Precolumn derivatization, HPLC

K Hayakawa, T Schilpp, K Imai, T Higuchi, OS Wong. Determination of aspartic acid, phenylalanine and aspartylphenylalanine in aspartame-containing samples using a precolumn derivatization HPLC method. J Agric Food Chem 38(5) 1256-1260, 1990. 

The identification and quantification of potentially cytotoxic carbonyl compounds (e.g. aldehydes such as pentanal, hexanal, traw-2-octenal and 4-hydroxy-/mAW-2-nonenal, and ketones such as propan- and hexan-2-ones) also serves as a useful marker of the oxidative deterioration of PUFAs in isolated biological samples and chemical model systems. One method developed utilizes HPLC coupled with spectrophotometric detection and involves precolumn derivatization of peroxidized PUFA-derived aldehydes and alternative carbonyl compounds with 2,4-DNPH followed by separation of the resulting chromophoric 2,4-dinitrophenylhydrazones on a reversed-phase column and spectrophotometric detection at a wavelength of378 nm. This method has a relatively high level of sensitivity, and has been successfully applied to the analysis of such products in rat hepatocytes and rat liver microsomal suspensions stimulated with carbon tetrachloride or ADP-iron complexes (Poli etui., 1985). 

Santagati and associates (2002) reported a method for the determination of amphetamine and one of its metabolites, 4-hydroxynorephedrine by RP-HPLC with precolumn derivatization and amperometric electrochemical detection. The derivatization was performed with 2,5-dihydroxybenzaldehyde as the electroactive reagent. The compounds were separated on a Hypersil ODS RP-18. The detector oxidation was set at +0.6 volts. 

Several other methods have been published using RP-HPLC for the determination amphetamines and related derivatives. Studies have shown the determination of amphetamine and related derivatives in plasma, urine, and hair by RP-HPLC with precolumn derivatization and either UV/VlS or fluorescence detection. Various methods are employed by SPE technologies using Cl8 cartridges for sample cleanup prior to derivatization. The derivatized compounds were separated on analytical columns of various Cl 8 bonded phase materials. The methods generally used water/acetonitrile mobile phases operated in gradient mode. All studies reported extraction recoveries of 85-102% for all the analytes, with LLOQs ranging from 5 to 60 ng/ml (Tedeschi et al., 1993 Ealco et al., 1996 Hernandez et al., 1997 Al-Dirbashi et al., 1997 Al-Dirbashi et al., 2000 Soares et al., 2001). 

Three approaches can be employed to separate peptide stereoisomers and amino acid enantiomers separations on chiral columns, separations on achiral stationary phases with mobile phases containing chiral selectors, and precolumn derivatization with chiral agents [111]. Cyclodextrins are most often used for the preparation of chiral columns and as chiral selectors in mobile phases. Macrocyclic antibiotics have also been used as chiral selectors [126]. Very recently, Ilsz et al. [127] reviewed HPLC separation of small peptides and amino acids on macrocyclic antibiotic-based chiral stationary phases. 

Diverse analytical methods have been proposed for the analysis of amino acids, including GC, HPLC, and capillary electrophoresis. The preferred method at present is RP-HPLC with precolumn derivatization, which has the advantages of requiring short analysis time, simple instrumentation, and low cost [192]. 

There are two major approaches to achieve enantiomeric separation of d- and L-amino acids. The first involves precolumn derivatization with a chiral reagent, followed by RP-HPLC [226], while the second involves direct separation of underivatized enantiomers on a chiral bonded phase [227], Weiss et al. [209] determined d- and L-form of amino acids by applying derivatization with OPA and chiral /V-isobutyryl-L-cysteine. 

Moret et al. [280] applied precolumn derivatization with Dns-Cl and OPA to the same sample (vegetables) extracts and compared the results. Figure 19.6 reports HPLC traces obtained for a spinach sample (Kromasil C18 column, 250x4.6mm i.d., 5[tm particle size). 

Postcolumn photochemical reactions are another approach to the detection problem. High-intensity UV light, generally provided by a Hg or Zn lamp, photolyzes the HPLC effluent, which passes through a Teflon (47) or quartz tube. The photolysis reaction determines the nature of the subsequent detection. If the compound has a UV chromophore, such as an aromatic ring, and an ionizable heteroatom, such as chlorine, then the products of the reaction can be detected conductometrically. Busch et al. (48) have examined more than 40 environmental pollutants for applicability to detection with photolysis and conductance detection. Haeberer and Scott (49) found the photoconductivity approach superior to precolumn derivatization for the determination of nitrosoamines in water and waste water. The primary limitation of this detection approach results from the inability to use mobile phases that contain ionic modifiers, that is, buffers and... 

Since then, there have been a number of reversed-phase separations employing precolumn derivatization. Interestingly, fluorescamine (not frequently employed for RP-HPLC of amino acids with precolumn reaction) has been reported for taurine analysis in milk (197) and human plasma (198). Precolumn derivatization with OPA/2-mercaptoethanol has been reported for the analysis of infant formula and human breast milk (199). Although not the principal focus of the study, Carratu et al. (200) report taurine values in parenteral solutions as determined by FMOC. In an excellent article, Woollard and Indyk (201) report the dansylation of taurine for its determination in a wide variety of dairy-related products. Subsequently, the same authors report the results of a large collaborative study (202) for the determination of taurine (again, by dansylation) in milk and infant formula. This study afforded an overall interlaboratory RSD of 7.0% and established a lower limit for determination at 5 mg taurine per 100 g of product. 

U Butikofer, D Fuchs, JO Bosset, W Gmur. Automated HPLC-amino acid determination of protein hydrolysates by precolumn derivatization with OPA and FMOC and comparison with classical ion exchange chromatography. Chromatographia 31 441-447, 1991. 

Cyclamate can also be determined after its oxidation to cyclohexylamine. Cyclohexy-lamine can be quantified by HPLC with trinitrobenzenesulfonic acid precolumn derivatization and UV detection (72). Cyclohexylamine can also be converted into a fluorescent derivative, separated on a Cl8 reversed-phase column with acetonitrile Na2HP04 buffer (64 36, v/v), and quantified at 350 and 440-650 nm of excitation and emission, respectively (73). 

Colistin (COL) is a multicomponent antibiotic (polymyxins E) that is produced by strains of inverse Bacillus polymyxa. It consists of a mixture of several closely related decapeptides with a general structure composed of a cyclic heptapeptide moiety and a side chain acetylated at the N-terminus by a fatty acid. Up to 13 different components have been identified. The two main components of colistin are polymyxins El and E2 they include the same amino acids but a different fatty acid (216). A selective and sensitive HPLC method was developed for the determination of COL residues in milk and four bovine tissues (muscle, liver, kidney, and fat). The sample pretreatment consists of protein precipitation with trichloracetic acid (TCA), solid-phase purification on Cl 8 SPE cartridges, and precolumn derivatization of colistin with o-phthalaldehyde and 2-mercaptoethanol in borate buffer (pH 10.5). The last step was performed automatically, and the resulting reaction mixture was injected into a switching HPLC system including a precolumn and the reversed-phase analytical column. Fluorescence detection was used. The structural study of El and E2 derivatives was carried out by HPLC coupled with an electrospray MS. Recoveries from the preseparation procedure were higher than 60%. 

P Kubalec, E Brandsteterova, A Bednarikova. Determination of oxacillin, cloxacillin and dicloxacillin in milk, meat and cheese samples using HPLC and precolumn derivatization. Z Lebensm Unters Forsch 205 85-88, 1997. 

Since BAs occurring in food do not exhibit satisfactory absorbance or fluorescence in the visible or ultraviolet range, chemical derivatization, either pre- (35-37) or postcolumn (38), is usually used for their detection in HPLC. The most frequently employed reagents for precolumn derivatization are fluorescamine, aminoquinolyl-lV-hydroxysuccinimidyl carbamate (AQC) (39, 40), 9-fluorenylmethyl chloroformate (FMOC) (41-43), 4-dimethylaminoazobenzene-4 -sul-fonyl chloride (dabsylchloride, DBS) (44), N-acetylcysteine (NAC) (45,46), and 5-dimethyl-amino-1-naphthalene-1-sulfonyl chloride (dansylchloride, DNS) (47,48), phthalaldehyde (PA), and orf/to-phthaldialdehyde (OPA) (49-51), together with thiols such as 3-mercaptopropionic acid (MPA) (37) and 2-mercaptoethanol (ME) (35,49). 

HPLC with precolumn derivatization (PA) and fluorescence detection at 440 nm (Acx = 230 nm). 

Ellman s reagent has been used not only for the determination of sulfhydryls in proteins and other molecules, but also as a precolumn derivatization reagent for the separation of thiol compounds by HPLC (Kuwata et al., 1982), in the study of thiol-dependent enzymes (Masamune etal., 1989 Tsukamoto and Wakil, 1988 Alvear et al., 1989), and to create sulfhydryl-reactive chromatography supports for the coupling of affinity ligands (Jayabaskaran etal., 1987). Another important use of the compound... 

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