Determination of NG,NG-dimethyl-L-arginine, an endogenous NO synthase inhibitor, by gas chromatography-mass spectrometry

A fully validated gas chromatographic-mass spectrometric (GC-MS) method for the accurate and precise quantification of NG,NG-dimethyl-L-arginine (asymmetric dimethylarginine, ADMA), an endogenous inhibitor of the NO synthase, in cell culture supernatants and in small volumes of plasma is described. ADMA was concentrated by solid phase extraction and converted to its methyl ester pentafluoropropionic amide derivative. The derivatives were analyzed without any further purification. Using gas chromatography-chemical ionization mass spectrometry, fragment ions at m/z 634 and m/z 640 were obtained for ADMA and for NG,NG-[2H6]-dimethyl-L-arginine ([2H6]-ADMA) as internal standard, respectively. [2H6]-ADMA was synthesized by reaction of L-ornithine fastened at bromcyan-agarose with dimethylamine. The limit of detection of the method was 2 fmol, while the limit of quantitation for cell culture supernatants was 0.05 microM. The method was validated in a concentration range of 0-1.2 microM in cell culture medium and 0-2 microM in 50 microl aliquots of human plasma. The precision was > or =97% and the accuracy was determined to be > or =94%. This method is fast, rugged and an alternative to high performance liquid chromatography (HPLC) analysis of ADMA in cell culture supernatants and small volumes of human plasma.     

Detection and determination of reticuline and N-methylcoculaurine in the Annonaceae family using liquid chromatography-tandem mass spectrometry

In Guadeloupe, the French West Indies, there is a high incidence of atypical parkinsonism or progressive supranuclear palsy, and all of the investigated patients had taken herbal tea or tropical fruits of the Annonaceae family. Local inhabitants consume the fruits, and also drink tea made from the leaves. In the present study, we used liquid chromatography-tandem mass spectrometry (LC/MS/MS) with multiple reaction monitoring (MRM) to detect low-molecular-weight neurotoxic benzylisoquinoline derivatives in the Annonaceae family. We detected reticuline and N-methylcoculaurine in every Annona muricata sample examined, except for pulp and seed. They were not detected in sweetsop fruits. Norreticuline was not detected in any sample. These three compounds were toxic to SH-SY5Y neuroblastoma cells and inhibited mitochondrial respiratory complex I. It is possible that uptake of the benzylisoquinoline derivatives reticuline and N-methylcoculaurine and their accumulation in the brain may be related to the pathogenesis of the local endemic disease.     

Kinetic characteristics and toxic effects of benzalkonium chloride following intravascular and oral administration in rats

Kinetic characteristics and toxic effects of benzalkonium chloride (BZK) following injection via jugular vein (JV), femoral artery (FA) and oral administration (PO) were experimentally investigated using rats. The BZK concentrations in blood and tissues (lung, liver and kidney) were determined by high-performance liquid chromatography with solid phase extraction. Toxic doses of 15 and 250 mg/kg of BZK were used for intravascular (JV and FA) and PO administration, respectively. The fatal effects appeared soon after the dose in JV-rats, while delayed in FA- or PO-rats. The blood BZK concentrations and the elimination half-lives were similar between JV- and FA-rats, while the distribution of BZK in tissues was slightly different. In PO administration, the rats that aspirated BZK into their lungs had some symptoms, while the rats that did not aspirate BZK appeared to be normal. The BZK concentrations in blood and tissues were significantly higher in the aspirated PO-rats. The toxic degree of BZK was correlated with the BZK concentration in orally dosed rats. Lung and kidney had higher BZK concentrations compared to blood or liver, and they could be the target organs of BZK.Keyword: Benzalkonium chloride     

Stir bar sorptive extraction and thermal desorption-gas chromatography-mass spectrometry for the measurement of 4-nonylphenol and 4-tert-octylphenol in human biological samples

Alkylphenols, 4-nonylphenol (NP) and 4-tert-octylphenol (OP), in human urine and plasma samples were analyzed using stir bar sorptive extraction (SBSE) in combination with thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). The method involved correction by stable isotopically labeled surrogate standards, 4-(1-methyl)octylphenol-d5 (m-OP-d5) and deuterium 4-tert-octylphenol (OP-d). A biological sample was extracted for 60 min at room temperature (25 degrees C) using a stir bar coated with a 500 microm thick polydimethylsiloxane (PDMS) layer. Then, the stir bar was analyzed by TD-GC-MS in the selected ion monitoring (SIM) mode without any derivatization step. The average recoveries in human urine and plasma samples spiked with NP and OP at levels of 0.5 and 10 ng ml-1 were between 95.8 and 99.8% with correction using the added surrogate standards. The limits of quantitation were 0.2 ng ml-1 for NP and 0.02 ng ml-1 for OP. We measured the background levels of NP and OP in five human urine and three human plasma samples from healthy volunteers. NP and OP were not detected in all human urine samples (N.D. < 0.2 ng ml-1 for NP, and N.D. < 0.02 ng ml-1 for OP). However, 0.2-0.3 ng ml-1 for NP and 0.1-0.2 ng ml-1 for OP in human plasma samples were observed by this method.     

Synthesis of [(18)O(2)]valproic acid and its use as an internal standard for the quantitative measurement by gas chromatography-electron ionization mass spectrometry

A specific method for the quantitative determination of valproic acid in human plasma is presented. Valproate was extracted from acidified plasma by hexane extraction and converted to its trimethylsilyl derivative without sample concentration. The derivatives were analyzed without any further purification. Using gas chromatography-electron ionization mass spectrometry, diagnostic useful fragment ions at m/z 201 and 205 were obtained for valproic acid and [(18)O(2)]valproic acid internal standard, respectively. [(18)O(2)]Valproic acid was synthesized from unlabeled valproate by acid-catalyzed exchange reaction in H(2)(18)O. The method was validated in the expected concentration range of a pharmacokinetic study. Thus, calibration graphs were linear within a range of 0.47-120 microgram/ml plasma. Intra-day precision was 2.29% (0.47 microgram/ml), 2.93% (4 microgram/ml), 3.22% (20 microgram/ml) and 4.40% (80 microgram/ml), inter-day variability was found to be 1.49% (0.47 microgram/ml), 3.79% (20 microgram/ml), 2.74% (40 microgram/ml) and 3.03% (80 microgram/ml). Inter-day accuracy showed deviations of 1.94% (0.47 microgram/ml), 0.53% (4 microgram/ml), -0.32% (20 microgram/ml) and 0.06% (80 microgram/ml). The method is rugged and robust and has been applied to the batch analysis of valproate during pharmacokinetic profiling of the drug.     

Growth and development in school-age children from Rostov region,Russia: Comparison between urban and rural settings

The purposes of the current study were: (1) to describe growth and physical development and establish norms for schoolchildren from Rostov region in Russia; (2) to compare major characteristics of development between urban and rural children by sex and age.Nearly 200,000 children (198,712) aged between 7 and 17 years from 232 urban and rural schools of Rostov region (Southern Federal District of Russia) participated in the study. School age is a period of intensive growth and physiological and psychological development. Irregularities of personal development are caused by a multitude of factors, such as sex differences, heredity, socio-economic status of a family, standard of living, particular environmental conditions, and lifestyle.It has been established that children from the Southern Federal District of Russia had body mass index values higher than age-appropriate norms for all Russians (Total Russian, Rudnev et al., 2014) and World Health Organization charts. Children from urban settings were taller and heavier than children from rural settings.Sex is one of the most influential factors which play key role in determining specific characteristics of growth and personal development. According to our results, boys and girls both had similar age-related changes in weight and height, but their respective dynamics differed. Girls’ height and weight values accelerated at the age 10 to 12 years and plateaued after the age fourteen, whereas in boys height and weight steadily increased with age, showing slight acceleration at the age 12 to 13 years, and reached a plateau by the age of seventeen.     

Simultaneous determination of formate and acetate in whole blood and urine from humans using gas chromatography-mass spectrometry

We devised a sensitive and simple method for simultaneous determination of formate and acetate in whole blood and urine from humans using gas chromatography-mass spectrometry. Formate and acetate were alkylated with pentafluorobenzyl bromide in the mixture of acetone and phosphate buffer (pH 6.8). The derivatives obtained were analyzed using gas chromatography-mass spectrometry in positive-ion electron ionization (EI) mode. The lower limit of detection for both compounds was 0.02mM. The calibration curves for formate and acetate were linear over the concentration range from 0.05 to 5.0mM. Accuracy and precision of the method were evaluated and the coefficients of variation were within 10%. With use of this method, levels of formate and acetate in whole blood can be determined in forensic cases.     

Evaluation of the contribution of D9S1120 to anthropological studies in Native American populations

The D9S1120 locus exhibits a population-specific allele of 9 repeats (9RA) in all Native American and two Siberian populations currently studied, but it is absent in other worldwide populations. Although this feature has been used in anthropological genetic studies, its impact on the evaluation of the structure and genetic relations among Native American populations has been scarcely assessed. Consequently, the aim of this study was to evaluate the anthropological impact of D9S1120 when it was added to STR population datasets in Mexican Native American groups. We analyzed D9S1120 by PCR and capillary electrophoresis (CE) in 1117 unrelated individuals from 13 native groups from the north and west of Mexico. Additional worldwide populations previously studied with D9S1120 and/or 15 autosomal STRs (Identifier kit) were included for interpopulation analyses. We report statistical results of forensic importance for D9S1120. On average, the modal alleles were the Native American-specific allele 9RA (0.3254) and 16 (0.3362). Genetic distances between Native American and worldwide populations were estimated. When D9S1120 was included in the 15 STR population dataset, we observed improvements for admixture estimation in Mestizo populations and for representing congruent genetic relationships in dendrograms. Analysis of molecular variance (AMOVA) based on D9S1120 confirms that most of the genetic variability in the Mexican population is attributable to their Native American backgrounds, and allows the detection of significant intercontinental differentiation attributed to the exclusive presence of 9RA in America. Our findings demonstrate the contribution of D9S1120 to a better understanding of the genetic relationships and structure among Mexican Native groups.     

Effects of atrazine on osmoregulation in the Chinese mitten crab,Eriocheir sinensis

This study integrates results from acute contamination with atrazine of isolated perfused gills, and from in vivo chronic contamination of euryhaline Chinese mitten crabs, Eriocheir sinensis, acclimated to freshwater. Atrazine 1 mg/l in contact with the basolateral membrane (IN) increases the transepithelial potential difference (TEP) from -20.8 +/- 4.9 to -29.7 +/- 3.8 mV in isolated perfused posterior gills (P < 0.01). This effect is only partially explained by a modification of Na(+) and Cl(-) active influxes. No TEP modification is detected when atrazine is added (OUT) indicating that molecular mechanisms located on the basolateral membrane are likely to be the only ones affected. Another explanation would be that cuticular barrier prevents atrazine penetration into the gill. Haemolymph osmolarity, Na(+) and Cl(-) concentrations of crabs living in freshwater contaminated with atrazine 1 mg/l during 14 days are not significantly modified. We conclude that although atrazine can disturb osmoregulatory mechanisms of isolated gills, this pollutant would be of minor importance in affecting osmoregulatory capacities of the Chinese mitten crab in natural conditions.     

Liquid chromatographic method for detection and quantitation of STI-571 and its main metabolite N-desmethyl-STI in plasma, urine, cerebrospinal fluid, culture medium and cell preparations

An isocratic online-enrichment HPLC-assay was developed allowing for the simple and fast separation and quantitation of STI-571 and its main metabolite N-desmethyl-STI (N-DesM-STI) in plasma, urine, cerebrospinal fluid (CSF), culture media and cell preparations in various concentrations using UV-detection at 260 nm. The analytical procedure consists of an online concentration of STI-571 and N-DesM-STI in the HPLC system followed by the elution on a ZirChrom-PBD analytical column. Time of analysis is 40 min including the enrichment time of 5 min. The detection limit is 10 ng/ml in plasma, CSF, culture medium (RPMI) and 25 ng/ml in urine for both STI-571 and N-DesM-STI. The intra-day precision, as expressed by the coefficient of variation (CV), in plasma samples ranges between 1.74 and 8.60% for STI-571 and 1.45 and 8.87% for N-DesM-STI. The corresponding values for urine measurements are 2.17-7.54% (STI-571) and 1.31-9.51% (N-DesM-STI). The inter-day precision analyzed over a 7-month time period was 8.31% (STI-571) or 6.88% (N-DesM-STI) and 16.45% (STI-571) or 14.83% (N-DesM-STI) for a concentration of 1000 ng/ml in plasma and 750 ng/ml in urine, respectively. Moreover, we demonstrate that with an alternative, but more time and labor consuming sample preparation and the implementation of electrochemical detection, a detection limit < 10 ng/ml can be achieved. The method described was used to perform pharmacokinetic measurements of STI-571 and N-desmethyl-STI in patient samples and for kinetic measurements of intracellular STI-571 and N-DesM-STI following in vitro incubation.     

Analysis of N,N-diethyl-m-toluamide in porcine skin perfusates using solid-phase extraction disks and reversed-phase high-performance liquid chromatography

N,N-Diethyl-m-toluamide (DEET) is frequently used as an insect repellent by military and civilian populations. Because dermal exposure has resulted in several cases of DEET toxicosis, there is a need to rapidly and reliably determine DEET concentrations in biological matrices. An improved method for the analysis of DEET was developed for determining transdermal diffusion of low levels of DEET following application to an in vitro porcine skin flow-through diffusion cell system. The technical improvement involved the use of disk solid-phase extraction (SPE) instead of packed-bed SPE. The disk SPE method required small volumes of preconditioning, wash, and elution solvent (0.5-1 ml) to extract DEET from perfusate samples containing bovine serum albumin (BSA). The limit of quantitation (LOQ) was estimated as 0.08 micro g/ml DEET and recoveries from BSA media samples spiked with DEET ranged from 90.1 to 117% with relative standard deviation (RSD) ranging from 2.0 to 13.1%. This method was used to analyze perfusate samples from skin (n=4) topically exposed to DEET-ethanol formulations. The data from these analyses determined that DEET permeability in porcine skin was 2.55 x 10(-5)+/-0.54 x 10(-5) cm/h.     

Determination of bromide in whole blood and urine from humans using gas chromatography-mass spectrometry

We devised a sensitive and simple method for determination of bromide in whole blood and urine from humans using gas chromatography-mass spectrometry. Bromide was alkylated with pentafluorobenzyl p-toluenesulphonate in the mixture of acetone and phosphate buffer (pH 6.8). The derivative obtained was analyzed using gas chromatography-mass spectrometry with the positive-ion EI mode. The lower limit of detection for the compound was 1 mg/l. The calibration curve for bromide was linear over the concentration range from 2 to 100 mg/l. With use of this method, levels of bromide in whole blood and urine were determined in cases of poisoning by inhaled brominated hydrocarbons.     

Sex-specific reliability and multidimensional stability of responses to tests assessing neuromuscular function

The objective of this study was to estimate sex-specific effects in the test–retest cross-reliability of peripheral and central changes in nonlinear and linear measures of a surface electromyography signal during a brief (5 second) and sustained (2 minute) isometric maximal voluntary contraction, combined with superimposed electrical stimulation involving the ankle plantar flexors over five identical trials. In this study, we repeated the testing protocol used in our previous study of 10 women (age 20.9, SD = 0.3 years) (Bernecke et al., 2015) in a group of 10 men (age 21.2, SD = 0.4 years). Despite the central (sex effect; p < 0.05, η_p² > 0.71, SP > 70%) and peripheral fatigability (sex effect; p < 0.01, η_p² > 0.8, SP > 90%) during sustained isometric maximal voluntary contraction, and lower reliability for central activation ratio during brief (intraclass correlation coefficient [ICC] = 0.95 for men and ICC = 0.82 for women) and sustained maximal voluntary contraction (ICC > 0.82 for men and ICC > 0.66 for women) over ankle plantar flexors expressed in women more than in men, all the ICCs of all indices measured by tests assessing neuromuscular function across the five identical test–retest trials were found as meaningful (correlation significance of p < 0.05 was reached) and no significant differences were found between trials for any of the measured variables. In conclusion, the present study demonstrated greater central and peripheral fatigue for female participants following sustained (2 minute) isometric maximal voluntary contraction of the plantar flexor muscles for all repeated trials and indicated an acceptable agreement between measurements of the characteristic variables made using the three different devices (dynamometry, electrical stimulation, and surface electromyography) over time for both sexes.     

Chiral liquid chromatography resolution and stereoselective pharmacokinetic study of the enantiomers of a novel anticonvulsant, N-(4-chlorophenyl)-1-(4-pyridyl)ethylamine, in rats

A selective chiral high performance liquid chromatographic (HPLC) method was developed and validated to separate and quantify the enantiomers of a novel anticonvulsant agent, N-(4-chlorophenyl)-1-(4-pyridyl)ethylamine (AAP-Cl), in rat plasma. After extraction of the plasma samples with ethyl acetate, the separation was accomplished by an HPLC system consisting of a Chirex chiral column (250 mm x 4.6 mm i.d.) and a mobile phase of hexane:ethanol:tetrahydrofuran (280:20:40 (v/v)) containing trifluroacetic acid (0.3% (v/v)) and triethylamine (0.018% (v/v)) at a flow rate of 0.8 ml/min with UV detection. Male Sprague-Dawley rats were given (+)-AAP-Cl (10 and 20 mg/kg), (-)-AAP-Cl (10 mg/kg) or the racemic mixture (20 mg/kg) by i.v. bolus injection and serial blood samples were collected at different times after drug administration. (+)-AAP-Cl and (-)-AAP-Cl were separated with a resolution factor, Rs, of at least 1.4, and a separation factor, alpha, greater than 1.09. Linear calibration curves were obtained over the concentration range of 0.5-30 microg/ml in plasma for both (+)-AAP-Cl and (-)-AAP-Cl (R2 > or = 0.996) with a limit of quantitation of 100 ng/ml and the recovery was greater than 80% for both enantiomers. The accuracy and precision for both enantiomers ranged from 96 to 102% (+/-0.2-7%) at upper and lower concentrations. The plasma concentration-time profiles of the enantiomers of AAP-Cl were best described by a two-compartment open model with a mean terminal half-life of about 5h, volume of distribution at steady state of 3 l/kg and clearance of about 0.6l/(hkg) in rats. There was no significant difference between the pharmacokinetic parameters of (+)-AAP-Cl and (-)-AAP-Cl, suggesting that the disposition of AAP-Cl in rats is not enantioselective. In addition, no chiral inversion of (+)-AAP-Cl to (-)-AAP-Cl or vice versa was observed. The results of this investigation have shed some light on the mechanism of action and disposition of AAP-Cl in rats.     

Antioxidant,anticancer, apoptosis properties and chemical composition of black truffle Terfezia claveryi

Desert truffles are seasonal and important edible fungi that grow wild in many countries around the world. Truffles are natural food sources that have significant compositions. In this work, the antioxidant, chemical composition, anticancer, and antiangiogenesis properties of the Terfezia claveryi truffle were investigated. Solvent extractions of the T. claveryi were evaluated for antioxidant activities using (DPPH, FRAP and ABTS methods). The extracts cytotoxicity on the cancer cell lines (HT29, MCF-7, PC3 and U-87 MG) was determined by MTT assay, while the anti-angiogenic efficacy was tested using ex-vivo assay. All extracts showed moderate anticancer activities against all cancer cells (p < 0.05). The hexane extract inhibited the brain cell line (U-87 MG) with an IC₅₀ of 50 μg/ml and significantly promoted cell apoptosis through the mitochondrial pathway and DNA fragmentation p < 0.001. The ethanol extract demonstrated potent antioxidants; DPPH, FRAP, and ABTS with an IC₅₀ value of 52, 48.5 and 64.7 μg/ml, respectively. In addition, the hexane and ethyl acetate extract significantly (p < 0.001) inhibited the sprouting of microvessels by 100% and 81.2%, at 100 μg/ml, respectively. The GC analysis of the most active extract (hexane) showed the presence of several potent phytochemicals such as stigmasterol, beta-Sitosterol, squalene, lupeol, octadecadienoic acid, and oleic acid.