Comparison of a capillary electrophoresis method with high-performance liquid chromatography for the determination of biogenic amines in various food samples

A capillary electrophoresis (CE) and a high-performance liquid chromatography (HPLC) method to analyze biogenic amines in food were compared. An automated precolumn derivatization with o-phthaldialdehyde (OPA) allows for the determination of aliphatic amines and amino acids by HPLC. In contrast, for the measurement of histamine and tyramine by CE, no laborious sample pretreatment was necessary. The biogenic amines were separated by CE or HPLC in less than 9 or 20 min, respectively. The calibration curves were linear to at least 100 mg/kg (r=0.999) and 1,000 mg/kg for HPLC and CE, respectively, with detection limits for histamine of 0.5 mg/kg (fluorescence detector) or 1 mg/kg (diode array detector) with HPLC and 2 mg/kg with CE. The detection limits for tyramine were 1.5 mg/kg with HPLC and 6 mg/kg with CE and for further amines (e.g., putrescine, spermidine, cadaverine, agmatine) ranging from 1.0 to 8.5 mg/kg with HPLC. There was a good correlation between CE and HPLC (correlation coefficient for histamine: 0.994).     

A rapid and validated HPLC method to quantify sphingosine 1-phosphate in human plasma using solid-phase extraction followed by derivatization with fluorescence detection

We describe the development and validation of analytical methodology for the determination of sphingosine 1-phosphate (S1P) in plasma. It uses solid-phase extraction (SPE) followed by an automated reversed-phase gradient HPLC column-switching system with a pre-column derivatization with o-phthalaldehyde (OPA) and fluorescence detection. The limit of quantification was determined at 100 ng/ml exogenous sphingosine 1-phosphate with a relative standard deviation for precision and accuracy <15%. The within- and between-day relative standard deviation for precision and accuracy were also less than 15%. This validated method should be suitable to quantify plasma concentration of sphingosine 1-phosphate in relatively large numbers of samples.     

Simultaneous fluorometric measurement of histamine and tele-methylhistamine levels in rodent brain by high-performance liquid chromatography

An improved high-performance liquid chromatography (HPLC) method was developed for simultaneous analysis of histamine (HA) and tele-methylhistamine (tele-MHA) levels in mouse and rat brain. The method consists of a solid-phase extraction (SPE) and subsequent HPLC with postcolumn derivatization of the amines with o-phthalaldehyde. The recovery rates of HA and tele-MHA during the SPE procedure were 82.8+/-3.4 and 86.0+/-1.7%, respectively. The detection limits for HA and tele-MHA were 8 and 12pg, respectively, with sufficient linearity up to 30pg. Using this newly developed system, we observed that the brain tele-MHA levels in H3 receptor knockout mice were significantly higher than those of wild-type mice by 2.1-fold. Furthermore, we also observed that the brain HA and tele-MHA levels in Zucker rats were significantly lower than those of lean rats by 76.6+/-5.3 and 77.8+/-5.0%, respectively. These observations coincided well with those of previous studies using radioimmunoassay or HPLC with precolumn OPA derivatization, confirming the utilization of the assay system.     

Liquid chromatographic determination of 1-adamantanamine and 2-adamantanamine in human plasma after pre-column derivatization with o-phthalaldehyde and 1-thio-beta-D-glucose

We investigated high-performance liquid chromatographic (HPLC) determination of 1-adamantanamine hydrochloride (1-ADA) and 2-adamantanamine hydrochloride (2-ADA) in human plasma after the derivatization with o-phthalaldehyde (OPA) and 1-thio-beta-D-glucose (TG). Extracted human plasma samples were mixed with OPA and TG at room temperature for 6 min and injected onto HPLC. Retention times of 1-ADA and 2-ADA derivatives were 12.6 and 14.1 min, respectively. The lower limits of detection of 1-ADA and 2-ADA were 0.02 and 0.008 microg/ml, and the lower limits of quantitation of 1-ADA and 2-ADA were 0.025 and 0.01 microg/ml, respectively. The coefficients of variation for intra-day and inter-day assay of 1-ADA and 2-ADA were less than 4.4 and 6.0%, respectively. L-Dopa and dopamine were not found to interfere with the peaks of 1-ADA and 2-ADA derivatives. Human plasma unbound fraction (f(p)) values of 1-ADA varied between 0.32 and 0.48, while those of 2-ADA varied between 0.38 and 0.68. These results indicate that HPLC assay of 1-ADA and 2-ADA by derivatization with OPA and TG is simple, rapid, sensitive and reproducible for determining 1-ADA and 2-ADA in human plasma.     

High-performance liquid chromatographic resolution of (R,S)-α-alkyl-α-amino acids as diastereomeric derivatives

Summary A number (27) of racemic-alkyl--amino acids (AAA) were derivatized either witho-phthaldialdehyde (OPA) in combination withN-t-butoxycarbonyl-L-cysteine (Boc-Cys) orN-acetyl-L-cysteine (Ac-Cys), or withN ²-(5-fluoro-2,4-dinitrophenyl)-L-alanine amide (Marfey's reagent). The resolution of the diastereoisomers formed was investigated by reversed-phase (C₁₈) high-performance liquid chromatography (HPLC) using gradient elution conditions employing sodium phosphate buffers of pH 7.2 together with acetonitrile, and fluorescence detection at 344 nm (excitation) and 443 nm (emission) for the OPA/Boc-Cys or OPA/Ac-Cys derivatives. For the diastereomers formed by derivatization with Marfey's reagent triethylammonium phosphate buffers of pH 3.0 (pH 7.2 for acidic AAA) together with acetonitrile, and u.v. detection at 340 nm were used. Whereas with Marfey's reagent all diastereomers of AAA showed complete, or almost complete, resolution, only 8, or 11, respectively of the diastereomers formed by derivatization with OPA/Boc-Cys or OPA/Ac-Cys were resolved under the chromatographic conditions used.     

High‐performance liquid chromatography evaluation of the enantiomeric purity of amino acids by means of automated precolumn derivatization with ortho‐phthalaldehyde and chiral thiols

The use of ortho‐phthalaldehyde (OPA) for the derivatization of amino acids (AA) is well known. It enables the separation of the derivatives on common reversed phase columns and improves the sensitivity with fluorescence detection. With the use of a chiral thiol an indirect enantioseparation of chiral amines and AAs is feasible. The major drawback of the OPA‐derivatization is the poor stability of the products. Here, a method with an in‐needle derivatization procedure is optimized to facilitate a quantitative conversion of the AA with OPA and the chiral thiols N‐acetyl‐L‐cysteine or N‐isobutyryl‐L‐cysteine, followed by a subsequent analysis, eluding the stability issue. Both enantiomers of a single AA were separated as OPA‐derivatives with a pentafluorophenyl column and a gradient program consisting of 50 mM sodium acetate buffer pH = 5.0 and acetonitrile. Fluorescence detection is commonly used to achieve sufficient sensitivity. In this study, the enantiomeric impurity of an AA can be detected indirectly with common UV spectrophotometric detection with a limit of quantitation of 0.04%. Seventeen different L‐AAs were tested and the amount of D‐AA for each individual AA was calculated by means of area normalization, which ranged from not detectable up to 4.29%. The recovery of the minor enantiomer of L‐ and D‐AA was demonstrated for three AAs at a 0.04% level and ranged between 92.3 and 113.3%, with the relative standard deviation between 1.7 and 8.2%.     

Determination of cellular thiols and glutathione-related enzyme activities: versatility of high-performance liquid chromatography–spectrofluorimetric detection

A high-performance liquid chromatography (HPLC) method to determine the most important cellular thiols [reduced glutathione (GSH), cysteine, γ-glutamylcysteine and cysteinylglycine] is described. Separation relies upon isocratic ion-pairing reversed-phase chromatography and detection is operated by spectrofluorimetry coupled with post-column derivatization reactions using either N-(1-pyrenyl)maleimide (NPM) or ortho-phthalaldehyde (OPA). When OPA is used without co-reagent, only GSH and γ-glutamylcysteine are detected (heterobifunctional reaction). However, either the OPA reaction in the presence of glycine in the mobile phase (thiol-selective reaction) or NPM allows the detection of all the cited thiols. The HPLC system has been validated as concerning linearity, accuracy and precision. The low detection limits reached (in the pmol range for each thiol injected) allow the screening and the quantification of thiols (as NPM derivatives) in V79cl and V79HGGT cells as well as the measurement of two cytosolic enzymes related to the glutathione synthesis, using the heterobifunctional OPA reaction.     

HPLC determination of fumonisin mycotoxins in maize: a comparative study of naphthalene-2,3-dicarboxaldehyde and o-phthaldialdehyde derivatization reagents for fluorescence and diode array detection

Fumonisins are mycotoxins produced by various species of Fusarium and occur naturally in contaminated maize and maize-based foods. Ingestion of fumonisins has considerable health implications for humans and animals. Since fumonisins lack a useful chromophore or fluorophore, their determination in maize is routinely achieved via HPLC with fluorescence detection (FLD) after precolumn derivatization. This study optimized naphthalene-2,3-dicarboxaldehyde (NDA) derivatization of fumonisins in naturally contaminated maize following strong anion exchange (SAX) solid phase extraction (SPE) clean-up and utilizing diode array detection (DAD) as a practical alternative simultaneously to FLD. The limit of detection (LOD) for fumonisin B(1) (FB(1)), fumonisin B(2) (FB(2)) and fumonisin B(3) (FB(3)) with FLD was 0.11 ng, 0.50 ng and 0.27 ng, respectively, and with DAD it was 13.8 ng, 12.5 ng and 6.6 ng, respectively injected on column. The coefficient of variation (CV, n = 6) for FB(1), FB(2) and FB(3) in a naturally contaminated samples obtained with FLD was 2.6%, 1.8% and 5.3%, respectively, compared to 6.0%, 3.4% and 9.5%, respectively, obtained with DAD. Subsequently the optimized NDA derivatization was compared to the widely used o-phthaldialdehyde (OPA) derivatization agent as well as alternative sample clean-up with immunoaffinity column (IAC) by analyzing naturally contaminated maize samples (n = 15) ranging in total fumonisin (TFB = FB(1)+FB(2)+FB(3)) levels from 106 to 6000 μg/kg. After immunoaffinity column clean-up of extracted samples, the recoveries of spiked maize samples for NDA-FLD of FB(1), FB(2) and FB(3) were 62%, 94% and 64%, respectively. NDA proved to be an effective derivatization reagent of fumonisin in naturally contaminated maize samples following IAC clean-up, except for DAD at TFB levels below 1000 μg/kg. In contrast NDA derivatization following SAX clean-up produced results comparable to OPA only for levels below 1000 μg/kg. Aside from the difference in detection limits, FLD and DAD produced comparable results irrespective of the clean-up method or the derivatization agent.     

Determination of fumonisins in maize by HPLC with ultraviolet detection of <Emphasis Type="Italic">o</Emphasis>-phthaldialdehyde derivatives

Fumonisins are mycotoxins that are produced by various Fusarium species and occur naturally in maize and maize-based foods. Fumonisins are carcinogenic, causing liver cancer in rats, and are associated with oesophageal cancer and neural tube defects in humans. Analytical methods for individual fumonisin analogues in maize rely on reversed-phase high-performance liquid chromatographic (HPLC) separation after suitable extraction and clean-up. As fumonisins lack a useful chromophore or fluorophore, HPLC detection is achieved by suitable derivatization and sensitive, specific fluorescence detection. A widely used and validated method involves extract clean-up on strong anion exchange solid phase extraction cartridges and pre-column derivatization with o-phthaldialdehyde (OPA). However, many laboratories requiring infrequent fumonisin analysis are only equipped with HPLC with ultraviolet (UV) detection. A HPLC system equipped with both UV and fluorescence detectors connected in series was used to determine the extent to which UV offers an alternative to fluorescence detection in the above analytical method. Comparison of the detection systems using fumonisin standards indicated that fluorescence is about 20-times more sensitive than UV. Analysis of maize samples with differing fumonisin contamination levels indicated that, at fumonisin B₁ levels above 1,000 μg/kg, the two detection systems were comparable and gave repeatabilities equal or less than 10% on six replicate analyses. Although a sensitive fumonisin analysis requires fluorescence detection, UV may offer an alternative in certain circumstances.     

Liquid chromatography method for determination of bivalirudin in human plasma and urine using automated ortho-phthalaldehyde derivatization and fluorescence detection

A high-performance liquid chromatographic (HPLC) method was developed using solid-phase extraction, o-phthalaldehyde (OPA) derivatization and fluorescence detection for the determination of the direct thrombin inhibitor bivalirudin in human plasma and urine. The use of this assay will facilitate the study of the pharmacodynamics of bivalirudin in studies of special patient populations. A C(18) bioanalytical column at a flow rate of 1 ml/min with an aqueous trifluoroacetic acid (0.1% TFA in deionized water, pH 2.2, v/v) mobile phase and methanol gradient was used. The assay demonstrated linearity from 3 to 20 microg/ml bivalirudin in plasma, with a detection limit of 1 microg/ml. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of bivalirudin in patients undergoing percutaneous coronary interventions (PCIs).     

A rapid method for the determination of taurine in biological tissue

A rapid method for the determination of taurine is described. Based on high performance liquid chromatography (HPLC) separation, it avoids all transfers, precleaning treatments and the use of internal standards. The method consists of precolumn derivatization with O-phthaldialdehyde (OPA) followed by separation on a reversed phase styrene column using an acetonitrile-citrate buffer mixture as the eluting solvent. Naturally occurring substances, often quoted to co-migrate with taurine, are separated and the method described has been used to determine the taurine content of various tissues and blood fractions. These determinations indicate that the only satisfactory determination of blood taurine is that of whole blood.     

On-line coupling of sequential injection with liquid chromatography for the automated derivatization and determination of gamma-aminobutyric acid in human biological fluids

The principle of sequential injection analysis (SIA) was exploited to develop a rapid fully automated and efficient pre-column derivatization procedure coupled on-line to liquid chromatography (HPLC). Using the SIA-HPLC derivatization protocol gamma-aminobutyric acid (GABA) was determined fluorimetrically in human biological fluids with o-phthaldialdehyde (OPA) as derivatization reagent and minimum sample pretreatment. A lab-built SIA system was used to handle samples, standard solutions and OPA reagent. Appropriate volumes of the reagents were introduced in the holding coil of the SIA system and were mixed on propulsion to the HPLC loop through a suitable reaction coil. The chemical (pH, c(OPA), c(mercaptoethanol)) and instrumental variables (volumes of sample and reagent, reaction time) of the reaction were studied and optimized in terms of maximum sensitivity. The chromatographic variables (gradient composition of the eluent and flow rate) were studied for optimum selectivity and peak characteristics. The developed experimental configuration facilitated fully-automated operation thus minimizing errors in handling. Additionally the method as a whole provided very satisfactory sensitivity, precision and accuracy. Direct determination of GABA in human urine and cerebrospinal fluid (CSF) at microg L(-1) (ppb) levels was accomplished, with minimum sample pretreatment.     

Femtomole peptide mapping by derivatization, high-performance liquid chromatography, and fluorescence detection.

A highly sensitive peptide mapping method using derivatization and fluorescence detection is described. Bovine cytochrome c was digested using a buffer compatible with the derivatization that followed. The derivatization was performed with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The peptide mapping of the tagged digest was conducted with both HPLC and capillary LC (CLC) systems. A capillary LC-electrospray ionization mass spectrometer (MS) was set up for measuring the molecular weights of the tagged peptides. Optimization was made of the conditions used for digestion, derivatization, and mapping. MS measurements of the tagged peptides suggested that there was only one derivatization product produced from all peptides (except one) and that all the identified peptides were fully tagged. Peptide mapping of the tagged digest reviews a larger number of peptides, covering almost the entire sequence. Peptide mapping of a 20 fmol amount of tagged digest was readily performed with the CLC system. By using derivatization and fluorescence detection, the sensitivity of peptide mapping could be improved 2000 times compared to that observed with uv detection of untagged peptides.     

Quantitation of nitric oxide-derived nitrite from activated macrophages using microdialysis sampling

An HPLC method for detecting nitrite in microdialysis samples obtained from activated RAW 264.7 macrophages in cell culture has been developed. Nitrite was quantified using a pre-column derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) followed by HPLC-UV analysis of the azide product. For dialysates, the detection limit of nitrite was 750 nM and the quantitation limit was 2.5 microM. The microdialysis relative recovery of nitrite in the macrophage cell culture medium was determined to be 86+/-2% (n=3) at a flow rate of 0.7 microl/min. Nitrite produced from activated macrophages was measured immediately after lipopolysaccharide (LPS) stimulation using microdialysis sampling.     

Determination of S-nitrosoglutathione in human and rat plasma by high-performance liquid chromatography with fluorescence and ultraviolet absorbance detection after precolumn derivatization with o-phthalaldehyde.

An analytical method is described for the quantification of S-nitrosoglutathione (GSNO), a potent physiological vasodilator and inhibitor of platelet aggregation, in the presence of a high excess of reduced glutathione (GSH). The method is based on the quantitative elimination of GSH by N-ethylmaleimide, the conversion of GSNO by 2-mercaptoethanol to GSH, its reaction with o-phthalaldehyde (OPA) to form a highly fluorescent and UV-absorbing tricyclic isoindole derivative, and subsequent high-performance liquid chromatographic (HPLC) separation with fluorescence and/or UV absorbance detection. The OPA derivatives of GSH and GSNO obtained by this method were found to be identical by mass spectrometry. GSH (up to 50 microM) did not interfere with the analysis of GSNO (up to 1000 nM). The limits of detection of the method for buffered aqueous solutions of GSNO were determined as 3 nM using fluorescence and 70 nM using UV absorbance detection. Isolation of GSNO by HPLC analysis (pH 7.0) of plasma ultrafiltrate samples (200 microl) prior to derivatization allows specific and artifact-free quantification of GSNO in human and rat plasma. Reduced and oxidized glutathione, nitrite, and cysteine did not interfere with the measurement of GSNO in human and rat plasma. The limit of quantitation (LOQ) of the combined method was determined as 100 nM of GSNO in human plasma ultrafiltrate using fluorescence detection. No endogenous GSNO could be detected in ultrafiltrate samples of plasma of 10 healthy humans at concentrations exceeding the LOQ of the method. After iv infusion of GSNO (125 micromol/kg body wt) in a rat for 20 min GSNO and GSH were detected in rat plasma at 60 and 130 microM, respectively. The method should be useful to investigate formation, metabolism, and reactions of GSNO in vitro and in vivo at physiologically relevant concentrations.     

High-performance liquid chromatographic method for quantification of busulfan in plasma after derivatization by tetrafluorothiophenol

A high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of busulfan in plasma. Busulfan was extracted in toluene, derivatized by 2,3,5,6-tetrafluorothiophenol to obtain di-TFTP-butane, the derivatization product was then re-extracted in toluene and injected into the HPLC system with ultraviolet detection (wavelength: 275 nm). Recovery from extraction was 80%, the limit of quantification was 50 ng/ml and linearity ranged from 50 to 2000 ng/ml. In addition, forty-two samples obtained from pediatric patients treated with busulfan were analyzed by the HPLC and GC–MS assays based on the same derivatization procedure. The correlation between the di-TFTP-butane concentrations was highly significant (p<0.0001), demonstrating that the two methods were in good agreement.     

Micellar electrokinetic chromatography of hydroxyproline and other secondary amino acids in biological samples with laser-induced fluorescence detection

Micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) was used for the rapid and sensitive detection of hydroxyproline in serum and hydrolyzed urine that were pre-column derivatized with 9-fluorenylmethyl chloroformate (FMOC). The application of the combined o-phthalaldehyde (OPA)/FMOC derivatization in MEKC for the selective detection of secondary amino acids in biological samples is investigated.     

Liquid chromatography method for detecting native fluorescent bioamines in urine using post-column derivatization and intramolecular FRET detection

Liquid chromatography (LC) with fluorescence detection is described for simultaneous determination of native fluorescent bioamines (indoleamines and catecholamines). This is based on intramolecular fluorescence resonance energy transfer (FRET) in an LC system following post-column derivatization of native fluorescent bioamines' amino groups with o-phthalaldehyde (OPA). OPA fluorescence was achieved through an intramolecular FRET process when the molecules were excited at maximum excitation wavelength of the native fluorescent bioamines. Bioamines separated by reversed-phase LC on ODS column were derivatized with OPA and 2-mercaptoethanol. This method provides sufficient selectivity and sensitivity for the determination of normetanephrine, dopamine, tyrosine, 5-hydroxytryptamine, tryptamine, and tryptophan in healthy human urine without prior sample purification.     

Simple method for the simultaneous isolation and determination of fumonisin B1 and its metabolite aminopentol-1 in swine liver by liquid chromatography--fluorescence detection

An analytical method based on high-performance liquid chromatography (HPLC) combined with fluorescence detection (FL) has been developed for the simultaneous determination of fumonisin B1 (FB1) and its totally hydrolized metabolite aminopentol-1 (AP1) in pig liver. The sample preparation is based on a single solid phase extraction (SPE). o-Phthalaldehyde (OPA) was used for pre-column derivatization before the programmed reversed-phase analysis on phenylhexyl column. The developed method shows good repeatibility for inter- and intra-day precision as well as adequate linearity of calibration curves (r2 was 0.9855 for FB1 and 0.9831 for AP1). Average recoveries from the matrix were 93.6% for FB1 and 95.3% for AP1. The limit of quantification (LOQ) in swine liver was 75 microg/kg for FB1 and 42 microg/kg for AP1.     

A Highly Sensitive Assay for Histamine Using Ion-Pair HPLC Coupled with Postcolumn Fluorescent Derivatization: Its Application to Biological Specimens

A simple and highly sensitive method for the determination of histamine (HA) was developed using ion-pair, reversed-phase HPLC coupled with postcolumn o-phthalaldehyde derivatization fluorometry, and it was applied to the unpurified extracts of human and rat plasma, and brains of rats and mice. The HA concentrations both in the plasma and brains determined by the present method were well consistent with the values obtained by cation-exchange HPLC with postcolumn fluorescent derivatization currently in use. The present method was more advantageous than the assay using cation-exchange HPLC: (1) it was three to four times more sensitive (the detection limit was 0.5 pg of HA), and (2) it enabled the measurement of HA in samples containing (R)alpha-methylhistamine, a potent and specific H3-receptor agonist, which could not be separated from HA by cation-exchange chromatography. Using the present method coupled with intracerebral microdialysis, we found in the rat hypothalamus that (R)alpha-methylhistamine (5 mg/kg i.p.) markedly decreased the extracellular concentration of HA with a maximal effect (83% reduction) during 30-60 min after injection, suggesting that most of HA in the microdialysate fraction is neuronal in origin.