Composite Nanofibers as Novel Sorbents for On-Line and Off-Line Solid-Phase Extraction in Chromatographic System: A Comparison for Detection of Free Biogenic Monoamines and Their Metabolites in Plasma

Two different pretreatment approaches have been used for the enrichment and separation of biogenic monoamines and metabolites in plasma for high performance liquid chromatography (HPLC) determination. The first approach, based on on-line packed-fiber solid-phase extraction (PFSPE) coupled with HPLC, allows for the simultaneous detection of epinephrine (E), norepinephrine (NE), dopamine (DA), 3-methoxyl epinephrine (MN), norepinephrine (NMN), 3-methoxytyramine (3-MT), and 5-hydroxytryptamin (5-HT). Using this developed on-line PFSPE–HPLC method, the limit of detections (LODs) of the seven analytes ranged from 1 ng/mL (NMN and MN) to 2 ng/mL (NE, E, DA, 3-MT and 5-HT). The reportable ranges were 5–300 ng/mL for NE and DA, 5–100 ng/mL for E, and 5–200 ng/mL for NMN, MN, 3-MT and 5-HT. The off-line PFSPE–HPLC was employed in the second approach and could provide simultaneous detection of NE, E, DA, NMN, and MN. The linearity was verified in the range of 0.5–20 ng/mL (NE, E, and DA) and 20–250 ng/mL (NMN and MN). The LODs of the five analytes ranged from 0.2 ng/mL (NE, E, and DA) to 5 ng/mL (NMN and MN). This study verified the possibility of using nanofibers as an adsorbent in an on-line PFSPE–HPLC system for the determination of biogenic monoamines and their metabolites in human plasma. Compared with the off-line PFSPE approach, the on-line PFSPE method deserves attention mainly due to its greener character, derived from the automation of the process and high-throughput with less operators’ handling.     

Enantiomeric separation of d‐ and l‐serine using high‐performance liquid chromatography with electrochemical detection following pre‐column diastereomer derivatization

Enantiomeric separation of d ‐ and l ‐serine on an octadecylsilica column was investigated using (2R)‐2,5‐dioxopyrrolidin‐1‐yl‐2,5,7,8‐tetramethyl‐6‐(tetrahydro‐2H‐pyran‐2‐yloxy)chroman‐2‐carboxylate (R‐NPCA), which was developed for a pre‐column derivatization reagent for electrochemical detection. In addition, (2S)‐2,5‐dioxopyrrolidin‐1‐yl‐2,5,7,8‐tetramethyl‐6‐(tetrahydro‐2H‐pyran‐2‐yloxy)chroman‐2‐carboxylate (S‐NPCA) was newly synthesized from (S)‐(?)‐6‐hydroxy‐2,5,7,8‐tetramethylchroman‐2‐carboxylic acid (S‐α‐CA), and the enantiomeric separation of d ‐ and l ‐serine using S‐NPCA was also examined. The enantiomeric separation of d ,l ‐serine was achieved using the R‐ or S‐NPCA as a chiral derivatization reagent, and the elution orders of the enantiomers were reversed between R‐ and S‐NPCA. The elution orders of d ‐ and l ‐serine unexpectedly reversed between the phosphate buffer at pH 4.0 and pH 2.2, both of which were used in the mobile phase. Separation factors obtained using R‐ and S‐NPCA were similar—1.09 and 1.07, respectively. The detection limit was approximately 940 fmol on the column (signal‐to‐noise ratio 3) when the applied voltage was +650 mV. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of monoamine and amino acid neurotransmitters in rat endbrain tissues by pre-column derivatization with high-performance liquid chromatographic fluorescence detection and mass spectrometric identification

A sensitive and efficient method for simultaneous determination of glutamic acid (Glu), gamma-amino-butyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) in rat endbrains was developed by high-performance liquid chromatography (HPLC) with fluorescence detection and on-line mass spectrometric identification following derivatization with 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC). Different parameters which influenced derivatization and separation were optimized. The complete separation of five neurotransmitter (NT) derivatives was performed on a reversed-phase Hypersil BDS-C(18) column with a gradient elution. The rapid structure identification of five neurotransmitter derivatives was carried out by on-line mass spectrometry with electrospray ionization (ESI) source in positive ion mode, and the BCEOC-labeled derivatives were characterized by easy-to-interpret mass spectra. Stability of derivatives, repeatability, precision and accuracy were evaluated and the results were excellent for efficient HPLC analysis. The quantitative linear range of five neurotransmitters were 2.441-2x10(4) nM, and limits of detection were in the range of 0.398-1.258 nM (S/N=3:1). The changes of their concentrations in endbrains of three rat groups were also studied using this HPLC fluorescence detection method. The results indicated that exhausting exercise could obviously influence the concentrations of neurotransmitters in rat endbrains. The established method exhibited excellent validity, high sensitivity and convenience, and provided a new technique for simultaneous analysis of monoamine and amino acid neurotransmitters in rat brain.     

Determination of catecholamines and serotonin by micellar electrokinetic chromatography with laser-induced fluorescence detection

6-Oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein, a new synthesized fluorescent reagent, was established for the first time as a label for the sensitive analysis of catecholamines (CAs) and serotonin (5-HT) by micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection. After careful study on the derivatization conditions such as pH value, reagent concentration, temperature and reaction time, the labeling reaction was accomplished as quickly as 7 min with stable yield. The separation parameters for the CAs and 5-HT were also optimized in detail. The derivatives were baseline separated in a running buffer containing 30 mM boric acid and 15 mM sodium dodeculsulfate at pH 9.0. The detection limits ranged from 5 x 10(-10) to 2 x 10(-9) M (signal-to-noise ratio = 3). The rapid and sensitive method was also applied to the determination of the CAs and 5-HT of urine samples.     

Determination of levodopa and biogenic amines in urine samples using high-performance liquid chromatography

A chromatographic system is developed for the separation and determination of levodopa, biogenic amines, and their metabolites from the catecholamines group: dopamine (DA), epinephrine (E), normetanephrine (NMN), metanephrine (MN), 3,4-dihydroxyphenylacetic acid (DOMA), 3-metoxy-4-hydroxyphenyl-glycol (MHPG), and homovanillic acid (HVA); and indoloamines group: serotonin (5HT) and 5-hydroxyindole-3-acetic acid (5HIAA) in urine. The limit of detection (LOD) and limit of quantitation (LOQ) are determined for all compounds with signal-to-noise ratio (S/N) of 3 and 10, respectively. LOD 10 (ng/mL) and LOQ 30 (ng/mL) are determined for L-DOPA, DOMA, E, NMN, DA, MN, and MHPG, as well as LOD 8 (ng/mL) and LOQ 24 (ng/mL) for HVA, 5HT, and 5HIAA. A fluorescence detector is used. Gradient elution with acetate buffer (pH=4.66) with methanol is applied. In urine samples from patients treated with levodopa, the following concentrations (microg/mL) of analytes are determined: L-DOPA 3.73-46.80, DOMA 1.43-28.43, E 0.83-13.57, NMN 2.58-8.81, DA 24.07-62.11, MN 0.89-66.20, MHPG 6.72-63.64, 5HT 22.96-95.27, 5HIAA 1.45-14.77, and HVA 0.21-15.07.     

High-performance liquid chromatographic analysis of arachidonic acid metabolites by pre-column derivatization using 9-anthryldiazomethane

Arachidonic acid (AA) metabolites produced by washed human platelets and rat macrophages were analyzed by high-performance liquid chromatography (HPLC) using a pre-column derivatization method. The reagent, 9-anthryldiazomethane, used in this study and AA metabolites derivatized by the reagent were purified by gel permeation chromatography (PG-pak C column), prior to normal-phase HPLC analysis. A sample containing eleven derivatives (12-, 15- and 5-hydroxyeicosatetetraenoic acid, 12-L-heptadecatrienoic acid, leukotriene B4, prostaglandins B2, D2, E2 and F2 alpha, thromboxane B2 and 6-keto-prostaglandin F1 alpha) was separated on a normal-phase column (PG-pak B); the detection limit is better than 100 pg for all components.     

Simultaneous determination of serotonin and 5-hydroxyindole-3-acetic acid in human urine by automated precolumn derivatization and semi-microbore column liquid chromatography with fluorescence detection.

An improved high-performance liquid-chromatographic (HPLC) assay for the simultaneous determination of serotonin (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) in human urine is reported. Following the automated precolumn derivatization of urinary 5-HT and 5-HIAA with benzylamine, the derivatives are separated by isocratic elution on a reversed-phase C18 semi-microbore column, and are fluorometrically detected at an excitation wavelength of 345 nm and an emission wavelength of 480 nm. The detection limits of 5-HT and 5-HIAA are 7 and 5 nmol/l in urine (0.7 and 0.5 fmol/20-microl injection). The proposed automatic method permits a highly selective and sensitive determination of 5-HT and 5-HIAA in human urine without any sample purification.     

Determination of hyaluronic acid by high-performance liquid chromatography of the oligosaccharides derived therefrom as 1-(4-methoxy)phenyl-3-methyl-5-pyrazolone derivatives

Hyaluronic acid (HA) was digested with various kinds of depolymerizing enzymes and the products were analysed by high-performance liquid chromatography (HPLC) after derivatization with 1-(4-methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP). As hyaluronate 4-glycanohydrolase (EC 3.2.1.35) from sheep testis showed a high efficiency for depolymerization, giving the tetra- and hexasaccharides abundantly, and is inexpensive, a method for the specific determination of HA was established, based on digestion by this enzyme followed by determination of the tetra- or hexasaccharide derived therefrom as the PMPMP derivatives by HPLC with UV detection. This method allowed the determination of HA in the range 0.5–50 μg with high reproducibility.     

Derivatization chemistries for determination of serotonin, norepinephrine and dopamine in brain microdialysis samples by liquid chromatography with fluorescence detection

The present paper provides an overview on currently developed derivatization chemistries and techniques for determination of monoamine neurotransmitters serotonin (5-HT), norepinephrine (NE) and dopamine (DA) in microdialysis samples by microbore liquid chromatography with fluorescence detection. In mild alkaline conditions, 5-hydroxyindoles and catecholamines react with benzylamine (BA), forming highly fluorescent 2-phenyl-4,5-pyrrolobenzoxazoles and 2-phenyl(4,5-dihydropyrrolo) [2,3-f]benzoxazoles, respectively. However, for derivatization of DA a higher fluorescence intensity was achieved for reaction with 1,2-diphenylethylenediamine (DPE) rather than with BA, therefore for simultaneous determination of 5-HT, NE and DA in brain microdialysates, a two-step derivatization with BA followed by DPE was developed. The detection limits for 5-HT, NE and DA were 0.2, 0.08 and 0.13 fmol, respectively, in an injection volume of 20 microL, which corresponds to concentrations of 30, 12 and 19.5 pm, respectively in standard solution prior to derivatization. The experimental data presented demonstrate the ability of the technique to simultaneously monitor neuronally releasable pools of monoamine neurotransmitters in the rat and mouse brains at basal conditions and following pharmacological treatments or physiological stimuli. These techniques play an important role in drug discovery and clinical investigation of psychiatric and neurological diseases such as depression, schizophrenia and Parkinson's disease.     

A fully automated amino acid analyzer using NBD-F as a fluorescent derivatization reagent

A fully automated amino acid analyzer using NBD-F (4- fluoro-7-nitro-2,1,3-benzoxadiazole) as a fluorescent derivatization reagent was developed. The whole analytical process was fully automated from derivatization, injection to HPLC separation and quantitation. The derivatization reaction conditions were re-evaluated and optimized. Amino acids were derivatized by NBD-F for 40 min at room temperature in the borate buffer (pH 9.5). The derivatives were separated within 100 min and fluorometrically detected at 540 nm with excitation at 470 nm. The detection limits for amino acids were in the range of 2.8-20 fmol. The calibration curves were linear over the range of 20 fmol to 20 pmol on column with the correlation coefficients of 0.999. The coefficients of variation were less than 5% at 3 pmol injection for all amino acids. Amino acids in rat plasma were determined by the proposed HPLC method.     

Analysis of dideoxyadenosine triphosphate by CE with fluorescence detection. I. Derivatization through the phosphate group

A CZE method was developed, which separates 2',3'-dideoxyadenosine-5'-triphosphate (ddATP) from other metabolites and endogenous nucleotides at high concentrations (20-200 microg/mL) to allow UV detection. To enhance sensitivity, fluorescence detection which requires prior derivatization of compounds was examined. Precapillary derivatization of ddATP in the presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) with dansyl ethylenediamine (dansyl EDA) was faster and stable compared to that of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylenediamine (BODIPY FL EDA). Reaction conditions, reagent concentrations and detection parameters were optimized and highest derivatization efficiency was achieved in 0.1 M 1-methylimidazole buffer (pH 8.0) with 140 mM EDAC in 1-methylimidazole buffer and 30 mM dansyl EDA in DMF for 90 min at 60 degrees C. Dansyl EDA derivatives of ddATP, 2'-deoxyadenosine-5'-triphosphate (dATP) and ATP were comigrating with the CZE method; therefore, a MEKC method was developed and optimized for repeatable separations. Upon dansylation, sensitivity of ddATP with fluorescence detection (LOQ = 12 ng/mL) was 160 times higher than UV detection (LOQ = 1.9 microg/mL).     

Enhancement of atmospheric pressure chemical ionization for the determination of free and glycine-conjugated bile acids in human serum

A highly sensitive and accurate method based on the precolumn derivatization of bile acids (BA) with a high ionization efficiency labeling reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl-benzenesulfonate (BDEBS) coupled with LC/MS has been developed. After derivatization, BA molecules introduced a weak basic nitrogen atom into the molecular core structure that was readily ionized in commonly used acidic HPLC mobile phases. Derivatives were sufficiently stable to be efficiently analyzed by atmospheric pressure chemical ionization (APCI)-MS/MS in positive-ion mode. The MS/MS spectra of BA derivatives showed an intense protonated molecular ion at m/z [M + H]+. The collision-induced dissociation of the molecular ion produced fragment ions at [MH-H2O]+, [MH-2H2O]+, [MH-3H2O]+. The characteristic fragment ions were at m/z 320.8, 262.8, and 243.7 corresponding to a cleavage of N-CO, O-CO, and C-OCO, respectively, and bonds of derivatized molecules. The selected reaction monitoring, based on the m/z [M+H]+ --> [MH-H2O]+, [MH-2H2O]+, [MH-3H2O]+, 320.8, 262.8, and 243.7 transitions, was highly specific for the BA derivatives. The LODs for APCI in a positive-ion mode, at an S/N of 5, were 44.36-153.6 fmol. The validation results showed high accuracy in the range of 93-107% and the mean interday precision for all standards was <15% at broad linear dynamic ranges (0.0244-25 nmol/mL). Good linear responses were observed with coefficients of > 0.9935 in APCI/MS detection. Therefore, the facile BDEBS derivatization coupled with mass spectrometric analysis allowed the development of a highly sensitive and specific method for the quantitation of trace levels of the free and glycine-conjugated BA from human serum samples.     

6-Oxy-(acetyl ethylenediamine) fluorescein, a novel fluorescent derivatization reagent for carboxylic acids and its application in HPLC

A novel fluorescent derivatization reagent for carboxylic acids, 6-oxy-(acetyl ethylenediamine) fluorescein (AEF), was well designed, synthesized, and applied to HPLC. The derivatization reaction with 12 fatty acids, including n-valeric acid (C5), n-hexanoic acid (C6), n-heptanoic acid (C7), n-octanoic acid (C8), n-nonanoic acid (C9), n-decanoic acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid (C16), stearic acid (C18), oleic acid (C18:1), and linoleic acid (C18:2), was completed at 55 degrees C within 40 min. The derivatives of fatty acids were separated on a C18 RP column and detected by fluorescence detection. The LODs attained were 0.4-1.2 nM (S/N of 3). It has been demonstrated that AEF is a prominent derivatization reagent for carboxylic acids which is suitable for HPLC.     

新型荧光衍生试剂用于脂肪胺的反相高效液相色谱-荧光检测分析

利用新型荧光试剂4-(1H-菲并[9,10-d]咪唑-2-)苯甲酸(PIBA)进行柱前衍生并经荧光检测对脂肪胺进行了高效液相色谱(HPLC)分离和在线质谱定性。激发和发射波长分别为ex=261nm,em=443nm。80℃下在吡啶溶剂中用N-乙基-N’-[(3-二甲氨基)丙基]碳二亚胺盐酸盐(EDC)做催化剂,衍生反应10min后获得稳定的荧光产物。在EclipseXDB-C8色谱柱(4.6150mm,5mm)上,梯度洗脱对12种游离脂肪胺衍生物进行了优化分离。采用大气压化学电离源(APCI)正离子模式,实现了各种脂肪胺衍生物的测定。多数脂肪胺的线性回归系数大于0.9999,检测限为10.5～53.4fmol。     

Determination of phytohormones in plant samples based on the precolumn fluorescent derivatization with 1,3,5,7-tetramethyl-8-aminozide-difluoroboradiaza-s-indacene by HPLC for routine use

Six phytohormones including indole butyric acid (IBA), naphthalene acetic acid (NAA), 2,4-dichloro-phenoxy acetic acid (2,4-D), indole-3-acetic acid (IAA), abscisic acid (ABA), and salicylic acid (SA) in crude plant extractions have been quantitated by means of high-performance liquid chromatography (HPLC) with fluorescence detection based on the precolumn derivatization using 1,3,5,7-tetramethyl-8-aminozide-difluoroboradiaza-s-indacene (BODIPY-aminozide), a fluorescent reagent synthesized in our lab recently. The optimization of derivatization conditions was carefully studied by an L(25) (5(6)) orthogonal array design (OAD) with five factors at five levels that are important influence parameters in the improvement of derivatization efficiency. The separation conditions were also studied in detail. Under the optimal conditions, the detection limits (S/N=3) of the six phytohormones were found from 0.12 to 0.75 nM. The proposed method was the first investigation of aminozide for the analysis of phytohormones and has been successfully applied to the determination of phytohormones in plant samples such as cucumber and tomato with recoveries of 94-105%.     

Analysis of urinary neurotransmitters by capillary electrophoresis: sensitivity enhancement using field-amplified sample injection and molecular imprinted polymer solid phase extraction

Capillary electrophoresis (CE) has been investigated for the analysis of some neurotransmitters, dopamine (DA), 3-methoxytyramine (3-MT) and serotonin (5-hydroxytryptamine, 5-HT) at nanomolar concentrations in urine. Field-amplified sample injection (FASI) has been used to improve the sensitivity through the online pre-concentration samples. The cationic analytes were stacked at the capillary inlet between a zone of low conductivity - sample and pre-injection plug - and a zone of high conductivity - running buffer. Several FASI parameters have been optimized (ionic strength of the running buffer, concentration of the sample protonation agent, composition of the sample solvent and nature of the pre-injection plug). Best results were obtained using H₃PO₄–LiOH (pH 4, ionic strength of 80 mmol L⁻¹) as running buffer, 100 μmol L⁻¹ of H₃PO₄ in methanol–water 90/10 (v/v) as sample solvent and 100 μmol L⁻¹ of H₃PO₄ in water for the pre-injection plug.In these conditions, the linearity was verified in the 50–300 nmol L⁻¹ concentration range for DA, 3-MT and 5-HT with a determination coefficient (r²) higher than 0.99. The limits of quantification (10 nmol L⁻¹ for DA and 3-MT, 5.9 nmol L⁻¹ for 5-HT) were 500 times lower than those obtained with hydrodynamic injection. However, if this method is applied to the analysis of neurotransmitters in urine, the presence of salts in the matrix greatly reduces the sensitivity of the FASI/CE–UV method.Therefore, a solid phase extraction (SPE) on a dedicated imprinted polymer (MIP) was developed to extract specific neurotransmitters, catecholamines, metanephrines and indolamines, from urine. Matrix salts were thus discarded after sample extraction on AFFINIMIP™ Catecholamine & Metanephrine (100 mg) cartridge.Therefore, lower limits of quantification were determined in artificial urine (46 nmol L⁻¹ for DA, 11 nmol L⁻¹ for 3-MT and 6 nmol L⁻¹ for 5-HT).The application of this protocol MIP-SPE/FASI–CE–UV analysis of neurotransmitters in human urine gave rise to electropherograms with a very good base line and signal to noise ratios above 15.     

Analysis of primary aromatic amines using precolumn derivatization by HPLC fluorescence detection and online MS identification

2-(2-phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA) and 2-(9-acridone)-acetic acid (AAA), two novel precolumn fluorescent derivatization reagents, have been developed and compared for analysis of primary aromatic amines by high performance liquid chromatographic fluorescence detection coupled with online mass spectrometric identification. PPIA and AAA react rapidly and smoothly with the aromatic amines on the basis of a condensation reaction using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) as dehydrating catalyst to form stable derivatives with emission wavelengths at 380 and 440 nm, respectively. Taking six primary aromatic amines (aniline, 2-methylaniline, 2-methoxyaniline, 4-methylaniline, 4-chloroaniline, and 4-bromoaniline) as testing compounds, derivatization conditions such as coupling reagent, basic catalyst, reaction temperature and time, reaction solvent, and fluorescent labeling reagent concentration have also been investigated. With the better PPIA method, chromatographic separation of derivatized aromatic amines exhibited a good baseline resolution on an RP column. At the same time, by online mass spectrometric identification with atmospheric pressure chemical ionization (APCI) source in positive ion mode, the PPIA-labeled derivatives were characterized by easy-to-interpret mass spectra due to the prominent protonated molecular ion m/z [M + H](+) and specific fragment ions (MS/MS) m/z 335 and 295. The linear range is 24.41 fmol-200.0 pmol with correlation coefficients in the range of 0.9996-0.9999, and detection limits of PPIA-labeled aromatic amines are 0.12-0.21 nmol/L (S/N = 3). Method repeatability, precision, and recovery were evaluated and the results were excellent for the efficient HPLC analysis. The most important argument, however, was the high sensitivity and ease-of-handling of the PPIA method. Preliminary experiments with wastewater samples collected from the waterspout of a paper mill and its nearby soil where pollution with aromatic amines may be expected show that the method is highly validated with little interference in the chromatogram.     

超高效液相色谱-串联质谱法定量分析尿液中色氨酸及其代谢产物

基于超高效液相色谱-串联质谱(UPLC-MS/MS)建立定量分析色氨酸(Trp)及代谢产物3-OH-犬尿氨酸(3-OH-Kyn)、3-OH-邻氨基苯甲酸(3-OH-AA)、黄尿酸(XA)、犬尿氨酸(Kyn)、5-羟基吲哚乙酸(5-HIAA)、犬尿喹啉酸(KA)和5-羟色胺(5-HT)的方法,应用该方法分析其在尿样中的含...     

Determination of sulphonamides in animal tissues by high performance liquid chromatography with pre-column derivatization of 9-fluorenylmethyl chloroformate

A novel approach for simultaneous determination of 12 sulphonamides (sulphadiazine, sulphamethazine, sulphathiazole, sulphadimethoxine, sulphamerazine, sulphapyridine, sulphamethoxazole, suphamethizole, sulphaquinoxaline, sulphameter, sulphamonomethoxine, and sulphachloropyridazine) in animal tissues (swine muscle and liver, chicken muscle, beef muscle) by HPLC with UV detection has been developed. A pre-column derivatization of the sulphonamide compounds with 9-fluorenylmethyl chloroformate (FMOC-Cl) has been proposed and the reaction conditions have been optimized. The FMOC-sulphonamide derivatives were purified by SPE with silica gel as solid support prior to HPLC separation. The limits of detection for the sulphonamide compounds were greatly improved after the derivatization and purification step for the derivatives. Sulphonamide residues in animal tissues were extracted by acetonitrile and purified by solid phase extraction with C(18) as the solid support. The method developed has high sensitivity and good repeatability, and the average recoveries for most of the sulphonamides at various spiking levels were above 70% with relative standard deviations below 13.7%. The limits of detection for most sulphonamides can reach 3-5 microg/kg.     

High-performance liquid chromatography with sequential injection for online precolumn derivatization of some heavy metals

HPLC was coupled with sequential injection (SI) for simultaneous analyses of some heavy metals, including Co(II), Ni(II), Cu(II), and Fe(II). 2-(5-Nitro-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (nitro-PAPS) was employed as a derivatizing reagent for sensitive spectrophotometric detection by online precolumn derivatization. The SI system offers an automated handling of sample and reagent, online precolumn derivatization, and propulsion of derivatives to the HPLC injection loop. The metal-nitro-PAPS complexes were separated on a C(18)-muBondapak column (3.9x300 mm(2)). Using the proposed SI-HPLC system, determination of four metal ions by means of nitro-PAPS complexes was achieved within 13 min in which the parallel of derivatization and separation were processed at the same time. Linear calibration graphs were obtained in the ranges of 0.005-0.250 mg/L for Cu(II), 0.007-1.000 mg/L for Co(II), 0.005-0.075 mg/L for Ni(II), and 0.005-0.100 mg/L for Fe(II). The system provides means for automation with good precision and minimizing error in solution handling with the RSD of less than 6%. The detection limits obtained were 2 microg/L for Cu(II) and Co(II), and 1 microg/L for Ni(II) and Fe(II). The method was successfully applied for the determination of metal ions in various samples, including milk powder for infant, mineral supplements, local wines, and drinking water.