Dabsyl Chloride Research Articles

Tronchuda cabbage (Brassica oleracea L. var. costata DC) leaf free amino acid profile

Free amino acid profile of eighteen samples of tronchuda cabbage (Brassica oleracea L. var. costata DC) leaves was determined by a reversed-phase HPLC/UV-Vis methodology, after pre-column derivatization with dabsyl chloride. Samples were harvested in three collection months and internal and external leaves were separated.

Production of hypotaurine from l-cysteinesulfinate by rat liver mitochondria

Hypotaurine is the precursor of taurine production from L-cysteinesulfinate. It is recognized that hypotaurine production in the liver occurs in cytosol. In the present study, hypotaurine production from L-cysteinesulfinate in rat liver mitochondria was investigated. The mitochondrial preparation prepared according to the method of Hogeboom and washed repeatedly with 0.25 M sucrose solution was incubated with L-cysteinesulfinate. Products were derivatized with dabsyl chloride and dabsylated amino acids were analyzed by RP-HPLC. Presence of a peak corresponding to dabsyl-hypotaurine was confirmed. The peak of dabsyl-hypotaurine was converted quantitatively to dabsyl-taurine by the treatment with H(2)O(2). Optimum pH of the reaction was shown to be broad between 6.0 and 7.8 and Km for L-cysteinesulfinate was 0.11 mM. Results indicate the presence of L-cysteinesulfinate decarboxylase activity in liver mitochondria. Mitochondrial cysteine metabolism was summarized and possible antioxidant roles of cysteine metabolites including hypotaurine in mitochondria are discussed.

Differential Binding of Calmodulin Domains to Constitutive and Inducible Nitric Oxide Synthase Enzymes

Calmodulin (CaM) is a Ca2+ signal transducing protein that binds and activates many cellular enzymes with physiological relevance, including the mammalian nitric oxide synthase (NOS) isozymes: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). The mechanism of CaM binding and activation to the iNOS enzyme is poorly understood in part due to the strength of the bound complex and the difficulty of assessing the role played by regions outside of the CaM-binding domain. To further elucidate these processes, we have developed the methodology to investigate CaM binding to the iNOS holoenzyme and generate CaM mutant proteins selectively labeled with fluorescent dyes at specific residues in the N-terminal lobe, C-terminal lobe, or linker region of the protein. In the present study, an iNOS CaM coexpression system allowed for the investigation of CaM binding to the holoenzyme; three different mutant CaM proteins with cysteine substitutions at residues T34 (N-domain), K75 (central linker), and T110 (C-domain) were fluorescently labeled with acrylodan or Alexa Fluor 546 C5-maleimide. These proteins were used to investigate the differential association of each region of CaM with the three NOS isoforms. We have also N-terminally labeled an iNOS CaM-binding domain peptide with dabsyl chloride in order to perform FRET studies between Alexa-labeled residues in the N- and C-terminal domains of CaM to determine CaM's orientation when associated to iNOS. Our FRET results show that CaM binds to the iNOS CaM-binding domain in an antiparallel orientation. Our steady-state fluorescence and circular dichroism studies show that both the N- and C-terminal EF hand pairs of CaM bind to the CaM-binding domain peptide of iNOS in a Ca2+-independent manner; however, only the C-terminal domain showed large Ca2+-dependent conformational changes when associated with the target sequence. Steady-state fluorescence showed that Alexa-labeled CaM proteins are capable of binding to holo-iNOS coexpressed with nCaM, but this complex is a transient species and can be displaced with the addition of excess CaM. Our results show that CaM does not bind to iNOS in a sequential manner as previously proposed for the nNOS enzyme. This investigation provides additional insight into why iNOS remains active even under basal levels of Ca2+ in the cell.

HPLC Determination of Free Amino Acids Profile of Dão Red Wine: Effect of Dekkera bruxellensis Contamination

A reversed‐phase HPLC/UV‐Vis methodology for determination of twenty free amino acids in Dão red wine is described. The sample preparation was simple, involving only a pre‐column derivatization with dabsyl chloride. The HPLC/UV‐Vis procedure is sensitive, reproducible, and accurate. The detection limit values for free amino acids were low, between 0.01 and 0.07 µg/mL, and the method was precise. As a general rule, the recovery values were high. This methodology was applied to the evaluation of the effect of the spoilage wine yeast Dekkera bruxellensis on the Dão red wine free amino acids profile. This yeast seems to influence the free amino acids composition of the wine, including aspartic and glutamic acids, asparagine, glutamine, and isoleucine as the main affected compounds.

Aggregation behavior of dabsylated poly(ε-L-lysine) in aqueous DMSO solution

Side chain amino groups of microbial Poly(e-L-lysine) (e-PL) were chemically modified with an amino labeling reagent, 4-dimethylaminoazobenzene-4’-sulfonyl chloride (dabsyl chloride) to give dabsylated e-PL (Dabs-e-PL). Aggregation behavior of Dabs-e-PL in aqueous DMSO solution was studied through UV-Vis and CD spectra. In DMSO solution, Dabs-e-PL shows a symmetric visible absorption at 450 nm, corresponding to the azobenzene chromophore in the monomeric state. Upon addition of water to the DMSO solution, the λmax is shifted to around 400 nm. CD spectra were detected in the region of the blue shifted band: a pair of CD signals of opposite sign appears with a crossover point related to the shifted λmax. Similar trends were also observed for UV-Vis and CD spectra of dabsylated oligo(e-L-lysine)mers. These spectroscopic results indicate that side chain azo chromophores of Dabs-e-PL form H-aggregates and are disposed according to a chiral arrangement with right handedness in aqueous DMSO solution.

γ-Glutamyl transpeptidase acylation with peptidic substrates: free energy relationships measured by an HPLC kinetic assay

gamma-Glutamyl transpeptidase (GGT, EC 2.3.2.2) is a highly glycosylated heterodimeric enzyme linked to the external cellular membrane that catalyzes the hydrolysis of glutathione as well as the transfer of its gamma-glutamyl group to amino acids and dipeptides in a transpeptidation reaction. The measurement of both the hydrolytic and transpeptidation activity of this important enzyme is a challenge, since its native substrates are not highly chromogenic. We have developed an HPLC-based method for the quantitative photometric detection of numerous enzyme substrates and products, after their pre-column derivation with dabsyl chloride. The broad applicability of this method was demonstrated in the kinetic investigation of transpeptidation reactions of rat kidney GGT with glutathione, its native substrate, as well as a series of pertinent glutathione analogues. The pH-rate profile constructed for glutathione confirmed the dependence on the ionisation state of at least two residues. Analysis of the free-energy relationships in the series of synthetic peptidic substrate analogues revealed the importance of enzyme-substrate interactions unrelated to amine leaving group basicity during the acylation step. These results are further interpreted in the context of the recently published structure for a similar GGT.

Structural investigation of microbial poly(?-L-lysine) derivatives with azo dyes by solid-state 13C and 15 N NMR

Solid-state high-resolution 13C and 15N NMR spectra of microbial poly(e-lysine) (e-PL) derivatives with azo dyes have been measured. Chemically modified derivatives of e-PL, e-PL/MO and e-PL/DC were prepared through reactions with methyl orange (MO) and dabsyl chloride (DC), respectively. Side chain α-amino groups of e-PL in e-PL/MO are involved in ionic bonds with methyl orange to form poly-ion complexes, (e-PL)-NH3+SO3--(MO). On the other hand, e-PL is allowed to react with dabsyl chloride in e-PL/DC to form covalent sulfonamide bonds, (e-PL)-NH-SO2-(DC). These chemically modified e-PL’s exhibit 15N NMR signals characteristic of the binding mode at the α-amino groups. The spectral analysis reveals that the e-PL/DC sample contains a small amount of ion complexes with MO. It has been shown that 15N solid-state NMR is a useful tool for structural determination of e-PL and its derivatives.

Argpyrimidine, a methylglyoxal-derived advanced glycation end-product in familial amyloidotic polyneuropathy.

FAP (familial amyloidotic polyneuropathy) is a systemic amyloid disease characterized by the formation of extracellular deposits of transthyretin. More than 80 single point mutations are associated with amyloidogenic behaviour and the onset of this fatal disease. It is believed that mutant forms of transthyretin lead to a decreased stability of the tetramer, which dissociates into monomers that are prone to unfolding and aggregation, later forming beta-fibrils in amyloid deposits. This theory does not explain the formation of beta-fibrils nor why they are toxic to nearby cells. Age at disease onset may vary by decades for patients with the same mutation. Moreover, non-mutated transthyretin also forms the same deposits in SSA (senile systemic amyloidosis), suggesting that mutations may only accelerate this process, but are not the determinant factor in amyloid fibril formation and cell toxicity. We propose that glycation is involved in amyloidogenesis, since amyloid fibrils present several properties common to glycated proteins. It was shown recently that glycation causes the structural transition from the folded soluble form to beta-fibrils in serum albumin. We identified for the first time a methylglyoxal-derived advanced glycation end-product, argpyrimidine [N(delta)-(5-hydroxy-4,6-dimethylpyrimidin-2-yl)-L-ornithine] in amyloid fibrils from FAP patients. Unequivocal argpyrimidine identification was achieved chromatographically by amino acid analysis using dabsyl (4-dimethylaminoazobenzene-4'-sulphonyl) chloride. Argpyrimidine was found at a concentration of 162.40+/-9.05 pmol/mg of protein in FAP patients, and it was not detected in control subjects. The presence of argpyrimidine in amyloid deposits from FAP patients supports the view that protein glycation is an important factor in amyloid diseases.

Interrelationships among Microbiological, Physicochemical, and Biochemical Properties of Terrincho Cheese, with Emphasis on Biogenic Amines

Changes in the microbiological, physicochemical, and biochemical characteristics of Terrincho cheese as represented by native microflora, pH, water activity, soluble nitrogen fractions, free amino acids, and biogenic amines (e.g., ethylamine, dimethylamine, tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, cystamine, and spermine) during ripening were monitored. Terrincho is a traditional Portuguese cheese manufactured from raw ewe's milk. The main groups of microorganisms (lactococci, lactobacilli, enterococci, pseudomonads, staphylococci, coliforms, yeasts, and molds) were determined following conventional microbiological procedures. Free amino acids and biogenic amines were determined by reverse-phase high-performance liquid chromatography, following extraction from the cheese matrix and derivatization with dabsyl chloride. The total content of free amino acids ranged from 1,730 mg/kg of dry matter at the beginning of the ripening stage to 5,180 mg/kg of dry matter by day 60 of ripening; such an increase was highly correlated with the increase of water-soluble nitrogen in total nitrogen, 12% trichloroacetic acid–soluble nitrogen in total nitrogen, and 5% phosphotungstic acid–soluble nitrogen in total nitrogen throughout ripening. Histamine was consistently present at very low levels, whereas putrescine, cadaverine, and tryptamine were the dominant biogenic amines and increased in concentration during ripening. Ethylamine, tryptamine, phenylethylamine, and cystamine reached maxima by 30 days of ripening and decreased thereafter. Significant correlations between amino acid precursors and corresponding biogenic amines, as well as between biogenic amines and microbial viable numbers, were observed.

Detector supports: application to aliphatic amines in wastewater

Solid support assisted derivatization coupled to diffuse reflectance spectroscopy (DRS) was proposed and proved useful for the detection and quantification of aliphatic amines in water as an example. Dabsyl chloride (DBS), ninhydrin and sodium 1,2-naphtoquinone 4-sulphonate (NQS) were assayed as derivatization reagents. C 18 and SDB-XC disks and C 18 cartridges were tested for amine retention and after that derivatization. The decrease of the orange colour of dabsyl chloride on SBD-XC disks produced by the formation of its derivative with methylamine in the support (10 min at 100 °C) allowed the selective determination of the amine at concentration level equal or higher than 0.5 mg L −1. Ninhydrin can be used for methylamine, ethylamine, propylamine, butylamine and pentylamine (between 5 and 15 mg L −1) by measuring the diffuse reflectance produced by the brown derivative formed in C 18 extraction disks after 15 min at 100 °C. NQS and C 18 SPE columns can be also employed to estimate amines, but the detection limits were higher than those provided by DBS and Ninhydrin, around 10 mg L −1. As an example, found concentration of methylamine or total amines (expressed as –NH 2–N mg L −1) in a wastewater sample is given employing dabsyl chloride or ninhydrin reagents, respectively with satisfactory results.

Simple quantitative HPLC method for measuring physiologic amino acids in cerebrospinal fluid without pretreatment.

Because the cerebral spinal fluid (CSF) bathes the whole central nervous system, its contents reflect changes occurring in the central neurons. Quantitative analysis of amino acids present in CSF has been helpful in improving our understanding of disease conditions associated with pain (1)(2)(3). Amino acids in CSF can be determined either by postcolumn derivatization with ninhydrin or o -phthalaldehyde (OPA) by commercially available amino acid analyzers or by precolumn derivatization with different reagents, such as dansyl chloride, phenylisothiocyanate, fluorenylmethyl chloroformate, dabsyl chloride, and OPA, followed by HPLC separation (4)(5)(6). Precolumn derivatization with OPA has been used predominantly for analysis of amino acids in CSF (1)(2)(7). However, OPA reacts only with primary amines, and the OPA adducts are unstable (4)(8). The preanalytical processes, including sample storage conditions and the pretreatment used for amino acid analysis in physiologic fluids, have not been standardized, making it difficult to compare results among laboratories (5)(9)(10)(11)(12). Pretreatment with different deproteination methods, including strong acids (3)(4)(9)(10), organic solvents (13)(14)(15), or ultrafiltration (2)(16)(17), has been shown to adversely affect the quantitative results for amino acids (16)(18). Dabsyl chloride has been used to convert primary and secondary amines to their colored derivatives with subsequent separation by HPLC (19). This method has been used for analysis of amino acids and other compounds from physiologic fluid and tissue extracts (8)(15)(16)(20). However, use of this method for CSF samples has not been reported. We report an improved method using dabsyl chloride in the presence of the nonionic neutral surfactants Triton X-100 (Sigma) or Tween 20 (Bio-Rad), which …

Dityrosine as a product of oxidative stress and fluorescent probe.

Dityrosine can be a natural component of protein structure, a product of environmental stress, or a product of in vitro protein modification. It is both a cross-link and a fluorescent probe that reports structural and functional information on the cross-linked protein molecule. Diverse reactions produce tyrosyl radicals, which in turn may couple to yield dityrosine. Identification and quantitation of dityrosine in protein hydrolysates usually employs reversed phase high pressure liquid chromatography (RP-HPLC) or gas chromatography. RP-HPLC of protein hydrolysates that have been derivatized with dabsyl chloride gives a complete amino acid analysis that includes dityrosine and 3-nitrotyrosine. Calmodulin, which contains a single pair of tyrosyl residues, undergoes both photoactivated and enzyme-catalyzed dityrosine formation. Polarization measurements, employing the intrinsic fluorescence of dityrosine, and catalytic activity determinations show how different patterns of inter- and intramolecular cross-linking affect the interactions of calmodulin with Ca(2+) and enzymes.

Determination of taurine and hypotaurine in animal tissues by reversed-phase high-performance liquid chromatography after derivatization with dabsyl chloride.

Taurine and sulfate are major end products of L-cysteine metabolism in mammals1, and they play various roles as tissue constituents and physiologically important compounds. Taurine is found in high concentrations in skeletal and heart muscle and brain2. It is utilized for the synthesis of taurine conjugates of bile acids. It is believed that most taurine is produced via the cysteinesulfinate pathway3and cysteamine pathway4. In both pathways, hypotaurine is an obligatory precursor leading to taurine. Hypotaurine content in animal tissues is far less than the taurine content. However, it has been reported that hypotaurine is found in rat brain5, accumulates in the liver of cysteine-administered rats3and in the regenerating rat liver6, increases in blood of rats injected with L-cysteine7, and is excreted in the urine of rats fed L-cystine8. When hypotaurine was injected into rats, urinary taurine excretion increased in a dose-dependent manner9.KeywordsPeak Area RatioSulfonyl ChlorideTaurine ContentAutomatic Amino Acid AnalyzerTaurine ConjugateThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Characterization of selected Spanish table wine samples according to their biogenic amine content from liquid chromatographic determination.

Pattern recognition techniques, such as principal component analysis, cluster analysis, and linear discriminant analysis, have been applied to samples of red, white, and rosé wines to determine whether some biogenic amines could be considered as chemical descriptors. Eight amines (tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine) were determined by RP-HPLC, after derivatization with dabsyl chloride. However, only putrescine, cadaverine, histamine, tyramine, spermidine, and spermine were found in the wines analyzed. From the association between variables obtained by principal component analysis and clustering and from the relationship found by linear discriminant analysis, it can be deduced that the amines generated during malolactic fermentation (putrescine, histamine, and tyramine) could be used as chemical descriptors to characterize table wine samples.

Synthesis and Characterization of a Collagen Model δ-O-Phosphohydroxylysine-Containing Peptide

The existence of δ-O-phosphohydroxylysine (HylP) residues in collagen has been reported. However, such phosphorylated residues have not been isolated nor has their location within the protein sequence been identified. To develop the analytical chemistry necessary for the identification of HylP in proteins, a model HylP-containing peptide, Phe-dl-HylP-Gly-Gln-Pro-Ala-Ile-Gly-Phe (I), was synthesized. The peptide was assembled using 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase synthesis; Nα-Fmoc-dl-hydroxy-dl-Lys(Nε-tert-butyloxycarbonyl)-OH was used to incorporate Hyl, and global phosphitylation/oxidation was used to introduce the phosphate group. The pKa2 of the phosphate group, as determined using 31P NMR, was 5.6. Phosphoamino acid analysis of I was performed using either dabsyl chloride or phenylisothiocyanate derivatization followed by microbore reversed-phase HPLC separation of Nα,ε-didabsyl-HylP or Nα,ε-diphenylthiocarbamyl-HylP. HylP was found to be relatively resistant to acid hydrolysis, allowing for its quantitation. Solid-phase Edman degradation of I was used for positive identification of Nα-phenylthiohydantoin-Nε-phenylthiocarbamyl-HylP. The masses of the phenylthiohydantoin and dabsyl derivatives of HylP were confirmed by electrospray ionization triple-quadrupole (ESI-MS). Peptide I was positively identified as a phosphopeptide by ESI-MS/precursor-ion scanning. Low-energy ESI-MS/MS confirmed the position of HylP within the sequence of I. Phosphorylation of Hyl led to complete resistance of I to lysine-specific endopeptidases.

Procedure for the determination of biogenic amines by derivatization with dabsyl chloride and reversed phase liquid chromatography

Existing procedures for the determination of biogenic amines by derivatization with dabsyl chloride and separation by RPLC differ very little from those employed in amino acid analysis. Since some difficulties attributable to the higher hydrophobicity of dabsyl-amides were detected on using these procedures, important modifications are proposed, including extraction of the reaction mixture with an organic solvent, drying of the extract and redissolving the residue in acetone before injecting. Dabsylation and chromatographic conditions were also studied. Although recovery from spiked meat extracts is very good, significant matrix effects are detected when extraction is carried out from spiked whole meat samples. These adverse effects can be overcome by means of standard addition.

Effect of temperature on evolution of free amino acid and biogenic amine contents during storage of Azeitão cheese

Abstract A study on the evolution of free amino acids and biogenic amines in Azeitao cheese during 4 weeks at different temperatures of storage (4 and 25°C) was performed. Free amino acids and biogenic amines were determined by RP-HPLC with visible detection, following extraction from the cheese and derivatization with dabsyl chloride. The method presented a linear relation between peak area and concentration from 2–200 mg/l. The detection limit value was less than 1.5 mg/l. The average repeatability was less than 4%. The major free amino acids were proline, valine, isoleucine and leucine and the major amines were tyramine, cadaverine and histamine. Room temperature (25°C) promoted a significant increase of the contents of valine, leucine, tyramine and putrescine, expressed as g/kg of dry matter. These two free amino acids and two biogenic amines may serve as indicators of temperatures changes in ripened cheese.

Determination of amphetamine and its metabolite p‐hydroxyamphetamine in rat urine by reversed‐phase high‐performance liquid chromatography after dabsyl derivatization

The consumption of amphetamine is illicit and controlled due to both the elicited behavioural deviations and the toxicity effects reported in abusers. Thus, amphetamine levels in biological samples must be monitored in several clinical and forensic circumstances. In spite of the interspecies differences in the preferred route of biotransformation, benzylmethylketone, benzoic acid and 4-hydroxyamphetamine are the principal metabolites of amphetamine. However, the clinical and forensic studies are focused in the parent compound and in 4-hydroxyamphetamine since benzylmethylketone is a minor metabolite in human and benzoic acid is also an endogenous compound. In the present study amphetamine and its metabolite, 4-hydroxyamphetamine, are quantified in urine by HPLC after derivatization with 4-dimethylaminoazobenzene-4'-sulfonyl chloride (dabsyl chloride). This derivatization procedure transforms amphetamine and its hydroxylated metabolite in compounds with similar lipofilicity, enabling their quantitative and simultaneous extraction with an organic solvent. The precision of the HPLC technique was 7.3 and 10.0% for amphetamine and 4-hydroxyamphetamine derivatives, respectively. For the overall procedure, including enzymatic hydrolysis, derivatization and extraction of the derivatives, the obtained values were 9.3 and 6.2%. Recoveries obtained from spiked urines for amphetamine and 4-hydroxyamphetamine were better than 97% and 94% (mean value), respectively. The detection limits of the method was 10 ng for both compounds. The principal advantages of the present proposed method are the stability of the dabsyl derivatives at room temperature and the detection carried out in the visible region, reducing the interferences detected.

Analysis of urinary N-acetyl- S-( N-methylcarbamoyl)cysteine, the mercapturic acid derived from N, N-dimethylformamide

Human biotransformation of the industrial solvent N, N-dimethylformamide gives raise to N-acetyl- S-( N-methylcarbamoyl)cysteine (AMCC) which has the longest half-life (about 23 h) among urinary metabolites of N, N-dimethylformamide. It could be used for monitoring industrial exposure over several workdays, by measuring it in urine samples collected at the end of the working week. This is consistent with the suggestions of the American Conference of Governmental Industrial Hygienists, which established a limit of 40 mg/l for the year 2000. An easy, cheap and user-friendly method has been developed for determination of urinary AMCC. Unlike currently available methods, it requires neither a time-consuming preparation phase nor gas chromatographic analysis with a nitrogen-phosphorus or mass detector. The method uses high-performance liquid chromatography (HPLC), with an UV detector at 436 nm. A 10-μl volume of urine is added to a carbonate–hydrogen carbonate buffer and mixed with a dabsyl chloride solution in acetonitrile. The reaction between AMCC and the reagent is performed at 70°C for 10 min. The ‘dabsylated’ product is stable for at least 12 h. After brief centrifugation, the solution is ready for HPLC analysis using a C 18 column (250×4.6 mm, 5 μm). The method is sensitive (detection limit 1.8 mg/l) and specific. It identified urinary AMCC in urine of 40 subjects not exposed to N, N-dimethylformamide with a median concentration of 3.9 mg/l. In urine samples from 20 workers exposed to N, N-dimethylformamide (5–40.8 mg/m 3), AMCC concentrations ranged from 16 to 170 mg/l. Industrial toxicology laboratories with limited instrumentation will be able to use it in the biological monitoring of workers exposed to N, N-dimethylformamide.

Initial evaluation of quantitative performance of chromatographic methods using replicates at multiple concentrations

The introduction of a novel analytical method must be supported by consistent information about its quantitative potentialities; this is critical for whoever considers its utilization for an specific application. Unfortunately, literature abounds in papers proposing excellent chromatographic methods of analysis that have been subjected to comparatively poor quantitative evaluation. The methodology proposed in the present work makes use of some of the performance characteristics whose measurement is recommended in validation protocols; pertinent to this stage of method development are the detection and quantitation limits, the linear range and the repeatability. All this information can be calculated from the results of a calibration with several replicates at each analyte level. Replicates enable the calculation of reproducibility at several analyte levels and the estimation of the linear range; more important, replicates are necessary to detect changes in peak area standard deviation with analyte amount. Regression of calibration data by means of unweighted least-squares (ULSR) can only be performed in those cases in which homoscedasticity has been previously verified; heteroscedastic calibration data demand regression by means of weighted least-squares (WLSR), since ULSR results in gross overestimation of prediction limits at low analyte concentration. The proposal is used for the preliminary quantitative evaluation of a method for the determination of nine biogenic amines by means of pre-column derivatization with dabsyl chloride and separation of derivatives by RPLC. Limits of detection are calculated by a regression approach and by the classical signal-to-noise ratio method ( S/ N approach). No significant difference was detected for the amines limits of detection estimated by WLSR and by the S/ N approach; ULSR estimated limits of detection are between 7 and 78 times larger than those obtained by the other two methods, as a consequence of the heteroscedasticity of calibration data.