Fluorometric determination of streptomycin in serum by high-performance liquid chromatography using mobile phase containing fluorogenic reagent

A new postcolumn derivatization method for the fluorometric determination of streptomycin in serum by high-performance liquid chromatography is described. The serum was treated with 3.5% perchloric acid to precipitate proteins and the supernatant was directly injected into the chromatograph. Streptomycin was separated by reversed-phase, ion-pair chromatography with a mobile phase containing ninhydrin as a fluorogenic reagent, octanesulfonate, and 1,2-ethanedisulfonate as counterions, and was detected by fluorescence using continuous-flow, postcolumn derivatization in an alkaline stream with ninhydrin in the mobile phase. This method is sensitive to 1.0 microgram/ml using only 100 microliter of serum. Comparison with a fluorescence polarization immunoassay gave a good correlation coefficient of 0.976.     

Blood drugs and other analytical challenges (Methodological Surveys in Biochemistry,Vol. 7) : Edited by E. Reid,Ellis Horwood (Wiley), Chichester, 1978, X + 355 pp., price £ 19.50, ISBN 0-85312-124-9

A high-performance liquid chromatographic procedure has been developed for the separation and fluorometric detection of guanidino compounds in physiologic fluids. All guanidino compounds were separated on a 17 × 0.46 cm cation-exchange column using a stepwise pH gradient. The chromatographic system was designed to enable the use of the specific reagent 9,10-phenanthrenequinone as a means of monitoring the guanidino compounds of physiologic fluids. This new analytical method is so sensitive that it enables the analysis at the picomole level. Our automatic guanidino-compound analyzer was successfully applied to the quantitative determination of all guanidino compounds in physiologic fluids from normal controls and uremic patients.     

Fluorometric quantitation of amino sugars in the picomole range.

A fluorometric method has been developed for the convenient and quantitative assay of amino sugars over the concentration range of 10 nm to 6 mm. Linear results are obtained for reaction mixtures containing 6 pmol to 60 nmol hexosamine. The procedure involves the condensation of amino sugars with the fluorogenic reagent o-phthalaldehyde, at alkaline pH in the presence of 2-mercaptoethanol. Relative fluorescence intensities are then determined using excitation and emission wavelengths of 340 and 455 nm, respectively. The presence of 2-mercaptoethanol in reaction mixtures not only enhanced sensitivity of the assay, but also defined the excitation/emission spectra. Under the conditions described, amino acids were also found to react with o-phthalaldehyde, yielding fluorescence intensities similar to those of amino sugars. These results suggest the applicability of fluorescence techniques in automated amino sugar analyses, as well as the potential interference of other compounds containing primary amines.     

Simultaneous quantification of dopamine, 5-hydroxytryptaine and four metabolically related compounds by means of reversed-phase high-performance liquid chromatography with electrochemical detection

A method for simultaneously quantifying dopamine, 5-hydroxytryptamine (5-HT) and four metabolically related compounds has been developed, permitting more efficient neurochemical examination of these often interrelated biogenic amine systems. The method uses high-performance liquid chromatographic separation of these compounds on a C₁₈ reversed-phase column with a buffered mobile phase containing methanol as an organic modifier and heptanesulfonate as an ion-pair reagent. Using 5-hydroxy-N-methyltryptamine as an internal standard and electrochemical detection, chromatography time is less than 12 min. Sample preparation simply involves the addition of internal standard, homogenization in the mobile phase, centrifugation and injection of the supernatant into the chromatograph. The method is sensitive to a tissue content of these compounds of less than 1 ng. The utility of this method for neuropharmacological—neurochemical studies is illustrated with studies using inhibitors of monoamine oxidase (pargyline) and aromatic amino acid decarboxylase (RO 4-4602).     

Determination of inhibitors’ potency (IC50) by a direct high-performance liquid chromatographic method on an immobilised acetylcholinesterase column

An immobilised acetylcholinesterase (AChE) stationary phase was prepared by using an in situ AChE immobilisation procedure. A stainless steel column packed with epoxide silica was connected to the HPLC system and the enzyme solution at pH 5.8 was recycled through the column at a flow-rate of 0.5 ml/min for 24 h. The activity of the immobilised AChE was determined by injecting the substrate acetylthiocholine, using as mobile phase 0.1 M phosphate buffer (pH 7.4) containing Ellman’s reagent [5,5′-dithio-bis(2-nitrobenzoic acid)] and measuring the area of the obtained peak with UV detection at 412 nm. The effect of AChE inhibitors tacrine, edrophonium and donepezil were evaluated by the simultaneous injection of each inhibitor with the substrate. The resulting decrease in the AChE activity, as expressed by the decrease of the peak area detected at 412 nm, was related to the concentration and potency of the solutes. The obtained IC₅₀ values were compared with those derived by the conventional spectrophotometric method. This immobilized enzyme reactor, included in a chromatographic system, can be used for the rapid screening for new inhibitors allowing for the on-line determination of a compound’s inhibitory potency. The advantages over the conventional methods are the increased enzyme stability and system automation which allows a large number of compounds to be analysed continuously.     

Comparison of the chiral separation of amino-acid derivatives by a teicoplanin and RN-beta-CD CSPs using waterless mobile phases: Factors that enhance resolution

A variety of compounds containing amines (i.e., amino acids, amino alcohols, etc.) were chemically derivatized with a variety of electrophilic tagging reagents to elucidate the chiral recognition sites on a teicoplanin-bonded chiral stationary phase (CSP) and on R-naphthylethylcarbamate-beta-cyclodextrin (RN-beta-CD)-bonded CSP. Solutes were separated under optimum chromatographic conditions on teicoplanin and RN-beta-CD CSPs for comparison using an acetonitrile-based mobile phase. It was noted that the size of the analyte or tagging reagent exerted a greater influence on compounds separated on teicoplanin than on RN-beta-CD when using the polar organic mode. This suggests that chiral recognition on teicoplanin CSP is more sensitive to size and indicates that the hydrophobic pocket of teicoplanin plays a significant role in chiral recognition in this mode. However, the type of functional groups had a greater impact than the size of analyte on separations obtained from RN-beta-CD phase in the polar-organic mode. Specifically, the pi-pi interaction was enhanced by derivatizing the aromatic ring of the tagging reagent with electron-withdrawing groups and thus altered the resolution substantially. For both phases, chiral recognition is most pronounced when the stereogenic center of the analyte is near the tagging moiety and surrounded by functional groups (e.g., carboxylic, etc.) which are favorable for hydrogen bonding.     

Cysteic- and homocysteic acid-S-(aminoiminomethyl) amides: a new class of modified arginines useful in peptide synthesis.

A novel, practical synthesis of the title compounds and their derivatives are described. The protecting groups for amino, guanidino and carboxylic functions of substituted amides of cysteic and homocysteic acid were selected with the aim of making the amino acid derivatives synthons for peptide synthesis both in solution and by the solid phase method. Studying the structure-activity relationship some new kyotorphin, [Leu] kyotorphin and MIF-1 analogues, containing the unusual amino acid cysteic acid-S-(aminoiminomethyl) amide (sArg) in position two, have been prepared. It is a very promising compound, a structural analogue of arginine and an efficient antagonist in its metabolism.     

Determination of cycloserine in human plasma by high-performance liquid chromatography with fluorescence detection,using derivatization with p-benzoquinone

A new method for determining cycloserine in plasma samples is described. This method is based on the derivatization of cycloserine with p-benzoquinone, a reaction that takes place at the same time as the process of plasma deproteinization due to the presence of ethanol as solvent in the solution of the derivatization reagent. Four derivatives are obtained from this reaction. The main derivative is well correlated with the cycloserine concentration. The ratio between the volumes of the plasma sample and the reagent solution is 1:2 for a p-benzoquinone concentration of 1000 μg/mL. Elution from a C₁₈ column was isocratic, using a mobile phase containing (v/v) 85% aqueous 0.1% formic acid solution, and 15% (v/v) of a mixture of methanol and acetonitrile (1:1), with a flow-rate of 1 mL/min, at 25°C. Determinations by fluorescence detection were achieved with excitation at 381 nm and emission at 450 nm, with a detection limit of 10 ng/mL for an injection volume of 5 μL. This method was validated and applied to the determination of cycloserine in blood plasma samples of several healthy volunteers.     

Formation of bioorganic compounds in aqueous solution induced by plasma

When plasma jet of Ar-arc plasma was blown into an aqueous solution containing organic compounds, oxidation reactions were induced in the solution. The plasma-induced reaction was a powerful oxidation which could convert a methyl to a carboxyl group and cleave a carbon-carbon bond without using any oxidizing reagent. This reaction could be regarded as a model for the solar plasma-induced reaction in the primitive hydrosphere.     

Formation of bioorganic compounds in aqueous solution induced by plasma

When plasma jet of Ar-arc plasma was blown into an aqueous solution containing organic compounds, oxidation reactions were induced in the solution. The plasma-induced reaction was a powerful oxidation which could convert a methyl to a carboxyl group and cleave a carbon-carbon bond without using any oxidizing reagent. This reaction could be regarded as a model for the solar plasma-induced reaction in the primitive hydrosphere.     

Analysis of O-linked reducing oligosaccharides released by an in-line flow system

Reducing O-linked oligosaccharides from bovine submaxillary mucin, bovine fetuin, and porcine gastric mucin were recovered by nonreductive alkaline beta-elimination from an in-line flow system. Glycoproteins where attached to a solid support using hydrophobic interaction with alkali-resistant Poros reversed phase beads and a flow of alkali released the oligosaccharides. The alkali was subsequently neutralized by a continuous flow through cation exchange resin. The released oligosaccharides in the flow were trapped in a cartridge filled with graphitized carbon. Salt-free oligosaccharides could be recovered as a concentrated solution by elution with organic solvents from the cartridge. The glycosylation pattern of the released oligosaccharides was compared with the conventionally released and reduced oligosaccharides recovered from alkaline beta-elimination in the presence of borohydride. In general, the recovery from the in-line release was sometimes lower than from the reductive elimination method, but it was shown that alkaline degradation of reducing oligosaccharides was limited in this system. Liquid chromatography using graphitized carbon packing and high pH mobile phases together with negative ion electrospray mass spectrometry showed that both neutral and acidic reducing oligosaccharides could be analyzed in a single run. Reducing O-linked oligosaccharides could also be recovered in this way from human glycophorin separated by SDS-PAGE. The polyacrylamide was sufficient to retain the glycoprotein in the gel while the flow of alkali released the oligosaccharides. It was also shown that the alkaline conditions for releasing O-linked oligosaccharides from fetuin would partially release some N-linked oligosaccharides, particularly in the presence of reducing agent.     

Simultaneous analysis of disopyramide and quinidine in plasma by high-performance liquid chromatography

A new reversed-phase high-performance liquid chromatographic method allowing simultaneous measurement of plasma concentrations of disopyramide and quinidine is described. Disopyramide and quinidine were separated on a reversed-phase column using 0.05 M phosphate buffer (pH 3.0)—acetonitrile (73:27, v/v), as mobile phase and the peaks were monitored by UV absorbance at the wavelengths of 254 and 325 nm. The drugs were extracted from alkaline plasma with chloroform containing the internal standard. The organic phase was evaporated to dryness and the residue was redissolved in a small volume of the mobile phase before analysis by high-performance liquid chromatography. The method is convenient and reliable in routine monitoring of both drugs.     

Specific interactions of quercetin and other flavonoids with target proteins are revealed by elicited fluorescence

The fluorogenic properties of quercetin and similar flavonoids common in plants were exploited to analyse their interaction with target proteins. Quercetin produced a strong fluorescent signal upon binding to bovine serum albumin (BSA) and insulin. The fluorescent signal showed saturation kinetics with increasing flavonoid concentrations indicating the presence of defined peptide binding motifs. Other tested proteins showed no fluorescence with the flavonoids. In a comparative study including 22 flavonoids the compounds with fluorogenic properties were identified using our model proteins BSA and insulin and the structural requirements for the fluorogenic property were defined. Only flavones with a high degree of hydroxylation were able to elicit fluorescence. The emitted fluorescence was strongly enhanced at alkaline pH. Finally, an attempt was made to identify intracellular target molecules in live cells. Drosophila follicles showed a distinct staining pattern thus giving evidence that high concentrations of quercetin binding proteins are present in the nuclei and are associated with the ring canals. The presented biochemical and cytological data show that the interaction of the studied flavonoids with target proteins is specific and this finding opens up new experimental possibilities to systematically identify the cellular proteins with specific binding motifs for quercetin or other fluorogenic compounds of medical interest.     

Stereoselective high-performance liquid chromatographic analysis of ketoprofen and its acyl glucuronides in chronic renal insufficiency

A rapid, sensitive method was developed for the quantification of the R- and S-enantiomers of ketoprofen and their acyl glucuronide conjugates in the plasma and dialysate of hemodialysis-dependent anephric patients. Unconjugated R- and S-ketoprofen plasma concentrations were determined directly by liquid chromatography using a S,S-Whelk-O1 chiral stationary phase. R- and S-Ketoprofen glucuronide for use as standards were resolved using a C₁₈ reversed-phase HPLC column with a mobile phase containing the ion-pair reagent tetrabutylammonium hydrogen sulfate. Plasma glucuronides, however, could not be directly quantified due to matrix interference. Therefore, the glucuronides were isolated using reversed-phase HPLC and quantified after alkaline hydrolysis using the S,S-Whelk-O1 chiral stationary phase column.     

Fluorescence derivatisation of urinary corticosteroids for high-performance liquid chromatographic analysis

Corticosteroids containing a C₂₁ primary hydroxyl group were derivatised with 9-anthroyl cyanide. The reagent was prepared as a solution in acetonitrile, containing 0.1% triethylamine, at a concentration of 2 mg/ml. Approximately 1 μg of corticosteroid was reacted with 100 μl of this reagent, at 45°C for 2 h. The fluorescent derivatives were separated by HPLC on a silica column, 250×4.6 mm I.D., by stepwise elution, with a mobile phase of 2-propanol–hexane (2:98) for 20 min, followed by 2-propanol–hexane (7:93) from 20 to 40 min. The fluorescence detector was set to 370-nm excitation and 470-nm emission. The relatively low temperature for derivatisation avoided reaction with secondary hydroxyl groups and also prevented thermal degradation of the corticosteroids.     

Simultaneous determination of amodiaquine and its active metabolite in human blood by ion-pair liquid chromatography-tandem mass spectrometry

A sensitive and selective ion-pair liquid chromatography-tandem mass spectrometric method (IP-LC-MS/MS) for the simultaneous determination of amodiaquine (AQ) and its active metabolite, N-desethylamodiaquine (AQm), in human blood has been developed and validated. Pentafluoropropionic acid (PFPA) was applied as ion-pairing reagent in reversed-phase chromatographic separation. The effects of PFPA concentrations and the volume fraction of acetonitrile in the mobile phase on the retention of analytes were investigated on a Venusil MP-C(18) column, and the mobile phase was finally optimized as acetonitrile:water (23:77, v/v) with 0.0667% PFPA in the aqueous phase. The results proved that PFPA as an ion-pairing reagent could provide desirable chromatographic performance in the IP-LC-MS/MS determination of 4-aminoquinoline compounds. Blood samples were protein precipitated with acetonitrile using hydroxychloroquine (OHCQ) as the internal standard. The detection was carried out in multiple reaction monitoring (MRM) mode via positive atmospheric pressure chemical ionization (APCI) interface. The lower limits of quantification were established at 0.150 and 1.50 ng/mL for AQ and AQm, respectively. The validated IP-LC-MS/MS method was applied to a clinical pharmacokinetic study of AQ and AQm in human blood after an oral administration of 600 mg AQ hydrochloride (45 9mg base).     

Comparative Study of Guanidino Compounds in Serum and Brain of Mouse, Rat, Rabbit, and Man

Abstract: The levels of 12 guanidino compounds were determined in the serum and brain of mouse, rat, rabbit, and man using cation-exchange column chromatography with the fluorescence ninhydrin detection method. A comparative study of these compounds was made in the four groups studied, for serum and brain. In rabbit and man the different guanidino compounds were also determined in various brain regions. This study provides basic analytical data that could facilitate the interpretation of further biochemical and neurochemical studies dealing with guanidino compounds that are identified as being toxins in hyperargininemia and uremia.     

Polyproline derivatives as chiral selectors in high performance liquid chromatography: chromatographic and conformational studies

A proline oligopeptide-derived chiral selector (CS), containing 3,5-dimethylphenylcarbamate residues on the 4 position of the pyrrolidine rings, was bonded to a silica gel chromatographic matrix by the N-terminal group. The chromatographic behaviour of the resulting chiral stationary phase (CSP) was compared with that of a CSP containing the analogous monomeric CS and that resulting from bonding of the polyproline-derived CS by the carboxy-terminal group using several solvents as mobile phase. The CSs were also studied from the conformational point of view in solution using circular dichroism and 13C NMR. A relationship was found between the presence of an ordered conformation in the particular conditions used and increased enantioselectivity.     

Evaluation of fused-core and monolithic versus porous silica-based C18 columns and porous graphitic carbon for ion-pairing liquid chromatography analysis of catecholamines and related compounds

This paper evaluates the performances of reversed-phase (RPLC) and ion-pairing chromatography (IPLC) coupled with UV detection for the analysis of a set of 12 catecholamines and related compounds. Different chromatographic columns (porous C18-silica, perfluorinated C18-silica, porous graphitic carbon, monolithic and fused-core silica-based C18 columns) were tested using semi-long perfluorinated carboxylic acids as volatile ion-pairing reagents. Much more promising results were obtained by IPLC than by RPLC and important improvements in analytes peak symmetry and separation resolution were observed when using the "fast chromatography" columns (monolithic and fused-core C18) under IPLC conditions. For UV detection, a satisfactory separation of the 12 selected analytes was achieved in less than 20 min by using a fused-core particles column (Halo C18) and a mobile phase composed of a 1.25 mM nonafluoropentanoic acid aqueous solution and methanol under gradient elution mode. The chromatographic method developed can be directly coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS) in positive ionization mode and 10 solutes among those selected can be observed. The presence of the acidic ion-pairing reagent in the mobile phase makes this system incompatible with negative ionization mode and thus unable to detect the two acidic compounds that only responded in negative mode. In terms of MS detection, Monolithic C18 column proved to be the best one to reach the lowest detection limits (LODs) (from 0.5 ngmL(-1) to 10 ngmL(-1) depending on the neurotransmitter). The applicability of the optimized LC-MS/MS method to a "real world" sample was finally evaluated. The presence of the matrix leads to signal suppression for several solutes and thus to higher LODs.     

Quantitative analysis of methylguanidine and guanidine in physiologic fluids by high-performance liquid chromatography—fluorescence detection method

A rapid and sensitive high-performance liquid chromatographic method has been developed for the quantitative analysis of methylguanidine and guanidine in physiological fluids. These guanidino compounds are separated on a 6 × 0.23 cm cation-exchange column with 0.5 M sodium hydroxide solution. The guanidino compounds are detected with a fluorometer, which monitors the fluorescent guanidine derivatives produced by the reaction of the eluted constituents with 9,10-phenanthrenequinone. Sensitivity to sub-nanomole levels of methylguanidine and guanidine is demonstrated. The method was successfully applied to physiological fluids such as serum and cerebrospinal fluid from uremic patients.