Reversed-phase HPLC Separation Research Articles

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.

Reverse-phase HPLC Separation of D-Amygdalin and Neoamygdalin and Optimum Conditions for Inhibition of Racemization of Amygdalin.

In boiling aqueous solution, D-amygdalin usually begins to convert into neoamygdalin in 3 min and more than 30% of the initial D-amygdalin is found as neoamygdalin after 30 min. In this report, we establish methods for simple HPLC analysis and the inhibition of D-amygdalin conversion. D-Amygdalin and its conversion product, neoamygdalin, were clearly separated on reverse-phase column chromatography by an optimized eluent of 10 mM sodium phosphate buffer (pH 3.8) containing 6% acetonitrile. Linearity for analyzing D-amygdalin and neoamygdalin was observed in the range from 0.05 to 0.5 mM. The detection limits for D-amygdalin and neoamygdalin were ca. 5 microM per injected amount. We found that D-amygdalin conversion was completely inhibited by adding 0.05% citric acid to the aqueous solution before boiling. To prevent the loss of pharmaceutical potency of Tonin, we applied this method to measure the conversion rate of D-amygdalin. We confirmed that D-amygdalin conversion in Tonin is effectively inhibited by acidic boiling solution with 0.1% citric acid.

Peroxynitrite reacts with 8-nitropurines to yield 8-oxopurines.

Peroxynitrite reacts with 2'-deoxyguanosine to yield several major products, including 8-oxo-2'-deoxyguanosine (8-oxodG) and 8-nitroguanine (8-nitroGua). While the terminal products formed during the reaction of 8-oxodG with peroxynitrite have been previously characterized, those formed from 8-nitroGua have not. To identify these products, 9-ethyl-8-nitroxanthine was used as a model for 8-nitroGua, since the former could be easily synthesized in high yield, and facilitated reversed-phase HPLC separation of the resulting products. Using this model substrate, the products formed during the peroxynitrite reaction were identified as the ethyl derivatives of oxaluric acid, 5-iminoimidazolidin-2,4-dione, III, [N-nitro-N'-[2,4-dioxo-imidazolidine-5-ylidene]-urea, V, dehydroallantoin, parabanic acid, cyanuric acid, and uric acid. Upon the basis of the previous studies with 8-oxodG, these products were recognized as those expected to arise from peroxynitrite-mediated uric acid oxidation. Furthermore, the presence of uric acid in the reaction mixture led us to propose a model in which the 8-nitropurine is first converted to the 8-oxopurine which is further oxidized by peroxynitrite to give the observed final products. We have also provided evidence suggesting that the peroxynitrite anion, acting as a nucleophile, might be responsible for the initial conversion of the 8-nitropurine to the 8-oxopurine and that a hydroxyl radical or oxidative process is less likely to explain this conversion.

The Combined Effects of pH and Percent Methanol on the HPLC Separation of Benzoic Acid and Phenol

The effect of varying mobile-phase pH and percent methanol on the reversed-phase HPLC separation of benzoic acid and phenol is investigated. Both analytes are of interest owing to their classification as moderate environmental and health hazards. Baseline separation of the analytes is observed in five of the nine mobile-phase compositions studied. At low levels of methanol, as pH increases, the elution order of the benzoic acid and phenol peaks reverses. A similar trend is observed at intermediate levels of methanol. At high levels of methanol, as pH increases, the two-component sample begins to resolve, but baseline resolution is never achieved. In general, as percent methanol increases, the retention time of both analytes decreases. This is independent of mobile-phase pH.

Characterization of a New Decahydropyridoquinoline from Indigofera longeracemosa Boiv. Ex Baill. (Fabaceae)

Repeated silica gel chromatography of the dichloromethane extract of air-dried stems of Indigofera longeracemosa followed by reverse-phase HPLC separation yielded a new decahydropyridoquinoline, rel-(3S, 5R, 6S, SR, 8aR, 12aR)-8-acetoxy-6-butyl-3-isothiocyanatodecahydropyrido (2,1) quinoline.

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

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.

The determination and distribution of nucleotides in dairy products using HPLC and diode array detection

Abstract A method for the determination of adenosine, cytidine, uridine, guanosine and inosine 5′-monophosphates in milk and dairy products was optimized and its performance evaluated. The technique was based on ion-pair reversed-phase HPLC separation of the nucleotides and diode array detection. The chromatographic separation was achieved using a C18 column and a gradient elution with a mixture of two solvents: solvent A, water/glacial acetic acid/tetrabutilammonium hydrogensulphate (TBAHS) and solvent B, methanol/glacial acetic acid/TBAHS. The effluent was monitored using a Diode Array detector set at 260 nm. Validation of the proposed method was carried out by standard additions method, with recoveries of 98.3%. The precision of the method was also evaluated and reported a coefficient of variation (CV) as less than 3.2. Upon development the technique was applied on different dairy products in order to study the distribution of nucleotides therein. The samples included bovine, ovine and caprine milks, the corresponding manually manufactured cheeses, whey cheese and whey. Three commercial cheeses made from bovine, ovine and caprine milks, respectively, and 20 infant formulae were also analysed. In contrast to their absence in cheese upon preparation nucleotides were present in cheese at the end of ripening; this observation led to the extraction of DNA in order to evaluate whether nucleotides were released from degradation of nucleoproteins.

Validation study of assay method for DX-8951 and its metabolite in human plasma and urine by high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry.

A new liquid chromatographic/mass spectrometric assay has been developed for the determination of DX-8951, a new anti-tumor drug, and its 4-hydroxymethyl metabolite (UM-1) in human plasma and urine. Solid-phase extractions were used for sample preparation. A gradient reverse-phase HPLC separation was developed with mobile phases consisting of trifluoroacetic acid and methanol. The detection was conducted using atmospheric pressure chemical ionization tandem mass spectrometry in the selected reaction monitoring mode. A structural analog, camptothecin (CPT), was used as the internal standard. The assay was validated for the determination of DX-8951 and UM-1 in human plasma and urine. The lower limits of quantitation of DX-8951 and UM-1 were 0.1 ng/mL in plasma and 1 ng/mL in urine. The method showed a satisfactory sensitivity, precision, accuracy, recovery and selectivity.

Detection of aflatoxins (G(1-2), B(1-2)), sterigmatocystin, citrinine and ochratoxin A in samples contaminated by microbes.

A method is described for the simultaneous determination of common aflatoxins (G1, G2, B1, B2) and their precursor sterigmatocystin, and also citrinine and ochratoxin A. The method was applied to a building material matrix artificially contaminated with mycotoxin-producing fungi. The method includes extraction, sample pre-treatment and reversed-phase HPLC separation with tandem mass spectrometric identification and quantification using electrospray ionisation on a quadrupole ion trap mass analyser (ESI-MS-MS). Aqueous methanol was used in the initial extraction and solvent partitioning and solid phase extraction in the purification of samples. The HPLC separation was run on-line with the ESI-MS-MS detection. The limit of quantification of the procedure was 200 ng for all compounds. Recoveries of the sample pre-treatment varied from 28 to 99%. The average compound- and concentration-dependent accuracy and precision (RSD) were 21 and 113%, respectively. The method includes small sample volumes (approximately 1 g in 20 ml) and few, non-labour intensive, sample treatment steps. It should allow for a high throughput of samples with good prospects of automation.

Hyaluronate depolymerization following thermal decomposition of oxytetracycline.

Depolymerization of sodium hyaluronate (HA) by tetracyclines was investigated. Reduction in HA molecular weight was followed by size exclusion chromatography with a low angle laser light scattering detector. On mixing with oxytetracycline hydrochloride (OTC) solution and incubating at 37 degrees C, HA was gradually depolymerized. OTC, a representative antibiotic, is known as a photosensitizer, and phototoxic side effects relevant to radicals have been reported. However, HA depolymerization required no irradiation. As time passed, OTC solution incubated at 37 degrees C got colored reddish brown, even in the dark. With reversed-phase HPLC separation, several peaks derived from decomposed OTC appeared. One of the peaks had an absorbance in the visible range. A quantitative correlation between the discoloration and the HA depolymerization rate was obtained. On the other hand, when samples were incubated below 25 degrees C, change of color was slight, and practically no HA depolymerization was observed after up to 4 h. Oxygen depletion by nitrogen saturation or addition of mannitol also prevented the depolymerization. Under anaerobic conditions, the color of the solution did not change, whereas it turned red under aerobic conditions in the presence of mannitol. The mannitol did not inhibit the OTC decomposition, but it preserved HA from damage. On the basis of the known decomposition of OTC and the results of HPLC separation, anhydrooxytetracycline can be proposed as the derivative causing HA depolymerization.

Determination of sucralose in foods by anion-exchange chromatography and reverse-phase chromatography

Simple and rapid methods for the determination of sucralose in foods were developed using anion-exchange chromatography (AEC) with pulsed amperometric detection and reverse-phase HPLC with refractive index detection. Sucralose was extracted with water or methanol, and the extracted solution was cleaned up on a Sep-Pak C18 cartridge and a Sep-Pak Alumina N cartridge. The AEC separation was performed on a CarboPac PA1 column (4.0 mm i.d x 250 mm) using 100 mmol/L sodium hydroxide-50 mmol/L sodium acetate solution as the mobile phase at a flow rate of 1.0 mL/min. The recoveries of sucralose from foods were 80.6-102.0%, and quantitation limits from foods except chewing gum were 0.5 microgram/g (2 micrograms/g from chewing gum). The reverse-phase HPLC separation was performed on an Inertsil ODS-3V column (4.6 mm i.d x 150 mm) using methanol-water (25:75) as the mobile phase at a flow rate of 1.0 mL/min. The recoveries of sucralose from foods were 80.2-121.2%, and quantitation limits from foods except chewing gum were 5 micrograms/g (20 micrograms/g from chewing gum).

Degradation pathway of homoserine lactone bacterial signal molecules by halogen antimicrobials identified by liquid chromatography with photodiode array and mass spectrometric detection

The degradation pathway of acylated homoserine lactone bacterial signaling molecules by oxidizing hypochlorite and stabilized hypobromite antimicrobials has been characterized. A reversed-phase HPLC separation using a cyano column was developed to detect the parent lactones, lactone-hydrolysis products, and halogenation products. Elucidation of the structures of the reaction products was done with the aid of online photodiode array UV spectroscopy and atmospheric pressure chemical ionization mass spectrometry. Quantitative output of the HPLC method was also used to estimate the kinetics of the degradation pathway. The results of this work found that only β-keto-amide signal molecules are halogenated, where normal amide signals are not, and may represent one possible mechanism for control of industrial biofilms.

REVERSED PHASE HPLC OF OXYETHYLATES OF N-METHYLANILINE AND N-ETHYLANILINE

The conditions were selected for the reversed phase HPLC separation (phosphate buffer pH = 7.4 / methanol) of N-oxyethylates of N-methylaniline and N-ethylaniline on C18 columns, depending on the oxyethylene chain length. The use of the “mixed-mode” C18/anion column provided the possibility for simultaneous separation of oxyethylates, as well as, N,N-dimethylaniline and N,N-diethylaniline. UV spectra were taken for the compounds studied in order to find the optimum wavelengths for the detection procedure.

Stereoselective Metabolism of Anthracene, 9-Methylanthracene, 9,10-Dimethylanthracene, 9-Chloroanthracene, and 9-Nitroanthracene by Liver Microsomes of Neonatal Male B6C3F1 Mice

Stereoselective metabolism of anthracene, 9-methylanthracene (9-M-A), 9, 10-dimethylanthracene (9, 10-DMA), 9-chloroanthracene (9-Cl-A), and 9-nitroanthracene (9-nitro-A) by liver microsomes of neonatal (15-day old) male B6C3F1 mice in the presence of a NADPH regenerating system was conducted. After incubation, reversed-phase HPLC separation, and spectral analyses the following were identified: anthracene trans-1,2-dihydrodiol, 9-M-A trans-1,2- and -3,4-dihydrodiol, 9,10-DMA trans-1,2-dihydrodiol, 9-Cl-A trans-1,2- and -3,4-dihydrodiol, and 9-nitro-A trans-1,2- and -3,4-dihydrodiol. Circular dichroism (CD) spectral analysis indicated the major enantiomers of the anthracene, 9-M-A, 9,10-DMA, and 9-Cl-A trans-dihydrodiols all had R,R absolute configurations. These results indicate that these compounds are metabolized in a stereoselective manner by liver microsomes of neonatal male B6C3F1 mice.

15-Lipoxygenase expression and 15(S)-hydroxyeicoisatetraenoic acid release and reincorporation in induced sputum of asthmatic subjects

Background: Recent evidence shows that 15(S)-hydroxy-eicoisatetraenoic acid (15[S]-HETE) can be released and rapidly reincorporated into cellular lipids. These mechanisms exert several immunoregulatory functions that may be relevant in airway inflammation. Objective: Our purpose was to evaluate the levels of both soluble and cell-associated 15(S)-HETE and to examine 15-lipoxygenase (15-LO) messenger RNA (mRNA) expression in sputum samples obtained from 10 control and 18 asthmatic subjects. Methods: Levels of 15(S)-HETE were measured by reverse-phase HPLC separation followed by RIA in supernatants and in cell membrane–extracted phospholipids after acid hydrolysis. 15-LO mRNA was evaluated by primed in situ hybridization (PRINS). Combined immunocytochemistry and PRINS was used to identify the phenotype of cells bearing 15-LO transcripts. Results: Levels of both soluble and cell-associated 15(S)-HETE were higher in asthmatic than in control subjects (P < .0001). The percentage of cells expressing 15-LO mRNA was higher in asthmatic than in control subjects (P < .01). On double staining for specific cell-type markers and 15-LO mRNA, macrophages were the major source for 15-LO. Conclusion: This study shows that the induced sputum technique allows the evaluation of 15-LO activity and that soluble, cell-associated 15(S)-HETE and 15-LO levels are higher in asthmatic than in control subjects. In addition, this study indicates that, in induced sputum, airway macrophages are the major source of 15(S)-HETE in asthma. (J Allergy Clin Immunol 2000;105:711-6.)

A simple HPLC method for analysing diaminopimelic acid diastereomers in cell walls of Gram-positive bacteria.

A simple and sensitive method for separating and detecting the LL, DD and meso diastereomers of the dibasic amino acid diaminopimelic acid (DAP) in the peptidoglycan of Gram-positive bacteria is described. This method is based on reverse phase HPLC separation of chiral derivatives of DAP followed by fluorescence detection of the o-phthaldehyde derivatives. Its application to the analyses of cell walls of several Gram-positive bacteria is described, where 10 mg or less of dry cells is required.

Description and validation of an analytical method for the determination of paromomycin sulfate in medicated animal feeds.

A procedure for the extraction of paromomycin from different animal feed matrices (rabbit, chicken, pig feeds) and its subsequent determination via a reversed-phase ion-pair HPLC separation coupled with pulsed amperometric detection is described. The procedure optimised in terms of the extracting solvent and the solid phase extraction stationary phase allows the total recovery of the aminoglycoside antibiotic. The criteria used for the validation of the analytical method applied to the cited matrices are the linear dynamic range of the response, the detection limit, the repeatability, the intermediate repeatability and the accuracy. A comparison with a method described in the literature for the bulk analysis of this antibiotic is made.

Determination of Amphetamine by HPLC After Acetylation

An analytical procedure has been developed for the HPLC determination of amphetamine by off-line pre-column derivatization. The proposed procedure consists of sample preparation by acetylation of amphetamine with acetic anhydride and a subsequent reversed-phase HPLC separation on an octadecyl silica stationary phase with salt-free mobile phase (tetrahydrofuran, acetonitrile, 0.1% triethylamine in water, 15:15:70 v/v) applying UV-detection. The applicability of the elaborated procedure is demonstrated with results obtained by analysis of real samples seized in the Hungarian black market.

Assignment of the disulfide bonds of Ole e 1, a major allergen of olive tree pollen involved in fertilization.

The most prevalent allergen from olive tree pollen, Ole e 1, consists of a single polymorphic polypeptide chain of 145 amino acids which includes six cysteine residues at positions 19, 22, 43, 78, 90 and 131. By using an homogeneous form of the allergen expressed in Pichia pastoris, the array of the disulfide bridges has been elucidated. Specific proteolysis with thermolysin and reverse-phase HPLC separation of the peptides allowed the determination of the disulfide bond between Cys43 and Cys78. Another thermolytic product, which contained three peptides linked by the remaining four cysteines, was digested with Glu-specific staphylococcal V8 protease and the products isolated by reverse-phase HPLC. Amino acid compositions and Edman degradation of the peptide products indicated the presence of the disulfide bonds at Cys19-Cys90 and Cys22-Cys131. These data can help in the analysis of the three-dimensional structure of the protein as well as in studies of its allergenic determinants.

Analysis of Nicotine, 3-Hydroxycotinine, Cotinine, and Caffeine in Urine of Passive Smokers by HPLC-Tandem Mass Spectrometry

A method is described for the simultaneous analysis of nicotine and two of its major metabolites, cotinine and 3-hydroxycotinine, as well as for caffeine from urine samples. The method was developed to assess exposure of restaurant and hotel workers to environmental tobacco smoke. The method includes sample pretreatment and reversed-phase HPLC separation with tandem mass spectrometric identification and quantification using electrospray ionization on a quadrupole ion trap mass analyzer. Sample pretreatment followed standard protocols, including addition of base before liquid-liquid partitioning against dichloromethane on a solid matrix, evaporation of the organic solvent using gaseous nitrogen, and transferring to HPLC vials using HPLC buffer. HPLC separation was run on-line with the electrospray ionization-tandem mass spectrometric detection. The detection limits of the procedure were in the 1 microg/L range, except for nicotine (10 microg/L of urine). Still lower detection limits can be achieved with larger sample volumes. Recoveries of the sample treatment varied from 99% (cotinine) to 78% (3-hydroxycotinine). The method described is straightforward and not labor-intensive and, therefore, permits a high throughput of samples with excellent prospects for automation. The applicability of the method was demonstrated in a small-scale study on restaurant employees.