Determination of amino acids in apple extracts by high performance liquid chromatography

Summary A rapid and sensitive method for the simultaneous determination of primary amino acids in apple is described. After sample preparation, amino acids were derivatized with o-phthaldialdehyde/2-mercaptoethanol and separated on a reversed phase column with a gradient of phosphate buffer-tetrahydrofuran-methanol as the mobile phase. Detection was carried out with a fluorescence detector at excitation and emission wavelengths of 340 nm and 425 nm respectively. Recovery studies showed good results for all substances (91–109%) (with coefficients of variation ranging, from 0.1 to 9.0%). This method was applied to the monitoring of amino acids during the ripening of apples.     

Measurement of polyunsaturated fatty acids of myocardial lipids by high performance liquid chromatography

Summary This report describes a modified method for the separation and analysis of polyunsaturated fatty acids such as 20:4, 20:5, and 22:6, using HPLC. The results show that these fatty acids are well separated from the saturated acids. Since the unsaturated fatty acids elute earlier than saturated acids, and this method does not require the fractionation of free fatty acids using thin layer chromatography, a necessary step for the gas chromatographic analysis, the recoveries of polyunsaturated fatty acids were significantly higher as compared to those from gas chromatography. Furthermore, HPLC and gas chromatographic methods gave identical results for the acyl chain composition of phosphatidylserine. The advantages of using HPLC over gas chromatography in determining the acyl chain composition of free fatty acids and phospholipids are also discussed.     

HPLC analysis of flavonoids and phenolic acids and aldehydes inEucalyptus spp.

Summary Standards of the polyphenols occurring in wood, bark and leaf extracts ofEucalyptus spp. (i.e. flavonoids and phenolic acids and aldehydes) have been analyzed by HPLC using reversed phase columns, gradient elution and diode-array detection. The conditions used are reported.     

Simultaneous isocratic separation of phenolic acids and flavonoids using micellar liquid chromatography

The simultaneous isocratic separation of a mixture of five phenolic acids and four flavonoids (two important groups of natural polyphenolic compounds with very different polarities) was investigated in three different RPLC modes using a hydro‐organic mobile phase, and mobile phases containing SDS at concentrations below and above the critical micellar concentration (submicellar LC and micellar LC (MLC), respectively). In the hydro‐organic mode, methanol and acetonitrile; in the submicellar mode methanol; and in the micellar mode, methanol and 1‐propanol were examined individually as organic modifiers. Regarding the other modes, MLC provided more appropriate resolutions and analysis time and was preferred for the separation of the selected compounds. Optimization of separation in MLC was performed using an interpretative approach for each alcohol. In this way, the retention of phenolic acids and flavonoids were modeled using the retention factors obtained from five different mobile phases, then the Pareto optimality method was applied to find the best compatibility between analysis time and quality of separation. The results of this study showed some promising advantages of MLC for the simultaneous separation of phenolic acids and flavonoids, including low consumption of organic solvent, good resolution, short analysis time, and no requirement of gradient elution.     

Determination of phenazopyridine in human plasma by high performance liquid chromatography

Summary A simple, low-cost, sensitive and selective HPLC method was developed for the determination of phenazopyridine in human plasma. The method employs UV detection of phenazopyridine and of the internal Standard at 2 different wavelengths. Calibration curves were linear over a large dynamic range, i.e., within 0.05–10.0 μg mL⁻¹ with limit of quantification of 0.05 μg mL⁻¹, and a limit of detection of 0.01 μg mL⁻¹.     

Determination of phenolic acids and flavonoids of apple and pear by high-performance liquid chromatography

A new HPLC stationary phase has been applied to the analysis of phenolic acids and flavonoids with diode array and mass spectrometric detection. The separation of 26 standard compounds was achieved within 1 h. The stationary phase displayed excellent resolution especially of flavonol glycosides. The analytical system has been used for the determination of phenolic compounds in apple pomace and apple juice, and in extracts of pear fruits of different cultivars. Apple pomace was found to be a promising source of phenolics. However, yields are affected by the drying conditions applied. Furthermore, the applicability of the analytical system for the authenticity control of apple and pear juice was demonstrated by determination of characteristic quercetin and isorhamnetin glycosides, and dihydrochalcones, respectively. Since isorhamnetin-3-glucoside was present in all pear cultivars investigated, the usefulness of arbutin as a specific marker of pear products appears to be doubtful.     

Determination of monensins A and B in the fermentation broth ofStreptomyces cinnamonensis by high performance liquid chromatography

Summary High performance liquid chromatography on an octadecyl silica column has been used to determine both the monensin A: monensin B ratio and by the method of standard addition, the concentration of both in the fermentation broth ofStreptomyces cinnamonensis. Refractive index detection was preferred to ultraviolet owing to the presence of UV-absorbing components which could not be completely separated from the substances of interest. A linear relationship was obtained from the calibration data. The coefficients of variation for the estimation both of the ratio and the concentrations of the compounds were better then 5%. The estimated limit of detection for both substances was about 1 g/ml. The results obtained from the determination of the ratios of monensins were compared with those obtained by chemical ionization mass spectrometry. Chromatographic separation of monensins on the silica gel column is also described.     

Determination of D- and L-amino acids in biological samples by two-dimensional column liquid chromatography

Summary A two-dimensional, column liquid chromatographic system is used for the determination of the D- and L-enantiomers of amino acids in biological samples. Separation of the amino acids is first on ion-exchange column by gradient elution with a sodium citrate-sodium chloride buffer. Enantioseparation is by subsequent injection of 3 μl heart-cuts of the individual amino acids onto a second column with a chiral crown ether stationary phase. Finally, fluorescence detection is after post-column labelling of the amino acids using ano-phthalaldehyde-2-mercaptoethanol reagent solution. The high separation power and selectivity of the system allow processing of complex samples with hardly any additional treatment and the determination of small quantities of D-amino acids in the presence of excess L-form. Applicability of the system is illustrated by the determination of D- and L-aspartate, serine, glutamate and alanine in various complex biological samples, such as protein hydrolysates, urine and biotechnological and food samples. Data are given on detectability, repeatability and linearity.     

Determination of D- and L-amino acids in biological samples by two-dimensional column liquid chromatography

Summary A two-dimensional, column liquid chromatographic system is used for the determination of the D- and L-enantiomers of amino acids in biological samples. Separation of the amino acids is first on ion-exchange column by gradient elution with a sodium citratesodium chloride buffer. Enantioseparation is by subsequent injection of 3 l heart-cuts of the individual amino acids onto a second column with a chiral crown ether stationary phase. Finally, fluorescence detection is after post-column labelling of the amino acids using ano-phthalaldehyde-2-mercaptoethanol reagent solution. The high separation power and selectivity of the system allow processing of complex samples with hardly any additional treatment and the determination of small quantities of D-amino acids in the presence of excess L-form. Applicability of the system is illustrated by the determination of D- and L-aspartate, serine, glutamate and alanine in various complex biological samples, such as protein hydrolysates, urine and biotechnological and food samples. Data are given on detectability, repeatability and linearity.     

Quantitative analysis of flavonoids and phenolic acids in Arnica montana L. by micellar electrokinetic capillary chromatography

Arnica montana preparations have been used in Europe for centuries to treat skin disorders. Among the biologically active ingredients in the flower heads of the plant are sequiterpenes, flavonoids and phenolic acids. For the simultaneous determination of compounds belonging to the latter two groups a micellar electrokinetic capillary chromatography (MEKC) method was developed and validated. By using an electrolyte solution containing 50 mM borax, 25 mM sodium dodecyl sulfate and 30% of acetonitrile the separation of seven flavonoids and four caffeic acid derivatives was feasible in less than 20 min. The optimized system was validated for repeatability (σ_rel ≤ 4.4%), precision (inter-day σ_rel ≤ 8.13%, intra-day σ_rel ≤ 4.32%), accuracy (recovery rates from 96.8 to 102.4%), sensitivity (limit of detection (LOD) ≤ 4.5 μg mL⁻¹) and linearity (R² ≥ 0.9996), and then successfully applied to assay several plant samples. In all of them the most dominant flavonoid was found to be quercetin 3-O-glucuronic acid, whereas 3,5-dicaffeoylquinic acid was the major phenolic acid; the total content of flavonoids and phenolic acids varied in the samples from 0.60 to 1.70%, and 1.03 to 2.24%, respectively.     

Analysis of terpenes fromGinkgo biloba L. extracts by reversed phase high-performance liquid chromatography

Summary The main terpenes ofGinkgo biloba L. extracts (bilobalide, ginkgolide A, ginkgolide B and ginkgolide C) have been separated by isocratic elution on a 3 μm C₁₈ Spherical column using 2-propanol:water (10∶90) as eluent.     

Separation ofAstragalus saponins by reversed phase high performance liquid chromatography and evaporative light scattering detection

Summary A new HPLC method permitted the separation of 13 triterpene lycosides isolated from differentAstragalus species within 40 min. A water/acetonitrile gradient was used as eluent and 5 μm RP-18 material as stationary phase. By using an evaporative light scattering (ELS) detector, the main saponins ofA. membranaceus could be detected at levels as low as 20.0 μg·mL⁻¹. This method facilitated distinction of differentAstragalus species as well as the analyses of market products containingA. membranaceus. The results showed variations from 0.019 to 0.184% in the total saponin content of the market products.     

Determination of cortisol in guinea pig urine by high performance thin-layer chromatography, high performance liquid chromatography, and thin-layer chromatography/radioimmunoassay

Summary In order to collect urinary samples from unrestrained guinea pigs, animals were kept in their familiar home cages with wood shavings for bedding. Cortisol was removed from shavings by a simple washing step, and an attempt was made to measure its concentrations by high performance thin-layer chromatography (HPTLC), high performance liquid chromatography (HPLC), or thin layer chromatography/radioimmunoassay (TLC-RIA). After intramuscular administration of 25 mg cortisol, cortisol excretion increased from about 20–30 μg/day to 400–500 μg/day (HPTLC: 531 μg/day, HPLC: 493 μg/day; TLC-RIA: 394 μg/day). Similarly, the treatment of the animals with 20 IU ACTH resulted in an augmented cortisol excretion, with mean values of 294 μg/day (HPTLC), 256 μg/day (HPLC) and 143 μg/day (TLC-RIA), respectively. The present study shows, for the first time, that cortisol excretion in unrestrained laboratory animals can be determined. Whilst the cortisol values measured by HPTLC and HPLC agree, the amounts measured by TLC-RIA were significantly lower. These differences are probably due to the presence of substances in urine or shavings which interfere with the radioimmunological determination. Hence, cortisol should be determined either by HPTLC or HPLC. Beside having a desirable specificity, these methods are more suited than TLC/RIA for steroid analysis since they confer the possibility of measuring additional steroids (e.g. precursors and/or metabolites of cortisol) in a single urine extract. This is especially the case for the HPTLC method since substances can be transformed into fluorescent derivatives.     

Determination of 8-hydroxyquinoline sulfate in tuberculin solutions by planar and high performance liquid chromatography

Summary TLC and HPLC methods for the determination of the preservative, 8-hydroxyquinoline sulfate in PPD-T tuberculin solution were developed. The planar chromatography method involved separation of 8-hydroxyquinoline sulfate on a TLC plate using a butyl-acetate: formic acid: 2-propanol mobile phase, detection and quantitation by densitometric scanning. The HPLC method was on a LiChrosorb RP-18 column with acetonitrile-water (65:35 v/v) mobile phase, adjusted to pH 3.05 by phosphoric acid. Linearity, reproducibility and accuracy were found to be satisfactory. Under selected conditions, the limit of detection (LOD) of both methods was similar-about 25 ng. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Separation of epigallocatechin and flavonoids from Hypericum perforatum L. by high-speed counter-current chromatography and preparative high-performance liquid chromatography

High-speed counter-current chromatography (HSCCC) and preparative high-performance liquid chromatography (prep-HPLC) were successively used for the separation of epigallocatechin and flavonoids from Hypericum perforatum L. The two-phase solvent system composed of ethyl acetate–methanol–water (10:1:10, v/v) was used for HSCCC. About 900 mg of the crude extract was separated by HSCCC, yielding 7.8 mg of quercitrin at a purity of over 97%, 12.6 mg of quercetin at a purity of over 93%, and 38.9 mg of a mixture of hyperoside, isoquercitrin and miquelianin constituting over 97% of the fraction. A mixture of epigallocatechin and avicularin pooled from three HSCCC runs, a total amount of 54.3 mg, was further separated by prep-HPLC yielding 23.4 mg of epigallocatechin and 15.3 mg of avicularin each at a purity of over 97%.     

Temperature-dependent separation of bryostatin 18 and 10 by high performance liquid chromatography

Summary The temperature-dependent separation of bryostatins by HPLC was examined on an octadecyl bonded stationary phase, using column temperatures between 0 and 40°C and mobile phase temperatures from 0 to 25°C. The retention time and resolution of bryostatins changed drastically and separation improved with decreasing temperature. A column temperature of less than 5°C and a mobile phase temperature of less than 15°C is recommended for a good resolution of bryostatins for routine work.     

Reverse-phase liquid chromatography ofp-tert-butylcalixarenes

Summary Thep-tert-butylcalix[n]arenes (n=4,6,7,8) andp-tert-butyldihomooxacalix[4]arene have been separated by reverse phase liquid chromatography. Optimum conditions have been obtained on a Spherisorb ODS1, 5 μm C18 column by isocratic ambient elution with acetonitrile-methyltert-butyl ether. Calibration plots have been obtained from purified calixarenes and the reliability of the method is confirmed from test mixtures of calixarenes of known composition.     

Correlation between hydrophobicity of amino acids and retention data in reversed-phase liquid chromatography with micellar eluents

Summary Hydrophobic character is usually expressed in terms of the partition coefficient in 1-octanol-water (log P_O/W). However, measurement of this coefficient is often problematic. Retention in micellar liquid chromatography is mainly due to hydrophobic interactions and can also be used as an index of hydrophobicity. A hydrophobicity scale was established with retention data foro-phthalaldehyde (OPA)-N-acetyl-L-cysteine (NAC) amino acid derivatives, using the glycine derivative as reference. Since the OPA-NAC derivatives only differ in the nature of R₁ in the amino acid (R₁CH(COOH)NH₂), in the absence of electrostatic interactions the hydrophobic character of the substituent was responsible for retention. Linear relationships were obtained between log of the ratiok′ of amino acid derivatives:k′ of the glycine derivative for a given mobile phase, and logP _O/W for the R₁ substituent. Good correlations were also found for phenylthiohydantoin amino acid derivatives.     

Determination of trans-fatty acids in food samples based on the precolumn fluorescence derivatization by high performance liquid chromatography

Trans-fatty acids are unsaturated fatty acids that are considered to have health risks. 1,3,5,7-Tetramethyl-8-butyrethylenediamine-difluoroboradiaza-s-indacene is a highly sensitive fluorescent labeling reagent for carboxylic acids developed by our lab. In this study, using this precolumn fluorescent derivatization reagent, a rapid and accurate high-performance liquid chromatography with fluorescence detection method was developed for the determination of two trans-fatty acids in food samples. Under the optimized derivative conditions, two trans-fatty acids were tagged with the fluorescent labeling reagent in the presence of 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide at 25°C for 30 min. Then, the baseline separation of trans- and cis-fatty acids and their saturated fatty acid with similar structures was achieved with less interference using a reversed-phased C₁₈ column with isocratic elution in 14 min. With fluorescence detection at λ_ex/λ_em = 490 /510 nm, the linear range of the TFAs was 1.0-200 nM with low detection limits in the range of 0.1–0.2 nM (signal-to-noise ratio = 3). In addition, the proposed approach was successfully applied for the detection of trans-fatty acids in food samples, and the recoveries using this method ranged from 96.02 to 109.22% with low relative standard deviations of 1.2–4.3% (n = 6).     

Correlation between hydrophobicity of amino acids and retention data in reversed-phase liquid chromatography with micellar eluents

Summary Hydrophobic character is usually expressed in terms of the partition coefficient in 1-octanol-water (log P_o/w). However, measurement of this coefficient is often problematic. Retention in micellar liquid chromatography is mainly due to hydrophobic interactions and can also be used as an index of hydrophobicity. A hydrophobicity scale was established with retention data foro-phthalaldehyde (OPA)-N-acetyl-L-cysteine (NAC) amino acid derivatives, using the glycine derivative as reference. Since the OPA-NAC derivatives only differ in the nature of R₁ in the amino acid (R₁CH (COOH)NH₂), in the absence of electrostatic interactions the hydrophobic character of the substituent was responsible for retention. Linear relationships were obtained between log of the ratiok′ of amino acid derivatives:k′ of the glycine derivative for a given mobile phase, and logP _o/w for the R₁ substituent. Good correlations were also found for phenylthiohydantoin amino acid derivatives.