, and 8.0 mg/kg PAB to rats, the t 1/2 were (16.1 ± 5.6), (30.0 ± 13.7), and (27.4 ± 5.3) min, respectively. The pharmacokinetics ( C 2 min , AUC) of PAB within the used dosage range were in accordance with linear pharmacokinetic characteristics. [ABSTRACT FROM AUTHOR]

The metabolism of gentiopicroside in vivo was studied by LC/MS following 2,4-dinitrophenylhydrazine derivatization for the first time. The ionization efficiency of the major metabolites erythrocentaurin and gentiopicral were greatly enhanced by the new analytical method developed, and they were successfully detected in rat plasma after oral administration of gentiopicroside. Methyl 4-formylbenzoate was used as the internal standard to quantify erythrocentaurin and gentiopicral in rat plasma in negative mode by UPLC–TOF-MS. It was found that erythrocentaurin reached the maximum plasma concentration of 625.2 ± 246.3 ng/mL at about 2 h and gentiopicral reached the maximum plasma concentration of 157.6 ± 86.6 ng/mL at about 4 h after oral administration of gentiopicroside at a dose of 200 mg/kg. The metabolic pathway of gentiopicroside to erythrocentaurin and gentiopicral was proposed. The monoterpene compound gentiopicroside was found to be metabolized to dihydroisocoumarin in vivo which may be responsible for the pharmacological effect of gentiopicroside. The results may shed light on clinical efficacy of gentiopicroside and the new analytical method developed may assist in studies for the metabolism of other natural iridoids and secoiridoids in vivo . [ABSTRACT FROM AUTHOR]

A simple and selective specific high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for determination of isobavachalcone (IBC) in rat plasma was developed. Neobavaisoflavone was used as an internal standard (IS). After protein precipitation with acetonitrile (2:1, v/v), the analyte and IS were separated on a 2.6 μm Kinetex C 18 column (100 mm × 2.1 mm i.d., Phenomenex) by isocratic elution with acetonitrile:water (60:40, v/v) as the mobile phase at a flow rate of 0.2 mL/min. An electrospray ionization (ESI) source was applied and operated in the negative ion mode; multiple reactions monitoring (MRM) mode was used for quantification, and the target fragment ions m / z 323.0 → 118.9 for IBC and m / z 321.1 → 265.0 for the IS were chosen. Good linearity was observed in the concentration range of 3.79–484.5 ng/mL for IBC in rat plasma. The recovery of IBC in plasma was in the range of 81.2–89.8%. Intra-day and inter-day precision were both lower than 10%. This method was suitable for pharmacokinetic studies after oral administration of 80 mg/kg IBC in rats. We also obtained pharmacokinetic parameters and concentration–time profiles for IBC after oral administration of IBC in rats. [ABSTRACT FROM AUTHOR]

Neuroactive metabolites in the kynurenine pathway of tryptophan catabolism are associated with neurodegenerative disorders. Tryptophan is transported across the blood–brain barrier and converted via the kynurenine pathway to N-formyl- l -kynurenine, which is further degraded to l -kynurenine. This metabolite can then generate a group of metabolites called kynurenines, most of which have neuroactive properties. The association of tryptophan catabolic pathway alterations with various central nervous system (CNS) pathologies has raised interest in analytical methods to accurately quantify kynurenines in body fluids. We here describe a rapid and sensitive reverse-phase HPLC–MS/MS method to quantify l -kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxy- l -kynurenine (3HK) and anthranilic acid (AA) in rat plasma. Our goal was to quantify these metabolites in a single run; given their different physico-chemical properties, major efforts were devoted to develop a chromatography suitable for all metabolites that involves plasma protein precipitation with acetonitrile followed by chromatographic separation by C18 RP chromatography, detected by electrospray mass spectrometry. Quantitation range was 0.098–100 ng/ml for 3HK, 9.8–20,000 ng/ml for KYN, 0.49–1000 ng/ml for KYNA and AA. The method was linear ( r > 0.9963) and validation parameters were within acceptance range (calibration standards and QC accuracy within ±30%). [ABSTRACT FROM AUTHOR]

CAT ((+)-(13aS)-deoxytylophorinine) is a novel anticancer drug belonging to phenanthroindolizidine alkaloids. A sensitive and reliable liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous quantification of CAT and its pharmacologically active 3-O-desmethyl metabolite (S-4) was developed and validated in rat plasma using rotundine as the internal standard (IS). CAT, S-4 and IS were extracted by acetonitrile protein precipitation and separated on an Eclipse XDB-C18 column (1.8 μm, 4.6 mm × 50 mm) with acetonitrile–water (27:73, v/v) mobile phase containing 0.1% formic acid at a 0.4 mL/min flow rate. Positive ion electrospray ionization in multiple reaction monitoring mode was employed to measure CAT, S-4 and IS by monitoring the transitions m/z 364.2 → 70.1 for CAT, 350.1 → 70.1 for S-4 and 356.2 → 192.2 for IS. Good linear correlation ( r 2 > 0.991) was achieved for CAT and S-4 over the range of 0.214–128.16 and 0.044–11.00 ng/mL, respectively. The lower limit of quantification was 0.214 ng/mL for CAT and 0.044 ng/mL for S-4, using 50 μL rat plasma samples. The intra- and inter-day precisions were not exceed 15% and the accuracy ranged between 94.80% and 108.22%. The average extraction recoveries of both analytes were greater than 94.62%. The method was successfully applied to the pharmacokinetic study of CAT and S-4 in rats after oral administration. [ABSTRACT FROM AUTHOR]

Efficacy assessments using a combination of ibrutinib and lenalidomide necessitate the development of an analytical method for determination of both drugs in plasma with precision. A high performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of lenalidomide, ibrutinib, and its active metabolite PCI45227 in rat plasma. Extraction of lenalidomide, ibrutinib, PCI45227 and tolbutamide (internal standard; IS) from 50 μl rat plasma was carried out by liquid–liquid extraction with ethyl acetate:dichloromethane (90:10) ratio. Chromatographic separation of analytes was performed on YMC pack ODS AM (150 mm × 4.6 mm, 5 μm) column under gradient conditions with acetonitrile:0.1% formic acid buffer as the mobile phases at a flow rate of 1 ml/min. Precursor ion and product ion transition for analytes and IS were monitored on a triple quadrupole mass spectrometer, operated in the selective reaction monitoring with positive ionization mode. Method was validated over a concentration range of 0.72–183.20 ng/ml for ibrutinib, 0.76–194.33 ng/ml for PCI-45227 and 1.87–479.16 ng/ml for lenalidomide. Mean extraction recovery for ibrutinib, PCI-45227, lenalidomide and IS of 75.2%, 84.5%, 97.3% and 92.3% were consistent across low, medium, and high QC levels. Precision and accuracy at low, medium and high quality control levels were less than 15% across analytes. Bench top, wet, freeze–thaw and long term stability was evaluated for all the analytes. The analytical method was applied to support a pharmacokinetic study of simultaneous estimation of lenalidomide, ibrutinib, and its active metabolite PCI-45227 in Wistar rat. Assay reproducibility was demonstrated by re-analysis of 18 incurred samples. [ABSTRACT FROM AUTHOR]

Isobavachalcone (IBC) is a prenylated chalcone and belongs to the class of flavonoids, which is an active component isolated from Psoralea corylifolia L. IBC showed a range of significant pharmacological activities, including antibacterial, anticancer, anti-reverse transcriptase and antioxidant actions. In this research, the mass spectral fragmentation pattern of IBC was investigated to predict the in vivo metabolites, and five phase I metabolites and ten phase II metabolites of IBC in rat bile were elucidated and identified after oral administration using novel LC–ESI-MS n and LC–NMR method. The molecular structures of these metabolites were proposed on the basis of the characteristics of their precursor ions, product ions, MS/MS fragment behaviors and chromatographic retention time. The phase I metabolites were mainly biotransformed via the hydroxylation, reduction, cyclization and oxidative cleavage reactions. The phase II metabolites were mainly identified as the glucuronide conjugates and sulfated conjugates. All these findings were reported for the first time and would contribute to a further understanding of the in vivo intermediate processes and metabolic mechanism of isobavachalcone and its analogs. [ABSTRACT FROM AUTHOR]