Repeatability in column preparation of a reversed-phase C18 monolith and its application to separation of tocopherol homologues

This work investigated the repeatability of column preparation for a reversed-phase C18 monolith, namely stearyl methacrylate-co-ethylene glycol dimethacrylate (SMA-EDMA). The columns were thermally polymerised using three commonly available heating devices (GC oven, hot air oven and water bath) and their chromatographic performance evaluated using micro-liquid chromatography for separation of five test compounds. Precision in terms of %RSD of retention times were 9.0, 6.5, and 12.5 using GC oven, hot air oven and water bath, respectively. Between-batch precision for the hot air oven (n = 3 days) was less than 10.4% for retention time. The SMA-EDMA monolith was applied to the separation of tocopherol homologues by capillary electrochromatography. Usually tocopherol homologues cannot be completely separated by conventional reversed-phase C8- or C18-packed bed or C18-silica based monolithic columns. Polymer monolith has been shown to give remarkable selectivity towards the tocopherols compared to the conventional microparticulate phase and silica based monolith. Successful separation of the tocopherol isomers was achieved on the SMA-EDMA monolith without any column modification.     

Lipophilicity determination of N-( benzothiazol-2-yl )-α-amino alkyl phosphonic diesters by RP-HPLC and RP-HPTLC

Using methanol-water mixtures as the mobile phase,the chro-matographic retention parameters k' and Rf were determined by reversed-phase high-performance liquid chromatography(RP-HPLC)and reversed-phase high-performance thin-layer chromatography(RP-HPTLC)for N-(benzothiazol-2-yl)-o) amino alkyl phosphonic diesters and the correlation with lipophilicity parameter(ClogP)was established.Logkw values obtained from RP-HPLC and R values obtained from RP-HPTLC can be used to evaluate the lipophilicity of this kind of compounds.Chromatographic method is a good alternative for lipophilicity measurement.     

Separation of C27, C28 and C29 sterols by reversed-phase high-performance liquid chromatography on small particles.

The application of reversed-phase high-performance liquid chromatography to the analytical- and preparative-scale separation of sterols has been evaluated. The capacity factors, k', for a number of compounds chromatographed on a muBondapak C18 (LESS THAN 10 MUM) COLUMN ARE PRESENTED. C27, C28 and C29 sterols and also sterols differing in degree of unsaturation could be readily separated as their acetates in this system. The present reversed-phase chromatographic method is apparently not as selective as silver nitrate-silica gel thin-layer chromatography for the position of unsaturation in the sterol molecule.     

Analysis of amino acid 13C abundance from human and faunal bone collagen using liquid chromatography/isotope ratio mass spectrometry

The scope of compound-specific stable isotope analysis has recently been increased with the development of the LC IsoLink which interfaces high-performance liquid chromatography (HPLC) and isotope ratio mass spectrometry (IRMS) to provide online LC/IRMS. This enables isotopic measurement of non-volatile compounds previously not amenable to compound-specific analysis or requiring substantial modification for gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), which results in reduced precision. Amino acids are an example of such compounds.We present a new chromatographic method for the HPLC separation of underivatized amino acids using an acidic, aqueous mobile phase in conjunction with a mixed-mode stationary phase that can be interfaced with the LC IsoLink for compound-specific delta13C analysis. The method utilizes a reversed-phase Primesep-A column with embedded, ionizable, functional groups providing the capability for ion-exchange and hydrophobic interactions. Baseline separation of 15 amino acids and their carbon isotope values are reported with an average standard deviation of 0.18 per thousand (n = 6). In addition delta13C values of 18 amino acids are determined from modern protein and archaeological bone collagen hydrolysates, demonstrating the potential of this method for compound-specific applications in a number of fields including metabolic, ecological and palaeodietary studies.     

Separation and identification of some common isomeric plant triterpenoids by thin-layer chromatography and high-performance liquid chromatography

Chromatographic separation of 10 triterpenoids (α-amyrin, β-amyrin, δ-amyrin, lupeol, lupenon, lupeol acetate, cycloartenol, cycloartenol acetate, ursolic acid, oleanolic acid) and 2 sterols (stigmasterol and β-sitosterol) was studied. The chromatographic techniques included silica gel and reversed-phase (C₁₈ RP) thin-layer chromatography (TLC) and C₁₈ RP high-performance liquid chromatography (HPLC) using UV and mass spectrometric (MS) detection with atmospheric pressure chemical ionization (APCI). The TLC separation of the isomeric triterpenols lupeol, α-amyrin, β-amyrin and cycloartenol was achieved for the first time using C₁₈ RP-HPTLC plates. Cycloartenol could be separated from related compounds only on C₁₈ RP-TLC but not on the C₁₈ RP-HPLC. δ-Amyrin isolated from the tomato fruit surface extract could be separated from other amyrins only by HPLC. Tandem mass spectrometry allowed discrimination between the isomers lupeol, α-amyrin, β-amyrin, δ-amyrin, cycloartenol and between lupeol acetate and cycloartenol acetate. The combination of 3 TLC methods and 2 HPLC methods enables qualitative determination of all 12 compounds and proves to be useful for the analysis of plant extracts. It is recommended that TLC screening on silica gel and C₁₈ RP be performed before HPLC analysis.     

Fluorescence determination of 5-fluorouracil and 1-(tetrahydro-2-furanyl)-5-fluorouracil in blood serum by high-performance liquid chromatography

Fluorescence derivatization of 5-fluorouracil (5-FU) and 1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur, FT) with 4-bromomethyl-7-methoxycoumarin using 18-crown-6 as a catalyst is studied with aim of developing a sensitive and selective liquid chromatographic method. 5-FU and FT form virtually substituted derivatives which possess maxima in their fluorescence emission spectra near 400 nm. These derivatives are separated by thin-layer chromatography and high-performance liquid chromatography to confirm the completion of reaction. For the determination of 5-FU and FT in serum, the reversed-phase high-performance liquid chromatographic separation of the derivatives is studied with a C13 column. This chromatography is of importance for the accurate determination of 5-FU and FT, which are, respectively, an important antitumour agent for the treatment of solid tumours in clinical medicine and a masked form of 5-FU generated in vivo.     

Separation of carotenol fatty acid esters by high-performance liquid chromatography

Employing isocratic and gradient-elution high-performance liquid chromatography (HPLC) a number of straight-chain fatty acid esters (decanoate, laurate, myristate, palmitate) of violaxanthin, auroxanthin, lutein, zeaxanthin, isozeaxanthin, and beta-cryptoxanthin, prepared by partial synthesis, have been separated on a C18 reversed-phase column. Several chromatographic conditions were developed that separated a mixture of di-fatty acid esters (dimyristate, myristate palmitate mixed ester, dipalmitate) of violaxanthin, auroxanthin, lutein, and zeaxanthin in a single chromatographic run. Hydroxycarotenoids such as lutein, zeaxanthin, and isozeaxanthin that are not easily separated by HPLC on C18 reversed-phase columns, can be readily separated after derivatization with fatty acids and chromatography of their esters. Chromatographic conditions for optimum separation of carotenoids from various classes are discussed.     

Development and validation of a method for measuring the glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography

We developed and validated a simple method for measuring the individual glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography with a C18 reversed-phase column using an isocratic solvent system of acidified methanol--potassium phosphate. Without preliminary derivatization or purification, complete separation of the ten major conjugated bile acids present in bile could be achieved in 65 min. Total bile acid concentrations were identical when measured enzymatically and by summing the individual bile acids determined by high-performance liquid chromatography. Bile acid composition determined by gas-liquid chromatography correlated with results by high-performance liquid chromatography. Finally, measurements of individual glycine and taurine conjugates in human bile and in mixtures of bile acid standards by high-performance liquid chromatography and thin-layer chromatography gave similar results. This high-performance liquid chromatographic system permits simultaneous quantification of total and individual bile acids and their glycine and taurine conjugates in bile.     

Simultaneous determination of retinol and tocopherols by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method to determine retinol and all four tocopherols (alpha-, beta-, gamma- and delta-) simultaneously was established using a reversed-phase column (YMC-PACK A-302 S-5 120A ODS). The HPLC conditions were mobile phase 65% isopropanol, sample solvent 99.5% methanol and temperature 30 degrees C. Retinol and tocopherols were measured in rat liver.     

Chromatography of bis-(3-sulfopropyl) disulfide and its breakdown products by HPLC coupled with electrochemical detection

A chromatographic method for the detection of bis-(3-sulfopropyl) disulfide (SPS), a common additive in acidic copper plating baths, and its breakdown products is demonstrated. The detection scheme involves a combination of solid-phase extraction for sample pre-treatment, C(18) reversed-phase high-performance liquid chromatography column for separation, and electrochemical sensor for detection of all non-fully oxidized sulfur-containing compounds. We were able to achieve an effective separation and accurately assign chromatographic peaks to all detectable species. Owing to a high sensitivity of the utilized electrochemical detector, detection in low parts per billion range was possible. This can prove crucial for plating bath control, since minute amounts of certain by-products significantly affect the bath performance.     

Separation and quantification of angiotensins and some related peptides by high-performance liquid chromatography

A high-performance chromatographic technique for the separation of angiotensins and some related peptides is described. Complete separation of angiotensin I, angiotensin II, tetradecapeptide and the tetrapeptide Leu-Val-Tyr-Ser is achieved in a single step, using reversed-phase high-performance liquid chromatography. The application of this technique for the detection of renin activity in crude biological samples, employing the artificial renin substrate tetradecapeptide, is demonstrated.     

Reversed-phase high-performance liquid chromatography of tenuazonic acid and related tetramic acids

A reversed-phase high-performance liquid chromatographic system for the determination of the fungal toxin, tenuazonic acid, (5S,8S)-3-acetyl-5-sec.-butyltetramic acid, is described. The system utilizes a column packed with deactivated end-capped C18 silica with a high carbon load to overcome the problem of poor chromatographic performance of this beta-diketone on reversed-phase liquid chromatography which previously necessitated the use of anion-exchange, ligand-exchange or ion-pairing methods. The reversed-phase system allows the separation of tenuazonic acid from its (5R,8S)-diastereomer, allo-tenuazonic acid and was applied to the detection of tenuazonic acid in cultures of Alternaria alternata and Phoma sorghina. By means of diode-array ultraviolet detection, (5S)-3-acetyl-5-isopropyltetramic acid was observed in extracts of culture material. This metabolite was purified using the analytical reversed-phase system and was identified by 1H and 13C nuclear magnetic resonance spectroscopy.     

Preparative isolation of tetra-, penta- and hexapurine oligonucleotides from partial hydrolysates of depyrimidinated herring sperm DNA

Herring sperm DNA is chemically degraded to a complex mixture of purine nucleotides. The oligonucleotides are separated from the partial hydrolysates by column chromatography. The resulting mixture of trimer to hexamer purine oligonucleotides is subsequently fractionated on QAE-Sephadex into different mixtures of sequence-isomeric purine oligonucleotides. In a final separation, which uses reversed-phase (Nucleosil C18) high-performance liquid chromatography, these mixtures are separated under isocratic conditions into 35 pure defined purine oligonucleotides with four to six monomer units, 14 defined mixtures of sequence-isomeric purine oligonucleotides and several unidentified products. Purity and sequence of the isolated oligonucleotides are determined by the "fingerprint" method. The results of the high-performance liquid chromatographic and the "fingerprint" methods of the isolated oligonucleotides are discussed.     

Analysis of triacylglycerols in fat body of bumblebees by chromatographic methods

Triacylglycerols (TAGs) from the fat body of several bumblebee species (Bombus lucorum, B. terrestris, B. lapidarius, B. hypnorum, B. hortorum, and B. confusus) were studied using chromatographic techniques. Semi-preparative thin-layer chromatography was used to isolate the TAGs from the tissue extract. Gas chromatography (GC) enabled us to identify the fatty acids (FAs) that form bumblebee TAGs and to quantify their relative proportions. The TAGs were subsequently analysed by high-performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry. Two chromatographic systems, including non-aqueous reversed-phase chromatography and silver ion chromatography on cation exchange resin in silver (I) ionic form, were optimised and their performance compared. The most abundant fatty acids in bumblebees TAGs contained 18 or 16 carbon atoms; oleic acid predominated in most samples. TAGs were found to be a complex mixture of isomers; some of them, e.g. OLnO, PLnO, PoPoO, PoPoP, POO, or OOO (where Po is palmitoleic, P is palmitic, Ln is linolenic, and O is oleic acid) were abundant in particular species. The composition of both FAs and TAGs was found to be species-specific. Only minor differences were found among specimens of the same species.     

Direct and indirect high-performance liquid chromatographic enantioseparation of beta-amino acids

Direct and indirect reversed-phase (RP) high-performance liquid chromatographic methods were developed for the separation of enantiomers of 18 unnatural beta-amino acids, including several beta-3-homo amino acids. The direct separations of the underivatized analytes were performed on chiral stationary phases (CSPs) containing macrocyclic glycopeptide antibiotic teicoplanin (Chirobiotic T column) and teicoplanin aglycone (Chirobiotic TAG column). The indirect method involved pre-column derivatization with a new chiral derivatizing agent (CDA), (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester ((S)-NIFE), and subsequent separation of diastereomers on Discovery C18 and Hyperpep 300 C18 columns. The different methods were compared in systematic chromatographic examinations. The effects of organic modifier, mobile phase composition, pH and flow rate on the separation were investigated.     

Analytical methods for determination of new fluoroquinolones in biological matrices and pharmaceutical formulations by liquid chromatography: a review

Fluoroquinolones are one of the most promising and intensively studied drugs of contemporary anti-infective chemotherapy. New fluoroquinolone antibacterials with improved pharmacokinetic properties and a broad spectrum of activity have been developed, opening new windows of opportunity for clinical use. To our knowledge, no comprehensive and critical review of the analytical methods for the determination of these agents, which correspond to the third- and fourth-generation quinolones, has yet been published. This work summarizes for the first time most of the liquid chromatographic methods reported in the literature for the separation and quantification of the new fluoroquinolones in biological matrices and pharmaceutical formulations. A systematic and detailed survey of physicochemical properties, sample preparation procedures, and chromatographic and detection conditions is presented herein. In the course of this review several liquid chromatographic methods are discussed: reversed-phase high-performance liquid chromatography (RP-HPLC), ion-exchange high-performance liquid chromatography (IEX-HPLC), hydrophilic interaction liquid chromatography (HILIC), high-performance thin-layer chromatography (HPTLC) and other chiral chromatographic methods. Their advantages, applicability and limitations are also examined.     

Simultaneous Quantification of Flavonoids and Biflavonoids in Ginkgo biloba Using RP-HPTLC Densitometry Method

JPC – Journal of Planar Chromatography – Modern TLC - A reversed-phase high-performance thin-layer chromatography (RP-HPTLC) method was developed for the determination of flavonoids...     

Preparative isolation of vitamin D2 from previtamin D2 by recycle high-performance liquid chromatography.

A preparative high-performance liquid chromatographic method, based on recycle chromatography, to separate vitamin D2 (ergocalciferol) from previtamin D is described. The method provides efficient separation by means of a mixture of methanol, acetonitrile and hexane as eluent on a reversed-phase C18 column. Scale-up to a 2-in. diameter column resulted in the collection of 100% pure fractions based on UV detection at 265 nm. The total throughput and the economics of the purification were also optimized.     

Determination of vitamin E isomers of grape seeds by high-performance liquid chromatography-UV detection

A simple analytical method for the determination of vitamin E isomers in grape seeds by reversed-phase high-performance liquid chromatography with UV detection is described. The method is based on a solid-liquid extraction separation on an ODS column, and the analytes are monitored at 295 nm with a UV detector. Tocopherols are extracted in n-hexane and directly injected onto the column without using any purification step, such as saponification, prior to the separation and determination. The chromatographic separation of tocopherols is achieved in 12 min with a mobile phase that consists of n-hexane and isopropyl alcohol (99.99:0.01, v/v). The method is reproducible and accurate, with respect to demonstrating a relative standard deviation between 2.57% and 3.30% (n = 10, for 500 ng/mL) and a relative error between 0.84% and 6.54% (n = 10, for 500 ng/mL), respectively. The theoretical limits are estimated as 25 ng/mL for α-tocopherol, 43 ng/mL for γ-tocopherol, and 83 ng/mL for δ-tocopherols. The method is then applied for the determination of tocopherols in grape seeds grown in Turkey. The amounts of tocopherols are calculated by using the standard addition method.     

Determination of aniracetam and its main metabolite, N-anisoyl-GABA, in human plasma by high-performance liquid chromatography

Two different reversed-phase high-performance liquid chromatographic methods for the determination of aniracetam (I) and its metabolite N-anisoyl-GABA (II) in human plasma are described. The procedure for I involves direct injection of plasma samples spiked with the internal standard on a clean-up column followed by reversed-phase chromatography on a C18 column. The limit of quantification was 5 ng/ml, using a 200-microliters specimen of plasma. The mean inter-assay precision of the method up to 800 ng/ml was 3%. The procedure for II involved liquid-liquid extraction of II and the internal standard from plasma with ethyl acetate, and reversed-phase chromatography on a C18 column. The limit of quantification was 50 ng/ml using a 0.5-ml plasma specimen. The mean inter-assay precision up to 50 micrograms/ml was 6%. The applicability and accuracy of the methods were demonstrated by the analysis of over 1000 plasma samples from two bioavailability studies in healthy volunteers.