Acid In Biological Specimens Research Articles

Forensic Toxicological Aspects of Misoprostol Use in Pharmacological Abortions.

The aim of this study was establishment of an UHPLC-QqQ-MS/MS method for the deter-mination of misoprostol acid in biological specimens in cases of pharmacological abortions. Forensic toxicological examination was performed in three different biological samples (whole blood, placenta and fetal liver). The validation parameters of the method were as follows: limit of detection: 25 pg/mL; limit of quantification: 50 pg/mL, coefficient of determination: >0.999 (R2), intra- and interday accuracy and precision: not greater than 13.7%. The recovery and matrix effect were in the range of 88.3–95.1% and from −11.7 to −4.9%, respectively. Toxicological analysis of the mother’s blood (collected two days after pregnancy termination) did not reveal any abortifacients; however, misoprostol acid was found in the placenta (793 pg/g) and fetal liver (309 pg/g). The second case involved a fetus found near a garbage container. The concentration of misoprostol acid in the placenta was 2332 pg/g. In the presented study, an extensive literature review of misoprostol pharmacokinetics studies was performed. To our knowledge, the UHPLC-QqQ-MS/MS technique presented in this paper is the first quantitative method applied for forensic toxicological purposes. In addition, postmortem concentrations of misoprostol acid in miscarried fetuses due to illegal abortions were reported for the first time.

Sensitive and Selective Determination of Orotic Acid in Biological Specimens Using a Novel Fluorogenic Reaction.

Orotic acid is an intermediate in the synthesis pathway of uridine-5'-monophosphate, and increases in body fluids of patients suffering from hereditary disorders such as orotic aciduria and hyperammonemia. In this study, we developed a spectrofluorometric method with or without high-performance liquid chromatography for the selective and sensitive quantification of orotic acid in human biological specimens, using 4-trifluoromethylbenzamidoxime (4-TFMBAO) as a fluorogenic reagent. This reagent provided intensive fluorescence for only orotic acid amongst 62 compounds including structurally related bio-substances such as nucleic acid bases, nucleosides, nucleotides, amino acids, vitamins, bilirubin, uric acid, urea, creatine, creatinine and sugars. Under optimized reaction conditions, orotic acid was reacted with 4-TFMBAO, K3[Fe(CN)6] and K2CO3 in an aqueous solution. The fluorescence produced from the orotic acid derivative was measured at an excitation of 340nm and an emission of 460nm. A concentration of 1.2μM orotic acid per 1.0mM creatinine in normal urine and 0.64nmol orotic acid per 5.0 × 10(5) HeLa cells were determined by this method. The present method permitted the facile quantification of orotic acid in healthy human urine and cultured HeLa cells by spectrofluorometry and/or high-performance liquid chromatography.

081 Ambient temperature stabilization of nucleic acids in human blood

Biospecimen stabilization during collection and transport is of critical importance in the fields of biomedical research and molecular diagnostics. The emergence of personalized medicine has placed an even greater emphasis on the significance of protecting the quality and reliability of clinical samples. The current methods for blood and saliva collection depend heavily on cold-chain logistics and are fraught with potential complications to specimen integrity that can result inconsistencies in sample analysis. Nucleic acids in biological specimens are threatened by nucleolytic attack, oxidative damage and hydrolysis. Such agents can severely disrupt sequencing, genotyping and expression profile analyses. In this report we describe the development of chemical stabilization formulations that address the problem of sampling induced inconsistencies in human blood and saliva. The ambient chemical Biostabilization describes the preserve of nucleic acids in these biospecimens even under extreme environmental shipping conditions, removing the need for cold-chain logistics during sample collection, transport or storage. Here we show that the genomic DNA in blood can be stabilized during extreme temperature fluctuations ranging from −20 °C to +45 °C. The analogous DNA stabilization technology for saliva specimens preserves sample integrity for over 12 months at ambient temperature. We also compare freezer storage with chemical stabilization of RNA in blood. Our results demonstrate that the complete transcriptome can be stabilized at ambient temperatures presenting an alternative to cold based preservation. Source of funding: None declared. Conflict of interest: None declared. [email protected]

Quantitative analysis of bile acids in human plasma by liquid chromatography-electrospray tandem mass spectrometry: a simple and rapid one-step method.

Bile acids play a pivotal role in the metabolism of cholesterol and lipids. Their blood concentrations are important prognostic and diagnostic indicators of hepatobiliary and intestinal dysfunction. This class of molecules comprises a heterogeneous group of compounds with a common cholesterol scaffold. Recently, the introduction of liquid chromatography coupled to tandem mass spectrometry methods has revealed an innovative path in the quantisation of specific bile acids in biological specimens. A robust and sensitive method has been developed based on high performance liquid chromatography separation coupled to an electrospray triple-quadrupole mass spectrometer. Human plasma samples were analysed on a C18 reverse-phase column. The elution profiles were monitored in multiple reaction-monitoring mode, quantifying and identifying each analyte by its own unique precursor to product patterns. A linear correlation over a broad range of bile acid concentrations (0.1-100 microM) was observed. The average recovery period for all of the analysed bile acids was 98 +/- 3%. Intra-day and inter-day precision averages were 2% and 5.4%, respectively. The determination was achieved within a single chromatographic run for all unconjugated, glycine- and taurine-conjugated isomeric forms of bile acids. As a proof of principle this method has been validated on a small subset of cholestatic patients (n = 7) and compared to appropriate clinical controls (n = 10). Based upon our encouraging experimental results, the described HPLC separation coupled to tandem mass spectrometry method for the analysis of bile acids in biological samples is deemed a robust and accurate procedure. Consequently, we propose this technique as a suitable candidate method for the identification and quantitation of bile acids in routine analysis.

Determination of Amino Acids by High-Performance Liquid Chromatography with Electrochemical Detection Using Ferrocene Derivatization Reagents

Abstract Two new reagents possessing ferrocene as an electrophore and isothiocyanate reactive toward the amino group were prepared and evaluated for pre-column derivatization of amino acids in high-performance liquid chromatography with electrochemical detection. The utilities of these reagents were investigated employing glycine as a model compound. Ferrocenylisothiocyanate was more favorable with respect to reactivity and electrochemical properties. The newly developed method was applied to the determination of 4-aminobutyric acid in biological specimens.

A reliable analysis of tissue free fatty acids by gas-liquid chromatography

A convenient and reliable gas-liquid chromatographic method for determining the free fatty acids in biological specimens is described. The free fatty acids were extracted with hexane in the presence of H 3PO 4 and then back-extracted from the hexane phase into a very small volume of trimethyl (α,α,α-trifluoro- m-tolyl)ammonium hydroxide solution. Direct injection of the resultant quaternary ammonium salts of the fatty acids into a gas-liquid chromatograph unit gave their methyl esters, with a high recovery. The presence of triglycerides, phospholipids, or cholesterol esters did not interfere with the determination of free fatty acids. This method was applied to determination of free fatty acids in the samples of serum or brain. The results were more precise and reliable than those reported with the conventional methods with TLC separation. This method should be a useful aid for providing precise information about the physiological or pathological roles of free fatty acids.

Analysis of volatile fatty acids in biological specimens by capillary gas chromatography

AbstractA method was developed to analyze and quantitate volatile fatty acids such as acetic, propionic, butyric, iso‐butyric, valeric, and iso‐valeric acid from samples of biological origin. A capillary column system including an automatic on‐column injection device as well as a precolumn of larger internal diameter than the analytical column was elaborated for this purpose.In order to obtain well resolved and correctly quantifiable chromatographic peaks it turned out to be essential to work under acidic/aqueous conditions. To achieve a better sample transfer into the chromatographic system an organic solvent had to be used together with the aqueous milieu, thus improving wetting properties of the liquid sample plug introduced into the column. Cold on‐column injection was applied in order to avoid discrimination of the various acids due to sample splitting and the automatic technique was chosen in view of the large number of samples from biological extractions which had to be analyzed.

Analysis of volatile fatty acids in biological specimens by capillary column gas chromatography.

A method was developed to analyze and quantify volatile fatty acids (VFA) such as acetic, propionic, butyric, isobutyric, valeric and isovaleric acids in biological specimens. To obtain good sample transfer into the chromatographic system an organic solvent had to be used together with an aqueous milieu, thus improving wetting properties of the liquid sample plug introduced into the column. Sample preparation was carried out under alkaline conditions in order to exclude or minimize sample losses due to sample transfer during the extraction and work-up procedure. A cold on-column injection was applied to avoid irregular discrimination of the various acids due to sample splitting and an automatic injection technique was used to accommodate the large number of samples generated from biological origin. Connection of a pre-column of wide internal diameter (0.53 mm) to the analytical column (0.32 m) was optimized and adapted to the nature of the injection solvent mixture consisting of acetonitrile, water and hydrochloric acid. To obtain well-separated and correctly quantifiable gas chromatographic peaks, it was essential to perform the chromatography under acidic aqueous conditions. Standard resolution conditions and response factors were evaluated. The chromatographic results of applying this method to biological specimens from both rats and humans are provided.

In Situ Localization of Ribonucleic Acids in Biological Specimens by Electron Energy Loss Spectroscopya

In Situ Localization of Ribonucleic Acids in Biological Specimens by Electron Energy Loss Spectroscopy<i>a</i>

The use of DNA probes in studies of human cytomegalovirus.

Hybridization assays provide a sensitive and rapid means for studying the molecular biology of viral replication and for identifying viral nucleic acid in biological specimens. Such assays are attractive because the detection of virus does not require intact virions or concomitant viral protein synthesis, both of which may be absent in a latently infected cell or in a virus-associated tumor. For molecular and clinical studies on human cytomegalovirus (HCMV), we have cloned and characterized subgenomic EcoRI fragments representative of the entire genome of HCMV strain AD169. To study the epidemiology of HCMV infections and to identify the presence of HCMV nucleic acid in urine, blood, Kaposi's sarcoma, and other tissues, we have used various hybridization techniques, including DNA dot/slot-blot hybridization, Southern blot hybridization, and in situ cytohybridization. These studies demonstrate how cloned molecular probes can be used to study the molecular biology, pathogenesis, and treatment of viral infections.

Detection of tuberculostearic acid in biological specimens by means of glass capillary gas chromatography-electron and chemical ionization mass spectrometry, utilizing selected ion monitoring.

Detection of tuberculostearic acid in biological specimens by means of glass capillary gas chromatography-electron and chemical ionization mass spectrometry, utilizing selected ion monitoring.

13] An improved method for measuring quinolinic acid in biological specimens

This chapter discusses the improved method for measuring quinolinic acid in biological specimens. In assay method, quinolinic acid is decarboxylated under acid conditions to nicotinic acid. It has been shown that this conversion is accomplished best, by using glacial acetic acid that results in a 97% conversion. Under the conditions used in this assay, the conversion is only 85% complete. It is important to maintain the oven temperature during decarboxylation at 105° to prevent decarboxylation from being any less complete. After decarboxylation to nicotinic acid, this compound can be measured, by the colorimetric method of Waisman and Elvehjem. When the reaction mixture of buffered sodium acetate solution (containing the nicotinic acid formed by the decarboxylation of quinolinic acid), ethanol, cyanogen bromide, and o -toluidine is incubated at 30°, the maximum color intensity is reached at 22 min. With another method, quinolinic acid is extracted from tissue by perchloric acid. It is absorbed onto charcoal, and then eluted with ammonium hydroxide. The dried ammonium hydroxide eluate is decarboxylated and measured. This method has been used effectively to measure quinolinic acid in urine and liver.

Rapid Determination of Valproic Acid in Biological Specimens

A now rapid method Is presented for the detection and quantltalion of valprolr acid (dipropyl acetic acid), VPA, In biological fluids. The method involves a single microextraction of the familiar acid-tungstate filtrate for the Isolation of VPA. No back extraction or cleanup steps are required. Onemicroliter portions of the microextract are Injected on a short, 3% OV-225 glass column, maintained isothermally at 110~ Detection is accomplished by a standard flame Ionization detector. Quantitation is achieved and precision is maintained through the use of an Internal standard (paramethadione) carried through the procedure.

Improved method for measuring quinolinic acid in biological specimens

A method for the determination of quinolinic acid in liver and urine has been described. The procedure involves extraction of the tissue with perchloric acid and absorption of the quinolinic acid with Norit-A followed by elution with ammonium hydroxide and decarboxylation in glacial acetic acid. The colorimetric method of Waisman and Elvehjem has been modified in order to increase the sensitivity of the method. With the above procedure as little as 20 μg of quinolinic acid can be measured in 25 gm of rat liver and as little as 10 μg of quinolinic acid in 50 ml of urine. We have found this procedure for the measurement of quinolinic acid to be more sensitive, reliable, and faster than the use of ion-exchange or thin-layer chromatography as described by others (8,9). With this assay we have been able to measure changes in the quinolinic acid level of the liver during physiological alterations (starvation and refeeding).