Pre-column Derivatization High-performance Liquid Chromatography Research Articles

Determination of total retronecine esters‐type hepatotoxic pyrrolizidine alkaloids in plant materials by pre‐column derivatization high‐performance liquid chromatography

A pre-column derivatization high-performance liquid chromatography method with diode array detection was developed and validated to determine the total retronecine esters-type hepatotoxic pyrrolizidine alkaloids (RET-HPAs) in herbs. The RET-HPAs reacted with o-chloranil in methanolic solution heated for 3 h, and an oxidative derivative was produced that could be detected at a maximal absorption of 223 nm. The analysis was performed using a C18 column with an isocratic elution of methanol and aqueous 0.01% triethylamine (adjusted to pH 4 with formic acid), and the detection was carried out with DAD at 223 nm. The validation of the method included linearity, sensitivity, recovery and stability. It showed a good linear regression (r(2) > 0.9900) in the range of 2.5-250 microM with a limit of detection (S/N = 3) of 0.5 microM. The method provided desirable repeatability with overall intra- and inter-day variations of less than 4.6%. The obtained recoveries for both of the extraction and derivatization process were between 94.6 and 100.7% (n = 3).

Pre-column Derivatization-High Performance Liquid Chromatography for the Determination of Aliphatic Amines with Fluorescence Detection and Mass Spectrometry Identification

A simple and highly sensitive method based on the derivatization of aliphatic amines with 2-(2-phenyl-1 H-phenanthro-[9,10- d]imidazole-1-yl)-acetic acid (PPIA) followed by high-performance liquid chromatography (HPLC) with fluorescence detection and on-line post-column mass spectrometric identification has been developed. Optimum derivatization, giving the corresponding stable fluorescent derivatives, was obtained by the reaction of aliphatic amines with PPIA at 60 ° for 15 min in the presence of N-ethyl- N′-(3-dimethylamino propyl)carbodiimide hydrochloride (EDC) catalyst in acetonitrile solvent. The fluorescence detection wavelength of PPIA derivatives was 380 nm (260 nm of excitation wavelength). Separation of the derivatized amines with a good baseline resolution in conjunction with a gradient elution was carried out on a reversed phase Eclipse XDB-C 8 column (4.6 mm × 150 mm, 5 μm), in this way, the contents of aliphatic amines in waste water of paper mill, telencephalon tissue of rat and acidophilus milk were determined. The identification of aliphatic amine derivatives was carried out by post-column online mass spectrometry with atmospheric pressure chemical ionization (APCI) source in positive ion mode. In virtue of the MS and MS/MS information of intermediates of derivatization reaction, the derivatization reaction mechanism was confirmed. The established method exhibits excellent reproducibility and recovery. Excellent linear responses were observed with coefficients >0.9996, and detection limits (at signal-to-noise of 3:1) were 3.1–18.2 fmol.

Determination of fluoxetine enantiomers in rat plasma by pre-column fluorescence derivatization and column-switching high-performance liquid chromatography.

A column-switching HPLC method employing both octadecylsilica (ODS) and chiral columns with fluorescence detection was developed for the determination of enantiomer of fluoxetine (FLX), an antidepressant drug, in rat plasma. Racemic FLX was derivatized with a fluorescent reagent, 4-(N-chloroformylmethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole (NBD-COCl) or 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl) and the enantiomeric separation of the resultant derivatives was examined on an amylose-based chiral column (CHIRALPAK AD-RH) in the reversed-phase mode. The derivative with NBD-COCl (NBD-FLX) showed a sufficient separation factor (a) and resolution (Rs) compared with that with DBD-COCl. Thus, FLX was derivatized with NBD-COCl and the resultant NBD-FLX was first quantified on the ODS column and then introduced to the CHIRALPAK AD-RH column via a six-port switching valve to examine the enantiomeric ratio. The intra- and inter-day accuracy (97.6-112.7%) and precision (1.47-10.60%) were satisfactory in the range 10-1000 nM FLX and the limit of quantification was approximately 10 nM. The absolute recoveries of FLX with hexane from rat plasma were in the range 87.5-92.2% (n = 3). The method was applied to determine FLX enantiomers in the plasma of rats administered FLX orally, and it was shown that the R-isomer was eliminated faster than the S-isomer.

Determination of sucralose in foods by HPLC using pre-column derivatization

The development of a sensitive pre-column derivatization high-performance liquid chromatography (HPLC) method for determination of sucralose is reported. Sucralose is converted into a strongly ultraviolet (UV)-absorbing derivative, possessing strong absorption at 260 nm, by treatment with p-nitrobenzoyl chloride (PNBCl). Homogenized samples were dialyzed and washed with a Bond Elut ENV cartridge, then the eluate was evaporated to dryness and the residue was derivatized. Subsequently, the sucralose derivative was purified with hexane-ethyl actate (9:1) in a silica cartridge, and then the sucralose derivative was eluted with acetone. HPLC was performed on a phenyl column, using acetonitrile-water (73:27) as a mobile phase with UV detection (260 nm). The calibration curve was linear in the range of 1 microgram/mL to 50 micrograms/mL of sucralose. The recoveries of sucralose from eight kinds of foods spiked at the levels of 0.20 and 0.05 g/kg of sucralose were more than 76.2% with SD values in the range from 0.90% to 4.31%. The quantitative limit of the developed method was 0.005 g/kg for sucralose in samples.

THE FRESHWATER CYANOBACTERIUM PLANKTOTHRIX SP. FP1: MOLECULAR IDENTIFICATION AND DETECTION OF PARALYTIC SHELLFISH POISONING TOXINS

A filamentous cyanobacterium, belonging to the Order of Oscillatoriales, was found to be responsible for a toxic algal bloom in Lake Varese, Italy, during the summer of 1997. Morphological characters, as well as near complete 16S rRNA gene sequencing, revealed that the dominant species of the bloom was most closely related to the genus Planktothrix. In addition, genetic analysis of the phycocyanin operon of Planktothrix sp. FP1 revealed a novel primary structure, previously undescribed within the cyanobacteria, which was used as a genetic marker for rapid detection and identification of this toxic strain. The occurrence of saxitoxin (STX), a principal toxin in paralytic shellfish poisoning (PSP), was confirmed in the natural bloom sample by both pre-column and post-column derivatization high-performance liquid chromatography (HPLC) analyses, and eventually by liquid chromatography/mass spectrometry (LC/MS). The toxicity of this field sample was also revealed by electrophysiological assays in which the extract inhibited 90% of the voltage-dependent Na+ current in human neuroblastoma cells at the STX concentration of 80 nM. The cultured strain showed a lower physiologic activity than the bloom sample (67% blockage of Na+ current at a toxin concentration of 200 nM), and STX was detected only by pre-column HPLC, indicating the presence of a compound structurally close to STX. Chemical and molecular genetic analyses performed here add Planktothrix sp. FP1 to the growing list of diverse cyanobacterial species capable of synthesizing STX and its related compounds.

A Sensitive and Fast Method for the Determination of Polyamines in Biological Samples. Benzoyl Chloride Pre-Column Derivatization High-Performance Liquid Chromatography

Abstract A sensitive, rapid pre-column derivatization HPLC procedure for determination of biologically important polyamines is described. Benzoylated putrescine, cadaverine, norspermidine, spermidine, norspermine and spermine were separated on reverse-phase column using 42:58 acetonitrile:water as mobile phase and detected by UV absorption at 198 nm. The detection limits were: 0.8 pmol for putrescine, 1 pmol for spermidine and 1.3 pmol for spermine. The method was successfully applied to the analysis of polyamines in small samples of unicellular organisms.

Investigation of "signal" constituents for the evaluation of animal crude drugs. I. Free amino acids and total amino acids.

The contents of free amino acids (FAA) and total amino acids (TAA; including peptide and protein forms) in 10 animal drugs were determined using precolumn-derivatization high performance liquid chromatography (HPLC). The results showed that the FAA and TAA content was characteristic for a particular animal crude drug. For example, Bezoar Bovis contained much more taurine relative to the FAA and TAA content compared with other crude drugs, and kokurozin contained much more TAA and much less secondary amino acids relative to TAA compared with koukuzin. FAA and TAA were proved to be "signal" constituents for the evaluation of animal crude drugs.

Quantitation of tryptophan and other plasma amino acids by automated pre-column o-phthaldialdehyde derivatization high-performance liquid chromatography: improved sample preparation

Pre-column derivatization with o-phthaldialdehyde is a rapid and sensitive method for the quantitation of amino acids in biological fluids. This method uses acetonitrile as a deproteinizing reagent which gives improved recovery of tryptophan compared with 5-sulfosalicylic acid and permits the measurement of aspartic acid which coelutes with 5-sulfosalicylic acid. The method is automated to increase reproducibility and convenience. Mean coefficients of variation for peak areas relative to internal standard were 3.2 and 5.2% for amino acid standards and plasma samples, respectively. The presence of nitrilotriacetic acid stabilized the o-phthaldialdehyde reagent which is important in an automated system. The method is suitable for the analysis of large numbers of plasma samples where total tryptophan and aspartic acid are of interest.

Comparison of automated pre-column and post-column analysis of amino acid oligomers

It has been shown that various amino acids will polymerize under plausible prebiotic conditions on mineral surfaces, such as clays and soluble salts, to form varying amounts of oligomers ( n = 2–6). The investigation of these surface reactions required a quantitative method for the separation and detection of these amino acid oligomers at the picomole level in the presence of nanomole levels of the parent amino acid. In initial high-performance liquid chromatography (HPLC) studies using a classical postcolumn o-phthalaldehyde (OPA) derivatization ion-exchange HPLC procedure with fluorescence detection, problems encountered included lengthy analysis time, inadequate separation and large relative differences in sensitivity for the separated species, expressed as a variable fluorescent yield, which contributed to poor quantitation. We have compared a simple, automated, pre-column OPA derivatization and reversed-phase HPLC method with the classical post-column OPA derivatization and ion-exchange HPLC procedure. A comparison of UV and fluorescent detection of the amino acid oligomers is also presented. The conclusion reached is that pre-column OPA derivatization, reversed-phase HPLC and UV detection produces enhanced separation, improved sensitivity and faster analysis than post-column OPA derivatization, ion-exchange HPLC and fluorescence detection.

In vitro release and electrophysiological effects in situ of homocysteic acid, an endogenous N-methyl-(D)-aspartic acid agonist, in the mammalian striatum

A potassium-induced, calcium-dependent release of endogenous homocysteic acid (HCA) from rat striatal slices was demonstrated. A precolumn derivatization high-performance liquid chromatography method was developed that allowed quantitative determination of sulfur-containing amino acids at the picomole level. Intracellular recordings from cat caudate neurons during simultaneous microiontophoretic application of drugs and electrical stimulation of the corticocaudate pathway showed that (L)-HCA evoked a depolarization pattern similar to that induced by N-methyl-(D)-aspartic acid (NMDA), and both these depolarizations could be selectively inhibited by a specific NMDA antagonist, (D)-2-amino-7-phosphonoheptanoic acid [(D)-AP-7]. A selective antagonism of (L)-HCA-induced depolarizations by (D)-AP-7 was confirmed in quantitative experiments with the frog hemisected spinal cord in vitro. Small quantities of iontophoretically applied (L)-HCA, but not of quisqualate, potentiated cortically evoked EPSPs in cat caudate neurons. These observations suggest that (L)-HCA might be a candidate as an NMDA-receptor-preferring endogenous transmitter in the caudate nucleus. One possible function for such transmitter systems could be the enhancement of EPSPs.