Liquid chromatography-tandem mass spectrometry analysis of nitazoxanide and its major metabolites in goat

A rapid, sensitive and specific liquid chromatography-electrospray ionization (ESI) tandem mass spectrometry (LC-MS-MS) method has been developed for the identification of nitazoxanide metabolites in goat plasma and urine. The purified samples was separated using an XTerra MS C8 column with the mobile phase consisted of acetonitrile and 10-mM ammonium acetate buffer (pH 2.5) followed a linear gradient elution, and detected by MS-MS. Identification and structural elucidation of the metabolites were performed by comparing their retention-times, full scan, product ion scan, precursor ion scan and neutral loss scan MS-MS spectra with those of the parent drug or other available standard. Four metabolites (tizoxanide, tizoxanide glucuronide, tizoxanide sulfate and hydroxylated tizoxanide sulfate) were found and identified in goat after single oral administration of 200mg/kg dose of nitazoxanide. In addition, the possible metabolic pathway was proposed for the first time. The results proved that the established method was simple, reliable and sensitive, revealing that it could be used to rapid screen and identify the structures of active metabolites responsible for pharmacological effects of nitazoxanide and to better understand its in vivo metabolism.     

Liquid chromatography-tandem mass spectrometry analysis of protocatechuic aldehyde and its phase I and II metabolites in rat

A method using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) analysis was established for the identification of metabolites in rat after oral administration of protocatechuic aldehyde, a major bioactive phenolic acid in the roots of Salvia miltiorrhiza. Eleven metabolites in rat plasma and urine were firstly identified as protocatechuic aldehyde, protocatechuic acid and their methylated, glucuronized or glycine conjugates on the basis of their MS fragmentation behaviors, while nine of these metabolites (except protocatechuic aldehyde and protocatechuic acid) were detected in rat bile. In addition, the possible metabolic pathway was proposed for the first time. In the phase I metabolism, protocatechuic aldehyde could be oxidized to protocatechuic acid. The conjugates would be formed in rat intestine, liver and kidney and excreted from rat urine and bile. Enthrohepatic circulation played an important role in the metabolism of protocatechuic aldehyde. The results proved that the established method was simple, reliable and sensitive, revealing that it could be used to rapid screen and identify the structures of active components responsible for pharmacological effects of protocatechuic aldehyde and to better understand its in vivo metabolism.     

Liquid chromatography-tandem mass spectrometry analysis of anisodamine and its phase I and II metabolites in rat urine

A sensitive and specific method for the analysis of anisodamine and its metabolites in rat urine by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) was developed. Various extraction techniques (free fraction, acid hydrolyses and enzyme hydrolyses) and their comparison were carried out for investigation of the metabolism of anisodamine. After extraction procedure the pretreated samples were injected on a reversed-phase C18 column with mobile phase (0.2 ml/min) of methanol/0.01% triethylamine solution (adjusted to pH 3.5 with formic acid) (60:40, v/v) and detected by MS/MS. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular masses (DeltaM), retention-times and full scan MS(n) spectra with those of the parent drug. At least 11 metabolites (N-demethyl-6beta-hydroxytropine, 6beta-hydroxytropine, tropic acid, N-demethylanisodamine, hydroxyanisodamine, anisodamine N-oxide, hydroxyanisodamine N-oxide, glucuronide conjugated N-demethylanisodamine, sulfate conjugated and glucuronide conjugated anisodamine, sulfate conjugated hydroxyanisodamine) and the parent drug were found in rat urine after the administration of a single oral dose 25mg/kg of anisodamine. Hydroxyanisodamine, anisodamine N-oxide and the parent drug were detected in rat urine for up 95 h after ingestion of anisodamine.     

Liquid chromatography-tandem mass spectrometry characterization of ergocristam degradation products

The UPLC method with diode array UV detection was developed for qualitative determination of ergocristine and ergocristam including degradation products. The mechanism of the ergocristam disruptive reaction was described based on MS/MS characterization of ammonolytic product, N-(d-lysergyl)-l-valinamide (A1) and two methanolytic products, methyl ester of N-(d-lysergyl)-l-valine (M2), and N-[N-(d-lysergyl)-l-valyl]-l-phenylalanyl-d-prolyl methyl ester (M1). The influence of extraction conditions on epimerization and degradation of ergocristine and ergocristam was tested and conditions for reproducible decomposition of ergocristam were found. The presented method could potentially be applied for ergot alkaloids determination in sclerotia, fermentation broth, mycelium, and possibly contaminated food products, i.e. corn, flour, bread, etc., and feeding stuffs containing ungrounded cereals.     

Role of flavonoids in controlling the phototoxicity of Hypericum perforatum extracts.

Hypericum perforatum extracts are used mainly as oral antidepressants. Depending on source the extracts contain various amounts of phenylpropanes, flavonol derivates, biflavones, proathocyanidines, xanthones, phloroglucinoles, some amino acids, naphtodianthrones (hypericines) and essential oil constituents. The therapeutic use of Hypericum perforatum extracts however is limited by their phototoxic potential. It was the aim of the present study to investigate the phototoxic potential of 3 Hypericum perforatum extracts from different sources as well as some of its main constituents. In order to systematically study the phototoxic potential we established a modified neutral red assay utilizing an immortalized human keratinocyte cell line (HaCaT cells) as substrate and UVA irradiation. This modified neutral red assay was found to be a simple and reliable method for detecting phototoxic effects of reference agents and plant extracts. The validity of this method was demonstrated with known phototoxic compounds like chloropromazine and psoralenes like 5-MOP. Hypericum perforatum extracts demonstrated cytotoxicity and photocytotoxicity in a dose and UVA-dose dependent manner. Hypericine itself also evoked severe phototoxic effects and was thus identified as the main phototoxic constituent. Among the tested flavonoids quercitrin was found to be cytotoxic, while rutin unexpectedly demonstrated phototoxicity whereas quercitrin was effective to control the phototoxic activity of Hypericum perforatum extracts.     

Identification of anti-inflammatory constituents in Hypericum perforatum and Hypericum gentianoides extracts using RAW 264.7 mouse macrophages

Hypericum perforatum (St. John’s wort) is an herb widely used as supplement for mild to moderate depression. Our prior studies established synergistic anti-inflammatory activity associated with 4 bioactive compounds in a fraction of a H. perforatum ethanol extract. Whether these 4 compounds also contributed to the ethanol extract activity was addressed in the research reported here. Despite the popularity of H. perforatum, other Hypericum species with different phytochemical profiles could have their anti-inflammatory potentials attributed to these or other compounds. In the current study, ethanol extracts of different Hypericum species were compared for their inhibitory effect on LPS-induced prostaglandin E2 (PGE2) and nitric oxide (NO) production in RAW 264.7 mouse macrophages. Among these extracts, those made from H. perforatum and H. gentianoides demonstrated stronger overall efficacy. LC–MS analysis established the 4 compounds were present in the H. perforatum extract and pseudohypericin in all active fractions. The 4 compounds accounted for a significant part of the extract’s inhibitory activity on PGE2, NO, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in RAW 264.7 as well as peritoneal macrophages. Pseudohypericin was the most important contributor of the anti-inflammatory potential among the 4 compounds. The lipophilic fractions of H. gentianoides extract, which did not contain the previously identified active constituents, decreased PGE2 and NO potently. These fractions were rich in acylphloroglucinols, including uliginosin A that accounted for a proportion of the anti-inflammatory activity observed with the active fractions. Overall, the current study established that a different group of major anti-inflammatory constituents were present in H. gentianoides, while showing that the previously identified 4 compound combination was important for H. perforatum’s anti-inflammatory potential.     

Liquid chromatography-tandem mass spectrometry assay for the quantitation of beta-dihydroartemisinin in rat plasma

Dihydroartemisinin (DHA) is a sesquiterpene used in the world as an antimalarial. To evaluate the pharmacokinetics of dihydroartemisinin in rats, a sensitive and specific liquid chromatography/tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantitation of dihydroartemisinin in rat plasma. For detection, a Sciex API 4000 LC-MS/MS with a TurboIonSpray ionization (ESI) inlet in the positive ion-multiple reaction monitoring (MRM) mode was used. The plasma samples were pre-treated by a simple liquid-liquid extraction with diethyl ether. The statistical evaluation for this method reveals excellent linearity, accuracy and precision for the range of concentrations 0.2-100.0 ng/mL. The method had a lower limit of quantification (LLOQ) of 0.2 ng/mL for beta-dihydroartemisinin in 100 microL of plasma. The method was successfully applied to the characterization of the pharmacokinetic profile of beta-dihydroartemisinin in rats after oral administration.     

In vitro photochemical and phototoxicological characterization of major constituents in St. John's Wort (Hypericum perforatum) extracts

Extracts from St. John's Wort (SJW: Hypericum perforatum) have been used for the treatment of mild-to-moderate depression. In spite of the high therapeutic potential, orally administered SJW sometimes causes phototoxic skin responses. As such, the present study aimed to clarify the phototoxic mechanisms and to identify the major phototoxins of SJW extract. Photobiochemical properties of SJW extract and 19 known constituents were characterized with focus on generation of reactive oxygen species (ROS), lipid peroxidation, and DNA photocleavage, which are indicative of photosensitive, photoirritant, and photogenotoxic potentials, respectively. ROS assay revealed the photoreactivity of SJW extract and some SJW ingredients as evidenced by type I and/or II photochemical reactions under light exposure. Not all the ROS-generating constituents caused photosensitized peroxidation of linoleic acid and photodynamic cleavage of plasmid DNA, and only hypericin, pseudohypericin, and hyperforin exhibited in vitro photoirritant potential. Concomitant UV exposure of quercitrin, an SJW component with potent UV/Vis absorption, with hyperforin resulted in significant attenuation of photodynamic generation of singlet oxygen from hyperforin, but not with hypericin. In conclusion, our results suggested that hypericin, pseudohypericin, and hyperforin might be responsible for the in vitro phototoxic effects of SJW extract.     

Liquid chromatography-tandem mass spectrometry method for the determination of tranexamic acid in human plasma

A new method for the determination of tranexamic acid (TA) in human plasma using high performance liquid chromatography with tandem mass spectrometric detection was described. TA and the internal standard, methyldopa, was extracted from a 200 l plasma sample by a one-step deproteination using perchloric acid. Chromatographic separation was performed on an Xtrra MS C18 Column (2.1 mm x 100 mm, 3.5 microm) with the mobile phase consisting of 10% acetonitrile in 2 mM ammonium acetate buffer (pH 3.5) at a flow rate of 0.15 ml/min. The total run time was 5 min for each sample. Detection and quantitation was performed by the mass spectrometer using the multiple reaction monitoring of the precursor-product ion pair m/z 158 --> 95 for TA and m/z 212 --> 166 for methyldopa, respectively. The method was linear over the concentration range of 0.02-10.00 g/ml with lower limit of quantification of 0.02 microg/ml for TA. The intra- and inter-day precision was less than 11% and accuracy ranged -10.88 to 11.35% at the TA concentrations tested. The present method provides a relatively simple and sensitive assay with short turn-around time. The method has been successfully applied to a clinical pharmacokinetic study of TA in 12 healthy subjects.     

Liquid chromatography-tandem mass spectrometric determination of ceramides and related lipid species in cellular extracts

A liquid chromatography-electrospray ionization tandem mass spectrometric (LC-MS/MS) method was developed for the simultaneous qualitative and quantitative determination of sphingolipid metabolites such as ceramides, sphingisine, sphinganine, sphingomyelins, and ceramide 1-phosphates in the extracts of human promyelocytic leukemia cells (HL-60). The assay uses C(4) ceramide as an internal standard; simple liquid extraction; a short XTerra MS C(18) (3 microm, 50 mm x2.0 mm) column; a gradient mobile phase of 5mM ammonium formate (pH 4.0)/methanol/tetrahydrofuran (5/2/3-->1/2/7); mass spectrometric detection using electrospray ionization. This LC-MS/MS method allowed the identification of 22 sphingolipid derivatives at pmol levels. In addition, this technique was successfully applied to analyze the changes of the sphingolipids profiles in cancer cells treated with apoptosis inducing agents, C(2) ceramide and H(2)O(2).     

Liquid chromatography-tandem mass spectrometry method for determination of N-acetylglucosamine concentration in human plasma

A simple, reliable and sensitive liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for quantification of N-acetylglucosamine in human plasma. Plasma samples were pretreated with acetonitrile for protein precipitation. The chromatographic separation was performed on Hypersil Silica column (150mmx2mm, 5microm). The deprotonated analyte ion was detected in negative ionization mode by multiple reaction monitoring mode. The mass transition pairs of m/z 220.3-->118.9 and m/z 226.4-->123.2 were used to detect N-acetylglucosamine and internal standard 13C6-N-acetylglucosamine, respectively. The assay exhibited a linear range from 20 to 1280ng/ml for N-acetylglucosamine in human plasma. Acceptable precision and accuracy were obtained for concentrations of the calibration standard and quality control. The validated method was successfully applied to analyze human plasma samples in a pharmacokinetic study.     

Determination of 8-methoxypsoralen in human plasma, and microdialysates using liquid chromatography-tandem mass spectrometry

The validation of a LC/MS/MS method for the determination of 8-methoxypsoralen (8-MOP) in human plasma and microdialysates after topical application is described. Plasma samples were extracted by liquid-liquid extraction with diisopropylether using 4,5',8-trimethylpsoralen (TMP) as internal standard. Chromatographic separation of plasma sample extracts was carried out using a short narrow-bore Nucleosil C18 column (30 mm x 2.0 mm i.d.) with acetonitrile/(2 mM ammonium acetate buffer, 2 mM acetic acid) (80:20, v/v). For mass spectrometric analysis an API 3000 triple quadrupole mass spectrometer was employed. The mass transitions used were m/z 217.2-->174.0 for 8-MOP and m/z 229.1-->142.1 for TMP. Microdialysis samples diluted with an equal amount of acetonitrile did not require any extraction and were analyzed directly on a narrow-bore Nucleosil C18 column (70 mm x 2.0mm i.d.) with acetonitrile/(2 mM ammonium acetate buffer, 2 mM acetic acid) (50:50, v/v) with the mass transition m/z 217.2-->174.0. The assays were validated over the concentration ranges of 0.5-50 ng/ml for plasma samples and 0.25-50 ng/ml for microdialysates, respectively.     

Cytotoxic activities of Hypericum perforatum L. extracts against 2D and 3D cancer cell models

Cytotechnology - Six extracts were obtained from plant species Hypericum perforatum L., collected at Samsun in Turkey. The aim of this study was to examine the mechanisms of the anticancer activity...     

Liquid chromatography-tandem mass spectrometry validated method for the estimation of indapamide in human whole blood

A highly precise and sensitive method for the estimation of indapamide in human whole blood using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) is described. The method developed is validated in human whole-blood matrix, with a sensitivity of 0.5 ng/ml as lower limit of quantification. The procedure for the extraction of indapamide and glimepiride as internal standard (IS) involves haemolysis and deprotienation of whole blood using ZnSO(4) followed by liquid-liquid extraction using ethyl acetate. The sample extracts after drying were reconstituted and analysed by LC-MS/MS, equipped with turbo ion spray (TIS) source, operating in the positive ion and selective reaction monitoring (SRM) acquisition mode to quantify indapamide in human whole blood. The mean recovery for indapamide was 82.40 and 93.23% for IS. The total run time was 2.5 min to monitor both indapamide and the IS. The response of the LC-MS/MS method for indapamide was linear over the range of 0.5-80.0 ng/ml with correlation coefficient, r>or=0.9991. The coefficient of variance (% CV) at 0.5 ng/ml was 4.02% and the accuracy was well within the accepted limit of +/-20% at 0.5 ng/ml and +/-15% at all other concentrations in the linear range. This method is fully validated for the accuracy, precision and stability studies and also applied to subject-sample analysis of bioequivalence study for 1.5mg sustained-release (SR) formulations.     

Liquid chromatography-tandem mass spectrometry method for determination of phencynonate in rat blood and urine

A sensitive and specific high-performance liquid chromatographic assay with electrospray ionization mass spectrometry detection (LC-ESI-MS) has been developed and validated for the identification and quantification of the novel anticholinergic drug phencynonate in rat blood and urine. The sample pretreatment involves basification and iterative liquid-liquid extraction with ethyl ether-dichloromethane (2:1, v/v) solution, followed by LC separation and positive electrospray ionization mass spectrometry detection. The chromatography was on BetaBasic-18 column (150 mm x 2.1mm i.d., 3 microm). The mobile phase was composed of methanol-water (85:15, v/v), containing 0.5 per thousand formic acid, which was pumped at a flow-rate of 0.2 ml/min. Thiencynonate was selected as the internal standard (IS). Simultaneous MS detection of phencynonate and IS was performed at m/z 358.4 (phencynonate), m/z 364 (thiencynonate), and the selected reaction ion monitoring (SRM) of the two compounds was at 156. Phencynonate eluted at approximately 5.25 min, thiencynonate eluted at approximately 5.10 min and no endogenous materials interfered with their measurement. Linearity was obtained over the concentration range of 1-100 ng/ml in rat blood and 1-500 ng/ml in rat urine. The lower limit of quantification (LLOQ) was reproducible at 1 ng/ml in both of rat blood and urine. The precision measured was obtained from 2.92 to 9.76% in rat blood and 4.17 to 9.76% in rat urine. Extraction recoveries were in the range of 69.57-79.49% in blood and 56.85-64.86% in urine. This method was successfully applied to the identification and quantification of phencynonate in pharmacokinetic studies.     

Liquid chromatography-tandem mass spectrometry method for routine measurement of oxalic acid in human plasma

A solid phase extraction (SPE)-LC-MSMS method for the routine determination of oxalic acid (OX) in plasma, a diagnostic marker of primary hyperoxaluria (PH), was developed and validated. The normal range of OX was found to be 3-11mumol/L (n=67), with no differences attributable to gender or age. The effect of pre-analytical factors on the in vitro production of OX was investigated, and plasma was found to be stable for 1-2h at room temperature, less after ingestion of vitamin C; the process was not completely stopped by preservation at either -20 or -70 degrees C.     

Liquid chromatography-tandem mass spectrometry method for determination of the pyridinium aldoxime 4-PAO in brain,liver, lung,and kidney

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was validated and applied to the in vitro determination of 4-[(hydroxyimino)methyl]-1-octylpyridinium cation (4-PAO), which can penetrate the blood–brain barrier and reactivate acetylcholinesterase (AChE) inhibited by alkylphosphonate in the brain, liver, lung, and kidney. The limit of detection (LOD) was 0.235 μg cation/g wet weight, and the quantification range and linearity of the calibration curve extended over a range of 0.470–941 μg cation/g wet weight. For the proof of applicability, when 4-PAO was administrated intravenously via the rat tail vein at 10% LD₅₀, we were able to quantify the 4-PAO concentration in the tissues: brain 7.60 ± 1.32 μg cation/g wet weight (mean ± SD, n = 5), liver 26.8 ± 2.82 μg cation/g, lung 76.4 ± 24.9 μg cation/g, and kidney 638 ± 266 μg cation/g. In addition, the methods for 4-[(hydroxyimino)methyl]-1-decylpyridinium bromide (4-PAD) and 4-[(hydroxyimino)methyl]-1-(2-phenylethyl) pyridinium bromide (4-PAPE) were partly validated referring to the findings of the 4-PAO full validation. Thus, the LC-MS/MS method described in this study can be useful for quantification of pyridinium aldoxime methiodide (PAM)-type oximes in biological samples.     

贯叶金丝桃叶中分泌细胞团的超微结构

随着贯叶金丝桃（Hypericum perforatum L.)叶中分泌细胞团的发育,其细胞中质体的数量和体积逐渐增大,但一些质体局部出现解体,大量的深色管状结构和小泡出现在退化质体的周围,有些小泡与液泡融合,并将其内容物释放至液泡中,导致液泡中出现大量的多泡结构,多膜结构和嗜锇滴。同时,高尔基体分泌小泡进入液泡。然而,当分泌细胞团发育成熟后,分泌细胞被含有灰色均匀的分泌物（金丝桃素）的大液泡所占据,嗜锇滴消失。表明嗜锇滴可能是金丝桃素的前体物,来源于退化的质体。出现于质体和嗜锇滴之间的内质网和高尔基体可能也参与了金丝桃素前体物的合成和细胞内的转运。     

贯叶金丝桃叶中分泌细胞团的超微结构

随着贯叶金丝桃( Hypericum perforatum L.)叶中分泌细胞团的发育,其细胞中质体的数量和体积逐渐增大,但一些质体局部出现解体,大量的深色管状结构和小泡出现在退化质体的周围,有些小泡与液泡融合,并将其内容物释放至液泡中,导致液泡中出现大量的多泡结构、多膜结构和嗜锇滴.同时,高尔基体分泌小泡进入液泡.然而,当分泌细胞团发育成熟后,分泌细胞被含有灰色均匀的分泌物(金丝桃素)的大液泡所占据,嗜锇滴消失.表明嗜锇滴可能是金丝桃素的前体物,来源于退化的质体.出现于质体和嗜锇滴之间的内质网和高尔基体可能也参与了金丝桃素前体物的合成和细胞内的转运.