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.     

Analysis of aldehydes by micro high-performance liquid chromatography with post-column derivatization on enzyme-immobilized glass beads

Summary Aldehydes in liquors were analyzed by micro high-performance liquid chromatography with post-column derivatization on the enzyme-immobilized glass beads. β-nicotinamide adenine dinucleotide (NAD) was converted to its reduced form (NADH) on the enzyme-immobilized column in the presence of an aldehyde and a thiol, followed by fluorometric detection. A detection limit of 2.5 ppm (50 pg) was achieved for acetaldehyde.     

Analysis of digoxin at therapeutic concentrations using high-performance liquid chromatography with post-column derivatization

A high-performance liquid chromatographic (HPLC) procedure has been developed for the analysis of digoxin in plasma at therapeutic concentrations. The assay method provides resolution of digoxin from its metabolites using a 15 cm X 4.6 mm HPLC column containing 3-micron octadecylsilane-bonded stationary phase. The effluent of the column is passed through a post-column reactor in which a fluorescent derivative is formed by the co-addition of hydrochloric acid and dehydroascorbic acid. Detection of the derivative is accomplished in a fluorometer with excitation at 336 nm and emission at 425 nm. The extraction efficiency for recovery of digoxin from plasma samples was 70% using chloroform-isopropanol (9:1) following a pre-wash with isooctane to remove endogenous substances. The calibration curve was linear (r = 0.9999) over the range 0.5-4 ng/ml digoxin in plasma using digitoxigenin as internal standard. The minimum detectable quantity of digoxin in plasma was 0.5 ng/ml at a signal-to-noise ratio of 4:1. Split-samples of digoxin control sera were assayed by the HPLC procedure and by the prescribed radioimmunoassay procedure. Excellent correlation was observed between the two methods (r = 0.999). No interference was noted when a selection of commonly co-prescribed drugs were evaluated for chromatographic co-elution or interference in detection with that of digoxin or the internal standard.     

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 sugars by liquid chromatography with post-column catalytic derivatization and fluorescence detection

Summary A method for the determination of reducing sugars such as fructose and glucose and nonreducing sugar such as sucrose by high performance liquid chromatography followed by an acidic hydrolysis and a derivatization with benzamidine has been developed. After separation of sugars on a gel column packed with a polymer-based cation exchange material (Sugar-Pak I, Waters-Millipore), the sucrose is first hydrolysed in a solid phase reactor to convert it into reducing subunits. A post-column fluorigenic reaction with benzamidine under alkaline condition allows the selective determination of both natural and converted reducing carbohydrates.This procedure has proven to be selective (fluorigenic detection) and highly sensitive (allowing detection as little as picomoles amounts), reproducible and linear over a broad range of concentrations: 5×10^–4 to 1.0×10^–2 M.The applicability of this method to natural matrices such as plant extracts and beverages is also described. The sugar content of a barley extract has been determined and compared with a specific enzymatic test. The determined sugar content of natural and commercial lemon juices as well as of Cola beverages has been compared with those found by the conventional LC refractive index analytical procedure. In all cases, the results were comparable and were within the experimental errors of the methods.     

Speciation of mercury by ion chromatography with post-column derivatization

The chromatographic behaviour of Hg(II), methylmercury, phenylmercury and Cu(II) on three different sorbents, strong acidic sulfobutyl cation-exchanger, weak basic aminopropyl anion-exchanger and silicagel has been studied. A dithizone solution in cetyltrimethylammonium hydrogensulfate water micellar medium was found as a useful post-column derivatization reagent for UV-VIS detection. The detection limits 1.1 ng, 2.2 ng, 6.2 ng and 1.6 ng were found in silicagel chromatographic system and dithizone detection system for Hg(II), methylmercury, phenylmercury and respectively for Cu(II).     

Analysis of alkyl organoiodide mixtures by high-performance liquid chromatography using electrochemical detection with a post-column photochemical reactor

A method for the analysis of six alkyl organoiodides (iodomethane, iodoethane, 1-iodopropane, 1-iodobutane, 1-iodopentane, 1-iodohexane) commonly found in acetic acid process was developed. In this method the target analytes were determined by high-performance liquid chromatography (HPLC) using a post-column photochemical reactor with electrochemical detection (ED) in less than 30 min. HPLC was performed in ODS C18 reversed-phase column (5 microm, 250 x 4.6 mm I.D.) under isocratic conditions with methanol-0.067 M acetate buffer (70:30, v/v), pH 6.2 as mobile phase at flow-rate 1.1 ml/min. Alkyl organoiodides, which are electrochemically inactive, were made oxidizable at potential of 120 mV after post-column irradiation with low-pressure mercury lamp in a knitted PTFE tube. The photoreactor was placedin an aluminum housing full of nitrogen in order to prevent from the interference of oxygen. The detection limit for most analytes was of the order of 1-2 microg/l. The HPLC-ED method with a post-column photochemical reactor has good precision and linearity and can be readily applied to the routine determination of alkyl organoiodides in real acetic acid samples.     

Determination of ampicillin in biological fluids by coupled-column liquid chromatography and post-column derivatization

A rapid and sensitive high-performance liquid chromatographic method has been developed for the determination of ampicillin in plasma and urine. The method involves a simple deproteinization step and separation on a coupled-column chromatographic system followed by post-column derivatization and fluorescence detection. The method has been used for the determination of ampicillin in various clinical studies. The high sensitivity makes it especially useful for small sample volumes, e.g. samples from pediatric patients.     

高效液相色谱柱后衍生法测定蜂王浆中的链霉素

采用高效液相色谱柱后衍生法测定蜂王浆中的链霉素。样品用庚烷磺酸钠的酸性溶液进行提取,通过LC-18柱和WCX柱两次固相萃 取达到净化的目的。该方法采用反相C8柱,以0.01 mol/L的庚烷磺酸钠溶液和乙腈为流动相,在碱性条件下以1,2-萘醌-4-磺酸钠溶液 为柱后衍生化试剂,用荧光检测器检测。该方法的线性范围为0.02～0.5 mg/L,相关系数为0.9958,方法的检出限和定量限分别为为 0.005 mg/kg和0.01 mg/kg,回收率范围为84.0%～104.0%,相对标准偏差不大于7.9%。结果表明,该方法能有效地减少蜂王浆中链霉素 检测的假阳性结果,符合目前检测工作的需要。     

Determination of tetracycline antibiotics in salmon muscle by liquid chromatography using post-column derivatization with fluorescence detection

A simple and accurate cleanup procedure using polymeric sorbent was developed for the determination of oxytetracycline (OTC) and tetracycline (TC) residues in salmon muscle. It was applied to the analysis of 20 salmon samples during a month period. The OTC and TC residues were extracted with ethylenediaminetetracetic acid (EDTA)-McIlvaine buffer acidified at pH 4.0 and cleaned up by solid-phase extraction with a polymeric sorbent. The advantages of the polymeric sorbent over the silica-based sorbent in the cleanup of salmon muscle samples are described. A liquid chromatographic method with post-column derivatization and fluorescence detection is proposed because of its sensitivity and specificity. The average recoveries of OTC and TC from muscle salmon tissue fortified at 50, 100, and 200 microg/kg levels, ranged from 83.9 to 93.4% with a coefficient of variation between 4.09 and 5.80%. The limit of quantitation for OTC and TC in salmon muscle was 50 microg/kg.     

Determination of zeolite-a in detergent powders as acid-soluble aluminum by high-performance liquid chromatography with post-column derivatization

A high-performance liquid chromatographic method is described for the rapid determination of zeolite-A in detergents as acid-soluble aluminum. Detergent samples are dissolved by digestion with nitric acid. Chromatographic separation on a strong cation exchanger in acidic ammonium sulfate media is combined with the highly selective color reaction of acid-soluble aluminum with pyrocatechol violet. Interferences of the chelating agents in detergents are thus eliminated. After the digestion, determinations take 8 min per sample. The relative standard deviation is less than 1% and quantitative recoveries are obtained. Several commercial detergents were analyzed successfully. The results obtained by the proposed method and by compleximetric titration agreed very closely for zeolite-A contents in the range 2–22%.     

Analysis of carbohydrates by HPLC with post-column derivatization

Post-column derivatization of oligosaccharides after HPLC separation with a variety of systems has been achieved either by reaction with thymol in concentrated sulfuric acid or after hydrolysis of saccharides with hydrochloric acid and derivatization of the resulting monosaccharides with p-aminobenzoic acid hydrazide. With both reactions the detection of reducing and non-reducing sugars is possible even at trace levels. The latter reaction, despite the two reaction steps required, is much more convenient and practical, whereas with the first reaction it is also possible to determine alkylpolyglucosides.     

Determination of amoxicillin in body fluids by reversed-phase liquid chromatography coupled with a post-column derivatization procedure

Quantitative methods for determination of amoxicillin in body fluids are described. They comprise separation by reversed-phase chromatography (LiChrosorb RP-8, 5 micron) of the aqueous supernatants obtained from plasma or urine after purification steps involving protein precipitation followed by extraction in the case of plasma, or a double extraction procedure in the case of urine, post-column derivatization with air segmentation, and finally measurement of the UV absorbance at 310 nm. The derivatization involves formation of the mercuric mercaptide of penicillenic acid and is specific for compounds with an intact penicillanic acid ring system. Detection limits achieved on injecting 200 microliter of plasma and 20 microliter of urine are about 25 ng/ml and 200 ng/ml, respectively, but it is possible to improve the sensitivity further by injecting larger volumes. Precisions (srel) obtained for determination of 0.10 and 0.45 migrogram/ml in plasma were 3.72 and 1.40%, respectively. Some problems regarding column stability originating from the injection of biological samples are discussed.     

柱后衍生高效液相色谱法测定虾中14种磺胺类药物残留量

建立了虾中14种磺胺类药物残留量的柱后衍生高效液相色谱检测方法。样品在加入内标物磺胺吡啶后用乙酸乙酯提取,提取液浓缩后用4 mL乙酸乙酯溶解残余物,用盐酸溶液反萃取,正己烷去脂,盐酸溶液经滤膜过滤后,加入乙腈、甲醇和3.5 mol/L乙酸钠溶液（体积比为5：5：20）的混合溶液混匀后,经高效液相色谱分离,用荧光胺衍生试剂进行柱后衍生,荧光检测器检测。采用基质标样添加法绘制标准曲线,内标法定量。对柱后衍生系统参数进行了优化,确定了荧光胺溶液的浓度、流速和反应温度分别为0.2 g/L、0.15 mL/min和50℃。14种磺胺类药物在5~200 μg/L范围内具有良好的线性。磺胺类药物的定量限（LOQ,S/N=10）为1.0~5.0 μg/kg。在1.0~100.0 μg/kg添加水平内,磺胺类药物的平均回收率为77.8%~103.6%,相对标准偏差（RSD）为2.9%~9.1%（n=6）。实验结果表明该方法灵敏、准确,重复性好,适用于虾中磺胺类药物的残留检测。     

Determination of sulfathiazole in type C medicated swine feed by reversed-phase liquid chromatography with post-column derivatization

A convenient method was developed for determination of sulfathiazole (STZ) in Type C medicated swine feed by reversed-phase liquid chromatography (LC) with post-column derivatization. Addition of extractant solution (0.2N HCl and 1.5% diethylamine in 25% methanol) and an internal standard (IS), sulfamethylthiazole (SMZ), to 5 g sample was followed by mechanical shaking for 1 h. The extract was clarified by chilling, centrifugation, and filtering before injection onto a C18 reversed-phase column. The mobile phase components were 2% acetic acid and 1:1 acetonitrile-methanol (83 + 17%, v/v). Run time was about 20 min. Determination and, largely, the method's selectivity were based on detection at 450 nm of the derivative formed by the post-column reaction of dimethylaminobenzaldehyde with the primary amine of the analyte and IS. The IS, SMZ, differs from STZ by a single substituent methyl group, is stable, and is readily resolved from STZ. Although SMZ is not commercially available, it can be synthesized with relative ease from purchased reagents and will be supplied by the authors to interested laboratories. In single-laboratory validation, linearity was demonstrated over the range of 0.055-550 microg/mL, well beyond the target concentration of 5.5 microg/mL. The estimated limit of detection was 0.04 microg/mL; the calculated limit of quantitation was 0.13 microg/mL (feed concentration of 2.4 g/T or 2.7 mg/kg). Wet-spiking trials with a variety of swine feed matrixes showed recovery to be 100-102% for the intended concentration range, 50-200 g/T, with coefficient of variation (CV) < 2%. The method ruggedness was verified with an overall CV of 2.9%.