A pre-column derivatization high-performance liquid chromatography method for simultaneous determination of short-chain and medium-chain fatty acids in a fecal sample

Short-chain and medium-chain fatty acids have plentiful biological functions, which play a crucial role in the diagnosis and therapy of many diseases. Herein, a new method for simultaneous quantifying 17 short-chain and medium-chain fatty acids with high-performance liquid chromatography coupled with an ultraviolet detector was developed and the pre-column derivatization by indole-3-acetic acid hydrazide was performed to improve the separation and detection. The conditions of the derivatization reaction were systematically investigated. Subsequently, the method was validated and the results showed a satisfactory linearity (linear regression coefficients > 0.9969), the limit of detection (4.0×10⁻³–1.9×10⁻² μmol/L), precision (0.9%–7.3% for intra-day and 2.0%–9.8% for inter-day), recovery (90.0%–109.1% with relative standard deviation <7.7%) and stability (0.1%–3.3% for standard solution and 0.2%–3.9% for fecal sample). Finally, the established method was successfully applied to quantify short-chain and medium-chain fatty acids in the feces of healthy control and diabetic rats. Eleven kinds of short-chain and medium-chain fatty acids were detected and six of them showed a significant difference between the control group and the model group.     

Rapid analysis of amino acids using pre-column derivatization

A new approach to the pre-column derivatization and analysis of amino acids is described. The method is based upon formation of a phenylthiocarbamyl derivative of the amino acids. The derivatization method is rapid, efficient, sensitive, and specific for the analysis of primary and secondary amino acids in protein hydrolyzates. The liquid chromatographic system allows for the rapid, bonded-phase separation with ultraviolet detection of the common amino acids with 12-min analysis time and a 1-pmol sensitivity.     

柱前衍生化-超高效液相色谱法快速测定酱油中的18种氨基酸

建立了一种6-氨基喹啉基-N-羟基琥珀酰亚氨基甲酸酯(AQC)柱前衍生,超高效液相色谱(UPLC)对酱油中18种氨基酸进行快速分离检测的方法。采用BEH C18色谱柱分离,在260 nm波长下检测,以乙酸铵-乙酸-乙腈-水和乙腈-乙酸为流动相,将流动相梯度和流速梯度相结合,在12 min内实现了18种氨基酸衍生物的分离。方法的线性回归系数(r2)均大于0.999,检出限为0.032～0.12 mg/L,日间相对标准偏差(RSD)为0.72%～4.05%,在酱油中18种氨基酸的加标回收率为90.2%～103.7%。该方法前处理过程简单,分离时间短,是检测酱油中氨基酸的有效手段,可用于酱油的质量评定。     

Separation and determination of B vitamins and essential amino acids in health drinks by CE-LIF with simultaneous derivatization

An efficient and sensitive method for the separation and determination of three essential amino acids and three B vitamins by CE-LIF with a simultaneous derivatization procedure was developed. The conditions for derivatization and separation of these micronutrients were investigated. FITC was used as the reagent for fluorescence tagging of arginine (Arg), valine (Val), tryptophan (Trp), folic acid (FA), and niacinamide (NA). Riboflavin (RF) was detected without derivatization. Derivatization of analytes dissolved in borate solution was performed by successive introduction of fluorescence reagent and analytes followed by water bathing at 43°C. The molar ratio of sample/reagent (S/R), derivatization temperature, and incubation time significantly influenced the efficiency of derivatization. To maximize the fluorescence yield, a high S/R (≥20) was required. The nonderivatized RF and five derivatized analytes were separated in the optimized CE-LIF system with the application of 22 kV voltage and 25 mM borate buffer at pH 9.85. Validation of the method showed good linearity for the corrected peak areas versus standard concentrations for the six analytes. The RSDs (n = 3) of the migration time and the peak area obtained for the analytes ranged from 0.4 to 1.1% and from 1.9 to 4.4%, respectively. The developed method, with the lowest LOD of 0.5 nM, was successfully applied for the efficient derivatization and determination of B vitamins in four health drink samples.     

Sensitivity enhancement by on-line preconcentration and in-capillary derivatization for the electrophoretic determination of amino acids

This study describes an application of on-line preconcentration by large-volume stacking in combination with in-capillary derivatization for enhancing spectrophotometric detection sensitivity in capillary electrophoresis. The method is illustrated by an example dealing with the determination of amino acids with 1,2-naphthoquinone-4-sulfonate as a labelling agent. Samples are dissolved in water in order to create a stacking process based on differences in the conductivity between this medium and a concentrated running buffer. The in-capillary derivatization is accomplished following a sandwich procedure in which the sample is inserted between two segments of reagent. Amino acid derivatives are obtained and separated in a fused-silica capillary with a sodium borate electrolyte buffer using 2-propanol as an organic modifier. The method is applied to the analysis of amino acids in pharmaceutical and feed samples. A good concordance between the predicted values and those obtained with the standard method is observed, with overall quantification error below 5%. The proposed procedure allows the detection limits sensitivity to be enhanced in 1000-fold with respect to conventional precapillary derivatization.     

高效液相色谱-柱前衍生化法测定饲料中的含硫氨基酸

发展了一种饲料中含硫氨基酸的检测方法。样品经过甲酸氧化,将其中的胱氨酸和蛋氨酸分别氧化为磺基丙氨酸和蛋氨酸砜;再经酸水解后,采用2,4-二硝基氟苯进行柱前衍生化,衍生物经高效液相色谱分析。使用Elite AAK C18色谱柱(250 mm×4.6 mm, 5 μm)分离;以0.05 mol/L乙酸钠和乙腈-水(50:50, v/v)为流动相,梯度洗脱,流速为1.2 mL/min;检测波长为360 nm;柱温为31 ℃。结果表明,胱氨酸在0.4～16.0 mg/L、蛋氨酸在0.7～29.6 mg/L范围内的线性相关系数分别为0.9999、0.9998;胱氨酸和蛋氨酸的定量限(信噪比(S/N)=10)分别为2.6 μg/kg和3.1 μg/kg;3次样品平行测定的胱氨酸和蛋氨酸含量的相对标准偏差(RSD)分别为0.79%和2.56%,加标回收率分别为100.28%～102.00%和105.72%～107.89%。该方法分析成本低,测定结果准确,灵敏度高,符合饲料中含硫氨基酸的检测要求。     

A new strategy for the selective determination of D-amino acids: enzymatic and chemical modifications for pre-column derivatization

A new strategy for the selective determination of D-amino acids (DAAs) employing a pre-column derivatization was designed with concepts based on both enzymatic and chemical modifications. Selective determination of DAAs was accomplished by following: DAA was enzymatically modified with D-amino acid oxidase (DAAO: EC 1.4.3.3) to form an alpha-keto acid. Subsequently, resulting alpha-keto acid was detected by high-performance liquid chromatography (HPLC) after chemical modification with o-phenylenediamine (PDA) in the presence of 2-mercaptoethanol (2ME) to give the corresponding quinoxalinol derivative (PDA-alpha-keto acid derivative). After optimizing the pre-column derivatization and HPLC separation, five peaks corresponding to DAAs (D-alanine, D-leucine, D-methionine, D-phenylalanine, D-valine (as the standard mixture of DAAs in this paper) were separately eluted and monitored by means of a conventional HPLC system with a gradient elution on octadecyl silica gel (ODS) column and a fluorescence detector (Ex.: 341 nm, Em.: 413 nm), respectively. It was confirmed that the present method was incapable of detecting L-amino acids (LAA) when a sample solution consisting of both LAAs and DAAs was examined. The linearity of the peak-area responses to their concentration range of DAAs from 10 to 500 microM is 0.994-1.000, and their detection limits were 0.2-1 microM (signal/noise = 3). When this method was applied to a methanolic extract of short-necked clams, Ruditapes philippinarum (in Japanese, Asari), a big peak, corresponding to D-alanine was detected, corresponding to 2.9 mg/g D-alanine. In this paper, we present an example of pre-column derivatization method that was newly configured to take into account both the biological and chemical properties of the substances in question.     

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.     

Continuous on-line derivatization and determination of amino acids by a microfluidic capillary electrophoresis system with a continuous sample introduction interface

An automatic, rapid and continuous on-line derivatization system coupled to microfluidic capillary electrophoresis (CE) for the determination of amino acids using o-phthaldialdehyde/N-acetyl-l-cysteine (OPA/NAC) as the derivative agents has been developed. By on-line derivatization, amino acids were automatically and reproducibly converted to the UV-absorbing derivatives, which were separated by capillary zone electrophoresis (CZE). Optimization of derivatization and separation condition was carried out to achieve both good sensitivity and separation efficiency. The separation could be achieved within 4 min and sample throughput rate can reach up to 16 h⁻¹. The repeatability (defined as relative standard deviation, R.S.D.) was 2.56, 2.85, 3.24 and 3.60% with peak area evaluation and 2.93, 3.12, 4.20 and 4.91% with peak height evaluation for arginine (Arg), phenylalanine (Phe), serine (Ser) and glycine (Gly), respectively. The limits of detection (S/N=3) were 10.46, 13.14, 34.39 and 44.79 μmol/l for Arg, Phe, Ser and Gly, respectively. Major advantages of the proposed method include improved precision and efficient automation of the derivatization by the FI system and the enhanced sampling frequencies by the combined FI-CE system.     

An automated high-performance liquid chromatographic method for the determination of aminoglycosides in serum using pre-column sample clean-up and derivatization

An automated high-performance liquid chromatographic method for the determination of the aminoglycosides amikacin, dibekacin, gentamicin, netilmicin, sisomicin and tobramycin is described. The procedure involves sample clean-up by adsorption of the aminoglycosides on a pre-column, subsequent derivatization with o-phthalaldehyde and on-line separation of derivatives by column switching. A short cation-exchange column serving concurrently as a guard column in combination with a reversed-phase column was used for separation. Except for the determination of netilmicin an internal standard consisting of an aminoglycoside was used in each assay. The signals of the aminoglycosides determined were linear within the range of 1-16 mg/l serum. The inter-assay imprecision (n = 10) calculated as coefficient of variation was less than 6%. The results were obtained within 20 min after injection of the serum sample. Easy performance and flexibility make the procedure feasible for therapeutic drug monitoring.     

HPLC of amino acids and oligopeptides by pre-column fluorescence derivatization with N-hydroxysuccinimidyl-α-(9-phenanthrene)-acetate

Summary A sensitive LC method for the detection of amino acids and oligopeptides with pre-column fluorescence derivatization has been developed. Glycine, glycylglycine, triglycine, glutathione, glutamic acid, and cysteine were separated on a reversed-phase C₁₈ column with methanol-water-triethylamine eluent, derivatization and chromatographic conditions were optimized. The six derivatives were eluted in 20 min with good reproducibility. The relative standard derviations (n=6) at an analytical concentration of 2×10⁻⁶ M are <5%. Detection limits (signal-to-noise ratio=3) for the six derivatives are 23–68 fmol.     

Evaluation of conditions for the HPLC determination of plasma amino acids using precolumn derivatization

For the determination of free amino acids in plasma, the conditions for precolumn derivatization of the amino acids and the chromatographic separation were examined. The isoindole products, formed by the reaction of the primary amino acids with orthophthalaldehyde (OPA), were readily separated by RPLC and detected spectrofluorometrically using an excitation wave-length of 300 nm and an emission cut-off filter of 440 nm. Since the sensitivity of this method permits determination of amino acids in the femtomole range, the analysis can be performed on samples as small as 10 μl of filtered plasma or serum. The separation is achieved in approximately 35 minutes with good precision for the majority of the amino acids.     

Continuous flow derivatization system coupled to capillary electrophoresis for the determination of amino acids

A derivatization system coupled to capillary electrophoresis for the determination of amino acids using 1,2-naphthoquinone-4-sulfonate as a labeling agent is described. In this system, amino acids are derivatized on-line in a three-channel flow manifold for sample, reagent and buffer solutions. The reaction takes place in a PTFE coil heated at 80 degrees C. The resulting solution, which contains the amino acid derivatives, is introduced into the electrophoretic system by means of an appropriate interface. Subsequently, amino acid derivatives are separated at 25 kV using a 40 mM sodium tetraborate aqueous solution with 30% (v/v) isopropanol solution as a running buffer. The electropherograms are monitored spectrophotometrically at 230 nm. The method has been applied to the determination of amino acids in feed samples and pharmaceutical preparations. A good concordance of the predicted values with those given by a standard amino acid analyzer is shown.     

HPLC enantioseparation of amino acids and their protected derivatives used in peptide synthesis with pre-column chiral derivatization

Summary Indirect chiral separation methods based on enantiomeric derivatizations were developed in order to monitor optical purity of uncoded amino acids and a new series of amino acid derivatives. Marfey's reagent was used for derivatization of amino groups: whilst boxyl groups were derivatised with (1R, 2R)-or (1S, 2S)-2-amino-1(4-nitrophenyl)-1,3-propanediol reagents were used, respectively. The separations of diastereomeric derivatives formed via derivatization were optimized in RP-HPLC and NP-HPLC systems. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

HPLC-based method using sample pre-column clean-up for the determination of methanethiol and ethanethiol in parenteral amino acid solutions

A method has been developed for the chromatographic determination of methanethiol (MT) and ethanethiol (ET) as contaminants in amino acid parenteral nutrition (PN) solutions. The clean-up of the samples before chromatographic analysis was investigated by solid-phase extraction (SPE) on pre-columns filled with polyethylene powder (PE), aluminium oxide (AlOx), silica (SiOx), or polyurethane foam (PUF) as adsorbents. The thiols were more efficiently separated from the matrices by SPE on PUF pre-columns. Simultaneous derivatization and elution with DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) enabled further discrimination between MT and ET by reversed-phase HPLC with spectrophotometric detection. The retention times for the derivatized MT and ET species were 12.5 and 23.0 min, respectively. Recoveries from spiked PN samples were calculated to be approximately 90%, and the MT and ET content of commercial PN solutions was determined using the methodology described. Detection limits of 15 and 10 microg L(-1) were calculated for MT and ET, respectively.     

HPLC-based method using sample pre-column clean-up for the determination of methanethiol and ethanethiol in parenteral amino acid solutions

A method has been developed for the chromatographic determination of methanethiol (MT) and ethanethiol (ET) as contaminants in amino acid parenteral nutrition (PN) solutions. The clean-up of the samples before chromatographic analysis was investigated by solid-phase extraction (SPE) on pre-columns filled with polyethylene powder (PE), aluminium oxide (AlOx), silica (SiOx), or polyurethane foam (PUF) as adsorbents. The thiols were more efficiently separated from the matrices by SPE on PUF pre-columns. Simultaneous derivatization and elution with DTNB (5,5′-dithiobis(2-nitrobenzoic acid)) enabled further discrimination between MT and ET by reversed-phase HPLC with spectrophotometric detection. The retention times for the derivatized MT and ET species were 12.5 and 23.0 min, respectively. Recoveries from spiked PN samples were calculated to be approximately 90%, and the MT and ET content of commercial PN solutions was determined using the methodology described. Detection limits of 15 and 10 μg L^–1 were calculated for MT and ET, respectively.     

Application of amino acid analysis by high-performance liquid chromatography with phenyl isothiocyanate derivatization to the rapid determination of free amino acids in biological samples.

An amino acid analysis by reversed-phase high-performance liquid chromatography after precolumn derivatization with phenyl isothiocyanate was adapted to the determination of free amino acids in plasma or other biological fluids and in tissue homogenates. Preparation of samples included deproteinization by 3% sulphosalicylic acid, and careful removal under high vacuum of residual phenyl isothiocyanate after derivatization. A Waters Pico-Tag column (15 cm long) was used, immersed in a water-bath at 38 degrees C. In rat or human plasma, separation of 23 individual amino acids, plus the unresolved pair tryptophan and ornithine, was obtained within 13 min. Including the time for column washing and re-equilibration, samples could be chromatographed at 23-min intervals. Variability was tested for each amino acid by calculating the coefficients of variation of retention times (less than 1% in the average) and peak areas (less than 4% for both intra-day and inter-day determinations). The linearity for each standard amino acid was remarkable over the concentration range 3-50 nmol/ml. The mean recovery of amino acid standards added to plasma prior to derivatization was 97 +/- 0.8%, except for aspartate (82%) and glutamate (81%). This method is rapid (almost three samples per hour can be analysed, more than in any other reported technique), with satisfactory precision, sensitivity and reproducibility. Therefore, it is well suited for routine analysis of free amino acids in both clinical and research work.