Flow injection chemiluminescence determination of l-cysteine in amino acid mixture and human urine with the BrO3 −–quinine system

In this paper, a novel flow injection chemiluminescence (CL) determination of l-cysteine is proposed. The method is based on the CL reaction of l-cysteine and KBrO₃ in acidic medium. The CL intensity was greatly enhanced in the presence of quinine. The CL intensity was linear with l-cysteine concentration in the range of 0.2–80 g L^–1, and the detection limit was 0.1 g L^–1 (3). A complete analysis, including sampling and injecting, could be performed in 1 min, giving a throughput of about 60 h^–1. The relative standard deviation was 1.6% for 0.8 g L^–1 l-cysteine (n=11). The proposed method was satisfactorily applied to the determination of cysteine in an amino acid mixture and human urine. The mechanism of the CL reaction is also discussed.     

Determination of the Enantiomeric Purity of l-Carnitine and Acetyl-l-Carnitine by Chiral Liquid Chromatography

A chiral liquid chromatographic method for determination of the enantiomeric purity of both l-carnitine and acetyl-l-carnitine is described. Separation of the enantiomers of dl-carnitine and acetyl-dl-carnitine was achieved on a commercial chiral column (Chiralcel OD-R) after derivatization with (alpha-bromo)methyl phenyl ketone. Introduction of this lipophilic UV chromophoric group to the carnitine and acetylcarnitine molecules improved their retention, resolution, and UV detection. The mobile phase was 74:26 (v/v) 0.5 mol L^-1 sodium perchlorate–acetonitrile, pH 3.8, and the flow rate was 0.4 mL min^-1. Detection was performed at 235 nm. The method is selective and reliable for determination of the enantiomeric purity of bulk drug substances l-carnitine and acetyl-l-carnitine.     

Mechanistic studies on the conjugation of methyl isocyanate with N-acetyl-cysteine in rats

In order to investigate the utility of selected thiols as scavengers of MIC, we first assessed the chemical stability of SMG, AMCC and SMC by measuring their rates of reaction in vitro with thiophan. The inital rates of carbamoylation of thiorphan (0.5 mM) by the above conjugates (0.5 mM) in aqueous buffer at pH 7.4 and 37°C were 2.51, 0.76 and 8.47 mol L^–1 min^–1, respectively, indicating that the mercapturate AMCC was the most stable of the three MIC conjugates.In light of these results, studies were conducted to examine the effect of pretreatment withN-acetyl-l-cysteine (l-NAC; 500 mg kg^–1, i.p.) on the urinary elimination of AMCC in rats dosed with MIC (15 mg kg^–1, i.p.). In separate experiments, groups of rats were pretreated with eitherN-acetyl-d-cysteine (d-NAC) orN-trideuteroacetyl-l-cysteine (d₃-l-NAC) in order to explore the mechanism by which MIC undergoes conjugation to AMCCin vivo. The results indicated that exogenous NAC effectively enhances the urinary excretion of MIC in the form of AMCC, and that it does so largely by direct conjugation with the isocyanate, rather thanvia biosynthesis to GSH.     

Determination of serum <Emphasis Type="SmallCaps">d</Emphasis>-lactic and <Emphasis Type="SmallCaps">l</Emphasis>-lactic acids in normal subjects and diabetic patients by column-switching HPLC with pre-column fluorescence derivatization

d-Lactic and l-lactic acids were simultaneously determined by means of a column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. As a fluorescence reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was employed for the fluorescence derivatization of lactic acid. The proposed HPLC system adopted both octylsilica (Cadenza CD-C8) and amylose-based chiral columns (CHIRALPAK AD-RH), which proved to give a sufficient enantiomeric separation of the lactic acid derivatives with a separation factor () of 1.32 and a resolution (R_s) of 1.98. Moreover, the features of the first elution of d-lactic acid peak in the proposed HPLC were convenient for the determination of trace amount of serum d-lactic acid, which is known to increase under diabetes. Intra-day and inter-day accuracies were in the range of 90.5–101.2 and 89.0–100.7%, and the intra-day and inter-day precisions were 0.3–1.2 and 0.4–4.8%, respectively. The proposed method was applied to determine d-lactic and l-lactic acids in human serum of normal subjects and diabetic patients, showing that both d-lactic and l-lactic acid concentrations were significantly increased in the serum of diabetic patients (n=31) as compared with normal subjects (n=21). This fact was found for the first time owing to the development of the proposed HPLC method which is able to determine d-lactic and l-lactic acid simultaneously. Finally, serum d-lactic acid concentrations determined by the proposed HPLC method were compared with those from a reported enzymatic assay, and the smaller p value between normal subjects and diabetic patients was shown by the proposed HPLC method.     

Quantitative Bestimmung von l-Carnitin mit Hilfe von Carnitindehydrogenase aus Pseudomonas putida

Zusammenfassung Es wird eine Methode beschrieben, die eine quantitative Bestimmung von l-Carnitin durch Einsatz von Carnitindehydrogenase aus Pseudomonas putida in einem einfachen optischen Test bei 340 nm ermöglicht. Bei Durchführung der Reaktion im pH-Bereich zwischen 9,95 und 10,05 kann man nach einer Inkubation bei 30° C für 30 min einen Umsatz von mehr als 99% erreichen, wenn mindestens 2 U Enzym eingesetzt werden. Eine Linearität zwischen l-Carnitinkonzentration und Extinktionsdifferenz ist allerdings nur bis zu einer Konzentration von 25–30 mol/l gegeben. Bei Kopplung der Reaktion mit Phenazinmethosulfat und Iodnitrotetrazoliumchlorid kann ein quantitativer Umsatz bereits im pH-Bereich von 8–9 erzielt werden. Die Konzentrationszunahme des sich bildenden Formazans kann bei 500 nm verfolgt werden. Es können so noch etwa 2 nmol l-Carnitin sicher nachgewiesen werden.

---

Enzymatic determination of l-carnitine with carnitine-dehydrogenase from Pseudomonas Putida

Summary The use of carnitine-dehydrogenase from Pseudomonas putida for the spectrophotometric determination (= 340 nm) of l-carnitine in amounts of 5–50 nmol is described. More than 99% of l-carnitine is oxidized in the presence of 3.2 mol NAD⁺ and 2U of the enzyme in glycine buffer pH 9.95–10.05. If the NADH formed is reoxidized by phenazine methosulfate and tetrazolium salt, the formazan production (=500 nm) is proportional to l-carnitine already at pH 8–9. It is possible to detect up to 2 nmol of l-carnitine in this test system.

     

Über die Raney-Nickel-Entschwefelung schwefelhaltiger cyclischer Dipeptide

Raney nickel desulfuration of 3.6-epidithio-l-prolyl-l-proline anhydride andcis-3.6-bis(ethylthio)-l-prolyl-l-proline anhydride yields racemiccis-prolyl-proline anhydride, whereas after the reaction of 3-ethylthio-l-prolyl-l-proline anhydride no inversion of configuration was observed.—During the action of Raney nickel on one asymmetric center the configuration is only retained if steric factors inhibit an inversion.

---

---

Herrn Prof. Dr.F. Hecht mit herzlichen Grüßen gewidment.     

Comparative study on the determination of cephalexin in its dosage forms by spectrophotometry and HPLC with UV-vis detection

This paper discusses the spectrophotometric determination of cephalexin as the intact cephalexin or as its acid-induced degradation product. Cephalexin can be determined in the range 1 × 10^–5–18 × 10^–5 M with relative standard deviations of 5-1%. The limits of quantitation and detection were 10^–5 and 0.3 × 10^–5 M, respectively. These procedures were compared with reversed-phase HPLC determination. No interference was observed in the presence of common pharmaceutical adjuvants. The H-point standard additions method was applied in order to correct for the possible presence of the cephalexin precursor, 7-aminocephalosporanic acid; this improves the selectivity of the UV-vis spectrophotometric method.     

Third-order derivative spectrophotometric determination of phosphate as ternary complex with molybdate and rhodamine 6G

This paper describes a third-order derivative Spectrophotometric procedure for the determination of traces of phosphate, based on the formation of a ternary ion-association complex by reaction of phosphate with rhodamine 6G in the presence of molybdate. The pink complex is readily formed in 0.08–0.16M H₂SO₄ medium and remained stable for 2 h. The influence of various instrumental parameters and reaction conditions for maximum colour development are discussed. The few interfering ions can be masked by the addition of appropriate reagents. The procedure is simple, rapid and reliable and allows the determination of 1.5 g l^–1 of phosphate. The relative standard deviation for the determination of 0.08 mg l^–1 of phosphate was 1.1%. The method is applicable to the determination of phosphate in river water, sea water, drinking water, milk powder and standard reference materials.     

Determination of pipemidic acid based on flow-injection chemiluminescence due to energy transfer from peroxynitrous acid synthesized on-line

A flow-injection chemiluminescence (CL) method for the determination of pipemidic acid is described. It is based on energy transfer from excited state peroxynitrous acid to pipemidic acid, in which the excited state peroxynitrous acid is synthesized on-line by the mixing of acid hydrogen peroxide with nitrite in a flow system and the CL is from two excited states of pipemidic acid. The proposed method allows the measurement of pipemidic acid over the range of 2.0×10^–7–2.0×10^–5 mol l^–1 . The detection limit is 6.3×10^–8 mol l^–1, and the relative standard deviation for 2.0×10^–6 mol l^–1 pipemidic acid (n= 9) is 0.9%. This method was evaluated by the analysis of pipemidic acid in pharmaceutical preparations.     

Enzymatische Bestimmung von l-Tryptophan mit Hilfe der Chorismatmutase aus Claviceps paspali

Zusammenfassung Eine enzymatische Methode zur genauen und empfindlichen Bestimmung von l-Tryptophan wird beschrieben, die auf der Aktivierung von Chorismatmutase beruht. Das über DEAE-Cellulose gereinigte Enzym wird durch l-Tryptophan schon in Konzentrationen von über 10^–7 Mol/l aktiviert. Der günstigste Meßbereich liegt zwischen 0,08 und 2,0 g l-Tryptophan/ml. Kleinere Versuchsansätze sind möglich. Der enzymatische Test wird zweckmäßig bei pH 8 ausgeführt, da bei diesem pH-Wert eine relativ große Aktivierung beobachtet wird.

---

Enzymatic determination of l-tryptophan by means of chorismate-mutase from claviceps paspali

An exact and sensitive enzymatic method for the determination of l-tryptophan is based on the activation of chorismatemutase. The enzyme purified by DEAE-cellulose chromatography is activated by l-tryptophan already in concentrations above 10^–7 Mol/l. The most favourable concentration is between 0.08 and 2.0 g of l-tryptophan/ml. Determinations can also be performed in smaller scale. The enzymatic test is performed at pH 8, as at this pH-value a relatively large activation can be observed.

     

Effect of sample preparation methods on the D,L-enantiomer ratio of extracted selenomethionine

Effects of the two most widespread sample preparation techniques on the d,l-enantiomer ratio of extracted selenomethionine were monitored through the analysis of the certified reference material selenium-enriched yeast and the isolated protein fraction of high selenium monkeypot nut. The extracted selenomethionine (SeMet) fractions were orthogonally cleaned up with anion exchange chromatography before carrying out the enantiomer-specific detection to increase the robustness and the efficiency of the subsequent o-phthal-aldehyde and n-isobutyril-cysteine-based derivatisation process and reversed phase-high-performance liquid chromatography-inductively coupled plasma mass spectroscopy (ICP-MS) detection. The two techniques, namely methanesulphonic acid (MSA) based digestion and proteolytic digestion with protease XIV, resulted in significantly different ratio of d,l-selenomethionine with the final results of 2.2–2.7% and 0.5–0.6% of d-SeMet, respectively. The study revealed significant differences in the ICP-MS-related sensitivity of the derivatised selenomethionine enantiomers, which calls attention to the quantification of this selenoamino acid after MSA hydrolysis.     

Sensitive spectrofluorimetric determination of ruthenium at nanotrace levels using 2-(α-pyridyl) thioquinaldinamide [PTQA]

A new spectrofluorimetric method for the determination of ruthenium with nonfluorescent 2-(α-pyridyl) thioquinaldinamide (PTQA) is described. The oxidative reaction of Ru(III) upon PTQA gives oxidised fluorescent product (λ_ex(max)=347 nm; λ_em(max)=486 nm). The sensitivity of the fluorescence reaction between ruthenium and PTQA is greatly increased in the presence of Fe (III). The reaction is carried out in the acidity range 0.01–0.075 M H₂SO₄. The influence of reaction variables is discussed. The range of linearity is 1–400 μg l⁻¹ Ru(III). The standard deviation and relative standard deviation of the developed method are ±1.210 μg l⁻¹ Ru (III) and 2.4%, respectively (for 11 replicate determinations of 50 μg l⁻¹ Ru (III)). The effect of interferences from other metal ions, anions and complexing agents was studied; the masking action is discussed. The developed method has been successfully tested over synthetic mixtures of various base metals and platinum group metals, synthetic mixtures corresponding to osmiridium, certified reference materials in spiked conditions and rock samples.     

Products and kinetics of the thermal decomposition of (l-(−)-menthyl)[2,2′-methylene-bis-(4-methyl-6-tert-butylphenyl)] phosphite ozonide

Two conformers (chair, boat) of [l-(–)-menthyl)]-[2,2-methylene-bis-(4-methyl-6-tert-butylphenyl)] phosphite ozonide have been obtained by the low temperature ozonization (–80 °C) of [l-(–)-menthyl)]-[2,2-methylene-bis-(4-methyl-6-tert-butylphenyl)] phosphite. It was determined that decomposition of the ozonide is first order with the rate constant logk ₀ = (10.92±1.10)–(14.02±1.25)/gq ( = 2.303RT, kcal mol^–1), leading to [l-(–)-menthyl)]-[2,2-methylene-bis-(4-methyl-6-tert-butylphenyl)] phosphate and oxygen (including singlet oxygen). Conformational transitions (chair-boat) for [l-(–)-menthyl)]-[2,2-methylene-bis-(4-methyl-6-tert-butylphenyl)] phosphate have been registered by³¹P NMR spectroscopy.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 10, pp. 1758–1761, October, 1994.This work was supported by Russian Foundation for Basic Research (Project No. 93-03-532l).     

Optical enzyme sensor for determining <Emphasis Type="SmallCaps">l</Emphasis>-lysine content using <Emphasis Type="SmallCaps">l</Emphasis>-lysine oxidase from the rockfish <Emphasis Type="BoldItalic">Sebastes schlegeli</Emphasis>

l-Lysine (l-Lys) in living bodies is critical for metabolism; therefore, determination of its levels in food is important. Most enzymatic methods for l-Lys analysis are performed using l-lysine oxidase (LyOx), but commercially manufactured LyOx is generally not highly selective for l-Lys among amino acids. We previously isolated LyOx as an antibacterial protein secreted from the skin of the rockfish Sebastes schlegeli. In the present study, we developed an optical enzyme sensor system for rapid and continuous determination of l-Lys using this LyOx. The system comprised an immobilized LyOx membrane, an optical oxygen probe, a flow system, and a personal computer. The amount of l-Lys was detected as a decrease in the oxygen concentration due to the LyOx reaction. The specificity of the sensor was examined against various amino acids. The sensor response was specific for l-Lys. Good reproducibility was obtained in 58 assays. The response of the sensor using commercially prepared LyOx was unstable compared with the response using LyOx isolated in our laboratory. Our sensor system could be used for 5 weeks without our having to change the enzyme membrane. The calibration curve for a standard l-Lys solution was linear from 0.1 to 3.0 mmol L⁻¹. One assay could be completed within 2 min. The sensor was applied to determine the l-Lys content in food samples such as bonito cooking water and scallop hepatopancreas. The values obtained using the sensor and conventional high-performance liquid chromatography methods were well correlated.     

Multiresidue analysis of phenylurea herbicides in environmental waters by capillary electrophoresis using electrochemical detection

A rapid multiresidue method has been developed for the analysis of seven phenylurea herbicides in the presence of two s-triazines in environmental waters. A simple end-column electrochemical detector was used in combination with a commercially-available capillary electrophoresis instrument with UV detection. The determination of phenylurea pesticides using micellar electrokinetic capillary chromatography with electrochemical detection represents the first such determination that has been reported. In both detection systems, linear ranges were obtained for the seven phenylurea herbicides at concentrations lower than 2.0×10^–5 mol l^–1, in 0.020 mol l^–1 phosphoric acid at pH 7.0 and containing 0.020 mol l^–1 of sodium dodecylsulfate, in order to obtain selectivity in the additional separation by a micellar distribution process. Under these conditions a detection limit lower than 5.0×10^–6 mol l^–1 (0.25 pmol of pesticide) was achieved for most of them. The pesticides were resolved in less than 30 min.     

A new method for the determination of free L-carnitine in serum samples based on high field single quantum coherence filtering 1H-NMR spectroscopy

The rapid and accurate determination of specific metabolites present in biofluids is a very demanding task which is essential in both medicine and chemistry. l-carnitine (3-hydroxy-4-N-trimethylammonium butyrate) is an important metabolite which participates in a series of biological paths and therefore its determination is of diagnostic importance. A single quantum coherence filtering ¹H NMR methodology was used for the accurate and rapid determination of l-carnitine in human serum samples. The methodology is based on spectral simplification, and specifically on the distinction of the N-methyl proton signal of l-carnitine that is greatly overlapped in the ¹H-NMR spectrum of serum. The quantitative results provided by the proposed method are in excellent agreement with those obtained by the enzymatic method, which is widely used. The proposed method is rapid (~20 min of experimental time), selective, sensitive, and has good analytical characteristics (accuracy, reproducibility). Selected protein precipitation methods were also investigated and sample pretreatment with EtOH is suggested.     

Thermal lens spectrometry as a tool for determination of stability constants of complex compounds

The potential of thermal lens spectrometry in the determination of stability constants of complex compounds was explored using copper(I) and iron(II) complexes with 1,10-phenanthroline and 2,9-dimethyl-1,10-phenanthroline as examples. Thermal lens spectrometry offers advantages over conventional spectrophotometry in the determination of stability constants both in aqueous and nonaqueous media. The overall and stepwise stability constants of iron(II) tris(1,10-phenanthrolinate), copper(I) bis(2,9-dimethyl-1,10-phenanthrolinate), and copper(I) bis(1,10-phenanthrolinate) were determined at levels as low as 10⁻⁸–10⁻⁶ mol L⁻¹.__________Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 123–133, January, 2005.     

Dimericd- anddl-dysprosium(iii) tartrates: paramagnetic birefringence,molecular mechanics,stereoselectivity

The molar constants of paramagnetic birefringence (PBR) for the dimeric dysprosium(iii)d- anddl-tartrates, Dy₂(d-Tart)(l-Tart)^2– (1) and Dy₂(d-Tart)₂ ^2– (2) have been determined by means of pH-metric and PBR measurements. The simulation of the structure of the ligand and solvate environment has been carried out using the method of molecular mechanics (Dashevsky-Plyamovaty model, the MIND program). In addition to the four oxygen atoms from the ligand, each Dy^III ion coordinates four molecules of water and a Na⁺ ion.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 6, pp. 1029–1032, June, 1994.     

Simple LC Method with Chemiluminescence Detection for Simultaneous Determination of Arbutin and l-Ascorbic Acid in Whitening Cosmetics

Simultaneous determination of arbutin (ART) and l-ascorbic acid (AA) by HPLC with chemiluminescence detection is proposed for the first time. This method is based on the CL reaction of acidic potassium permanganate with ART and AA in the presence of formaldehyde as enhancer. The separation was performed on a C₁₈ column with a 90:10 (v/v) mixture of 0.02 M phosphate buffer and methanol as mobile phase. The effects of several conditions on HPLC resolution and CL emission were studied systematically. The linear ranges were 0.5–50 and 1–200 μg mL⁻¹ for ART and AA, respectively. The detection limits were 0.2 and 0.3 μg mL⁻¹, respectively. The method was successfully applied to the determination of ART and AA in whitening cosmetics.     

Separation ofN-carbamoyl aspartate andl-dihydroorotate from serum by high-performance liquid chromatography with fluorimetric detection

Summary A high-performance liquid chromatographic method, with 9-anthryldiazomethane as derivatizing agent, has been developed for the simultaneous determination ofN-carbamoyl aspartate andl-dihydroorotate in serum. Sample preparation for 1 mL serum was by simple liquid-liquid extraction and then derivatization. The compounds were separated on a Luna C18(2) column by use of a gradient prepared from acetonitrile and 10 mM sodium acetate buffer, pH 6.0, and fluorimetric detection was performed at excitation and emission wavelengths of 365 nm and 412 nm, respectively. The response was found to be linearly dependent on concentration between 0.8 and 60 μg mL⁻¹ forl-dihydrooratate and between 0.9 and 90 μg mL⁻¹ forN-carbamoyl aspartate; the mean recovery rates were 50 and 51%, respectively. The limits of detection and quantification were 0.33 μg mL⁻¹ and 0.6 μg mL⁻¹, respectively, forl-dihydroorotate and 0.4 μg mL⁻¹ and 0.7 μg mL⁻¹ forN-carbamoyl aspartate. This method can be used to assess accumulation ofN-carbamoyl aspartate andl-dihydroorotate in body fluids in situations where cellular pyrimidine de novo synthesis is impaired.