Acetate Gradient Research Articles

Measurement of the Direct Oral Anticoagulants Apixaban, Dabigatran, Edoxaban, and Rivaroxaban in Human Plasma Using Turbulent Flow Liquid Chromatography With High-Resolution Mass Spectrometry

Direct oral anticoagulants (DOACs) are prescribed for systemic anticoagulation. Fixed doses are recommended, but dose individualization may be warranted. Functional coagulation assays may be available, but their use requires knowledge of the drug taken. To provide alternative methodology for guiding dosage, we have developed and validated a liquid chromatography-mass spectrometric assay for apixaban, dabigatran, edoxaban, and rivaroxaban at the concentrations attained during therapy. Samples, calibrators, and internal quality controls (100 μL) were mixed with internal standard solution (50 μg/L both dabigatran-13C6 and rivaroxaban-13C6 in acetonitrile) and, after centrifugation (16,400g, 4 minutes), supernatant (100 μL) was injected onto a Cyclone-C18-P-XL TurboFlow column. Analytes were focused onto an Accucore PhenylHexyl (2.1 × 100 mm, 2.6 μm) analytical column and eluted using a methanol + acetonitrile (1 + 1):aqueous ammonium acetate (10 mmol/L) gradient. Data were acquired using high-resolution mass spectrometry in full-scan mode (100-2000 m/z) with data-dependent fragmentation to confirm peak identity. Calibration was linear (1-500 μg/L all analytes). Total analysis time was 6 minutes. Intra-assay imprecision (% RSD) at 1 μg/L was 2.6%, 4.2%, 17.3%, and 9.5% for apixaban, dabigatran, edoxaban, and rivaroxaban, respectively. Mean recovery was 96%-101%. No signal suppression or enhancement was observed. Apixaban, dabigatran, and rivaroxaban were stable over 3 freeze-thaw cycles, after storage at room temperature, and at 2-8°C for up to 2 weeks. Edoxaban was stable over 3 freeze-thaw cycles but showed a mean deterioration of 16% if stored at 2-8°C (2 weeks) and of 18% and 70% (1 day and 2 weeks, respectively) at room temperature. The method is suitable for high-throughput therapeutic drug monitoring of DOACs. The acquisition of full scan data allows for the retrospective identification of metabolites. The method can be used to identify a particular DOAC if information on the drug taken is lacking.

Antimicrobial Metabolites from the Endophytic Fungus Aspergillus sp. of Eucommia ulmoides

Endophytic fungi are eukaryotic organisms that live inside plant tissues and are usually specific at the host species level [1]. They have proven to be a rich source of novel organic compounds with interesting biological activities and a high level of biodiversity [2]. In the course of our search for biologically active metabolites from endophytic fungi from Chinese medicinal plants, a subculture of an isolate of Aspergillus sp., obtained from roots of Eucommia ulmoides, was cultivated on potato dextrose agar (PDA). An ethyl acetate extract of the culture showed significant antimicrobial activity. This prompted us to carry out secondary metabolites studies on this fungus, which resulted in the isolation of seven compounds. Herein we describe the isolation, structural elucidation, and antimicrobial activities of its secondary metabolites. The fungal strain ER15 was isolated from the roots of Eucommia ulmoides, collected in the Qinling Mountains, Shaanxi Province, China, on September 10, 2010. The fungus was identified as a member of the genus Aspergillus by DNA amplification and sequencing of the ITS region. The fungal strain has been preserved at the Shanxi Datong University, Shanxi Province, China. Starter cultures were maintained on PDA medium at 28 C for 7 days. Plugs of agar supporting mycelial growth were cut and transferred aseptically to 1000 mL Erlenmeyer flasks containing 400 mL of liquid Czapek medium at 28 C on a rotary shaker set to 120 r/min for 15 days. The fungal culture (60 L) was filtered through cheesecloth. The filtrate was concentrated to 10 L below 60 C and then extracted five times with ethyl acetate (15 L). The dried mycelium (55 C, 95 g) was extracted three times with methanol (4 L). All extracts were concentrated at reduced pressure to afford 12.1 g of a crude extract. The crude extract was separated into eight fractions (A–H) on silica gel (300 g) using gradients of ethyl acetate–methanol (1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 0:1). Fraction A was separated by silica gel column chromatography eluting with petroleum ether– ethyl acetate (1:2) to give pure compound 1 (87 mg), together with crude compound 2. The crude compound 2 was then recrystallized from ethyl acetate–methanol to give the pure compound 2 (11 mg). Fraction B was separated by column chromatography on silica gel with a gradient of ethyl acetate in petroleum ether to give five subfractions (B1-5). Fraction B3 was further separated by silica gel column chromatography and eluted with a gradient of ethyl acetate in petroleum ether and Sephadex LH-20 with ethyl acetate–methanol (1:1) to give compounds 3 (118 mg), 4 (27 mg), and 5 (19 mg). Similarly, compounds 6 (10 mg) and 7 (37 mg) were isolated from fraction B4 after elution with a gradient of ethyl acetate in petroleum ether and Sephadex LH-20 with ethyl acetate–methanol (1:1). The secondary metabolites were identified as ergosterol (1) [3], cerevisterol (2) [4], 5-hydroxymethylfuran-3-carboxylic acid (3) [5], 5-methoxymethylfuran-3-carboxylic acid (4) [6], allantoin (5) [7], trypacidin (6) [8], and monomethylsulochrin (7) [9] by comparison of their spectral data with the reported data in the literature.

O25: HPLC-HRMS vs UPLC-MS/MS for a systematic toxicological screening in hair

Aims The aim of the present work was to compare the performances of HPLC-High Resolution Mass Spectrometry (HPLC-HRMS) in full scan and UPLC- Multiple mass spectrometry (UPLC-MS/MS) in multiple reaction monitoring (MRM) mode for the systematic, general unknown screening of drugs and pharmaceuticals in hair samples collected for clinical toxicology purposes at the local University Hospital. Methods 10 mg hair were washed and extracted by a rapid, micropulverized method already published. Extracts were analyzed in two different instruments, the former consisting of an Orbitrap coupled to a Finnigan Surveyor HPLC, the latter of a Xevo TQD triple quadrupole coupled to an Acquity UPLC. For HPLC, an Atlantis T3 (150 × 2,1 mm, 3 μm) was used while the UPLC employed a HSS C18 (150 × 2.1 × 1,8 μm), with a gradient of ammonium acetate and acetonitrile (formic acid 0.1%) at 300 μL/min and 400 μL/min respectively. The Orbitrap was operated in positive ion electrospray mode at a resolution of 60000 with mass locking, in full scan mode, with a in-house developed library of 600 compounds; the identificatiion criteria were 2 ppm accuracy of the exact mass of the protonated molecule, ± 0.1 min of the retention time, a minimum area count of 10000 arbitrary units; the Xevo TQD worked in switching ion polarity electrospray mode, and in MRM using a Waters toxicology library of 800 (untargeted screening) or 180 compounds (targeted screening), a recognition window of ±0.2 min and a minimum area count of 600 arbitraty units. Results Compounds belonging to drugs of abuse and/or to pharmaceutical drugs could be identified with both analytical approaches in the same hair samples. In more than 100 hair samples collected in the clinical settings in a year, drugs belonging to the following therapeutic categories were identified: anaesthetics, anti-epileptics, antidepressants, benzodiazepines, sedatives, hypnotics, opioids, anti-psychotics, neuroleptics, barbiturates, stimulants, non steroidal analgesics, steroids, beta-blockers, diuretics, antibiotics, antiarrhythmic agents, antihypertensive drugs, anti-coagulants, hypoglycaemic agents. The screening results were in every case confirmed by injection of the tentatively identified analyte(s) in methanol standard mixtures. When possible, the compliance with prescribed therapies or alleged use/abuse was verified. Conclusions The UPLC-MRM method turned to be more rapid than the HPLC-HRMS (14 min vs 30 min total run time); the possibility of polarity switching could be applied in routine only in the Xevo TQD allowing to easily identify acidic drugs such as non steroidal analgesics. The HPLC-Orbitrap, working in full scan, resulted superior for implementing the library with new molecules, since no adjustment of the acquisition method was needed, and for the postacquisition, retrospective screening of the raw files. In the UPLCMRM method, limits of identification with targeted analytes were in the order 0.1 – 0.2 ng/mg for basic drugs, with 10 mg hair samples, whereas in the HPLC-HRMS limits were generally lower.

Determination of 12 potential nephrotoxicity biomarkers in rat serum and urine by liquid chromatography with mass spectrometry and its application to renal failure induced bySemen Strychni

In previous nephrotoxicity metabonomic studies, several potential biomarkers were found and evaluated. To investigate the relationship between the nephrotoxicity biomarkers and the therapeutic role of Radix Glycyrrhizae extract on Semen Strychni-induced renal failure, 12 typical biomarkers are selected and a simple LC-MS method has been developed and validated. Citric acid, guanidinosuccinic acid, taurine, guanidinoacetic acid, uric acid, creatinine, hippuric acid, xanthurenic acid, kynurenic acid, 3-indoxyl sulfate, indole-3-acetic acid, and phenaceturic acid were separated by a Phenomenex Luna C18 column and a methanol/water (5 mM ammonium acetate) gradient program with a runtime of 20 min. The prepared calibration curves showed good linearity with regression coefficients all above 0.9913. The absolute recoveries of analytes from serum and urine were all more than 70.4%. With the developed method, analytes were successfully determined in serum and urine samples within 52 days. Results showed that guanidinosuccinic acid, guanidinoacetic acid, 3-indoxyl sulfate, and indole-3-acetic acid (only in urine) were more sensitive than the conventional renal function markers in evaluating the therapeutic role of Radix Glycyrrhizae extract on Semen Strychni-induced renal failure. The method could be further used in predicting and monitoring renal failure cause by other reasons in the following researches.

A quantitative LC–MS/MS method for determination of thiazolidinedione mitoNEET ligand NL-1 in mouse serum suitable for pharmacokinetic studies

Thiazolidinedione (TZD) compounds have shown promise as antidiabetic, antibiotics, antifungal and neuroprotective agents. The mitochondrial effect of a novel mitoNEET ligand, NL-1 {5-[(3,5-di-tert-butyl-4-hydroxyphenyl)methyl]-1,3-thiazolidine-2,4-dione}, and other TZD compounds, is a newly proposed mechanism for the neuroprotective action of these TZD compounds. In this work, a sensitive LC-MS/MS assay has been developed and validated for quantification of NL-1 in mouse serum. Sample preparation involved an acetonitrile protein precipitation procedure with addition of an internal standard NL-2 {5-[(4-hydroxy-3,5-dimethyl-phenyl)methyl]thiazolidine-2,4-dione}. LC-MS/MS analysis utilized a Columbus C-18 HPLC column (2mm×50mm, 5μm). Chromatography employed a multiple step gradient program that featured a steep linear gradient (25-95% in 0.5min) of 15μM ammonium acetate (additive for eliminating carry-over) in 2% methanol mixing with increasing proportions of 100% methanol. The HPLC was interfaced to a QTrap 5500 mass spectrometer (AB Sciex) equipped with an electrospray ionization source used in a negative ionization mode. Multiple reaction monitoring (MRM) of m/z 334→263 for NL-1 and m/z 250→179 for NL-2 was done. The method had a linear range of at least 1-100ng/mL in serum. The intra-assay and inter-assay percent coefficient of variation (%CV) were less than 4% and accuracies (%RE) ranged from -2.7% to 2.0%. The analytical procedure gave 96-115% absolute extraction recovery of NL-1. The relative matrix effect was measured and found to be insignificant. The analyte in serum was confirmed to be stable during storage and treatment. The method is suitable for pharmacokinetic (PK) studies of the parent drug NL-1 based on the preliminary serum results from dosed NL-1 mouse studies.

Probing influence of methodological variation on active loading of acetazolamide into nanoliposomes: Biophysical, in vitro, ex vivo, in vivo and rheological investigation

In the present work comparative evaluation of acetate and pH gradient techniques for effective drug loading in liposomes has been investigated. The acetazolamide (ACZ) loaded liposomes prepared by two methods were analyzed by vesicle size analysis, zeta potential, percent encapsulation efficiency, in vitro drug release studies and intraocular pressure lowering activity. ICH guidelines were followed for determining stability of the prepared liposomes. The superiority of acetate gradient method for active loading of acetazolamide has been established. The prepared acetate gradient positive liposomes showed extended hypotensive effect when compared to other liposomal formulations. Thus ACZ loaded liposomes prepared by acetate gradient technique may serve as promising ocular delivery system in the treatment of glaucoma. The current work emphasizes the fact that the techniques used for active drug loading into liposomes strongly influence the pharmaceutical performance of the final formulation.

Borate-aided anion exchange high-performance liquid chromatography of uridine diphosphate-sugars in brain, heart, adipose and liver tissues

In this paper we describe a method optimized for the purification of uridine diphosphate (UDP)-sugars from liver, adipose tissue, brain, and heart, with highly reproducible up to 85% recoveries. Rapid tissue homogenization in cold ethanol, lipid removal by butanol extraction, and purification with a graphitized carbon column resulted in isolation of picomolar quantities of the UDP-sugars from 10 to 30mg of tissue. The UDP-sugars were baseline separated from each other, and from all major nucleotides using a CarboPac PA1 anion exchange column eluted with a gradient of acetate and borate buffers. The extraction and purification protocol produced samples with few unidentified peaks. UDP-N-acetylglucosamine was a dominant UDP-sugar in all the rat tissues studied. However, brain and adipose tissue showed high UDP-glucose levels, equal to that of UDP-N-acetylglucosamine. The UDP-N-acetylglucosamine showed 2.3–2.7 times higher levels than UDP-N-acetylgalactosamine in all tissues, and about the same ratio was found between UDP-glucose and UDP-galactose in adipose tissue and brain (2.6 and 2.8, respectively). Interestingly, the UDP-glucose/UDP-galactose ratio was markedly lower in liver (1.1) and heart (1.7). The UDP-N-acetylglucosamine/UDP-glucuronic acid ratio was also constant, between 9.7 and 7.7, except in liver with the ratio as low as 1.8. The distinct UDP-glucose/galactose ratio, and the abundance of UDP-glucuronic acid may reflect the specific role of liver in glycogen synthesis, and metabolism of hormones and xenobiotics, respectively, using these UDP-sugars as substrates.

Strong larvicidal potential of Artemisia annua leaf extract against malaria (Anopheles stephensi Liston) and dengue (Aedes aegypti L.) vectors and bioassay-driven isolation of the marker compounds

Malaria and dengue are the two most important vector-borne human diseases caused by mosquito vectors Anopheles stephensi and Aedes aegypti, respectively. Of the various strategies adopted for eliminating these diseases, controlling of vectors through herbs has been reckoned as one of the important measures for preventing their resurgence. Artemisia annua leaf chloroform extract when tried against larvae of A. stephensi and A. aegypti has shown a strong larvicidal activity against both of these vectors, their respective LC50 and LC90 values being 0.84 and 4.91 ppm for A. stephensi and 0.67 and 5.84 ppm for A. aegypti. The crude extract when separated through column chromatography using petroleum ether-ethyl acetate gradient (0-100%) yielded 76 fractions which were pooled into three different active fractions A, B and C on the basis of same or nearly similar R f values. The aforesaid pooled fractions when assayed against the larvae of A. stephensi too reported a strong larvicidal activity. The respective marker compound purified from the individual fractions A, B and C, were Artemisinin, Arteannuin B and Artemisinic acid, as confirmed and characterized through FT-IR and NMR. This is our first report of strong mortality of A. annua leaf chloroform extract against vectors of two deadly diseases. This technology can be scaled up for commercial exploitation.

Antimicrobial, antityrosinase and brine shrimp lethality test of Bauhinia rufescens Lam (Fabaceae)

salina). Methods: The plant materials were extracted based on polarity gradient of petroleum ether, ethyl acetate and methanol. The extracts were investigated for their antimicrobial, antityrosinase and cytotoxicity potentials. Results: The antimicrobial results indicated that the extracts inhibited the microbial growth in the range of 6.00-12.57 mm (450-1 800 µg/mL) for bacterial strains and 7.33-14.03 mm (225-1 800 µg/mL) for fungal strains. While the tyrosinase enzyme inhibition assay of the extracts showed that both the petroleum ether leaves and stem bark extracts exhibited a moderate inhibition (39.60% and 39.89%) towards the action of mushroom tyrosinase enzyme on L-DOPA. In the brine shrimp lethality assay, ethyl acetate and methanol extracts of the leaves of the plant were found toxic to the Artemia salina with IC50 values of 0.059 mg/mL and 0.389 mg/mL. However, both the petroleum ether and ethyl acetate extracts of the stem bark were nontoxic to the larvae. Conclusions: The result of the present study suggest the presence of phytochemicals possessing antimicrobial and antityrosinase activity as well as cytotoxic agents.

Isolation and biological evaluation of secondary metabolites of the endophytic fungus Aspergillus fumigatus from Astragalus membranaceus

Endophytic fungi are eukaryotic organisms that live inside plant tissues and are usually specific at the host species level [1, 2]. Endophytic fungi show high potential as sources of novel antimicrobial, antiviral, anticancer, antioxidant, and insecticidal compounds [3]. In the course of our search for biologically active metabolites of endophytic fungi from Chinese medicinal plants, an endophytic fungus, Aspergillus fumigatus, was isolated from the root of Astragalus membranaceus. The EtOAc extract of the culture showed significant antimicrobial activity. This prompted us to carry out secondary-metabolite studies on the fungus, which resulted in the isolation of eight active compounds, ergosterol (1), cerevisterol (2), ergosterol peroxide (3), fumitremorgin B (4), fumitremorgin C (5), verruculogen (6), cyclotryprostatins B (7), and fumiquinazoline J (8). Herein we describe the isolation, structural elucidation, and biological test of its secondary metabolites. The fungal strain AR05 was isolated from the root of Astragalus membranaceus, collected in the Hengshan Mountains, Shanxi Province, China, on August 10, 2010. By classical microscopic analysis, the fungus was identified as a member of the genus Aspergillus. It was further identified as Aspergillus fumigatus according to a molecular biological protocol by DNA amplification and sequencing of the ITS region. The fungal strain has been preserved at the College of Agronomy and Life Science, Shanxi Datong University, Shanxi Province. Starter cultures were maintained on PDA medium at 28 C for 7 days. Plugs of agar supporting mycelial growth were cut and transferred aseptically to 1000 mL Erlenmeyer flasks containing 400 mL of liquid Czapek medium at 28 C on a rotary shaker set to 120 rmp for 15 days. The fungal culture (40 L) was filtered through cheesecloth. The filtrate was concentrated to 5 L below 60 C and then extracted five times with ethyl acetate (4.5 L). The dried mycelium (55 C, 95 g) was extracted three times with methanol (4 L). All extracts were concentrated at reduced pressure to afford 9.8 g of a dark brown crude extract. The crude extract was subjected to silica gel column chromatography eluting successively with ethyl acetate–methanol gradients (1:0, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 0:1) and yielded eight fractions (A–H). Fraction A was purified by column chromatography on silica gel with a gradient of ethyl acetate in petroleum ether to give five subfractions (A1-5). Fraction A1 was separated by silica gel column chromatography with elution with petroleum ether–ethyl acetate (2:1) to give pure compounds 1–3. Fraction B was separated by column chromatography on silica gel with a gradient of ethyl acetate in petroleum ether to give five subfractions (B1-5). Fraction B2 was repeatedly subjected to silica gel column chromatography with a gradient of ethyl acetate in petroleum ether and Sephadex LH-20 with ethyl acetate–methanol (1:1) to yield compounds 4 and 5. Fraction B3 was repeatedly subjected to silica gel column chromatography with a gradient of ethyl acetate in petroleum ether and Sephadex LH-20 with ethyl acetate–methanol (1:1) to yield compounds 6–8. The compounds were subjected to characterization using spectroscopic analysis and identified as ergosterol (1) [4], cerevisterol (2) [5], ergosterol peroxide (3) [6, 7], fumitremorgin B (4) [8, 9], verruculogen (5) [10], fumitremorgin C (6) [11, 12], cyclotryprostatins B (7) [13], and fumiquinazoline J (8) [14, 15] by comparison of their spectral data with the reported data in the literature. Ergosterol (1), C28H44O, colorless needles, mp 155–157 C. ESI-MS m/z 397.0 [M + H]+. Cerevisterol (2), C28H46O3, colorless needles, mp 240–242 C. ESI-MS m/z 431.0 [M + H]+.

Semi-preparative isolation of dihydroresveratrol-3-O-β-d-glucuronide and four resveratrol conjugates from human urine after oral intake of a resveratrol-containing dietary supplement

A method for semi-preparative isolation of major resveratrol metabolites from human urine after oral intake of a trans-resveratrol-containing dietary supplement was developed. Pretreatment of the urine (6L) by using solid-phase extraction gave a brown oily residue (9.3g), which was separated using a combination of normal phase column chromatography and reversed-phase flash column chromatography resulting in fractions containing 1.1g crude trans-resveratrol-3-O-sulfate (M1), 86mg of a crude mixture of trans-resveratrol-3,5-O-disulfate (M2) and trans-resveratrol-3,4'-O-disulfate (M3), and 568mg of a crude mixture of trans-resveratrol-3-O-β-d-glucuronide (M4) and dihydroresveratrol-3-O-β-d-glucuronide (M5). Purification of the crude metabolites was performed by semi-preparative reversed-phase HPLC using a gradient of aqueous ammonium acetate (2.5mmol/L, pH 6.7)/acetonitrile for purification of M1, M2 and M3 or trifluoroacetic acid in water (pH 2.5)/acetonitrile for purification of M4 and M5. From a part of the crude metabolites (50-75mg), 47mg M1 (purity 98.7%), 14mg M2 (purity 96.1%), 10mg M3 (purity 96.3%), 38mg M4 (purity 98.2%) and 18mg M5 (purity 97.8%) were obtained. The structures of all isolated resveratrol metabolites were elucidated by spectroscopic and spectrometric methods such as 1D and 2D NMR, UV, and LC-MS. This method represents a novel approach to obtain resveratrol metabolites being the first method describing the direct isolation of pure resveratrol metabolites from urine samples in quantities sufficient for full chemical characterization and testing in vitro and in preclinical trials.

Isolation of anti-tumor compounds from the stem bark of Zanthoxylum ailanthoides Sieb. & Zucc. by silica gel column and counter-current chromatography

Silica gel column chromatography combined with high performance counter-current chromatography (HPCCC) was employed for the separation of potential anti-tumor compounds from a petroleum ether fraction of a crude extract of Zanthoxylum ailanthoides Sieb. & Zucc. This traditional Chinese medicine was recently found to display high inhibitory activity against A-549 human cancer cells in vitro and Lewis lung cancer in vivo. A 75% aqueous ethanol extract of the stem bark of Z. ailanthoides was fractionated with petroleum ether, ethyl acetate and n-butanol. In this paper, the petroleum ether fraction was pre-separated by silica gel column chromatography with a petroleum ether-ethyl acetate gradient. Two fractions were further separated and purified by HPCCC using n-hexane-ethyl acetate-methanol-water (3:1:2:1, v/v) and petroleum-ethyl acetate-methanol-water (8:6:7:7, v/v). Finally, coumarins and lignans including luvangetin, xanthyletin, hinokinin and asarinin were isolated and identified by MS, (1)H and (13)C NMR. In total, 56mg of xanthyletin (1), 140mg of hinokinin (2), 850mg of luvangetin (3) and 74mg of asarinin (4) were obtained from approximately 50g of petroleum ether extract, in 96.0%, 94.0%, 99.0% and 94.0% purity, respectively, as determined by HPLC. The separation method proved to be efficient, especially for those minor components.

Preparation of bromfenac-loaded liposomes modified with chitosan for ophthalmic drug delivery and evaluation of physicochemical properties and drug release profile

The purpose of this study was to design a submicron-sized liposomal non-steroidal anti-inflammatory drug (NSAID) preparation that targets the retina via topical instillation of eye drops. Bromfenac (BRF)-loaded liposomes were prepared using the calcium acetate gradient method. Liposome sizes and encapsulation efficiencies were optimized by screening several liposome formulations of lipid, drug concentration, and buffer solution. BRF entrapment efficiency was greater than 90% using this method, and was low using conventional hydration methods. High initial BRF loading using the pH gradient method caused aggregation of liposomes. To circumvent aggregation, the negatively charged lipid dicetylphosphate was incorporated into liposomes, which formed anion layer preventing coalescence. Release of BRF from liposomes was sustained for several hours depending on lipid concentration, inner water phase, initial drug amounts, and surface properties. Surface modification with chitosan (CS), a mucoadhesive cationic polymer, was achieved using electrostatic interactions of negatively charged liposomes. The optimal concentration of CS for evasion of liposome aggregation was 0.15%.

Five new secondary metabolites from Monascus purpureus-fermented Hordeum vulgare and Sorghum bicolor

Long grains of Hordeum vulgare and Sorghum bicolor were individually fermented with Monascus purpureus MTCC 369 under solid state fermentation. The aqueous extract of Monascus which fermented H. vulgare and S. bicolor was found to contain five different new metabolites. Silica gel column chromatography of the aqueous extract with a linear gradient of ethyl acetate, acetonitrile and carbon tetrachloride (v/v) yielded five new metabolites named benzopranyl capriate (9H-1-isoprenyl-benzopyran-5-isopropanoic acid-6-ol-6-n-decanoate), shorghumoic acid (n-octadec-8,11-dien-7α-ol-1-oic acid) and sorghumflavin A (2-n-butyloxo-6-β-hydroxy-7-β-isoprenyl ankaflavin) from Monascus-fermented S. bicolor, while hordeumflavin B (2-n-undecanyloxo-7-β-isoprenyl ankaflavin) and vulgaredilone (2-dodecanyl-7-β isopranyl monoscodilone) from Monascus-fermented H. vulgare.

Determination of bromadiolone and brodifacoum in human hair by liquid chromatography/tandem mass spectrometry and its application to poisoning cases

Bromadiolone and brodifacoum, two common anticoagulant rodenticides, are involved in the majority of anticoagulant rodenticide poisoning cases in humans in China. Hair analysis can provide long-term information on drug exposure. A method using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) was developed and validated for the measurement of bromadiolone and brodifacoum in human hair. A 1 mL aliquot of a phosphate buffer solution (pH 6.8) was added to 20 mg of pulverized hair followed by ultrasonication and liquid-liquid extraction. Liquid chromatography was performed using a C(18) column with a mobile phase gradient of ammonium acetate (10 mM) and methanol. A tandem mass spectrometer in multiple reaction monitoring mode with a negative electrospray ionization source was employed for detection. Warfarin-d(5) was used as an internal standard for both analytes. The limits of detection (LODs) for bromadiolone and brodifacoum were 0.010 and 0.025 ng/mg, respectively. The calibration curves for both analytes were linear from 0.025 to 1 ng/mg. The accuracy ranged from 90.3 to 109.3%, and the intra-day and inter-day imprecisions were less than 15%. The established method was found effective when applied to the analyses of bromadiolone or brodifacoum in five cases, indicating that segmental hair analysis could be useful for clinical and forensic purposes by identifying the time of ingestion.

Simultaneous determination of metronidazole, chloramphenicol and 10 sulfonamide residues in honey by LC–MS/MS

A liquid chromatography–tandem mass spectrometric method was established for the simultaneous determination of residues of metronidazole, chloramphenicol and 10 sulfonamides (sulfadiazine, sulfathiazol, sulfapyridin, sulfamerazin, sulfameter, sulfamethazine, sulfamethoxypyridazine, sulfachloropyridazine, sulfamethoxazole and sulfadimethoxin) in honey samples. Samples were first dissolved in water. Sonication was carried out in an ultrasonic bath to extract the target analytes with acetonitrile and the extract was concentrated by rotary evaporation. Chromatographic separation was carried out on an MGIII-C18 column (2.1 mm × 150 mm, 5 μm) using a mobile phase made up of methanol and 50 mmol L−1 ammonium acetate aqueous solution (include 0.3% formic acid) and gradient elution. Metronidazole, chloramphenicol and the sulfonamides were monitored in negative ion or positive ion selected reaction monitoring modes and quantified by the external standard method. The limit of detection was 0.1 μg kg−1 for chloramphenicol and 1.0 μg kg−1 for metronidazole and each sulfonamide, respectively. The linear range for chloramphenicol was 0.1–25.0 μg kg−1, and for both metronidazole and the sulfonamides were 1.0–100.0 μg kg−1, and all the correlation coefficients were greater than 0.99. The average spike recoveries of the method ranged from 81.6% to 99.7% with relative standard deviations of 3.61–8.97%. Therefore this method is convenient, rapid, sensitive and reliable, and can be successfully applied for the simultaneous detection of metronidazole, chloramphenicol and 10 sulfonamide residues in commercial products.

Liposomal diclofenac eye drop formulations targeting the retina: Formulation stability improvement using surface modification of liposomes

An efficient liposomal formulation for targeting the retina was produced as an optimal means of distributing therapeutic agents to the retina. Diclofenac was used as a model compound for liposome encapsulation, and the release rate and distribution to the retina were investigated. The calcium acetate gradient method was found to be the optimal method for encapsulating diclofenac into liposomes. Entrapment efficiency using this method was greater than 97%, whereas conventional hydration method achieved 51.3%. The resultant formulation obtained with the gradient method caused aggregation and/or fusion of liposomes. To avoid inhibition of retinal delivery due to the aggregation of the carrier, surface modification was performed simultaneously with the gradient method. The increase in particle size of the liposomal formulation clearly was inhibited for a long time in the presence of polyvinyl alcohol or its derivative. This observation may be explained by surface modification of the liposomes by physisorption or anchoring effect of polymers on the surface of the lipid bilayer. Furthermore, the sustained release profile of the diclofenac formulation was retained after modification. An in vivo animal study revealed that concentration of the accumulated diclofenac in the retina-choroid was enhanced 1.8-fold by surface-modified liposome entrapment compared to that of the unaltered diclofenac solution.

Anticancer property of Bryophyllum pinnata (Lam.) Oken. leaf on human cervical cancer cells

BackgroundBryophyllum pinnata (B. pinnata) is a common medicinal plant used in traditional medicine of India and of other countries for curing various infections, bowel diseases, healing wounds and other ailments. However, its anticancer properties are poorly defined. In view of broad spectrum therapeutic potential of B. pinnata we designed a study to examine anti-cancer and anti-Human Papillomavirus (HPV) activities in its leaf extracts and tried to isolate its active principle.MethodsA chloroform extract derived from a bulk of botanically well-characterized pulverized B. pinnata leaves was separated using column chromatography with step- gradient of petroleum ether and ethyl acetate. Fractions were characterized for phyto-chemical compounds by TLC, HPTLC and NMR and Biological activity of the fractions were examined by MTT-based cell viability assay, Electrophoretic Mobility Shift Assay, Northern blotting and assay of apoptosis related proteins by immunoblotting in human cervical cancer cells.ResultsResults showed presence of growth inhibitory activity in the crude leaf extracts with IC50 at 552 μg/ml which resolved to fraction F4 (Petroleum Ether: Ethyl Acetate:: 50:50) and showed IC50 at 91 μg/ml. Investigations of anti-viral activity of the extract and its fraction revealed a specific anti-HPV activity on cervical cancer cells as evidenced by downregulation of constitutively active AP1 specific DNA binding activity and suppression of oncogenic c-Fos and c-Jun expression which was accompanied by inhibition of HPV18 transcription. In addition to inhibiting growth, fraction F4 strongly induced apoptosis as evidenced by an increased expression of the pro-apoptotic protein Bax, suppression of the anti-apoptotic molecules Bcl-2, and activation of caspase-3 and cleavage of PARP-1. Phytochemical analysis of fraction F4 by HPTLC and NMR indicated presence of activity that resembled Bryophyllin A.ConclusionsOur study therefore demonstrates presence of anticancer and anti-HPV an activity in B. pinnata leaves that can be further exploited as a potential anticancer, anti-HPV therapeutic for treatment of HPV infection and cervical cancer.

Simultaneous quantification of selective serotonin reuptake inhibitors and metabolites in human plasma by liquid chromatography–electrospray mass spectrometry for therapeutic drug monitoring

A simple and sensitive liquid chromatography–electrospray ionization mass spectrometry method was developed for the simultaneous quantification in human plasma of all selective serotonin reuptake inhibitors (citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline) and their main active metabolites (desmethyl-citalopram and norfluoxetine). A stable isotope-labeled internal standard was used for each analyte to compensate for the global method variability, including extraction and ionization variations. After sample (250μl) pre-treatment with acetonitrile (500μl) to precipitate proteins, a fast solid-phase extraction procedure was performed using mixed mode Oasis MCX 96-well plate. Chromatographic separation was achieved in less than 9.0min on a XBridge C18 column (2.1×100mm; 3.5μm) using a gradient of ammonium acetate (pH 8.1; 50mM) and acetonitrile as mobile phase at a flow rate of 0.3ml/min. The method was fully validated according to Société Française des Sciences et Techniques Pharmaceutiques protocols and the latest Food and Drug Administration guidelines. Six point calibration curves were used to cover a large concentration range of 1–500ng/ml for citalopram, desmethyl-citalopram, paroxetine and sertraline, 1–1000ng/ml for fluoxetine and fluvoxamine, and 2–1000ng/ml for norfluoxetine. Good quantitative performances were achieved in terms of trueness (84.2–109.6%), repeatability (0.9–14.6%) and intermediate precision (1.8–18.0%) in the entire assay range including the lower limit of quantification. Internal standard-normalized matrix effects were lower than 13%. The accuracy profiles (total error) were mainly included in the acceptance limits of ±30% for biological samples. The method was successfully applied for routine therapeutic drug monitoring of more than 1600 patient plasma samples over 9 months. The β-expectation tolerance intervals determined during the validation phase were coherent with the results of quality control samples analyzed during routine use. This method is therefore precise and suitable both for therapeutic drug monitoring and pharmacokinetic studies in most clinical laboratories.

Ultra-high performance liquid chromatography-tandem mass spectrometry for the analysis of phenicol drugs and florfenicol-amine in foods

A method based on ultra-high performance liquid chromatography (UHPLC) is proposed for the determination of chloramphenicol (CAP), its related compounds, thiamphenicol (TAP) and florfenicol (FF), and the polar metabolite florfenicol-amine (FFA), in animal-derived foods (chicken and pork meat, fish, prawns and honey). For the retention of FFA and its simultaneous analysis with the parent compounds a phenyl-hexyl column is proposed. A fast separation is achieved in less than 2 minutes using a methanol : acetic acid-ammonium acetate buffer (5 mM, pH 5) and gradient elution. Under these conditions, the FFA is retained at more than twice the dead volume, as recommended by the legislation. For the coupling with mass spectrometry, heated-electrospray (H-ESI) is used as ionisation source improving vaporization efficiency. To prevent interferences using selected-reaction monitoring (SRM) both quantitation and confirmation transitions were carefully selected. Two different sample treatments based on solid-phase extraction with mixed-mode cartridges for fish and meat products and hydrophilic-lipophilic-balanced cartridges for honey are proposed, providing limits of quantitation (LOQs) below μg kg(-1) level.