Determination of paracetamol (acetaminophen) by HPLC with post-column enzymatic derivatization and fluorescence detection

Summary A novel method is described for the determination of paracetamol (acetaminophen;N-acetyl-4-aminophenol) in urine. After reversed-phase HPLC separation, paracetamol is oxidized by H₂O₂ with horseradish peroxidase catalysis. Detection is performed fluorimetrically at an excitation wavelength of 329 nm and an emission wavelength of 435 nm. Urine samples were spiked with paracetamol, diluted, and injected directly without further pretreatment. Under these conditions, the limit of detection was 2×10⁻⁸ molL⁻¹, and the limit of quantification was 7×10⁻⁸ molL⁻¹. The method was validated by two different approaches based on HPLC with UV-Vis detection.     

HPLC determination of colistin and aminoglycoside antibiotics in feeds by post-column derivatization and fluorescence detection

Summary Reversed-phase, HPLC methods employing post-column derivatization and fluorescence detection were developed for the determination of the peptide colistin and four aminoglycoside antibiotics in feeds. Extraction of the analytes was by sonication and shaking with dilute hydrochloric acid. Post-column derivatization was performed using orthophtaldialdehyde-2-mercaptoethanol chemistry. Assay of colistin was by using an acetonitrile-aqueous sodium sulphate-triethylammonium phosphate (pH 2.8) eluent. Aminoglycoside antibiotics:amikacin, kanamycin, gentamycin and neomycin were analyzed using a tetrahydrofuran-aqueous sodium sulphate-sodium pentanesulphonate-acetic acid mobile phase. The method was also applied to some pharmaceutical preparations. Preliminary results showed that the method can be adapted for the assay of the above antibiotics in meat and animal serum for residue and pharmacokinetic studies. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997.     

Identification of the major compounds in extracts ofVerbena officinalis L. (Verbenaceae) by HPLC with post-column derivatization

Summary A qualitative reversed-phase HPLC method has been developed for the analysis of 50% MeOH extracts ofVerbena officinalis L. (Verbenaceae) leaves. The method enables separation of the main constituents: iridoids, flavonoids and phenolic acid derivatives. Simultaneous detection at different wavelengths, measurement of the UV spectrum of each separated compound during elution and co-injection of reference substances facilitated easy and rapid identification of verbenalin, hastatoside and verbascoside. As some of these compounds, mainly flavonoids, have closely related structures, however, characterization by derivatization with reagents inducing a shift of UV absorption maxima was required. This furnished additional structural information. The reagents were adapted for compatibility with the solvent system used for the chromatographic separation.     

Determination of α-aminonitriles, α-amino acid amides and α-amino acids by means of HPLC,post-column reaction and fluorescence detection

Summary An HPLC method was developed for the simultaneous assay of intermediate (α-aminonitrile and α-amino acid amide) and end products (α-amino acid) in process streams of α-amino acid synthesis. Applications are given for Ala, Val and Leu. α-Aminonitriles were stable in a phosphate buffer pH 3, which was subsequently used for sample handling and chromatography. The α-aminonitrile, the corresponding acid amide and α-amino acid were separated using a buffered ion-pair mobile phase on an RP column and were detected fluorimetrically after post-column reaction with fluorescamine. Linearity and precision of the method are given.     

Determination of acetylcholine and choline in the femtomole range by means of HPLC,a post-column enzyme reactor,and electrochemical detection

Summary The measurement of choline and acetylcholine by means of HPLC, a post-column enzyme reactor, and electrochemical detection has been simplified and optimised. The use of a cation exchanger and enzyme reactor fitted in a cartridge holder appeared to result in reproducible, sensitive, and selective measurement of endogenous choline and acetylcholine with a lower detection limit of 50 fmole.     

Nucleotide pools and energy charges of smooth molluscan muscles analysed by reversed-phase,ion-pair,high-performance liquid chromatography

Summary A method is described for the analysis of the 5 mono-, di- and triphosphates of adenosine, guanosine, uridine and cytidine, as well as uridinediphosphate-glucose and cyclic AMP. Separation is achieved by reversed-phase ion-pair chromatography with linear gradient elution. Application of this method to the analysis of nucleotides in smooth molluscan muscles is described, including the determination of cAMP-levels and the calculation of energy charges for all of the four nucleotide systems.     

Comparison of different methods for the determination of several quinolonic and cinolonic antibiotics in trout muscle tissue by HPLC with fluorescence detection

Summary Quinolonic and cinolonic derivatives are mainly used as antibacterials in fish-farms. In this paper we describe a careful revision of the treatment procedures of samples and a prodedure for the determination of residues of these compounds. Because of the complexity and duration of these procedures, several studies have been carried out and these have lead to a simpler and shorter method. Three approaches have been examined: lyophilization followed by extraction with chloroform, solid-liquid extraction with chloroform and solid-liquid extraction with sodium hydroxide solution, followed by liquid-liquid partition in chloroform. Some previous studies into the partition equilibrium are also included. As a result of our studies we propose a procedure with a lower number of steps than those previously described in the literature. This method has been applied to the analysis of nalidixic, 7-hydroxymethylnalidixic and oxolinic acids and cinoxacin in trout muscle. These analysis have been carried out using an HPLC system equipped with a C₁₈ column and fluorimetric detection. The mobile phase was acetonitrile:oxalic acid. The recoveries obtained were: 70–97% for 7-hydroxymethylnalidixic acid, 75–78% for nalidixic acid, 71–95% for oxolinic acid and 72–85% for cinoxacin.     

HPLC separation of opium alkaloids on porous and non-porous stationary phases

Summary A new reversed-phase (RP) HPLC method has been developed and validated for the separation of the main opium alkaloids morphine, codeine, thebaine, papaverine and noscapine on a non-porous (micropellicular) stationary phase. On this phase quantification of the compounds by internal standardization with brucine was achieved extremely rapidly, in ca 1.5 min, only. Thus, the analysis time for the opium alkaloids was approximately one tenth of that on porous stationary phases. Different opium samples were investigated using non-porous and porous packings. The correlation between the results was excellent. Presented at Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Enhanced sensitivity by laser-induced fluorescence for the determination of calcitriol and other vitamin D3 metabolites in plasma

Summary A sensitive method for the determination of hydroxymetabolites of vitamin D₃, parficularly calcitriol (1,25-(OH)₂-D₃), in human plasma is reported. The method is based on the use of laser-induced fluorescence detection as an alternative to conventional fluorimetry in an integrated cleanup/preconcentration, HPLC separation and post-column derivatization system. The derivatization step is based on a dehydration reaction which takes place in secosteroid structures at high temperature in a strong-acid medium. A LOD of 0.01 pg mL⁻¹ (SNR=3) was obtained for each analyte with a linear dynamic range over 4 order of magnitude with excellent regression coefficients (≥0.9922) in all cases. The precision was studied at two concentration levels and the RSDs values (for n=5) were acceptable (between 2.6 and 4.7%). The method was also checked by applying it to human plasma spiked with the target analytes and excellent recoveries were obtained. This is the first time that these species have been determined at the sub-pg mL⁻¹ level.     

Comparative analysis of the underground parts ofGentiana species by HPLC with diode-array and mass spectrometric detection

Summary The aim of this work was to compare the chemical composition of the underground parts (roots and rhizomes) ofGentiana cruciata L.,Gentiana pneumonanthe L., andGentiana asclepiadea L.— the three gentians native to Hungary—with that of the widely used stomachicGentiana lutea L., to determine which of the three Hungarian species could be used as a substitute forGentiana lutea in pharmaceutical preparations. The four gentians were compared by means of RPHPLC with diode-array detection (DAD) and electrospray ionization-mass spectrometric detection (ESI-MSD). The quantities of the lead compounds, the secoiridoid-glycosides, in 220 samples of the underground parts of gentians originating from several locations in Hungary, were determined by a more economical RPHPLC-DAD method. The occurrence of the characteristic compounds investigated—bitter principles and xanthones—in the underground parts of the speciesGentiana asclepiadea L. suggest it might be a potential replacement forGentiana lutea L. in pharmaceutical products. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Simultaneous Determination of Phytohormones in Plant Extracts using SPME and HPLC

Solid-phase microextraction followed by HPLC was used for the determination of indole-3-acetic acid (IAA), abscisic acid (ABA), indole-3-butyric acid (IBA) and 1-naphthylacetic acid (NAA) in plant samples. Parameters influencing performance, including pH, salinity, extraction time, fiber coating and temperature, were optimized. A Carbowax-coated fiber was chosen for determination due to much higher extraction efficiency compared to polyacrylate fibers. The dynamic ranges spanned over three orders of magnitude. The LOD/(LOQ) values of the target compounds in pure water were 0.149(0.497), 0.442(1.472), 0.121(0.403), 0.058(0.193) μg L⁻¹ for IAA, ABA, IBA and NAA respectively. The method was successfully applied to the analysis of xylem fluid from Musa basjoo stem obtaining recoveries of 98.85% (IAA), 94% (IBA) and 94.30% (NAA). The method was also successfully applied to the analysis of these four target compounds in the hyperaccumulating plant, Viola baoshanensis. The results matched quite well with ones obtained by solid phase extraction followed by HPLC. The method developed was superior when applied to liquid samples because matrix effects could be eliminated.     

Electrochemically modulated liquid chromatographic separation of triazines and the effect of pH on retention

Electrochemically modulated liquid chromatography (EMLC) manipulates analyte retention by changing the potential applied (E_app) to a conductive stationary phase. This paper applies EMLC to the separation of a set of seven triazines which are commonly used but environmentally hazardous herbicides. Experiments herein examine the influence of E_app and the pH of the mobile phase on triazine retention. The results are discussed in terms of: (1) retention of triazines of dissimilar acid strengths and by correlations with the pH of the mobile phase; (2) how changes in E_app and acid–base equilibria modulate elution; (3) qualitative insights into EMLC-based retention; and (4) potential merits of EMLC in realizing the rapid separation of the seven-component triazine mixture.     

Improved cleanup method for the multiresidue analysis of N-methylcarbamates in grains,fruits and vegetables by means of HPLC with postcolumn reaction and fluorescence detection

Summary An improved and quick cleanup method has been developed for the liquid chromatographic determination of 21 N-methylcarbamate pesticides and 10 of their metabolites in grains, fruits and vegetables. Various types of solid phase extraction columns have been tested for the cleanup step in order to replace the time-consuming cleanup method found in the literature. Aminopropyl-bonded silica columns proved to be superior to unbonded silica or octyl, octadecyl, cyanopropyl and diol-bonded silica. For all 16 types of matrices tested, a single column cleanup step appeared to be satisfactory. The final determination was performed by separation of the N-methylcarbamates via high-performance liquid chromatography followed by postcolumn reaction, fluorogenic labelling of the hydrolytically formed methylamine with the orthophthalaldehyde/2-mercaptoethanol reagent, and fluorescence detection. Recovery data for 256 carbamate/commodity combinations will be given. The results of the routine analysis of real samples will also be presented. Finally, possibilities for confirming positive samples by an independent, second method will be discussed.     

Effect of drying temperature and blanching on the degradation of chlorophyll a and b in mint (Mentha spicata Huds.) and basil (Ocimum basilicum): Analysis by high performance liquid chromatography with photodiode array detection

Summary The effect of drying conditions on the preservation of chlorophyll pigments in mint and basil have been investigated in order to determine the effects of drying temperature and whether or not there was a prior blanching.Pigments extracted from fresh and dried samples were analyzed by reversed phase high performance liquid chromatography coupled to a photodiode array detector; isocratic separation was performed on a Zorbax ODS C18 column.The purity of the chromatographic peaks of chlorophylls and breakdown products was investigated. The visible spectra of standard samples of chlorophylls and pheophytins were compared, using least squares normalization with those of peaks from the extracts of fresh and dried mint and basil. The study has shown that chlorophylls were better preserved when drying was preceded by a short blanching; if samples were not blanched before drying, the degradation of chlorophylls a and b was best prevented by drying at low temperatures.     

Determination of carbendazim,thiabendazole, and<Emphasis Type="Italic"> o-</Emphasis>phenylphenol residues in lemons by HPLC following sample clean-up by ion-pairing

An efficient analytical method is presented involving effective sample clean-up with solid-phase extraction and HPLC-UV analysis for the simultaneous determination of carbendazim, thiabendazole, and o-phenylphenol residues in lemons. Sample preparation involves extraction with acetonitrile acidified with trifluoroacetic acid and an ethyl acetate/petroleum ether mixture. Purification of the crude extract was carried out with liquid–liquid partitioning after addition of an aqueous ammonia solution. Final clean-up was performed on polymeric reversed-phase cartridges pretreated with sodium dodecyl sulfate. Chromatographic analysis was performed on a reversed-phase HPLC column isocratically eluted with an acetonitrile/water/ammonia mixture and UV detection at 254 nm. The chromatographic method is repeatable, reproducible, and sensitive. Fungicide recoveries from lemon samples fortified at levels of 5 and 1 mg kg^–1 were 81–85% for carbendazim, 96–98% for thiabendazole, and 81–106% for o-phenylphenol with coefficients of variation of 2.5–7.4%. Detection limits for carbendazim, thiabendazole, and o-phenylphenol in lemons were 0.21, 0.27, and 0.51 mg kg^–1, respectively.     

High-performance liquid chromatographic separation of unusual amino acid enantiomers derivatized with (1S,2S)-1,3-diacetoxy-1-(4-nitrophenyl)-2-propyl-isothiocyanate

Summary A new chiral derivatizing agent (CDA), (1S,2S)-1,3-diacetoxy-1-(4-nitrophenyl)-2-propylisothiocyanate, (S,S)-DANI, was applied to the separation of the enantiomers of unusual amino acids containing two chiral centers. Different β-methyl-α-amino acids (β-MePhe, β-MeTyr and β-MeTrp) and β-amino acids with cycloalkane skeletons (2-aminocyclopentanecarboxylic acid and 2-aminocyclohexanecarboxylic acid) were derivatized and the thiourea derivatives produced were separated by reversed-phase high-performance liquid chromatography. The applicability of this new CDA in the separation of unusual amino acids is demonstrated. The four stereoisomers of the investigated amino acids (except β-MePhe) could be separated in one chromatographic run. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Study on the chromatographic behavior of water-soluble vitamins on p-tert-butyl-calix[8]arene-bonded silica gel stationary phase by HPLC

In this paper, the chromatographic behavior of some water-soluble vitamins was studied on a new p-tert-butyl-calix[8]arene-bonded silica gel stationary phase (CABS, 5 μm particle size, the bonded amount 0.071 mmol g⁻¹) by using vitamin standards as probes for HPLC. The comparative study of the separation of these compounds was done by using CABS and ODS as stationary phases under the same chromatographic conditions. The better separation of six vitamins including: B₁, B₂, B₆, B₁₂, C, and nicotinic acid (B₅), on CABS can be achieved by using isocratic mode with methanol-phosphate buffer (25:75, (v/v)) as mobile phase within 20 min. The results show that the calix[8]arene-bonded phase exhibits high selectivity for water-soluble vitamins. We found that the elution order of B₂ (12.08 min) and B₁₂ (16.42 min) on CABS was very different from that of B₁₂ (7.76 min) and B₂ (18.47 min) on ODS, which indicate that different retention mechanisms exist in the chromatographic processes of the two stationary phases. According to the chromatographic data, it can be concluded that various chromatographic retention mechanisms are responsible for the separation of above compounds on CABS, such as hydrophobic interaction, hydrogen bonding interaction, and π-π interaction. The new packing has two advantages over ODS. On one hand, the polar and ionized analytes, such as C and B₅, exhibited stronger affinities to CABS because of hydrogen bonding interaction. On the other hand, the retention of B₂ and B₁₂ became shorter on CABS with weaker hydrophobicity in comparison with ODS. The new material exhibits the promising application in the separation of water-soluble vitamins.     

Determination of thiol by high-performance liquid chromatography and fluorescence detection with 5-methyl-(2-(<Emphasis Type="Italic">m</Emphasis>-iodoacetylaminophenyl)benzoxazole

A new high-performance liquid chromatographic (HPLC) method for measuring low molecular weight (LMW) thiol-containing compounds, including cysteine (CysH), glutathione (GSH), N-acetylcysteine (Nac), penicillamine (PA), and 2-mercaptoethanol (2-ME), has been developed by using 5-methyl-(2-(m-iodoacetylaminophenyl)benzoxazole (MIPBO) as fluorescence-labeling reagent. The derivatization and separation conditions have been investigated in detail. Detection limits ranging from 3.5 to 15.0 fmol were achieved for the thiols investigated in a 16 min separation with detection wavelengths 310 and 375 nm for the excitation and emission, respectively. The utility of the proposed method has been validated by measuring CysH in human urine samples.     

Fingerprint Analysis of Eucommia Bark by LC-DAD and LC-MS with the Aid of Chemometrics

Chromatographic fingerprints of 46 Eucommia Bark samples were obtained by liquid chromatography-diode array detector (LC-DAD). These samples were collected from eight provinces in China, with different geographical locations, and climates. Seven common LC peaks that could be used for fingerprinting this common popular traditional Chinese medicine were found, and six were identified as substituted resinols (4 compounds), geniposidic acid and chlorogenic acid by LC-MS. Principal components analysis (PCA) indicated that samples from the Sichuan, Hubei, Shanxi and Anhui—the SHSA provinces, clustered together. The other objects from the four provinces, Guizhou, Jiangxi, Gansu and Henan, were discriminated and widely scattered on the biplot in four province clusters. The SHSA provinces are geographically close together while the others are spread out. Thus, such results suggested that the composition of the Eucommia Bark samples was dependent on their geographic location and environment. In general, the basis for discrimination on the PCA biplot from the original 46 objects× 7 variables data matrix was the same as that for the SHSA subset (36 × 7 matrix). The seven marker compound loading vectors grouped into three sets: (1) three closely correlating substituted resinol compounds and chlorogenic acid; (2) the fourth resinol compound identified by the OCH₃ substituent in the R4 position, and an unknown compound; and (3) the geniposidic acid, which was independent of the set 1 variables, and which negatively correlated with the set 2 ones above. These observations from the PCA biplot were supported by hierarchical cluster analysis, and indicated that Eucommia Bark preparations may be successfully compared with the use of the HPLC responses from the seven marker compounds and chemometric methods such as PCA and the complementary hierarchical cluster analysis (HCA).     

Determination of the molecular weight and other physicochemical properties of poly(bubylene glycol) 1000 by gradient RPHPLC and measurement of the ELSD, UV, and fluorescence responses

Summary Molecular weight and other physicochemical data for poly(butylene glycol) 1000 (PBG 1000) have been determined by use of a new, highly efficient, gradient reversed-phase high-performance liquid-chromatographic (RPHPLC) procedure. Separation of the native material or its α, ω-bis(1-naphthylurethane) derivative was achieved on a C₁₈ stationary phase with a ternary acetonitrile-water-tetrahydrofuran mobile phase. Detection was achieved by measurement of the signal response from evaporative light scattering (ELSD), UV, and fluorescence (FD) detection. Proof that all the oligomers contained in the sample had been separated by the method was obtained by liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOFMS). It was also confirmed by this technique that di(butylene glycol) is the lowest homologue in the sample. Although the dimer was also observable in the HPLC-UV trace of the PBG 1000 α, ω-bis(1-naphthylurethane) derivative, it was obviously too valatile to be seen in the ELSD trace; tri(butylene glycol) was, nevertheless, still recognizable with sufficient signal intensity. Because all the homologues were separated to baseline, the method was used to calculate the number- and weight-average molecular weights,M _n andM _w, both from peak areas and from peak heights. The best fit to data obtained from end-group titration were obtained from calculations based on the HPLC-UV response;M _n values of 948 and 937 were obtained from peak heights and areas, respectively.M _n andM _w values calculated from the ELSD trace obtained from native PBG 1000 were substantially (ca 10%) lower that those calculated from the UV trace obtained from the α, ω-bis(1-naphthylurethane) derivative. Similar differences were also discovered by comparing theM _n andM _w values obtained from UV and FD-values were approximately 20% higher for FD. When the retention times of individual oligomers, from either ELSD of the native sample or from UV-FD of the α,ω-bis(1-naphthylurethane) derivative, were plotted against the number of repeat units,P, the ELSD curve approaches the UV-FD curve at values ofP of approximately 60. This observation can be explained by the pronounced contribution of the hydrophobic end-groups to the overall retention of PBG 1000; the effect of this decreases with increasing chain length and becomes nearly independent ofP only for a very high degree of polymerization. Papers dedicated to Professor Dr. Heinz Engelhardt on the Occasion of his 65th birthday