Simultaneous determination of amoxicillin and ampicillin in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection using pre-column derivatization

A sensitive and robust method is presented for the simultaneous determination of amoxicillin (AMO) and ampicillin (AMP) in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD). This method used a simple liquid-liquid extraction of the samples with acetonitrile and dichloromethane as precipitation of proteins and extraction solvent. AMO and AMP reacted with salicylaldehyde to form fluorescent derivatives, which were then analyzed with RP-HPLC-FLD. Separation was carried out on an Athena C18 column with a mobile phase consisting of 0.01 M potassium dihydrogen phosphate, adjusted to pH 5.5 by 2M potassium hydroxide and acetonitrile. The detector response was linear over the tested concentration range from 5.0 to 800 ng/mL for AMO and AMP. The recovery values ranged from 78.4 to 88.7% for AMO and from 77.6 to 82.0% for AMP. The limits of detection were 1.2 for AMO and 0.4 μg/kg for AMP. The limits of quantification were 3.9 for AMO and 1.5 μg/kg for AMP. The corresponding intra-day and inter-day variation (relative standard deviation) were found to be less than 9.6 and 14.8%, respectively.     

Improved determination of the bisphosphonate pamidronate disodium in plasma and urine by pre-column derivatization with fluorescamine, high-performance liquid chromatography and fluorescence detection.

An improved method for the determination of 1-hydroxy-3-aminopropylidene-1,1-bisphosphonate (pamidronate) in human urine and plasma is described. The procedure is based on a co-precipitation of the bisphosphonates (pamidronate and 6-amino-1-hydroxypentilidene-bisphosphonate, used as internal standard) with calcium phosphate. After centrifugation the precipitate is redissolved in hydrochloric acid, followed by a second precipitation. Then the bisphosphonates are dissolved in ethylenediaminetetraacetic acid, derivatized with fluorescamine, and separated by high-performance liquid chromatography. Using fluorescence detection, the limit of quantitation for pamidronate was 0.8 mumol/l in plasma and 0.7 mumol/l in urine.     

Separation and determination of sugars by reversed-phase high-performance liquid chromatography after pre-column microwave-assisted derivatization

In this study the possibility of derivatizing sugars using microwave irradiation was investigated. The amount of reagent, irradiation intensity, and derivatization time were optimized. In the derivatization of sugars with p-nitroaniline the reaction is complete within 5 min at 600 W when the p-nitroaniline-to-sugar and NaBH₃CN-to-sugar mole ratios were above 1.4 and 3.1, respectively. A Doehlert design was used to optimize the mobile phase for separation of p-nitroaniline-labeled sugars; and the best separation was obtained by use of 0.01 mol L⁻¹ acetate buffer at pH 4.40 containing 11.0% acetonitrile. Analysis using this method was highly sensitive and analysis time was short. Finally, a food sample was analyzed using the proposed method.     

Simple, rapid and sensitive determination of plasma taurine by high-performance liquid chromatography using pre-column derivative formation with fluorescamine.

A simple, rapid and sensitive method for the determination of plasma taurine by high-performance liquid chromatography in the isocratic mode has been developed. The deproteinized plasma was treated with fluorescamine. These derivatives were separated on a LiChrospher 100 RP-8 column within 15 min. The detection limit for taurine was 0.2 microM. The plasma taurine contents of yellowtail fish, Seriola quinqueradiata, beef cattle, dairy cows and chickens were determined to be 125 +/- 54, 5.6 +/- 1.4, 2.2 +/- 0.7 and 20.0 +/- 9.6 micrograms/ml, respectively.     

High-performance liquid chromatographic determination of peptides in biological fluids by automated pre-column fluorescence derivatization with fluorescamine

Peptides containing a free alpha- or epsilon-amino group react with fluorescamine under mild alkaline conditions to generate a highly fluorescent but unstable reaction product and, consequently, practical high-performance liquid chromatographic (HPLC) approaches to analysis have typically involved the use of postcolumn derivatization. An automated precolumn approach is reported in which peptides are reacted with fluorescamine just prior to HPLC analysis by a commercially available autoinjector with derivatization capabilities. The autoinjector added base and fluorescamine reagent solutions to a sample vial containing peptide analytes, and the derivatization reaction was allowed to proceed for 5 min at room temperature prior to injection into the HPLC system. The derivatized peptides were analyzed by reversed-phase HPLC with fluorescence detection (excitation at 390 nm; emission 470-nm cut-off filter) on an octylsilica column. Optimization of the precolumn reaction conditions and the use of narrower HPLC columns (2 mm I.D.) resulted in a typical on-column detection limit of 30-50 fmol of peptide, which was substantially lower than that in previously reported post-column methods. This approach was applied to the HPLC of several naturally occurring and synthetic peptides containing alpha- and epsilon-amino groups. In combination with solid-phase extraction, prior to automated precolumn fluorescence derivatization and chromatographic analysis, the methodology was used for the determination of a synthetic growth hormone-releasing peptide in plasma samples.     

Fluorescence determination of N-acetylaspartic acid in the rat cerebrum homogenate using high-performance liquid chromatography with pre-column fluorescence derivatization

N-acetyl-L-aspartic acid (NAA) is an endogenous compound, and its brain concentration is suggested to be altered in neurological disorders. In the present study, a fluorescence determination method for NAA was developed by employing reversed-phase high-performance liquid chromatography (HPLC) with pre-column fluorescence derivatization using 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole (DBD-ED). Using methylsuccinic acid as the internal standard, a linear calibration curve for NAA was constructed in the range 125-1000 microM (n=3). The detection limit on the column was approximately 5.0 fmol (signal-to-noise ratio 3). The proposed HPLC method was applied to determine NAA in the rat cerebrum homogenate. Cerebrum NAA was successfully determined using 10 microL of the homogenate, and the validation data for the proposed HPLC method demonstrated satisfactory results. Intra- and inter-day precision and accuracy were within 1.1-7.0 and -8.1-6.3%, respectively. The concentration of NAA in the male rat cerebrum (13 weeks old) was 84+/-4.6 micromol/mg protein (n=3).     

Determination of beta-aspartylpeptidase activity in human faeces by high-performance liquid chromatography using pre-column derivatization with phenyl isothiocyanate

Bacterial enzymes are responsible for degradation of beta-aspartyl peptides in the intestinal tract. These peptides, especially the dipeptide beta-aspartylglycine, are useful as indicators of an impaired anaerobic intestinal microflora of antibiotic-treated patients. A method to separate the dipeptides beta-aspartylalanine, beta-aspartylglutamine, beta-aspartylglycine and beta-aspartylserine, using reversed-phase high-performance liquid chromatography and precolumn derivatization with phenyl isothiocyanate, was developed. This method was used to determine beta-aspartylpeptidase activity in faecal supernatants of healthy human volunteers and antibiotic-treated patients with beta-aspartylglycine as substrate. This activity was absent in the antibiotic-treated group, while in individuals with an intact intestinal flora it ranged from 16 to 100% degradation per 18 h. In addition, it was found that faecal enzyme preparations cleaved beta-aspartylglycine at a much lower rate than the other beta-aspartyl peptides.     

Fluorimetric detection of serum corticosterone using high-performance liquid chromatography.

A simple, sensitive, specific and reproducible method for the determination of corticosterone concentrations in rat serum using high-performance liquid chromatography (HPLC) with fluorimetric detection is described. Corticosterone is detectable down to 0.1 ng injected onto the HPLC column. Cortisol is used as an internal standard. Ethyl acetate was used for both initial serum corticosteroid extraction and the subsequent fluorophore extraction after sulfuric acid hydrolysis; thus sulfuric acid does not enter the HPLC system. The resultant fluorophores for both corticosterone and cortisol are stable for at least two weeks at ambient temperature not requiring storage at -20 degrees C. The procedure is highly suitable for use with HPLC systems utilising automatic sample injectors. The method is specific for corticosterone; dexamethasone, cortisone and gonadal steroids are not detectable and do not interfere in this assay.     

Fluorimetric determination of pantothenic acid in foods by liquid chromatography with post-column derivatization

A method to determine the content of free pantothenic acid in various foods by reverse phase liquid chromatography-fluorimetry is reported. It includes a purification of the samples by successive passages through anion and cation exchange cartridges and a post-column derivatization of pantothenic acid as the fluorescent 1-alkylthio-2-alkylisoindole (reaction of beta-alanin, formed by hot alkaline hydrolysis of pantothenic acid, with orthophthaldialdehyde in the presence of 3-mercaptopropionic acid). An enzymatic hydrolysis prior to the purification step (pepsin at 50 degrees C for 3 h, then pantetheinase and alkaline phosphatase at 20 degrees C for 18 h) made it possible to release the bound pantothenic acid and thus to obtain the total Vitamin B5 content of these foodstuffs. The method proposed for the determination of free and bound pantothenic acid gives a good recovery rate (96-101%) and a satisfactory repeatability (R.S.D.r less than 8%). Owing to its low detection limit (0.65 microg g(-1)) and the good resolution of the pantothenic acid peak, it could most probably be applied to the determination of this vitamin in any foodstuff.     

Determination of 3-methylhistidine in hydrolysed proteins by fluorescamine derivatization and high-performance liquid chromatography

A method is described for the determination of the 3-methylhistidine content in myofibrilar proteins (myosin and actin) using reversed-phase high-performance liquid chromatography with ultraviolet detection. Proteins were hydrolysed and free amino acids were derivatized with fluorescamine. Elution was performed isocratically with acetonitrile-water (24:76). This method allows the detection of picomole amounts of 3-methylhistidine in myosin and actin.     

Simultaneous determination of the sulphonylurea glimepiride and its metabolites in human serum and urine by high-performance liquid chromatography after pre-column derivatization

A sensitive and selective high-performance liquid chromatographic method has been developed for a new sulphonylurea, glimepiride, and its metabolites. The assay involves extraction with diethyl ether, thermolysis of the sulphonylureas at 100 degrees C and trapping of the resulting amines with 2,4-dinitrofluorobenzene. The derivatives were quantitated on a reversed-phase column by absorbance at 350 nm using a step gradient for the three compounds in serum and an isocratic run for the metabolites in urine. Analogous compounds were used as internal standards. The detection limit was 5 ng/ml for glimepiride and metabolite II and 10 ng/ml for metabolite I using 1 ml of serum. The method has been applied to the analysis of serum and urine samples from pharmacokinetic studies in humans.     

Rapid high-performance liquid chromatography method for determination of pregabalin in a pharmaceutical dosage form following derivatization with fluorescamine

A simple, fast, and sensitive HPLC method was developed and validated for the evaluation of pregabalin in a pharmaceutical dosage form using fluorescamine as a derivatization agent for the first time. After a precolumn derivatization (5 min, room temperature), the reaction mixture was chromatographed on a C18 column with isocratic elution using 0.2% of triethylamine in a mixture of methanol and water (10 + 90, v/v). 3-Aminopentanoic acid was used as the internal standard. Using fluorescent detection (lamdaex 395 nm, lamdaem 476 nm), a low detection limit of 0.02 microg/mL was reached. The method was linear (r > 0.999) over the lower (0.125-25 microg/mL) and higher (1.25-250 microg/mL) concentration range. The intraday and interday precision of the QC samples was < 4.3%, and the accuracy was 94.2-102.5%. The samples were stable for 24 h at 4 degrees C. The robustness study showed that the derivatization is more robust than the chromatography method. The method was applied for the analysis of pregabalin content in 25, 75, and 300 mg capsules, and a good agreement was found with the declared amount of pregabalin (the relative error did not exceed 3.2%). Finally, the method was successfully used for dissolution studies of pregabalin capsules.     

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.     

Determination of nateglinide in human plasma by high-performance liquid chromatography with pre-column derivatization using a coumarin-type fluorescent reagent

A sensitive and selective high-performance liquid chromatographic method has been developed and validated for the determination of nateglinide in human plasma. Nateglinide and the internal standard, undecylenic acid, were extracted from plasma by liquid-liquid extraction using a mixture of ethyl acetate-diethyl ether, 50:50 (v/v). Pre-column derivatization reaction was performed using a coumarin-type fluorescent reagent, N-(7-methoxy-4-methyl-2-oxo-2H-6-chromenyl)-2-bromoacetamide. The derivatization proceeded in acetone in the presence of potassium carbonate and catalyzed by 18-crown-6 ether. The fluorescent derivatives were separated under isocratic conditions on a Hypersil BDS-C8 analytical column (250.0 mm × 2.1 mm i.d., particle size 5 μm) with a mobile phase that consisted of 65% acetonitrile in water and pumped at a flow rate of 0.50 mL min⁻¹. The excitation and emission wavelengths were set at 345 and 435 nm, respectively. The assay was linear over a concentration range of 0.05-16.00 μg mL⁻¹ for nateglinide with a limit of quantitation of 0.05 μg mL⁻¹. Quality control samples (0.05, 4.50 and 16.00 μg mL⁻¹) in five replicates from five different runs of analysis demonstrated intra-assay precision (%coefficient of variation <6.8%), inter-assay precision (%coefficient of variation <1.6%) and an overall accuracy (%relative error) less than −3.4%. The method can be used to quantify nateglinide in human plasma covering a variety of pharmacokinetic or bioequivalence studies.     

Simultaneous determination of diastereoisomeric and enantiomeric impurities in SSS-octahydroindole-2-carboxylic acid by chiral high-performance liquid chromatography with pre-column derivatization

SSS-Octahydroindole-2-carboxylic acid (SSS-Oic) is a key intermediate used in the synthesis of some angiotensin-converting enzyme (ACE) inhibitors. The separation of diastereoisomers and enantiomers of Oic was performed using a pre-column derivatization chiral HPLC method. Phenyl isothiocyanate (PITC) was used as the derivatization reagent. Three PITC derivatives of Oic stereoisomers were separated on an Ultron ES-OVM chiral column (150 mm × 4.6 mm, 5 μm). Derivatization conditions such as reaction temperature, reaction time and derivatization reagent concentration were investigated. The chromatographic conditions for separation of the three PITC-Oic derivatives were optimized. The method was successfully applied in the diastereoisomeric and enantiomeric purity test of SSS-Oic.