Acid High-performance Liquid Chromatography Research Articles

Synthesis of novel chiral imidazolium stationary phases and their enantioseparation evaluation by high-performance liquid chromatography

Two novel chiral stationary phases (CSPs) were prepared by bonding chiral imidazoliums on the surface of silica gel. The chiral imidazoles were derivatized from chiral amines, 1-phenylethylamine and 1-(1-naphthyl)ethylamine. The obtained CSPs were characterized by Fourier Transform Infrared (FT-IR) spectroscopy and elemental analysis (EA), demonstrating the bonding densities of CSP 1 and CSP 2 were 0.43 mmol g−1 and 0.40 mmol g−1, respectively. These two CSPs could be used to availably separate 8 pharmaceuticals, 7 mandelic acid/its derivatives, 2 1-phenylethylamine derivatives, 1 1,1′-bi-2-naphthol, and 1 camphorsulfonic acid in high-performance liquid chromatography (HPLC). It is found that CSP 1 could effectively enantioseparate most chiral analytes, especially the acidic components, while CSP 2 could enantiorecognize all chiral analytes, although a number of components did not achieve baseline separation. Additionally, the effects of mobile phase composition, mobile phase pH and salt content, chiral selector structures, and analyte structures on the enantiorecognitions of the two CSPs were investigated. It is found that high acetonitrile content in mobile phases was conducive to enantiorecognition. Mobile phase pH and salt content could alter the retention behaviors of different enantiomers of the same chiral compound, resulting in better enantioresolution. Moreover, both chiral selector structures and substituted groups of analytes played a significant role in the separation of chiral solutes.

(S)-1-methyl-4-(5-(3-aminopyrrolidin-1-yl)-2,4-dinitrophenyl)piperazine as a novel chiral derivatization reagent for high-performance liquid chromatographic analysis of carboxylic acid enantiomers

A novel chiral derivatization reagent, (S)-1-methyl-4-(5-(3-aminopyrrolidin-1-yl)-2,4-dinitro-phenyl)piperazine (APy-PPZ) was developed for the enantiospecific determination of chiral carboxylic acids in high-performance liquid chromatography. APy-PPZ reacted with carboxylic acids within 10min at 30°C in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). Epimerization during the derivatization reaction was negligible. Diastereomers derived from non-steroidal anti-inflammatory drugs (NSAIDs) and N-acetylamino acids were completely separated by reversed-phase chromatography using a small particle (3.0μm) ODS column (Rs=1.20–4.12). The calibration curves were linear over the concentration range of 1–100μM for each enantiomer of IBU, FLU, and KET, 10–100μM for each enantiomer of NAc-Trp and NAC-Phe, and 50–200μM for each enantiomer of NAc-Leu and NAc-Met. The detection limits (S/N=3) of NSAIDs (1pmol for each enantiomer of IBU, FLU, KET) were one or two orders magnitude lower than that of N-acetylamino acids (10pmol for each NAc-Trp, NAC-Phe enantiomer, 100pmol for each NAc-Leu, NAc-Met enantiomer). The relative standard deviations (RSD, n=5) were less than 3.4% at 60μM for all carboxylic acid enantiomers. The proposed method was applied to the determination of (S)-IBU enantiomer in a pharmaceutical formulation.

Effect of the mobile phase on antibody-based enantiomer separations of amino acids in high-performance liquid chromatography

The effect of the mobile phase parameters flow rate, temperature, pH and ionic strength, as well as the addition of various organic modifiers on the enantiomer separation of various aromatic α-amino acids was investigated using two antibody-based chiral stationary phases that have opposing stereoselectivity. On both columns, a decrease in flow rate or temperature resulted in increased interaction with the retained enantiomer. It was found that the retention factor k 2 depends on the affinity between the analyte and the immobilized antibody and is not independent of the flow rate. Optimum separations of all amino acids investigated were obtained at pH 7.4 on both columns. While increased k 2 values were obtained at low ionic strength on the anti- d-amino acid antibody column, no such effect was observed on the anti- l-amino acid antibody column. The addition of organic modifiers did not improve separations. In all studies, the unretained enantiomer eluted with the void volume.

Effect of the mobile phase on antibody-based enantiomer separations of amino acids in high-performance liquid chromatography

The effect of the mobile phase parameters flow rate, temperature, pH and ionic strength, as well as the addition of various organic modifiers on the enantiomer separation of various aromatic alpha-amino acids was investigated using two antibody-based chiral stationary phases that have opposing stereoselectivity. On both columns, a decrease in flow rate or temperature resulted in increased interaction with the retained enantiomer. It was found that the retention factor k2 depends on the affinity between the analyte and the immobilized antibody and is not independent of the flow rate. Optimum separations of all amino acids investigated were obtained at pH 7.4 on both columns. While increased k2 values were obtained at low ionic strength on the anti-D-amino acid antibody column, no such effect was observed on the anti-L-amino acid antibody column. The addition of organic modifiers did not improve separations. In all studies, the unretained enantiomer eluted with the void volume.

A chromatographic approach to analyze dansyl amino acid-HP-beta-CD association using macrocyclic antibiotic as the stationary phase.

The retention mechanism for a series of D,L-dansyl amino acids in high-performance liquid chromatography is investigated using a teicoplanin stationary phase and hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as the mobile phase additive (0-16mM). A theoretical treatment is developed to determine the HP-beta-CD influence on the equilibrium between the teicoplanin phase and the aqueous medium, respectively. From the experimental data, the association constants of the D,L-dansyl amino acids-HP-beta-CD inclusion complexes are determined and discussed in relation to the enantiomer structure. A thermodynamic study confirms that both the retention and complexation mechanisms are independent of the dansyl amino acid molecular structure and its absolute carbon configuration.

NEW DETECTION METHOD OF α-, β-, AND γ-AMINO ACIDS COUPLED WITH AN ON-LINE PHOTO-CHEMICAL OXIDATION AND TRIS(2,2′-BIPYRIDINE)-RUTHENIUM(III) CHEMILUMINESCENCE

ABSTRACT A new postcolumn detection method for amino acids is proposed. The method is based on the oxidation of analytes to oxalate with subsequent chemiluminescent detection using tris(2,2′-bipyridine)ruthenium(III). A mixing solution of eluate and periodate as an oxidizing agent is delivered to a reaction coil, which is then irradiated with UV light to accelerate the oxidative reaction. The effects of pH, UV irradiation time and temperature on the oxidative reaction were examined. Detection limits of eleven kinds of amino acids in high-performance liquid chromatography ranged from 1 pmol of threonine and phenylalanine to 40 pmol of glycine at a signal-to-noise ratio of 3.

Enantioseparation of selected N- tert.-butyloxycarbonyl amino acids in high-performance liquid chromatography and capillary electrophoresis with a teicoplanin chiral selector

Enantioseparation of N- tert.-butyloxycarbonyl amino acids ( N- t-Boc-Aas) with teicoplanin chiral selector was performed in two different separation systems: A teicoplanin-based chiral stationary phase (CSP-TE) was used in reversed-phase HPLC, and the same chiral selector (CS) was added into a background electrolyte (BGE) in HPCE. The enantioselective interaction with the same CSP/CS can be influenced by several factors, such as mobile phase/background electrolyte composition: the buffer concentration, pH, the CS concentration, the presence of organic modifiers. In addition, the charge of the chiral selector related to the charge of the analyte and to EOF are important variables in CE. The effect of these parameters on enantioselectivity and enantioseparation of selected N- t-Boc-Aas was studied. The presence of a sufficient concentration (1% solution) of a triethylamine acetate buffer in the mobile phase was shown to be essential for enantioseparation of these blocked amino acids in HPLC. A certain concentration of teicoplanin aggregates (along with teicoplanin molecules) in the BGE is required to obtain enantioseparation of N- t-Boc-Aas in HPCE.

6-(N-(3-Propionohydrazino)thioureido)benzo(g) -phthalazine-1,4(2H,3H)-dione as a Derivatization Reagent for Fatty Acids in High-Performance Liquid Chromatography with Chemiluminescence Detection.

6-[N-(3-Propionohydrazino)thioureido]benzo[g]-phthalazine-1,4(2H,3H)-dione was found to be a sensitive chemiluminescence (CL) derivatization reagent for fatty acids in high-performance liquid chromatography. The reagent readily reacted selectively with fatty acids at room temperature in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and pyridine in aqueous solution to form the corresponding acid hydrazide derivatives, which produce chemiluminescence by reacting with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in an alkaline solution. The CL derivatives of seven fatty acids (myristic, linolenic, palmitoleic, linoleic, palmitic, oleic and stearic acids) were separated within 55 min by reversedphase (C18) liquid chromatography with isocratic elution, and were detected with CL detection after mixing with the oxidizing reagents. Calibration curves were linear up to at least 66.7 pmol per 20 µl injection volume (r=0.996 to 0.999). The detection limits (signal-to-noise ratio=3) for the acids were 11.8–64.4 fmol for an injection volume of 20 µl.

6,7-Methylenedioxy-1-methyl-2-oxo-1,2-dihydroquinoxalin-3-ylpropionohydrazide as a Highly Sensitive Fluorescence Derivatization Reagent for Carboxylic Acids in High-Performance Liquid Chromatography

6,7-Methylenedioxy-1-methyl-2-oxo-1,2-dihydroquinoxalin-3-ylpropionohydrazide as a Highly Sensitive Fluorescence Derivatization Reagent for Carboxylic Acids in High-Performance Liquid Chromatography

4-(5,6-Dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl Chloride as a Fluorescent Labeling Reagent for Determination of Amino Acids in High-Performance Liquid Chromatography and Its Application for Determination of Urinary Free Hydroxyproline

A highly sensitive fluorescent labeling reagent, 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride (DMS-Cl), for determination of amino acids by HPLC has been developed. DMS-Cl reacted with amino acids in the basic medium to produce the corresponding fluorescent sulfonamides (excitation and emission wavelength: 318 and 406 nm in aqueous acetonitrile, respectively). When the reactivity of DMS-Cl was investigated by means of reversed-phase HPLC using hydroxyproline (Hyp) and alanine (Ala) as model compounds, the reaction of Hyp was completed within 5 min at 25°C and that of Ala within 15 min at 70°C. The efficiency of conversion of Hyp into the fluorescent derivative was about 100%. The detection limits (signal-to-noise ratio = 3) of almost all amino acids were less than 5 fmol/injection. When the concentration of urinary free Hyp was measured with HPLC using DMS-Cl, the concentration (mean ± SD,n= 10) was 3.8 ± 2.76 μM (2.7 ± 1.71 nmol/mg creatinine).

N-(4-nitro-2,1,3-benzoxadiazoyl-7-yl)- N-methyl-2-aminoacetohydrazide (NBD-CO-Hz) as a precolumn fluorescent derivatization reagent for carboxylic acids in high-performance liquid chromatography

A new fluorescent reagent for carboxylic acids, N-(4-nitro-2,1,3-benzoxadiazoyl-7-yl)- N-methyl-2-aminoacetohydrazide (NBD-CO-Hz) was synthesized and its applicability as a precolumn derivatization reagent in high-performance liquid chromatography was examined. NBD-CO-Hz reacted with 2-arylpropionic acids (2-APAs), a group of non-steroidal antiinflammatory drugs (NSAIDs) in the presence of a condensing agent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and pyridine at room temperature for 2 h to give fluorescent adducts. The reaction solution was subjected to a reversed phase or a chiral stationary phase HPLC and the derivatives were detected fluorometrically at a wavelength of 530 nm with an exitation of 475 nm. The detection limits were in the fmol range on column.

4-(N-hydrazinoformylmethyl-N-methyl)-amino-7-N, N-dimethylaminosulphonyl-2,1,3-benzoxadiazole (DBD-CO-Hz) as a precolumn fluorescence derivatization reagent for carboxylic acids in high-performance liquid chromatography.

A new fluorescent reagent for carboxylic acids, 4-(N-hydrazinoformylmethyl-N-methyl)amino-7-N,N-dimethylaminosu lphonyl-2, 1,3-benzoxadiazole (DBD-CO-Hz) was synthesized and its applicability as a precolumn derivatization reagent in high-performance liquid chromatography (HPLC) was examined. DBD-CO-Hz reacted with saturated fatty acids in the presence of a condensing agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and pyridine at room temperature for 30 min to give fluorescent products. The reaction solution was subjected to a reversed-phase HPLC and detected fluorometrically at a wavelength of 550 nm with an excitation of 440 nm. The detection limits of the derivatives were at low fmol range on column.

2-(5-Hydrazinocarbonyl-2-oxazolyl)-5,6-dimethoxybenzothiazole as a precolumn fluorescence derivatization reagent for carboxylic acids in high-performance liquid chromatography and its application to the assay of fatty acids in human serum

A unique acid hydrazide, 2-(5-hydrazinocarbonyl-2-oxazolyl)-5,6-dimethoxybenzothiazole, that is characterized by the benzothiazole structure conjugated to an oxazoline moiety was synthesized, and its applicability as a precolumn derivation reagent for carboxylic acids in HPLC was examined in view of sensitivity and separability. The sensitivity of the hydrazide for carboxylic acids was determined using lauric acid to be 0.1 pmol (per 10-μ1 injection volume) at a signal-to-noise ratio of 3. The reagent allowed rapid assays of carboxylic acids within 20 min with satisfactory separability. The method was applied to the determination of fatty acids in human sera from healthy volunteers as well as from patients with diabetes or thyroid dysfunction.

2-(5-Hydrazinocarbonyl-2-furyl)-5,6-dimethoxybenzothiazole as a Precolumn Fluorescence Derivatization Reagent for Carboxylic Acids in High-Performance Liquid Chromatography and Its Application to the Assay of Fatty Acids in Human Serum.

A precolumn fluorescence derivatization reagent for carboxylic acids, 2-(5-hydrazinocarbonyl-2-furyl)-5, 6-dimethoxybenzothiazole, was developed and its applicability to fatty acids in biological samples was examined. This reagent was proved to be useful in the determination of fatty acids in high-performance liquid chromatography with the detection limit of approximately 50fmol per 10-μl injection volume. Fatty acids in human serum were assayed by the derivatization method using the reagent. The method was also applied to some prostaglandins.

Polymeric 6-aminoquinoline, an activated carbamate reagent for derivatization of amines and amino acids by high-performance liquid chromatography.

A new polymeric reagent containing the 6-aminoquinoline (6-AQ) tag was developed and applied for the off-line derivatization of amines and amino acids in high-performance liquid chromatography (HPLC). The synthesis and characterization of this polymeric reagent are described. An authentic external standard of a typical amine was synthesized and characterized for the determination of the derivatization efficiency. All amines had a derivatization efficiency higher than 50%; the derivatization of amino acids was performed under optimized phase-transfer catalysis reaction conditions. Derivatized amines and amino acids were separated under conventional reversed-phase conditions and determined by UV and FL detectors. To investigate the practical applications, this polymeric reagent was also used to derivatize protein hydrolysates.

2-(5-Hydrazinocarbonyl-2-oxazolyl)-5,6-methylenedioxybenzofuran as a Fluorescence Derivatization Reagent for Carboxylic Acids in High-Performance Liquid Chromatography

2-(5-Hydrazinocarbonyl-2-oxazolyl)-5,6-methylenedioxybenzofuran as a Fluorescence Derivatization Reagent for Carboxylic Acids in High-Performance Liquid Chromatography

4-(1-Methylphenanthro[9,10-d]imidazol-2-yl)-benzohydrazide as derivatization reagent for carboxylic acids in high-performance liquid chromatography with conventional and laser-induced fluorescence detection

4-(1-Methylphenanthro[9,10-d]imidazol-2-yl)-benzohydrazide has been developed as a highly sensitive and selective fluorescence derivatization reagent for carboxylic acids in high-performance liquid chromatography (HPLC). The reaction conditions were optimized with C16–C20 linear, saturated fatty acids. The derivatization reaction proceeded in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at mild temperatures. The resulting fluorescent derivatives were separated by reversed-phase (C18) liquid chromatography with aqueous methanol and were detected with conventional fluorescence detection at 460 nm with excitation at 325 nm. The detection limits (signal-to-noise ratio = 3) for the acids are 2–12 fmol for an injection volume of 10 µl. The fluorescent derivatives display an excitation maximum at 325 nm, which coincides closely with the light emission of the helium–cadmium laser. Hence, HPLC with the reagent was combined with helium–cadmium laser-induced fluorescence detection. Using this system, attomole detection limits (70–100 amol on-column) were achieved for various carboxylic acids.

4-(5,6-Dimethoxy-2-benzimidazoyl)benzohydrazide as Fluorescence Derivatization Reagent for Carboxylic Acids in High-Performance Liquid Chromatography

4-(5,6-Dimethoxy-2-benzimidazoyl)benzohydrazide as Fluorescence Derivatization Reagent for Carboxylic Acids in High-Performance Liquid Chromatography

3-Bromoacetyl-7-methoxycoumarin as a New Fluorescent Derivatization Reagent for Carboxylic Acids in High-Performance Liquid Chromatography

3-Bromoacetyl-7-methoxycoumarin (I) was synthesized as a highly reactive fluorescent derivatization reagent for use in high-performance liquid chromatographic analysis. Compound I readily reacted with carboxylic acids in acetone at room temperature for 30min in the presence of potassium bicarbonate and 18-crown-6(Method A), or an anion-exchange resin (Method B) as catalysts to give the corresponding fluorescent carboxylic acid coumarinacyl esters(IIa-o). When lauric acid as a test sample was derivatized with I by use of Methods A and B, the detection limits were 0.4 and 0.5pmol per injection (S/N=3), respectively. These methods were applicable to the measurements of free fatty acids in human plasma (10-100μl). Method B used a weakly basic anion-exchange resin instead of catalysts so in Method A, so it would be useful for the simplification of derivatization.

3-Bromomethyl-7-methoxy-1,4-benzoxazin-2-one as a highly sensitive fluorescence derivatization reagent for carboxylic acids in high-performance liquid chromatography

A fluorescence labelling reagent, 3-bromomethyl-7-methoxy-1,4-benzoxazin-2-one (BrMB), was synthesized from 2-amino-5-methoxyphenol and ethyl pyruvate by the Wislecenus reaction followed by bromination. Picomole to nanomole amounts of saturated aliphatic fatty acids were converted into the corresponding fluorogenic esters in the presence of anhydrous potassium carbonate and a crown ether as catalysts. BrMB derivatives of fatty acids were separated on a reversed-phase column and were sensitively detected fluorimetrically at 440 nm with excitation at 345 nm. Quantitative studies revealed that n-caproic acid was esterified completely under mild conditions and with sufficient reproducibility. The detection limit of the BrMB derivative of n-caproic acid was just below 2 fmol.