Amino acid analysis of physiological fluids by high-performance liquid chromatography with phenylisothiocyanate derivatization and comparison with ion-exchange chromatography

The suitability of pre-column derivatization with phenylisothiocyanate followed by high-performance liquid chromatography was investigated as a means of analyzing free amino acids in plasma and other physiological fluids. A comparison was made between this method and a conventional ion-exchange method. The correlation coefficient for all the amino acids tested was greater than 0.9, except for proline and tryptophan. Various forms of sample preparation were tried for plasma and amniotic fluid; it was finally decided that protein precipitation with acetonitrile was most suitable. Ultrafiltration was used for cerebrospinal fluid preparation while urine was treated the same as a standard mixture. The retention times relative to the internal standard (nor-leucine) are given for over 90 compounds. Some of these were chromatographed underivatized because they are known to be present in some physiological fluids and absorb at 254 nm because of their aromaticity. The imprecision for this method compared favourably with the standard ion-exchange method although each had specific amino acids for which the imprecision was poor. The technique is suitable for the same routine clinical analysis purposes as high-resolution ion-exchange chromatography. It also offers the advantages of speed of analysis, sensitivity and equipment versatility over the conventional ion-exchange methods.     

Determination of methylmalonic acid after diazonium derivatization by high-performance liquid chromatography

The use of a single prederivatization step in conjunction with high-performance liquid chromatography (h.p.l.c.) is described for the determination of methylmalonic acid (MMA). The method is based on the reaction of MMA and 4-diazobenzenesulfonic acid, which produces a derivative that has a molar absorptivity of about 9 × 10³ l mol^?1 cm^?1 at 353 nm. The derivatization reaction is optimized for various parameters. A reagent concentration of 3.3 mM at a reaction pH of 4.6 and a temperature of 100°C are optimal. The reaction product is separated from the excess of reagent and other interfering components by using a polystyrene-divinylbenzene column and a highly aqueous mobile phase. After a simple clean-up step, it is possible to quantify MMA in urine at about 0.8 mg l^?1 with linear response up to 32 mg l^?1.     

Simultaneous analysis of amino acid and biogenic polyamines by high-performance liquid chromatography after pre-column derivatization with N-(9-fluorenylmethoxycarbonyloxy)succinimide

A high-performance liquid chromatography method for the simultaneous analysis of amino acids and biogenic polyamines, using a new procedure for pre-column derivatization of amino groups with N-(9-fluorenylmethoxycarbonyloxy)succinimide is described. The separation of 20 amino acids and 4 biogenic polyamines was achieved within 32 min on a sequence of three short (50 mm) reversed-phase C18, 5 microm columns by elution buffers based on dibutylamine phosphate. The method linearity, calculated for each amino acid and polyamine, has a correlation coefficient higher than 0.991, in concentrations ranging from 0.2 to 50 microM, except for spermine and methionine, where the correlation coefficients were r = 0.984 and r = 0.979, respectively. The stability of derivatives in acidified samples at 4 degrees C and room temperature was demonstrated. The limit of quantitation was estimated to be around 50 pM in 50 microl sample injection. The repeatability of the method, expressed as R.S.D., ranged from 1.1 to 6.7%. The presented method was applied for the quantitation of amino acid and polyamine contents in beer, wine, and cell culture samples, using 2-aminoheptanoic acid or 1,7-diaminoheptane as internal standard.     

Simultaneous analysis of monosaccharides and oligosaccharides by high-performance liquid chromatography with postcolumn fluorescence derivatization

To develop a fluorimetric HPLC technique for the simultaneous microanalysis of reducing mono- and oligosaccharides, the technique of linear gradient elution was introduced into the postcolumn fluorimetric detemination system of reducing saccharides with benzamidine. Fluorescence measurement was performed at 288 nm for excitation and 470 nm for emission and an optimization study for this postcolumn fluorescence derivatization carried out. Under optimum conditions, the detection limits of D-glucose and maltohexaose were 1.78 and 2.59 pmol, respectively. The present method was successfully applied to saccharide analysis and should prove useful for automated simultaneous microanalysis of reducing mono- and oligosaccharides in foods.     

Determination of phosphonic acid breakdown products by high-performance liquid chromatography after derivatization

A new HPLC method for the determination of oxidative breakdown products of aminopolyphosphonates is presented. The phosphonate nitrilotrismethylenephosphonic (NTMP) acid undergoes catalytic oxidation by molecular oxygen in the presence of manganese(II). The two diphosphonates iminodimethylenephosphonic acid (IDMP) and formyliminodimethylenephosphonic acid (FIDMP) are formed. The analytical method employs the derivatization of the aldehyde group in FIDMP by 2,4-dinitrophenylhydrazine and of the imine group in IDMP by 9-fluorenyl methylchloroformate. The two derivatives are quantified in separate runs using the same acidic phosphate-acetonitrile eluent with detection at 370 nm for FIDMP and 260 nm for IDMP. The detection limit for FIDMP is 0.01 microM, for IDMP 0.02 microM. The method is suitable for the determination of the breakdown products in wastewater.     

Stereochemical analysis of betamethasone and dexamethasone by derivatization and high-performance liquid chromatography.

A simple and economical high-performance liquid chromatographic method has been developed for the simultaneous determination of betamethasone and dexamethasone. The method is based on the derivatization of the structural epimers of betamethasone and dexamethasone with a homochiral reagent, N-carbobenzoxy-L-phenylalanine. The derivatives obtained were easily recognized by a non-chiral silica column with n-hexane-dichloromethane-isopropanol (100:100:4, v/v/v) as a mobile phase and a good separation was obtained for quantitation. The method was satisfactorily applied to the determination of betamethasone and dexamethasone in tablets.     

Determination of alkylamines by high-performance liquid chromatography with post-column fluorescence derivatization

A styrene/divinylbenzene polymer column and an amino column are compared for the non-aqueous separation of primary, secondary and tertiary alkylamines. Post-column derivatization with o-phthalaldehyde/2-mercaptoethanol is selective for primary amines and derivatization with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is selective for secondary amines after on-line masking of primary amines. This procedure can tolerate 0.4 M butylamine. The limit of detection is 18.5 mM for dioctylamine (with NBD-Cl) and 0.18 mM for decylamine and tetraethylenepentamine (with o-phthalaldehyde/2-mercaptoethanol).     

Determination of citrulline and homocitrulline by high-performance liquid chromatography with post-column derivatization

A high-performance liquid chromatographic method was developed for the determination of citrulline and homocitrulline using a post-column colorimetric reaction with o-phthaladehyde and N-(1-naphthyl)-ethylenediamine. Citrulline and homocitrulline were determined with no interferences from protein amino acids. The results show that the level of citrulline in the plasma of patients with uremia on intermittent hemodialysis is higher than that in healthy human plasma, and that homocitrulline is excreted into the urine of healthy adults.     

Simultaneous determination of mycophenolic acid and valproic acid based on derivatization by high-performance liquid chromatography with fluorescence detection

A reliable and validated reversed-phase high-performance liquid chromatography (HPLC) method using fluorescence detection is reported for the simultaneous quantitation of mycophenolic acid (MPA) and valproic acid (VPA) in human plasma. The method is based on the pre-column derivatization of valproic acid with 4-bromomethyl-6, 7-dimethoxycoumarin (BrMMC) and online solvatochromism of MPA by pH adjustment. The linear calibration range was 0.50-30 microg/mL for MPA and 5.00-150 microg/mL for VPA. The relative standard deviations of the method of intra- and inter-day analyses (n = 6) were below 6.5 and 6.7% for MPA, and 5.8 and 6.3% for VPA, respectively. Dichloromethane was used for the simultaneous extraction of MPA and VPA from acidified plasma. This reliable method can be applied in the analysis of MPA and VPA in human plasma using only a small volume (100 microL).     

Monitoring dediazoniation product formation by high-performance liquid chromatography after derivatization

A derivatization protocol that exploits the rapid reaction between arenediazonium ions and a suitable coupling agent followed by high-performance liquid chromatography analyses of the reaction mixture was employed to determine the product distribution, the rate constants for product formation and the association constant of 4-nitrobenzenediazonium, PNBD, ion with beta-cyclodextrin, beta-CD. The derivatization of PNBD with the coupling agent leads to the formation of a stable azo dye that prevents by-side reactions of PNBD with the solvents of the mobile phase, including water, or the metallic parts of the chromatographic system that would eventually lead to erroneous identification and quantification of dediazoniation products. The results show that in the presence of beta-CD, nitrobenzene is formed at the expense of 4-nitrophenol, which is the major product in its absence. The observed rate constants for the interaction between PNBD and beta-CD increase upon increasing [beta-CD] showing a saturation profile indicative of the formation of an inclusion complex between PNBD and beta-CD. By fitting the experimental data to a simplified Lineaweaver-Burk equation, the corresponding association constant and the maximum acceleration rate of beta-CD towards PNBD were estimated. The protocol is applicable under a variety of experimental conditions provided that the rate of the coupling reaction is much faster than that of dediazoniation.     

Determination of fatty acids by high-performance liquid chromatography and fluorescence detection using precolumn derivatization with 9-fluorenylmethyl chloroformate

A new high-performance liquid chromatographic method is described for the determination of fatty acids in seed oils. The method was based on precolumn derivatization with 9-fluorenylmethyl chloroformate as a labeling agent and fluorescence detection. Fatty acids were extracted from the samples and subjected to derivatization with the reagent at 60°C for 10?min. The chromatographic separation of 14 fatty acids (C10–C22) was achieved on a combined loading compression octadecyl sulfate (CLC-ODS) column with a run time of 30?min. Three-step gradient elution of a mobile phase consisted of acetonitrile and water was used, and the signal was monitored at excitation and emission wavelengths of 265 and 315?nm, respectively. The method indicated favorable sensitivity and reproducibility for fatty acids’ derivatives. The detection limits, at a signal-to-noise ratio of 3, were 0.01–0.05?µg/ml and relative standard deviations (RSDs) were less than 0.27%. Excellent linear responses were observed with coefficients of 0.9995. This method was applied to quantify fatty acids in white, brown, and black sesame seeds’ oil.     

Determination of methyl methanesulfonate and ethyl methanesulfonate in methanesulfonic acid by derivatization followed by high‐performance liquid chromatography with ultraviolet detection

Methanesulfonic acid is routinely used in pharmaceuticals but can contain potentially genotoxic impurities such as methyl methanesulfonate and ethyl methanesulfonate. The aim of this study was to develop a simple high‐performance liquid chromatography with ultraviolet detection method for determining methyl methanesulfonate and ethyl methanesulfonate in methanesulfonic acid. Samples (250 mg) in water/acetonitrile (200 μL) were first combined with 10.0 mol/L sodium hydroxide solution (270 μL). Then they were mixed with 2.0 mg/mL N ,N‐diethyldithiocarbamate (500 μL), diluted to 5 mL with N ,N‐dimethylacetamide and allowed to react at 80°C for 1 h. The derivatives were analyzed using gradient high‐performance liquid chromatography with ultraviolet detection (277 nm) and structurally elucidated by liquid chromatography with mass spectrometry. With acetonitrile/5 mmol/L ammonium acetate solution as the eluent and 1 mL/min as the flow rate on a C18 column, the derivatives were eluted at 10.6 and 14.8 min. Good linearity (correlation coefficients > 0.999) and low limits of quantitation (0.6 ppm) were obtained. The recoveries were in the range of 80–115% with relative standard deviation < 5.0%. Finally, the established method was successfully used for the determination of methyl methanesulfonate and ethyl methanesulfonate in methanesulfonic acid.     

On-column endcapping and derivatization in reversed-phase high-performance liquid chromatography

Summary On-column endcapping and derivatization or regeneration of C₈ and C₁₈ reversed-phase HPLC columns with newly introduced reagents were studied. These treatments can increase column life expectancy by restoring retention times and original chromatographic characteristics of the columns. This is illustrated by examples. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Quantitative determination of quinic acid and derivatives by high-performance liquid chromatography after derivatization with p-bromophenacyl bromide

Quantitative determinations of quinic, shikimic and glycolic acide have been performed by reversed-phase high-performance liquid chormatography using p-bromophenacyl bromide as a visualizing reagent. The reactions are carried out in N,N-dimethylformamide (DMF) with KF as a catalyst. Linear calibration curves are obtained in the concentration range 0.1–2 mg acide per ml DMF. Spectroscopic and chromatographic properties of the pbromophenacyl bromide derivatives of quinic, shikimic, dehydroshikimic and glycolic acid are given. A quantitative analysis of quinic acid in plant material is demonstrated.     

Body fluid analysis of a phosphonic acid angiotensin-converting enzyme inhibitor using high-performance liquid chromatography and post-column derivatization with o-phthalaldehyde

A method is described for the extraction of a phosphonic acid angiotensin-converting enzyme inhibitor from either urine or plasma, and subsequent quantitation using high-performance liquid chromatographic (HPLC) analysis and post-column o-phthalaldehyde reagent derivatization. The compound cannot be quantitatively extracted from the body fluids, but use of a fluorinated internal standard allowed for the computation of accurate results. With the use of an internal standard, excellent precision, linearity, and recovery were obtained for analyte response in both urine and plasma. In urine a working range of 0.2-10 micrograms/ml was found, with a limit of detection of 0.1 micrograms/ml. For plasma the working range was found to be 2-500 ng/ml, and the limit of detection was established as 1 ng/ml. Due to the non-polar character of the analyte at low pH values, it was possible to use novel extraction (solid-phase C8 column) and HPLC [poly(styrenedivinyl benzene) HPLC column] conditions to separate and quantitate the compound from plasma and urine.