D-L amino acid analysis using automated precolumn derivatization with 1-fluoro-2,4-dinitrophenyl-5-alanine amide

Summary An automated procedure for the precolumn derivatization of enantiomeric amino acid mixtures with 1-fluoro-2,4-dinitrophenyl-5-alanine amide and a liquid chromatographic method for the separation of the derivatives with UV detection are reported. The system described allows to perform routine analyses using microbore columns with a sensitivity at the picomol level. Improvements for the use of this reagent in the protocol of a subtractive Edman degradation procedure of peptides to determine the sequence position of amino acid residues with concomitant identification of their chirality are also described.     

Amino acid analysis by reverse-phase high-performance liquid chromatography: precolumn derivatization with phenylisothiocyanate

Methods for the quantitative derivatization of amino acids with phenylisothiocyanate and for the separation and quantitation of the resulting phenylthiocarbamyl derivatives by reverse-phase high-performance liquid chromatography are described. Phenylthiocarbamylation of amino acids proceeds smoothly in 5 to 10 min at room temperature. Coupling solvents, reagent, and some byproducts are removed by rotary evaporation under high vacuum, and the phenylthiocarbamyl derivatives are dissolved in 0.05 M ammonium acetate, pH 6.8, for injection onto the octyl or octadecylsilyl reverse-phase column. Columns are equilibrated with the same solvent and the effluent stream is monitored continuously at 254 nm for detection of the amino acid derivatives. Elution of all of the phenylthiocarbamyl amino acids is achieved in about 30 min utilizing gradients of increasing concentrations of ammonium acetate and acetonitrile or methanol. This approach to amino acid analysis offers select advantages, both with respect to methods which employ reverse-phase separation of prederivatized samples and to the classical ion-exchange procedure. All amino acids, including proline, are converted quantitatively to phenylthiocarbamyl compounds and these are stable enough to eliminate any need for in-line derivatization. Furthermore, results comparable in sensitivity and precision to those obtained by state-of-the-art ion-exchange analyzers may be generated with equipment that need not be dedicated to a single application.     

Determination of vertilmicin in rat serum by high-performance liquid chromatography using 1-fluoro-2,4-dinitrobenzene derivatization

A procedure for the high-performance liquid chromatographic determination of vertilmicin in rat serum was described using pre-column derivatization. The serum proteins were precipitated with acetonitrile and vertilmicin in the supernatant was derivatized with 1-fluoro-2,4-dinitrobenzene. Etimicin was selected as the internal standard. The mobile phase consisted of methanol--20mM ammonium acetate (80:20, v/v), and flow-rate was 0.9 ml/min. Ultraviolet detection was set at 365 nm. The reaction products were chromatographed on a C(18) column kept at 40 degrees C. A good linearity was found in the range of 0.5-250 microg/ml. Both intra- and inter-day precisions of vertilmicin, expressed as the relative standard deviation, were less than 7.4%. Accuracy, expressed as the relative error, ranged from -0.1 to 3.6%. The mean absolute recovery of vertilmicin at three different concentrations was 92.5%. Serum volumes of 50 microl were sufficient for the determination of vertilmicin. The method was proved suitable for the pharmacokinetic study of vertilmicin in rats.     

Determination of amikacin in microlitre quantities of biological fluids by high-performance liquid chromatography using 1-fluoro-2,4-dinitrobenzene derivatization

Pre-column derivatization of amikacin with 1-fluoro-2,4-dinitrobenzene in 25 μl of guinea pig plasma or human serum produced a stable chromophore which was measured by UV detection after rapid separation on normal-phase or reversed-phase high-performance liquid chromatography systems. The reversed-phase system, selected for routine analysis due to instability of the normal-phase column, consisted of an Ultrasphere-ODS C18 column preceded by a guard column, and used acetonitrile—water (68:32) as the mobile phase. A high degree of linearity was found in the range of 2—64 μg/ml with a coefficient of variation averaging less than 5%.     

Amino acid analysis using 1-naphthylisocyanate as a precolumn high performance liquid chromatography derivatization reagent

A method which uses 1-naphthylisocyanate as an HPLC precolumn derivatization reagent for amino acid analysis is described. Derivatization is carried out by adding the isocyanate dissolved in dry acetone to a buffered amino acid solution followed by extraction of the excess reagent with cyclohexane. The resulting naphthylcarbamoyl amino acids are stable and highly fluorescent, with excitation maxima at 238 and 305 nm and an emission maximum at 385 nm, for most amino acids. Ultraviolet detection near 222 nm, the absorption maximum, can also be employed. HPLC procedures permitting the analysis of protein hydrolysates, brain extract, cerebrospinal fluid, and blood plasma are presented. The method is particularly suitable for auto-sampler procedures since samples can be derivatized and diluted in advance and stored at room temperature in the sampler while awaiting injection. Other advantages include high sensitivity, the possibility of recovering the derivatives from the column effluent, and the absence of a reagent peak in the chromatograms.     

Determination of ethambutol in plasma by high-performance liquid chromatography after pre-column derivatization

A new HPLC assay using UV detection (200 nm) was developed to determine ethambutol (EMB) concentrations in plasma. Following extraction (0.1 ml plasma) with chloroform, EMB and octylamine (used as internal standard) were derivatized with phenylethylisocyanate. Quantitation in plasma was achieved at 200 nm. There were no interferences from endogenous compounds. Intra- and inter-day variabilities were lower than 5.2 and 7.6%, respectively. The limit of quantitation of the method was 0.2 μg/ml. In plasma, ethambutol was found to be stable for at least one month when samples were stored at −20°C. This assay was applied to the therapeutic monitoring of EMB concentrations in 19 patients suffering from tuberculosis.     

Analysis of hydroxyproline in urine by high-performance liquid chromatography after dabsyl-chloride derivatization

Summary In order to analyse hydroxyproline (HYP) in urine, a high-performance liquid chromatographic method was modified. The primary amino groups were blocked with o-phthalaldehyde, and then the secondary amino groups were derivatized with 4-dimethylaminoazobenzene-4-sulphonyl chloride. In addition, the dabsylated samples were treated with ethyl acetate to obtain a simple elution profile in high-performance liquid chromatography. The dabsyl-HYP and -proline were eluted at 4.7 min and 8.0 min, respectively. The chromatographic analysis was completed within 10 min, including the time needed for reequilibration of the column. Using the present method, the concentration of HYP in urine was determined to 260 ± 6µmol/l.     

Bioanalysis of m-iodobenzylguanidine in plasma by high-performance liquid chromatography after derivatization with benzoin

A sensitive chromatographic assay has been developed for m-iodobenzylguanidine (MIBG) in human plasma based on the derivatization with benzoin. MIBG is first isolated from plasma using solid-phase extraction on a cyanopropyl-modified silica phase. After evaporation of the eluate, a fluorescent derivative is formed using benzoin. The derivative is analysed by reversed-phase liquid chromatography using a mixture 60% (v/v) acetonitrile, 30% (v/v) water and 10% (v/v) of the 0.5 M Tris buffer (pH 8.0) as the eluent and fluorescence detection at 320 nm for excitation and 435 nm for emission, respectively. In the evaluated concentration range (2–200 ng/ml) precisions 10% and accuracies in between 90 and 100% have been found, with 2 ng/ml being the lower limit of quantification using a 0.5-ml plasma sample volume. The assay can also be used without the internal standard benzylguanidine. The assay was successfully used to obtain a pharmacokinetic curve of MIBG.     

Determination of rat blood S-D-lactoylglutathione by a column-switching high-performance liquid chromatography with precolumn fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole

A method for the determination of S-d-lactoylglutathione (SLG), an intermediate metabolite of the glyoxalase system, in rat blood is described. After hemolysis and deproteinization of 30 microl of rat blood, SLG in the blood was determined by a column-switching HPLC system with precolumn fluorescence derivatization with a fluorogenic reagent, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). Calibration curves for the determination of SLG showed a good linearity (r2 > 0.999) over the range of 20-100 pmol SLG spiked in rat blood samples. The accuracy was in the range of 97-104% (20-100 pmol SLG spiked in rat blood sample, N = 4). The detection limit was 8.12 fmol, and the quantitation limit was 27.07 fmol, respectively. The intra- and interday coefficients of variance were 4.63% (N = 5) and 9.98% (N = 5), respectively. The concentrations of SLG in whole blood from male Wistar-Kyoto rats (12 weeks old) were 3.48+/-0.78 microM (N = 4, mean+/-SE). In streptozotocin-induced diabetic rats, the concentration of SLG was significantly increased, approx 5-fold, compared with normal rats, suggesting that the metabolic flux of the glyoxalase system increases in red blood cells during hyperglycemia.     

Determination of paromomycin in human plasma and urine by reversed-phase high-performance liquid chromatography using 2,4-dinitrofluorobenzene derivatization

A sensitive high-performance liquid chromatographic method for the determination of paromomycin in human plasma and urine was developed. Paromomycin was quantitated following pre-column derivatization with 2,4-dinitrofluorobenzene (DNFB). The chromatographic separation was carried out on a C₁₈ column at 50°C using a mobile phase consisting of 64% methanol in water adjusted to pH 3.0 with phosphoric acid. The eluents were monitored by UV detection at 350 nm. The linearity of response for paromomycin was demonstrated at concentrations from 0.5 to 50 μg/ml in plasma and 1 to 50 μg/ml in urine. The relative standard deviation of the assay procedure is less than 5%.     

Amino acid analysis of collagen hydrolysates by reverse-phase high-performance liquid chromatography of 9-fluorenylmethyl chloroformate derivatives

A recently described procedure for amino acid analyses has been modified and adapted for use in quantitating the unique mixture of products commonly found in hydrolysates of the collagens. The method involves precolumn derivatization of hydrolysates with 9-fluorenylmethyl chloroformate (FMOC-CL), chromatographic separation of the derivatives and excess reagent on a reverse-phase column, and quantitation based on the fluorescent properties of the derivatives. The method takes advantage of the ease with which stable derivatives are formed with the FMOC reagent. Using a ternary gradient system, a complete amino acid analysis with good resolution of all components can be performed within 35 min. The sensitivity of the method is comparable to levels attained by other derivatives and the fluorescence response of each derivative is linear over the total range of 1-800 pmol. Given these parameters, the method allows complete amino acid analyses to be performed on 100 ng of collagen corresponding to a single picomole of a collagen chain (Mr 100,000).     

Determination of penicillin G in bovine plasma by high-performance liquid chromatography after pre-column derivatization

A simple, selective, and sensitive liquid chromatographic method with ultraviolet detection was developed for the analysis of penicillin G in bovine plasma. The assay utilizes a simple extraction of penicillin G from plasma (with a known amount of penicillin V added as internal standard) with water, dilute sulphuric acid and sodium tungstate solutions, followed by concentration on a conditioned C₁₈ solid-phase extraction column. After elution with 500 μl of elution solution, the penicillins are derivatized with 500 μl of 1,2,4-triazole—mercuric chloride solution at 65°C for 30 min. The penicillin—mercury mercaptide complexes are separated by reversed-phase liquid chromatography on a C₁₈ column. The method, which has a detection limit of 5 ng/ml (ppb) in bovine plasma, was used to quantitatively measure the concentrations of penicillin G in plasma of steers at a series of intervals after the intramuscular administration of a commercial formulation of procaine penicillin G.     

Trace analysis of tobramycin in human plasma by derivatization and high-performance liquid chromatography with ultraviolet detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method is established for the trace determination of tobramycin in human plasma by derivatization. The method is based on the chemical derivatization of aminoglycoside antibiotic, tobramycin in human plasma, with 1-naphthyl isothiocyanate (NITC) in pyridine at 70 degrees C. After derivatization reaction, a methylamine/acetonitrile solution was added to the reaction mixture to eliminate the excess derivatizing agent and shorten the analysis time. The resulting derivative was separated using a Purospher STAR RP-18e column and a water-acetonitrile (50:50, v/v) mobile phase (detection at 230 nm). Optimization conditions for the derivatization of tobramycin were investigated by HPLC. The linear range for the quantitation of tobramycin in spiked plasma was over 0.93-9.34 mg/l; the detection limit (signal-to-noise ratio=3; injection volume, 10 microl) was about 0.23 mg/l. The relative standard deviation was less than 2.1% for intra-day assay (n=6) and 5.2% for inter-day assay (n=6) and relative recoveries were found greater than 99%.     

Fluorescence detection of cardenolides in reversed-phase high-performance liquid chromatography after post-column derivatization

Methods for the fluorescence derivatization of cardiac glycosides with concentrated acids from TLC are adopted to HPLC for post-column derivatization. The column effluent is blended with concentrated acids in a knitted tube reactor, which enables derivatization with negligible increase in chromatographic peak width. The selectivity of the reaction is temperature-dependent and influenced by the respective acid. Reactivity increases from H₃PO₄→CH₃SO₃H ≡ H₂SO₄. The conversion of digoxigenin, digitoxigenin and their digitoxosides is accelerated by Cu(II) acetate or Co(II) nitrate in H₂SO₄. Combined with a new two-mode, single-column solid-phase sample preparation, cardiac glycoside levels of less than 100 pg/glycoside in 1 ml plasma are detectable.     

Quantitative analysis of valienamine in the microbial degradation of validamycin A after derivatization with p-nitrofluorobenzene by reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatography method for the quantitative analysis of valienamine in the microbial degradation of validamycin A, using a procedure for pre-column derivatization of valienamine with p-nitrofluorobenzene is described. Valienamine in the broth was first isolated with the ion-exchange method. The optimized conditions for the derivatization were the reaction time 30 min and reaction temperature 100 degrees C. With the mobile phases consisting of acetonitrile-water (12:88) (eluent A) and methanol (eluent B), the gradient was carried out with 100% of A for 15 min and then 100% of B for another 10 min. The parameters in the process were the flow rate of the mobile phase 1.0 ml/min, the injection volume 20 microl, the column temperature 40 degrees C and wavelength of ultraviolet detection 398 nm in all runs. A good linearity was found in the range of 0.5-150.0 microg/ml. Both intra- and inter-day precisions of valienamine, expressed as the relative standard deviation, were less than 9.4%. Accuracy, expressed as the relative error, range from -0.5 to 2.7%. The mean absolute recovery of valienamine at three different concentrations was 94.2%. The method was proved suitable for the study on the process of microbial degradation of validamycin A to produce valienamine.     

The separation of D/L amino acid pairs by high-performance liquid chromatography after precolumn derivatization with optically active naphthylethyl isocyanate

A method for determining the optical purity of amino acids using HPLC and precolumn derivatization is described. (+)-1-(1-Naphthyl)ethyl isocyanate reacts with racemic amino acids, in high yield, to form naphthylethyl carbamoyl derivatives. The resulting diastereoisomeric pairs were separated on reversed-phase C18 columns and detected fluorometrically. Excitation maxima for naphthylethyl carbamoyl aspartic acid were 235 and 297 nm. The emission maximum was at 333 nm. Using a filter fluorometer with a zinc or cadmium lamp, less than 1 pmol of a D amino acid can be measured in the presence of 1000-fold excess of the L isomer. The column can also be monitored at lower sensitivity, using an ultraviolet detector operating at or near the absorption maximum of 222 nm. Chromatographic data are presented on the resolution of 17 amino acid pairs.     

Amino acid analysis by dinitrophenylation and reverse-phase high-pressure liquid chromatography

The determination of amino acids has been achieved by reverse-phase high-pressure liquid chromatography of their dinitrophenyl derivatives. The methods developed permit the quantitation of all amino acids commonly encountered in a protein hydrolysate and the effect of various parameters on this separation was systematically evaluated. The procedure eliminates the need for specialized postcolumn equipment as employed in conventional amino acid analysis and can be obtained by a simple gradient high-pressure chromatograph. The sensitivity obtained is comparable to that available by methods in common usage, being able to determine amino acids quantitatively in the low picomole range.     

Determination of urinary glucuronide conjugates by high-performance liquid chromatography with pre-column fluorescence derivatization

A simple and highly sensitive high-performance liquid chromatographic method for the direct determination of urinary glucuronide conjugates is described. The method is based on the direct derivatization of the glucuronic acid moiety in glucuronide conjugates with 6,7-dimethoxy-1-methyl-2 (1 H)-quinoxalinone-3-propionylcarboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 0–37°C. The resulting fluorescent derivatives are separated on a C₁₈ column using methanol—acetonitrile—0.5% triethylamine in water (1:1:2, v/v) as mobile phase, and are detected spectrofluorimetrically at 445 nm with excitation at 367 nm. The detection limits (signal-to-noise RATIO = 3) for the glucuronides are 13–48 fmol for an injection volume of 10 μl (130–480 fmol per 5 μl of human urine). The method was applied to the measurement of etiocholanorone-3-glucuronide and androsterone-3-glucuronide in human urine. The method is simple and rapid without conventional liquid—liquid extraction of the glucuronides from urine.     

Amino acid analysis by high-performance liquid chromatography of a single stained protein band from a polyacrylamide gel

A single stained band containing approximately 5 micrograms of protein was cut out of a polyacrylamide gel and subjected to hydrolysis together with the gel. The hydrolysate was subsequently analyzed for its amino acid content by high-performance liquid chromatography and postlabeling with o-phthalaldehyde. Bovine serum albumin, ribonuclease B, ovalbumin, pepsin, and chymotrypsinogen A were analyzed by this method, and their amino acid compositions were found to be in good agreement with the reported values. By this method, it is possible to quantitate 16 amino acids: Asx, Thr, Ser, Glx, Pro, Cys, Gly, Ala, Val, Ile, Leu, Tyr, Phe, His, Lys, and Arg. Thioglycolic acid is effective protection against the decomposition of Tyr, Cys, and Met; however, the recovery of Met is inconsistent. This method might be very helpful for the amino acid analysis of proteins of multicomponent systems, especially, those which can be resolved only by polyacrylamide gel electrophoresis.