Determination of kynurenine levels in rat plasma by high-performance liquid chromatography with pre-column fluorescence derivatization

Kynurenine (KYN), a tryptophan metabolite, is a crucial compound for modulating neurotransmission because it can be metabolized in vivo into both quinolinic acid and kynurenic acid, which are the agonist and antagonist, respectively, of N-methyl-d-aspartate receptor. For the highly sensitive detection of KYN by high-performance liquid chromatography (HPLC), a fluorescence derivatization of KYN with a benzofurazan-type fluorogenic reagent, 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was investigated in the present study. KYN was derivatized with DBD-F (DBD-KYN) at 60 °C for 30 min, and separated on an octadecylsilica column with a gradient elution of the mobile phase, which consists of 0.1% formic acid in acetonitrile/methanol/water. DBD-KYN was detected fluorimetrically at 553 nm with an excitation wavelength of 431 nm. The limits of detection and quantification were approximately 0.30 pmol [signal-to-noise ratio (S/N) 3] and 1.0 pmol (S/N, 10) on column, respectively. Plasma KYN levels were successfully determined using 10 μL of rat plasma with satisfactory precision and accuracy. Intra- and inter-day precisions and accuracies were 1.7-6.8%, and −10 to 9.6%, respectively. KYN levels in plasma of male Sprague-Dawley rats (7 weeks old) were approximately 2.4 ± 0.32 μmol L⁻¹ (n = 4). The proposed HPLC method was applied to determine KYN levels in the plasma of ketamine-treated rats—the animal model of schizophrenia.     

Gas analyzer for continuous monitoring of trace level methanethiol by microchannel collection and fluorescence detection

The highly odorous compound methanethiol, CH₃SH, is commonly produced in biodegradation of biomass and industrial processes, and is classed as 2000 times more odorous than NH₃. However, there is no simple analytical method for detecting low parts-per-billion in volume ratio (ppbv) levels of CH₃SH. In this study, a micro gas analysis system (μGAS) was developed for continuous or near real time measurement of CH₃SH at ppbv levels. In addition to a commercial fluorescence detector, a miniature high sensitivity fluorescence detector was developed using a novel micro-photomultiplier tube device. CH₃SH was collected by absorption into an alkaline solution in a honeycomb-patterned microchannel scrubber and then mixed with the fluorescent reagent, 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F). Gaseous CH₃SH was measured without serious interference from other sulfur compounds or amines. The limits of detection were 0.2 ppbv with the commercial detector and 0.3 ppbv with the miniature detector. CH₃SH produced from a pulping process was monitored with the μGAS system and the data agreed well with those obtained by collection with a silica gel tube followed by thermal desorption–gas chromatography–mass spectrometry. The portable system with the miniature fluorescence detector was used to monitor CH₃SH levels in near-real time in a stockyard and it was shown that the major odor component, CH₃SH, presented and its concentration varied dynamically with time.     

Sensitive determination of MDMA and its metabolite MDA in rat blood and brain microdialysates by HPLC with fluorescence detection

Simultaneous determination of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) in rat blood and brain microdialysates by high-performance liquid chromatography with fluorescence detection (HPLC-FL) was developed. Microdialysates were directly subjected to derivatization with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl). The DIB-derivatives of MDMA, MDA and the internal standard, 1-methyl-3-phenylpropylamine (MPPA), were isocratically separated on an ODS column using a mixture of 50 mm phosphate buffer (pH 7.0)-acetonitrile-methanol-2-propanol (50:45:5:2, v/v/v/v %) as an eluent at a flow rate of 1.5 mL/min. The calibration curves of MDA and MDMA spiked to blood and brain microdialysates were linear over the ranges 2.5-500 and 5.0-1000 ng/mL, respectively. The detection limits of MDA and MDMA were 1.2 and 4.2 for blood and 1.3 and 4.8 ng/mL for brain, respectively. Additionally, the intra- and the inter-assay precisions were lower than 5.6% for the blood and brain microdialysates (n = 4). The proposed method was successfully applied for the monitoring of MDMA and its metabolite MDA in rat blood and brain microdialysates, and the pharmacokinetic parameters of MDMA and MDA in the microdialysates after administration of MDMA (5 mg/kg, i.p.) with or without caffeine (20 mg/kg, i.p.) were evaluated.     

A sensitive fluorescence reagent for the determination of aldehydes from alcoholic beverage using high-performance liquid chromatography with fluorescence detection and mass spectrometric identification

A pre-column derivatization method for the sensitive determination of aldehydes using the tagging reagent 2-[2-(7H-dibenzo[a,g] carbazol-7-yl)-ethoxy] ethyl carbonylhydrazine (DBCEEC) followed by high-performance liquid chromatography with fluorescence detection and APCI-MS identification has been developed. The chromophore of fluoren-9-methoxy-carbonylhydrazine (Fmoc-hydrazine) reagent was replaced by 2-[2-(7H-dibenzo[a,g] carbazol-7-yl)-ethoxy] ethyl functional group, which resulted in a sensitive fluorescence tagging reagent DBCEEC. DBCEEC could easily and quickly labeled aldehydes. The maximum excitation (300 nm) and emission (400 nm) wavelengths did not essentially change for all the aldehyde derivatives. Derivatives were sufficiently stable to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z [M + (CH₂)_n]⁺ in positive-ion mode (M: molecular weight of DBCEEC, n: corresponding aldehyde carbon atom numbers). The collision-induced dissociation of protonated molecular ion formed fragment ions at m/z 294.6, m/z 338.6 and m/z 356.5. Studies on derivatization demonstrated excellent derivative yields in the presence of trichloroacetic acid (TCA) catalyst. Maximal yields close to 100% were observed with a 10 to 15-fold molar reagent excess. Separation of the derivatized aldehydes had been optimized on ZORBAX Eclipse XDB-C₈ column with aqueous acetonitrile as mobile phase in conjunction with a binary gradient elution. Excellent linear responses were observed at the concentration range of 0.01-10 nmol mL⁻¹ with coefficients of >0.9991. Detection limits obtained by the analysis of a derivatized standard containing 0.01 nmol mL⁻¹ of each aldehyde, were from 0.2 to 1.78 nmol L⁻¹ (at a signal-to-noise ratio of 3).     

Highly sensitive method for determination of N-nitrosamines using high-performance liquid chromatography with online UV irradiation and luminol chemiluminescence detection

A new method for the measurement of N-nitrosamines in part-per-trillion concentrations from water samples without preconcentration steps has been developed. This method is based on online UV irradiation after high-performance liquid chromatographic separation and subsequent luminol chemiluminescence detection without addition of an oxidant. It was confirmed that N-nitrosamines in basic aqueous solution were transformed to peroxynitrite by UV irradiation. The detection limits for this method were 1.5 ng/L, 2.9 ng/L, 3.0 ng/L, and 2.7 ng/L for N-nitrosodimethylamine, N-nitrosomorpholine, N-nitrosomethylethylamine, and N-nitrosopyrrolidine, respectively, at a signal-to-noise ratio of 3. The calibration graphs were linear in the range of 5–1000 ng/L for these N-nitrosamines. This method was used for the determination of N-nitrosamines in tap water, river water, and industrial plant effluent samples. The recoveries of N-nitrosodimethylamine, N-nitrosomorpholine, N-nitrosomethylethylamine, and N-nitrosopyrrolidine present in tap water sample at a concentration of 10 ng/L (mean ± standard deviation, n = 4) were (94.8 ± 2.7)%, (102.0 ± 6.9)%, (99.3 ± 3.9)%, and (102.8 ± 2.5)%, respectively. These results indicate that our proposed method can be applied satisfactorily to the determination of N-nitrosamines in water samples.     

Coupled liquid-liquid extraction and column switching LC for the determination of a new antihistaminic H1 drug in human urine

Summary Mizolastine (SL 85.0324) is a new antihistaminic H₁ benzimidazole derivative which is excreted into urine almost completely metabolized; about 2% of the unchanged drug is excreted as conjugated compound which requires enzymatic deconjugation before analysis. Since the existing methods for plasma samples do not work on deconjugated human urine due to interferences, a new method was developed. The method is based on a diethyl-ether extraction of mizolastine and an internal standard from alkalinized urine. The ether extract is back-extracted with an aqueous buffer (pH=2.6), this extract is neutralized (pH=6.5) and an aliquot injected into a C₁₈ pre-column where clean up and preconcentration take place. The analytes are then desorbed from the pre-column and transferred to the analytical column. The analytical column is a C₁₈ type specially seactivated for basic compounds with an eluent of acetonitrile/phosphate solution (pH=4.5), 40/60, v/v, at a flow rate of 1 ml min^–1. Detection is at 285 nm. The method is linear in the range 10–500 ng ml^–1 with a lower limit of detection of 10 ng ml^–1. The precision and accuracy, evaluated during intra-day and inter-day assays, are satisfactory for pharmacokinetic investigations.     

Quantification of biogenic amines by microchip electrophoresis with chemiluminescence detection

A highly sensitive microchip electrophoresis (MCE) method with chemiluminescence (CL) detection was developed for the determination of biogenic amines including agmatine (Agm), epinephrine (E), dopamine (DA), tyramine, and histamine in human urine samples. To achieve a high assay sensitivity, the targeted analytes were pre-column labeled by a CL tagging reagent, N-(4-aminobutyl)-N-ethylisoluminol (ABEI). ABEI-tagged biogenic amines after MCE separation reacted with hydrogen peroxide in the presence of horseradish peroxidase (HRP), producing CL emission. Since no CL reagent was added to the running buffer, the background of the CL detection was extremely low, resulting in a significant improvement in detection sensitivity. Detection limits (S/N = 3) were in the range from 5.9 × 10⁻⁸ to 7.7 × 10⁻⁸ M for the biogenic amines tested, which were at least 10 times lower than those of the MCE–CL methods previously reported. Separation of a urine sample on a 7 cm glass/poly(dimethylsiloxane) (PDMS) microchip channel was completed within 3 min. Analysis of human urine samples found that the levels of Agm, E and DA were in the ranges of 2.61 × 10⁻⁷ to 4.30 × 10⁻⁷ M, 0.81 × 10⁻⁷ to 1.12 × 10⁻⁷ M, and 8.76 × 10⁻⁷ to 11.21 × 10⁻⁷ M (n = 4), respectively.     

A simple and sensitive HPLC-fluorescence method for quantification of MDMA and MDA in blood with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label

A sensitive high-performance liquid chromatographic method with fluorescence detection to determine 3,4-methylenedioxymethamphethamine (MDMA) and 3,4-methylenedioxyamphethamine (MDA) in human and rat whole blood or plasma samples was developed by using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label. MDMA and MDA in a small amount of blood sample (ca 100 microL) were extracted by liquid-liquid extraction with ethyl acetate, and were derivatized with DIB-Cl under mild conditions (10 min at room temperature). A good separation of DIB-derivatives could be achieved within 45 min using a commercially available ODS column with an isocratic eluent of 10 mM citric acid-20 mM Na(2)HPO(4) aqueous buffer (pH 4.0)-CH(3)CN-CH(3)OH (50:45:5, v/v/v %). The calibration curves prepared with 1-methyl-3-phenylpropylamine (MPPA) as an internal standard showed good linearity (r = 0.999) with 0.36-0.83 ng/mL detection limit at a signal-to-noise ratio of 3. MDMA and MDA in rat whole blood could be monitored for 6 h after a single administration of MDMA (2.2 mg/kg, i.p.). The pharmacokinetic parameters for MDMA and MDA obtained by triplicate measurements were 426 +/- 23 and 39 +/- 6 ng/mL (C(max)), 20 +/- 5 and 100 +/- 10 min (T(max)), respectively.     

A highly sensitive method for the determination of adriamycin and adriamycinol in human plasma using HPLC with fluorimetric detection

Summary A simple and very sensitive HPLC method, for the simultaneous determination in human plasma of adriamycin and its metabolite adriamycinol, is described. Plasmas from patients were stored frozen. Thawed samples were extracted by absorption of anthracyclin onto a small C18 column. After evaporation of the eluate and reconstitution of the residue with methanol (100L), 30 to 40L of the mixture were injected into the chromatograph. Separation was obtained using an RP 8 column with a mobile phase of formate buffermethanol-acetonitrile (502327, v/v). A spectrofluorimeter was used as detector. The limit of sensitivity of the assay was 50 pcg/ml of plasma.     

Flow injection determination of cationic surfactants by using N-bromosuccinimide and N-chlorosuccinimide as new oxidizing agents for luminol chemiluminescence

Two highly sensitive chemiluminescence (CL) systems are described. The method is based on the CL generated during the oxidation of luminol by N-bromosuccinimide (NBS) and N-chlorosuccinimide (NCS) in alkaline medium. The emission intensity is reduced by the presence of some surfactants at concentrations lower than critical micelle concentration (cmc).A new, simple, rapid and selective flow injection CL method for the determination of cationic surfactants such as dodecyltrimethylammonium bromide (DTAB), cetyltrimethylammonium bromide (CTAB) and cetylpyridinium chloride (CPC) is proposed. Their determinations are based on the reducing effect on the emission intensity of NBS-luminol and NCS-luminol chemiluminescent reactions. The effect of analytical and flow injection analysis (FIA) variables on these CL systems and on the determination of the cationic surfactants are discussed. The optimum parameters for the determination of cationic surfactants were studied and were found to be the following: luminol, 1×10⁻⁶ M; NBS and NCS both, 5×10⁻² M; NaOH, 5×10⁻² M and flow rate, 3.5 ml min⁻¹.     

A novel fluorescent and chromogenic probe for cyanide detection in water based on the nucleophilic addition of cyanide to imine group

A fluorescent and colorimetric probe bearing salicylaldehyde hydrazone functionality has been prepared for cyanide sensing. The detection of cyanide was performed via the nucleophilic attack of cyanide anion on the imine group of the probe with a 1:1 binding stoichiometry, which could be confirmed by ¹H NMR and MS studies. The specific reaction results in a prominent fluorescence enhancement and a color change from colorless to yellow.     

Sensitive spectrophotometric methods for the determination of pindolol

Two simple, sensitive and reproducible visible spectrophotometric methods for the determination of Pindolol in amounts as low as 1 g are described. The first method is based on the oxidation of Pindolol by sodium nitrite under acidic conditions and the coupling of the oxidised intermediate withN-1-(naphthyl)ethylenediamine dihydrochloride (NED) to the colored product. The spectrum of the colored solution is strongly dependent on the length of the incubation period at room temperature during oxidation (3 min, _max 620 nm; 10 min or above _max 540 nm). Other couplers [diphenylamine (DPA) or 1-naphthylamine (-NA)] or indole derivatives (indole, tryptophan or isatin) when used instead of NED or Pindolol, respectively, also react to give a colored product with same maximum in each case irrespective of the length of the incubation period during oxidation. The second method is based on the coupling reaction involving Pindolol and 1,2-naphthaquinone-4-sulphonic acid sodium salt to form a coloured product (_max 660 nm). Both the methods have been employed to determine Pindolol content in bulk forms and pharmaceutical preparations.     

Development of a column-switching high-performance liquid chromatography for kynurenine enantiomers and its application to a pharmacokinetic study in rat plasma

Kynurenine (KYN), a tryptophan metabolite, is a precursor of kynurenic acid, which is an antagonist of N-methyl-d-aspartate receptor. In this study, an enantiomeric separation of d,l-KYN derivatized with the benzofurazan fluorescence reagent 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) (DBD-d,l-KYN) was first investigated by using a high-performance liquid chromatography (HPLC) with several chiral columns. As a consequence, DBD-d,l-KYN was enantiomerically separated on a cellulose-type chiral column (CHIRALCEL OJ-RH) with a mobile phase of H₂O/CH₃CN/MeOH (40/50/10) containing 0.1% acetic acid. Under this condition, the separation factor and resolution were 1.48 and 1.28, respectively. Next, a column-switching HPLC consisting of both octadecylsilica and chiral columns was developed and used to determine both d- and l-KYN enantiomers in 10 μL of rat plasma following the intraperitoneal administration of d,l-KYN to rats (10 mg kg⁻¹). The result revealed that the concentration of l-KYN was higher than that of d-KYN, suggesting that d-KYN was eliminated faster than l-KYN.     

Enantioselective HPLC method for quantitative determination of tramadol andO-desmethyltramadol in plasma and urine: Application to clinical studies

Summary A sensitive, enantioselective high-performance liquid chromatographic method has been developed for the separation and individual quantitative determination of (+)-and (-)-tramadol and (+)- and (-)-O-desmethyltramadol (M1) in plasma and urine. Extraction from plasma and urine was performed by solid-phase extraction (SPE) on disposable butyl silica (100 mg) extraction cartridges. Separation of the enantiomers of tramadol and M1 was achieved on a Chiralpak AD column containing amylose tris-(3,5-dimethylphenylcarbamate) as chiral selector. The mobile phase was isohexane-ethanol-diethylamine, 97:2.8:0.1 (v/v). Quinidine was used as internal standard. The analytes were detected by use of fluorescence detection. The limit of quantification for tramadol and M1 as 5 nM in plasma and 25 nM in urine. Recoveries were approximately 90% for tramadol and M1 in both plasma and urine. Linearity was observed for both enantiomers of tramadol and M1 in both plasma (r ²>0.999) and urine (r ²>0.997). The intra and inter-day precision (CV) did not exceed 6.0%. The applicability of the method was demonstrated by means of two clinical studies—a pharmacokinetic study in which a healthy volunteer received 150 mg tramadol hydrochloride as a single oral dose and a study in which poor and extensive CYP2D6 metabolizers received 50 mg tramadol hydrochloride as a single oral dose.     

Micellar electrokinetic chromatography-chemiluminescent detection of biogenic amines using N-(4-aminobutyl)-N-ethylisoluminol as derivatization reagent and trivalent copper chelate as chemiluminescence enhancer

A facile, sensitive and universal method was established for analysis of biogenic amines using micellar electrokinetic chromatography coupled with chemiluminescent (CL) detection. It was found that diperiodatocuprate (III) (K₅[Cu(HIO₆)₂], DPC), a transition metal chelate at unstable high oxidation state, could effectively enhance the reaction between luminol-type compound and hydrogen peroxide, to produce very strong CL signal. In addition, triethylamine was found to be able to effectively improve the yield of the derivatization reaction between biogenic amines and a luminol-type derivatization reagent, N-(4-aminobutyl)-N-ethylisoluminol (ABEI). Based on these facts, three biogenic amines were pre-column derivatized with ABEI, and post-column detected using high sensitive luminol-hydrogen peroxide-DPC CL system. Since the background was quite low, and the signal was quite strong, a considerable improved sensitivity was obtained. The presented method had been successfully applied to simultaneously analyze glycine, proline and phenylalanine with the detection limits (S/N = 3) of 0.030 μmol L⁻¹, 0.23 μmol L⁻¹ and 0.21 μmol L⁻¹, respectively. To evaluate its potential application value, glycine in saliva and urine samples was detected using this method, and satisfied results were obtained. This approach can be further extended to detection of many other compounds such as peptides and drugs by using luminol-type derivatization reagent.     

Claisen aromatic amino rearrangement in the series of fluorinated anilines

The effect ofmeta-substituents in the aromatic ring on the route of Claisen rearrangement ofN-(pent-3-en-2-yl)-3-fluoro(or 3-trifluoromethyl, 3,4-difluoro)anilines induced by ZnCl₂ was investigated. The formation of two possibleortho-alkenylated reaction products was observed. The ratio of these isomers depends on the nature of the acid catalyst. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 188–190, January, 1998.     

Determination of Free Fatty Acids in Bryophyte Plants and Soil by HPLC with Fluorescence Detection and Identification by Online MS

A method for the determination of long and short chain free fatty acids (FFAs), using 1-[2-(p-toluenesulfonate)-ethyl]-2-phenylimidazole-[4,5-f]-9,10-phenanthrene (TSPP) as labeling reagent, has been developed. Identification of FFA derivatives was carried out by HPLC-MS with atmospheric pressure chemical ionization (APCI) in positive ion mode. Gradient elution on an Agilent Eclipse XDB-C₈ column gave good separation of the derivatives. Excellent linear responses were observed and good compositional data could be obtained from as little as 200 mg of bryophyte plants and soil samples. Facile TSPP derivatization coupled with HPLC-APCI-MS analysis allowed the development of a highly sensitive method for the quantitative analysis of trace level of FFAs from biological and natural environmental samples.     

Liquid chromatography coupled to on-line post column derivatization for the determination of organic compounds: A review on instrumentation and chemistries

Analytical derivatization either in pre or post column modes is one of the most widely used sample pretreatment techniques coupled to liquid chromatography. In the present review article we selected to discuss the post column derivatization mode for the analysis of organic compounds. The first part of the review focuses to the instrumentation of post-column setups including not only fundamental components such as pumps and reactors but also less common parts such as static mixers and back-pressure regulators; the second part of the article discusses the most popular “chemistries” that are involved in post column applications, including reagent-less approaches and new sensing platforms such as the popular gold nanoparticles. Some representative recent applications are also presented as tables.     

A new method for the synthesis of [1,4]diazepino[6,5-b]indole derivatives

Reactions of 3-[(N-aryl-N-chloroacetyl)amino]-2-formylindoles with substituted anilines gave 1,4-diaryl-2-oxo-1,2,3,6-tetrahydro[1,4]diazepino[6,5-b]indol-4-ium chlorides and those with 4-aminopyridine yielded 4-amino-1-(1-aryl-2-oxo-2,5-dihydro-1H-pyrido[3,2-b]indol-3-yl)pyridinium chlorides. Reduction of 1,2,3,6-tetrahydrodiazepinoindol-4-ium chlorides afforded the corresponding hexahydro derivatives. An alternative synthesis of 1-(4-nitrophenyl)-3-oxo-4-phenyl-1,2,3,4,5,6-hexahydro[1,4]diazepino[6,5-b]indole from 3-[N-(4-nitrophenyl)amino]-2-[(phenylimino)methyl]indole was developed. The method involves the following sequence of transformations: reduction, chloroacetylation, and intramolecular alkylation. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2193–2199, December, 2006.     

Optimisation of the on-fibre derivatisation of volatile fatty acids in the simultaneous determination together with phenols and indoles in cow slurries

The on-fibre derivatisation of volatile fatty acids (VFAs) using N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) was optimised in the simultaneous determination of VFAs together with phenols and indoles by headspace solid-phase microextraction (SPME)–gas chromatography–mass spectrometry. Firstly, the nature of the SPME fibre was optimised and four different fibres were studied (100 μm polydimethylsiloxane, 85 μm Carboxen/polydimethylsiloxane, 5/30 μm divinylbenzene/Carboxen/polydimethylsiloxane and 85 μm polyacrylate). The optimum fibre (50/30 μm divinylbenzene/Carboxen/polydimethylsiloxane) was used to study the exposure time of the fibre to the derivatisation agent and the desorption time and temperature. Firstly, a factorial design was built but since the three variables had a significant effect, a central composite design was used to build the response surfaces. The best signals were obtained after the exposure of the fibre in the headspace of the MTBSTFA derivatisation reagent for 1 h and desorption at 300 °C for 9 min. The determination of underivatised phenols and indoles was not affected by the presence of the derivatisation reagent in the fibre.