Determination of sulfur amino acids in foods as related to bioavailability

During the processing of feedstuffs and foods, methionine can be oxidized to methionine sulfoxide and methionine sulfone, and cysteine can be oxidized to cysteic acid. Methionine sulfone and cysteic acid are nutritionally unavailable, but methionine sulfoxide can be utilized, at least to some degree. The degree of utilization depends on the levels of methionine, cysteine, and methionine sulfoxide in the diet, but there is no consensus in the literature on the quantitative impact of these dietary constituents on methionine sulfoxide utilization. Methionine and cysteine are most often determined after quantitative oxidation to methionine sulfone and cysteic acid, respectively, using performic acid oxidation prior to hydrolysis. However, this method may overestimate the methionine content of processed foods, as it will include any methionine sulfoxide and methionine sulfone present. A selection of analytical methods has been developed to allow the separate determination of the 3 oxidized forms of methionine, the merits of which are discussed in this review. An additional consideration for determining methionine and cysteine bioavailability is that not all dietary methionine and cysteine is digested and absorbed from the small intestine. Selected methods designed to determine the extent of digestion and absorption are discussed. Finally, a concept for a new assay for determining methionine bioavailability, which includes determining the digestibility of methionine and methionine sulfoxide as well as the utilization of methionine sulfoxide, is presented.     

Use of XAD-2 Macroreticular Resin for the Recovery of Aldicarb and its Metabolites in Drinking Water

Abstract

A simplified method for the determination of aldicarb and its oxidation products, aldicarb sulfoxide, and aldicarb sulfone, in water has been developed. Aldicarb and its metabolites are adsorbed on Amberlite XAD-2 polymer resin and then eluted with acetone. The eluate is analyzed for aldicarb and aldicarb sulfoxide by high performance liquid chromatography (HPLC) with UV detection at 254 nm. Total aldicarb residues can be determined by a colorimetric method. Typical detection limits in drinking water are 1 μg/1.     

Chromatographic determination of itopride hydrochloride in the presence of its degradation products

Two sensitive and reproducible methods are described for the quantitative determination of itopride hydrochloride (IH) in the presence of its degradation products. The first method is based on HPLC separation on a reversed phase Kromasil column [C18 (5-microm, 25 cm x 4.6 mm, ID)] at ambient temperature using a mobile phase consisting of methanol and water (70:30, v/v) adjusted to pH 4.0 with orthophosphoric acid with UV detection at 258 nm. The flow rate was 1.0 mL per min with an average operating pressure of 180 kg/cm2. The second method is based on HPTLC separation on silica gel 60 F254 using toluene:methanol:chloroform:10% ammonia (5.0:3.0:6.0:0.1, v/v/v/v) as mobile phase at 270 nm. The analysis of variance (ANOVA) and Student's t-test were applied to correlate the results of IH determination in dosage form by means of HPLC and HPTLC methods. The drug was subjected to acid and alkali hydrolysis, oxidation, dry heat, wet heat treatment, UV, and photodegradation. The proposed HPLC method was utilized to investigate the kinetics of the acidic, alkaline, and oxidative degradation processes at different temperatures and the apparent pseudo-first-order rate constant, half-life, and activation energy were calculated. In addition the pH-rate profile of degradation of IH in constant ionic strength buffer solutions in the pH range 2-11 was studied.     

HPLC separation and relative quantitation of kaempferol glycosides in soybean

Summary An HPLC method is described for the determination of kaempferol glycosides in soybean leaf extracts. The method is rapid and can provide qualitative and relative quantitative results for 9 kaempferol glycosides. The flavonol glycosides are extracted from soybean leaves by shaking the samples in 50% methanol; the extracts are filtered, evaporated to dryness and reconstituted in methanol before further purification through a C-18 Sep-Pak column. The samples are injected onto a C-18 HPLC column, separated by gradient elution with a 1% phosphoric acid: methanol mixture and detected on a UV/VIS diode-array detector. Flavonols were monitored at 265 and 348 nm and spectra from 180 to 400 nm were stored and used as an aid in identification. Relative quantities of the kaempferol glycosides among soybean cultivars were calculated from their proportion of peak area in the chromatograms. Total kaempferol concentration of the extracts was calculated after acid hydrolysis of the kaempferol glycosides to the aglycone and comparison of peak areas to kaempferol standards.     

Quantitative high-performance liquid chromatographic determination of sulfur amino acids in protein hydrolysates

A rapid, quantitative high-performance liquid chromatographic procedure for the determination of methionine and cystine after oxidation to methionine sulfone and cysteic acid is described. The Dns derivatives of the amino acids are separated by reversed-phase chromatography with a phosphate buffer-acetonitrile gradient and detected by UV absorption at 254 nm. The procedure is validated by confirming the methionine and cystine content of ribonuclease A. The average yields of cysteic acid and methionine sulfone from triplicate analyses of ribonuclease A were 98.1% (±3.3) and 106.1% (±2.4) of the theoretical values, respectively.     

A Stability-Indicating LC Assay Method for Pemetrexed Disodium

This present work describes the development of a stability-indicating high performance liquid chromatographic method for the quantitative determination of pemetrexed disodium. Pemetrexed disodium is an antifolate antineoplastic agent that exerts its action by disrupting folate-dependent metabolic processes essential for cell replication. The chromatographic separation was achieved on an ACE 3 C18 HPLC column using a mobile phase consisting of a mixture of buffer (solvent A) and organic modifier acetonitrile (solvent B). Forced degradation studies were performed on bulk sample of pemetrexed disodium using acid (0.5 N hydrochloric acid), base (0.5 N sodium hydroxide), oxidation (10% v/v hydrogen peroxide), heat (60 °C) and UV light (254 nm). Degradation of the drug substance was observed in base hydrolysis. Degradation product formed under acid and base hydrolysis was found to be starting material. The stressed samples were assayed using the developed LC method and the mass balance found was close to 99.5%, thus proving its stability-indicating power. The developed method was validated with respect to linearity, accuracy, precision and robustness.     

A Stability-Indicating LC Assay Method for Bicalutamide

A stability-indicating HPLC method for the quantitative determination of Bicalutamide is described. Bicalutamide is a nonsteroidal antiandrogen and is an oral medication that is used for treating prostate cancer. Separation was achieved on a Waters Symmetry shield RP18 HPLC column using a mobile phase consists of a mixture of phosphate buffer (Solvent A) and organic modifier acetonitrile (Solvent B). Degradation studies were performed on bulk samples of bicalutamide using acid (0.5 N methanolic hydrochloric acid), base (0.5 N methanolic sodium hydroxide), oxidation (10% v/v methanolic hydrogen peroxide), heat (60 °C) and UV light (254 nm). Degradation was observed under base hydrolysis to give the starting material used during the synthesis of bicalutamide. The degraded samples were assayed and gave a mass balance greater than 99.5%, thus proving the stability-indicating power of the developed method. The method was validated with respect to linearity, accuracy, precision and robustness.     

Nuclease P1 digestion/high-performance liquid chromatography, a practical method for DNA quantitation

We have developed a practical method for quantifying DNA. The method is practical in two ways. First, a single enzyme is used to digest the DNA to nucleotides that are then quantified by HPLC under ordinary conditions. Second, the method quantifies DNA even when it is impure. In our method, "nuclease P1/HPLC," the DNA is hydrolyzed by nuclease P1 and the resulting 2'-deoxynucleoside 5'-monophosphates are quantified by HPLC with UV detection. This method was applied to several kinds of genomic DNA in terms of origin and method by which it had been purified. Calf thymus DNA (purified by salt precipitation by the supplier), pig liver DNA (purified by phenolic extraction or by anion-exchange chromatography using a Genomic Tip from Qiagen) and mouse skin DNA (similarly purified) were tested. In some cases a given sample was purified by two of these methods. The values for the amount of DNA by our method were compared with those by three other methods: acid hydrolysis/HPLC (selected as a reference procedure), UV absorbance, and dye binding. Agreement for all DNA samples between the values by our method versus those provided by acid hydrolysis/HPLC was within 10% for amounts of DNA in the 19-54 microg range. In contrast, UV absorbance and the dye-binding assay gave differences up to 30-40% relative to the consistent values furnished by acid hydrolysis and our method. Overall, normalizing the concentrations of the DNA (thymus, liver, skin) by acid hydrolysis/HPLC in 10 samples to values of 1.0 gave the following, relative values and standard deviations: 1.01+/-.07 (nuclease P1/HPLC), 0.8+/-0.17 (dye binding), and 1.1+/-0.1 (UV). Since one cannot assume that any sample of DNA is pure, and determining purity of DNA is difficult, then nuclease P1/HPLC or acid hydrolysis/HPLC is recommended rather than the UV absorbance or dye binding for quantifying DNA whenever an accurate value is important.     

Validated stability-indicating methods for the determination of nizatidine in the presence of its sulfoxide derivative

Four new selective, precise, and accurate methods are described for the determination of nizatidine (NIZ) in the presence of its sulfoxide derivative in both the raw material and pharmaceutical preparations. Method A is based on zero-order (0D), first-derivative (1D), and second-derivative (2D) spectrophotometric measurement of NIZ in aqueous solution at the zero-crossing point of its sulfoxide derivative (at 314, 295-334, and 318-348 nm, respectively). Method B is a 1DD spectrophotometric method based on the simultaneous use of the first derivative of the ratio spectra and the measurement of peak amplitude at 297 nm. Method C uses a solvent-induced derivative-difference spectrophotometry with deltaD1 measurement from peak to peak at 315-345 nm. Method D involves quantitative densitometric evaluation of a mixture of the drug and its sulfoxide derivative after separation by high-performance thin-layer chromatography on silica gel plates with chloroform-methanol (9 + 1, v/v) as the mobile phase; Rf values for NIZ and its sulfoxide derivative were 0.4 and 0.2, respectively. The spot was scanned at 254 nm. The first-derivative spectrophotometric method was used to investigate the kinetics of the hydrogen peroxide degradation process at different temperatures. The apparent pseudo-first-order rate constant, half-life, and activation energy were calculated. The results obtained by the proposed methods were analyzed statistically and compared with those obtained by the official method. These methods are suitable as stability-indicating for the determination of NIZ in the presence of its oxidation-induced degradation product (sulfoxide derivative) either in the bulk powder or in pharmaceutical preparations.     

Comparison between HPLC and HPTLC densitometry for the determination of icariin from Epimedium koreanum extracts

Dry extracts of the aerial parts of Epimedium koreanum were quantified by HPLC and high performance TLC (HPTLC). A gradient HPLC method was used for the quantification of the prenylflavone glycoside icariin at 270 nm. A direct HPTLC assay was developed for the determination of icariin at 270 nm. The UV detection of both analytical assays were used to examine the purity of icariin peaks and compared with the standards. The assays provide good accuracy, reproducibility, and selectivity for the quantitative analysis of icariin. The icariin contents of five different dry extracts were compared by HPLC and HPTLC densitometry. The quantitative results of both analytical methods did not show any statistically significant differences between them, although a trend to slightly lower mean values could be found for the HPLC method.     

Simultaneous determination of triclabendazole and its sulphoxide and sulphone metabolites in bovine milk by high-performance liquid chromatography

A simple method has been developed for the simultaneous determination of triclabendazole and its metabolites (sulphoxide and sulphone) in bovine milk by reversed-phase high-performance liquid chromatography (HPLC). A milk sample was homogenized with sodium sulfate anhydrous and acetonitrile, and centrifuged. The supernatant was isolated, rinsed with n-hexane saturated with acetonitrile, and evaporated. The residue was dissolved with 0.1 M potassium dihydrogenphosphate, and 0.1 M sodium hydrogencarbonate, and then cleaned up on a Bond Elut C18 cartridge. The three compounds were separated on a Capcell Pak C18 UG 120 (5 microm, 150x4.6 mm I.D.) column and determined by UV detection at 295 nm. The mobile phase was acetonitrile-0.05 M ammonium acetate (50:50), and the flow-rate was 0.8 ml/min at 40 degrees C. The mean recoveries (n=4) were 89.1-95.0% with a relative standard deviation of 1.1-2.6%. The detection limits were 0.004-0.006 microg/g in milk. The proposed method was used to monitor raw milk samples for the market, and applied to the analysis of milk samples from 10 cows which had been administered with triclabendazole to control the liver fluke. The confirmation of the triclabendazole and its metabolites in the above milk sample has been achieved by electrospray LC-MS for the first time.     

Direct determination of free methionine in soy-based infant formula

A method was developed for the direct determination of free methionine in soy-based infant formula, with analyte separation and quantitation by reversed-phase liquid chromatography (LC), and UV absorbance at 214 nm, respectively. Sample preparation required only dilution with mobile phase and syringe filtration. Using a 0.02M KH2PO4 mobile phase (pH adjusted to 2.9 with 85% o-phosphoric acid) and 0.7 mL/min flow rate, methionine eluted at approximately 8 min, and total run time was 14 min after column regeneration with acetonitrile-water. System linearity was demonstrated as peak area versus analyte concentration, ranging from 80 to 120% of the formula specification for free methionine (r > 0.999, and all residuals < 0.45%). Intermediate precision relative standard deviation values were < 1.5% for ready-to-feed and reconstituted powder samples, and recoveries ranged from 98.0 to 103.5% for inter-method comparison with an amino acid analyzer method. The limit of quantitation was 3 mg methionine/L in the "as fed" infant formula. Despite the relatively weak UV absorptivity of methionine, the 214 nm signal was sufficiently intense in the 30-65 mg/L (201-436 microM) range to afford quantitation by peak area proportionation versus a 2-point external standard calibration. This direct UV detection after reversed-phase LC separation provides a simple and accurate method for determining free methionine without derivatization.     

The Use of Internal Surface Reversed-Phase Packing for the Solid Phase Extraction of Drugs from Serum

Abstract

The utilization of Internal Surface Reversed Phase (ISRP) packing as a solid phase extraction (SPE) matrix was investigated. Evaluation of the relative retention of nineteen medicinal agents on ISRP material was monitored using HPLC. The effects of altering pH and/or buffer concentration on retention were further studied using verapamil, phenelzine and tamoxifen as model compounds. Spiked serum samples containing the model compounds plus amitriptyline were also subjected to ISRP-SPE and HPLC. Verapamil, phenelzine, and tamoxifen were all strongly retained on ISPR as pH increased. The buffer concentration of the sample was not as critical on retention of these compounds as pH. Verapamil was quantitatively recovered from spiked serum samples (103 /pm 8.5%, n = 6), with a limit of detection of 10 ng/mL using fluorescent detection (γex = 280 nm, γem = 310 nm). Amitriptyline recovery was also quantitative (99.0/pm 5.3%, n = 6), and its limit of detection was 10 ng\mL employing short wavelength UV detection (γ = 214 nm). The ISRP packing as an SPE matrix failed to adequately disrupt the binding of either phenelzine or tamoxifen from serum proteins; therefore, recoveries of these compounds were poor (<50%). A comparison of ISRP to conventional C18 and C2 SPE columns indicated that the ISRP packing eliminated interferences as well or better than the conventional columns.     

HPLC/UV analysis of proteins in dairy products using a hydrophobic interaction chromatographic column.

High-performance liquid chromatography using a Chrompack P-300-RP column containing a polystyrene-divinylbenzene copolymer-based packing was examined to analyze bovine milk protein components. The separation of major raw-milk proteins could be performed rapidly and reliably with this HPLC/UV method. The determinations were performed in the linear ranges of 0.01-2.0 mg/ml for alpha-lactalbumin, 0.04-2.5 mg/ml for caseins and 0.02-2.0 mg/ml for beta-lactoglobulin. The validity of the method was verified. Since the chromatographic column enabled the quantification of only "native" milk proteins, the extent of denaturation and loss of milk proteins could be examined. Thus, evaluation of heat-induced proteins denaturation was carried out in raw milk heated for 5 min at pre-determined temperatures.     

维生素D包合物中维生素D的高效液相色谱测定

Vitamin D in the VDHCD inclusion complex was determined by HPLC on a Resolve silica column usingamobile phase of n-hexane:n-amylalcohol=95:5(V/V)and UV detection at 254 nm.The quantitative de-termination was performed with dimethyl phthalate as the internal standard.     

Electrochemical,fluorescence, and UV detection for HPLC analysis of various cysteine derivatives

Electrochemical (ECD), fluorescence (FLD), and UV spectrophotometric (UVD) detection were used to monitor various S-alk(en)yl-L -cysteines and their corresponding sulfoxide isomers following pre-column derivatization with o-phthaldialdehyde-tert-butylthiol and separation by reversed-phase HPLC. Recording of hydrodynamic voltammograms, FLD stop-flow scanning, and on-line captured UV spectra were methods used for establishing optimal detector settings which were defined as a compromise between favorable selectivity and high sensitivity. Optimal detector settings were found at: (A) 750 mV vs. Ag/AgCl for ECD; (B) excitation at 230 nm and emission at 420 nm for FLD; and (C) 337 nm for UVD. Various aspects of detector practicability such as selectivity, baseline disturbances due to excessive reagent, scanning possibilities, as well as detection limits were evaluated and compared. Minimal detectable amounts of the compounds were in the range of 130-160 fmol for ECD, and 2.5-3.5 pmol and 13-16 pmol for FLD and UVD. In addition, the possibilities and benefits of detector coupling were examined.     

Determination of protein oxidation by mass spectrometry and method transfer to quality control

Characterization and quantitative analysis of oxidation plays an important role in biopharmaceutical development. This study demonstrates an approach to the assessment of susceptible to oxidation methionine residues in monoclonal antibodies and recombinant proteins. A method for the determination of oxidation levels by peptide mapping with mass spectrometric (MS) detection is described and its advantages compared to the UV detection are presented. Good linearity and reproducibility for determination of oxidation with MS detection are demonstrated (R2 > 0.99; RSDs of 4-9%). Aspects of method transfer to quality control group (QC) are discussed. As well, a quick and easy flow injection/MS method is proposed to substitute for peptide map analysis. Peptide coverage, linearity, reproducibility, robustness, sensitivity and quantitative oxidation results are compared for the flow injection/MS and LC/MS approaches.     

Determination of lysinoalanine by high performance liquid chromatography

This work suggests an HPLC method for qualitative and quantitative determination of N^ε(2-amino-2-carboxyethyl)-L -lysine (LAL). LAL was released from total hydrolysates of various proteins of animal origin and derivatized with dansyl chloride. The performance of two different columns, Spherisorb 3S TG and μ-Bondapack C₁₈, was compared; better resolution and quantitative response were obtained with the former. The mobile phase was a mixture of 0.01 M phosphate buffer (pH 7) and acetonitrile. Linear response and quantitative repeatability were tested for both detectors used (UV-Vis set at 254 nm; fluorimetric set at λ_ex(max) = 360 nm and λ_em(max) = 525 nm). For LAL standard the minimum detectable amount was 0.05 ng, whereas for LAL in actual samples the amount was 0.5 ng (40 μg/g of analyzed proteins). Good analytical repeatability was obtained, resulting in CV % of 4.7 and 3.8 for UV and fluorimetric detectors, respectively. LAL recovery was determined using both detectors; the values obtained were 94 % (fluorimetric) and 92 % (UV). Greater noise levels were observed with the fluorimetric detector and its higher sensitivity could not, therefore, be fully utilized. The highest amounts of LAL were found in the casein (2816 μg/g) and cooked albumin (615 μg/g) samples.     

Simultaneous determination of three flavonoid aglycones in Elsholtzia blanda by adopting orthogonal test and high-performance liquid chromatography

A new HCl hydrolysis/HPLC method, by adopting L9(34) orthogonal test to optimize hydrolysis condition, has been developed for simultaneous determination of three flavonoid aglycones in Elsholtzia blanda benth. The HCl concentration, methanol concentration, hydrolysis temperature, hydrolysis time are taking as four inspecting foctors, and the contents of luteolin, apigenin, and 5-hydroxy-6,7-dimethoxyflavone in hydrolytic solution are used as the evaluation indexes. Agilent Zorbax SB-C18 is used as analytical column. The mobile phase is a mixture of methanol-0.2% phosphoric acid (70:30, v/v), and UV detector is set at 350 nm. The flow rate is 1.0 mL/min, the temperature of column is maintained at 30 degrees C. The optimal hydrolysis conditions are 3.0M HCl, 70% methanol, 85 degrees C hydrolytic temperature and 3 h hydrolytic time. Standard curves are linear over the concentration range 8.54-85.4 microg/mL, 1.2-12 microg/mL, 9.2-92 microg/mL, and their average recoveries are 96.8%, 98.0%, and 100.5% for luteolin, apigenin, 5-hydroxy-6, 7-dimethoxyflavone, respectively. Thus, the optimum hydrolysis condition is relatively gentle, and the HPLC method is proved to be simple, accurate, and sensitive, so it will be able be applied to quality control of medicinal plant of Elsholtzia blanda.     

Optimization for the determination of residual levomycetin (chloramphenicol) in cow milk by reversed-phase high-performance liquid chromatography

A sensitive, selective, accurate, and precise method is developed for determining residual levomycetin (chloramphenicol), in raw and pasteurized milk. It includes sample filtration, purification of sample by solid-phase extraction, purification of eluate by liquid-liquid extraction, preconcentration by evaporation under reduced pressure, and analysis by reversed-phase HPLC with UV detection at 278 nm. The mobile phase is an acetonitrile-water mixture 15: 85 with an addition of 1 vol % of isopropanol. The method detection limit is 0.45 μg/kg; the limit of quantification is 1.40 μg/kg; the repeatability limit is 6.93%.