Simple method for clinical determination of 13 carotenoids in human plasma using an isocratic high-performance liquid chromatographic method

We report a reversed-phase high-performance liquid chromatography method which resolves 13 identified carotenoids and nine unknown carotenoids from human plasma. A Nucleosil C₁₈ column and a Vydac C₁₈ column in series are used with an isocratic solvent system of acetonitrile–methanol containing 50 mM acetate ammonium–dichloromethane–water (70:15:10:5, v/v/v/v) as mobile phase at a flow-rate of 2 ml/min. The intra-day (4.5–8.3%) and inter-day (1.3–12.7%) coefficients of variation are suitable for routine clinical determinations.     

Thin-layer chromatographic determination of indolic tryptophan metabolites in human urine using Sep-Pak C18 extraction

Tryptophan and some of its indole metabolites were separated by thin-layer chromatography, stained with the Van Urk—Salkowski reagent, and quantitated by scanning densitometry. The application of this technique for the detection of the indoles in urine samples, employing Sep-Pak C₁₈ cartridges for extraction, was demonstrated. The proposed method is simple and accurate. The detection limits were 2 μg/ml 5-hydroxytryptophan, 1.75 μg/ml 5-hydroxyindolyl-3-acetic acid, 1.5 μg/ml tryptophan, 0.8 μg/ml indolyl-3-acetic acid, 0.9 μg/ml indolyl-3-butyric acid, 1.75 μg/ml serotonin, and 1.25 μg/ml tryptamine.     

Rapid and economical high-performance liquid chromatographic method for the determination of norfloxacin in serum using a microparticulate C18 guard cartridge

A rapid and economical high-performance liquid chromatographic assay is described for norfloxacin in serum. Samples (100 μl) containing N-ethylnorfloxacin as the internal standard were extracted into 1 ml of chloroform. Chromatography was performed at 30°C on a 40×3.2 mm I.D. C₁₈ guard cartridge (3 μm spherical particles) using a mobile phase of 11% (v/v) acetonitrile in 0.01 M phosphate buffer (pH 2.5) containing 0.001 M triethylamine, and pumped at 1 ml/min. Detection was at 279 nm. The retention times of norfloxacin and internal standard were 1.9 and 2.9 min, respectively. Calibration curves were linear (r>0.999) from 0.1 mg/l to at least 2.0 mg/l. Within-day and between-day precision (C.V.) were 8.6% or less, and accuracy was 5.3% or less. Absolute assay recovery of norfloxacin was over 70%.     

Reversed-phase high-performance liquid chromatographic identification of lutein and zeaxanthin stereoisomers in bovine retina using a C30 bonded phase

An efficient reversed-phase high-performance liquid chromatographic assay with ultraviolet detection at 450 nm for simultaneous determination of lutein and zeaxanthin stereoisomers in bovine retina is described. The procedure involves rapid and careful one-step hexane extraction of the carotenoids from the homogenized liquid and enrichment by on-line solid phase extraction on a polystyrene cartridge. The substances were eluted at a flow rate of 1 ml/min with acetone–water (85:15, v/v) on a C₃₀ reversed-phase column. C₃₀ phases exhibit superior shape selectivity for the separation of carotenoid stereoisomers compared with conventional C₁₈ phases. For comparison a mixture of iodine-isomerized lutein and zeaxanthin standards was separated.     

Enrichment of serum low-molecular-weight proteins using C18 absorbent under urea/dithiothreitol denatured environment

Serum low-molecular-weight proteins (LMWPs, molecular weight <30 kDa) are closely related to the body physiological and pathological situations, whereas many difficulties are encountered when enriching and fractionating them. Using C₁₈ absorbent (100 Å) enrichment and fractionation under urea/dithiothreitol (DTT) denatured environment followed by 60% acetonitrile (ACN) elution, serum LMWPs could be enriched more than 100-fold and were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE), and isotope-coded affinity tag (ICAT) labeling quantification. Proteins existing in human serum at low nanograms/milliliter (ng/ml) levels, such as myeloid-related proteins (MRPs), could be identified directly from 2-DE coupled with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and LTQ-Orbitrap MS. Sixteen proteins were confidentially identified and quantified using ICAT labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS). By virtue of its easy operation and high reproducibility to process large quantity complex serum samples, this method has potential uses in enriching LMWPs either in serum or in cell and tissue samples.     

Stereoselective determination of mepivacaine in human serum using a brush-type chiral stationary phase and solid-phase extraction

A stereoselective high-performance liquid chromatography assay method was developed for the quantitation of R-(+)- and S_-(−)-mepivacaine in human serum. The assay uses a Pirkle brush-type. ((S)-tert.-leucine, (R)-(-naphthyl)ethylamine stationary phase (Sumichiral OA-4700, 250×4 mm I.D.) at ambient temperature with a mobile phase of hexane-ethylenedichloride-absolutte methanol (85:10:5, v/v) for the separation of R-(+) and (S)-(−)-mepivacaine. The eluents were monitored using UV detection at 220 nm. Isolation of the analytes from serum was performed using a 1-ml C₁₈ solid-phase extraction cartridge with high recovery and selectivity. The detection limits were 100 ng/ml for each enantiomer and the limits of quantitation were 150 ng/ml for both enantiomers. Linear calibration curves in the 150–2400 ng/ml range showed good correlation coefficients (r>0.9994, N=3). Precision and accuracy of the method were within 2.1–5.3 and 2.0–3.6%, respectively, for (R)-(+)-mepivacaine and 2.7–5.7% and 1.7–4.2%, respectively, for S-(−)-mepivacaine.     

High pressure liquid chromatograph ic separation of C18 AND C19 steroids

High Pressure liquid chromatographic procedures for the resolution of mixtures of C₁₈ and C₁₉ steroids of biologic importance are described. On-line monitoring of the absorbance and refractive index of the eluate was found to be suitable for the quantitative measurement of the amounts of eluted steroids. Using a high sensitivity absorbance detector, reliable measurements of as little as 5 ng Δ⁴?3 keto steroids were possible.     

Functional properties of diapophytoene and related desaturases of C30 and C40 carotenoid biosynthetic pathways

The desaturation reactions of C₃₀ carotenoids from diapophytoene to diaponeurosporene was investigated in vitro and by complementation in Escherichia coli. The expressed diapophytoene desaturase from Staphylococcus aureus inserts three double bonds in an FAD-dependent reaction. The enzyme is inhibited by diphenylamine. In the complementation experiment diapophytoene desaturase was able to convert C₄₀ phytoene to some extend but exhibited a high affinity to ζ-carotene. Comparison to the reaction of a phytoene desaturase from Rhodobacter capsulatus catalyzing a parallel three-step desaturation sequence with the corresponding C₄₀ carotenes revealed that this desaturase can also convert C₃₀ diapophytoene. Other homologous bacterial C₄₀ carotene desaturases could also utilize C₃₀ substrates, including one type of ζ-carotene desaturase which converted diaponeurosporene to diapolycopene. Further complementation experiments including the diapophytoene synthase gene from S. aureus revealed that the C₃₀ carotenogenic pathway is determined by this initial enzyme which is highly homologous to C₄₀ phytoene synthases.     

Liquid chromatographic method for the determination of uridine in human serum

A validated gas chromatography (GC)-mass spectrometric (MS) method for the analysis of hydroxyproline in rat femur is reported. Hydroxyproline in bone hydrolysates was extracted with an anion exchange resin and the N(O)-tert-butyldimethylsilyl derivatives analyzed by GC-MS. The hydroxyproline concentration was estimated relative to pipecolic acid, 3,4-dehydroproline and n-tetracosane as internal standards. The mass-to-charge ratios (m/z) for the ions used for quantitation by single ion monitoring were 314 m/z for hydroxyproline, 198 m/z for pipecolic acid, 256 m/z for dehydroproline and 57 m/z for n-tetracosane. A coefficient of variation of 5.8% was achieved and the limit of detection was calculated to be 0.233 micromol/l bone hydrolysate.     

Efficient method for the quantitation of urinary leukotriene E4: extraction using an Empore C18 disk cartridge

We describe here an efficient procedure for the precise quantitation of leukotriene E₄ (LTE₄) in a small volume of urine, which was achieved mainly by the use of an Empore extraction disk cartridge. After addition of [³H]LTE₄ to 2 ml of urine, an Empore C₁₈ cartridge was used for initial extraction of the urine, which resulted in the extraction of LTE₄ in a small volume of solvent. The eluate could then be injected onto a high-performance liquid chromatography column without further concentration. After separation by high-performance liquid chromatography, LTE₄ was extracted from the effluent using an Empore C₁₈ cartridge. The concentration of LTE₄ was subsequently quantified by enzyme immunoassay. LTE₄ can be recovered from urine with sufficient efficiency (69.9±4.7%, mean±S.D., n = 101). The coefficient of variation of the assay procedure was less than 10%. When urine was spiked with different amounts of LTE₄, the recovery of LTE₄ added to the urine specimen was less than 120%. The concentration of LTE₄ in urine from normal healthy subjects was 48.0±15.3 pg/mg creatinine (n = 15).     

C3与C4植物的环境调控

环境条件决定着不同光合类型植物的地理分布范围和区域 ,一般来说 ,C4 植物分布于高温、强光的环境而 C3植物分布于阴凉、湿润的环境 ,且 C4 比 C3植物光合速率高。但环境条件影响着不同光合类型植物的光合潜能的发挥 ,C4 植物在高温、强光、干旱条件下所表现出来的优势在其它环境条件下未必就显现出来。环境条件甚至可以引起 C3、C4 光合途径间的相互转化 ,这使得目前几种鉴别植物光合类型的方法出现不一致的结果。因此 ,在判断植物的光合类型时 ,要注意多种手段的综合利用 ,同时注意植物所处环境条件的影响。     

High-performance liquid chromatographic separation of ondansetron enantiomers in serum using a cellulose-derivatized stationary phase and solid-phase extraction

R(−)-Ondansetron and S(+)-ondansetron in human serum were resolved and quantified using a stereospecific HPLC method. Each enantiomer and the internal standard prazosin were isolated from serum using a solid-phase extraction procedure on a cyanopropyl column. Recoveries of 97, 96 and 88% were obtained for the R(−)-enantiomer, the S(+)-enantiomer, and the internal standard, respectively. A cellulose-based chiral analytical column (Chiralcel OD) was used with a mobile phase consisting of hexane—95% ethanol—2-propanol—acetonitrile (65:25:10:1, v/v). Linear calibration curves were obtained for each enantiomer in serum in the concentration range 10–200 ng/ml. The limit of quantitation of each enantiomer was 10 ng/ml. The detection limit for each enantiomer in serum using UV detection at 216 nm was 2.5 ng/ml (signal-to-noise ratio of 3).     

Liquid chromatographic determination of the sphinganine/sphingosine ratio in serum

The fumonisin mycotoxins, which are worldwide contaminants of corn, inhibit de novo sphingolipid biosynthesis leading to elevation in the ratio of the sphingoid bases, sphinganine and sphingosine, in the serum of animals exposed to fumonisins. A new HPLC method for the determination of the ratio of these bases in serum has been developed involving lipid extraction, clean-up on a silica minicolumn and alkaline hydrolysis prior to precolumn o-phthaldialdehyde derivatisation and HPLC separation and quantification by fluorescence detection. Based on serum from both normal and fumonisin-exposed vervet monkeys, the method was shown to be reproducible (R.S.D.<10%).     

Surface lipid composition of C3 and C4 plants

The characteristic surface lipid compositions of several C₃ and C₄ plants are discussed. C₄ plants produce surface lipids (epicuticular waxes) made up of the ubiquitous classes of aliphatic compounds: free fatty acids, aldehydes, primary alcohols, alkanes and aliphatic linear esters. C₃ plants synthesize surface lipids comprising the ubiquitous classes and either of the two following groups of compound: (i) lβ-diketones, hydroxy lβ-diketones, alkan-2-ol esters; (il) ketones and secondary alcohols with the functional group in the middle of the hydrocarbon chain. These features are suggested to represent physioIogical characteristics of the plant and to be related to ecological adaptations. Wax class compositions might also be an ancillary method for defining the C₃ or C₄ mechanism of CO₂ assimilation in cases where uncertainty exists.     

Improved chromatographic performance of a modified human albumin based stationary phase

Derivatization of the free cys₃₄ in human serum albumin (HSA) anchored to a silica matrix has been performed by in situ reaction with ethacrynic acid. This modification, which is reported to occur under physiological conditions, gives rise in practice to a new column with different binding properties with respect to the column based on the native protein. Significant differences were observed in the binding of drugs known to bind to site I, (R)-(S)-warfarin and phenylbutazone, and to site II, 1,4-benzodiazepin-2-ones and nonsteroidal anti-inflammatory agents. In particular, the chromatographic retentions markedly decreased for most of the drugs, and, in the case of chiral compounds, significant differences were often observed in the behavior of the two enantiomers, with higher values of enantioselectivity obtained for some of the examined compounds. Furthermore, the noncovalent binding of ethacrynic acid to the protein modifies the binding properties of the albumin. Chirality 9:335–340, 1997. © 1997 Wiley-Liss, Inc.     

High-performance liquid chromatography determination of praziquantel enantiomers in human serum using a reversed-phase cellulose-based chiral stationary phase and disc solid-phase extraction

A sensitive HPLC method for the quantification of praziquantel enantiomers in human serum is described. The method involves the use of a novel disc solid-phase extraction for sample clean-up prior to HPLC analysis and is also free of interference from trans-4-hydroxypraziquantel, the major metabolite of praziquantel. Chromatographic resolution of the enantiomers was performed on a reversed-phase cellulose-based chiral column (Chiralcel OJ-R) under isocratic conditions using a mobile phase consisting of 0.1 M sodium perchlorate–acetonitrile (66:34, v/v) at a flow-rate of 0.5 ml/min. Recoveries for R-(−)- and S-(+)-praziquantel enantiomers were in the range of 84–89% at 50–500 ng/ml levels. Intra-day and inter-day precisions calculated as R.S.D. were in the ranges of 3–8% and 1–8% for both enantiomers, respectively. Intra-day and inter-day accuracies calculated as percent error were in the 0.2–5% and 0.3–8% ranges for both enantiomers, respectively. Linear calibration curves were in the concentration range 10–600 ng/ml for each enantiomer in serum. The limit of quantification of each enantiomer was 10 ng/ml. The detection limit for each enantiomer in serum using a UV detector set at 210 nm was 5 ng/ml (S/N=2).     

Liquid chromatographic determination of irbesartan in human plasma

A simple and sensitive method was developed for determination of irbesartan by liquid chromatography with fluorescence detection. Irbesartan and losartan (I.S.) in human plasma were extracted using diethyl ether:dichloromethane (7:3, v/v) followed by back extraction with 0.05 M sodium hydroxide. Neutralized samples were analyzed using 0.01 M potassium dihydrogen phosphate buffer (containing 0.07% triethylamine as peak modifier, pH was adjusted with orthophosphoric acid to pH 3.0) and acetonitrile (66:34, v/v). Chromatographic separation was achieved on an ODS-C-18 column (100 mm x 4.6 mm i.d., particle size 5 microm) using isocratic elution (at flow rate 1.25 ml/min). The peak was detected using a fluorescence detector set at Ex 259 nm and Em 385 nm, and the total time for a chromatographic separation was approximately 13 min. The validated quantitation ranges of this method were 15-4000 ng/ml with coefficients of variation between 0.75 and 12.53%. Mean recoveries were 73.3-77.1% with coefficients of variation of 3.7-6.3%. The between- and within-batch precision were 0.4-2.2% and 0.9-6.2%, respectively. The between- and within-batch relative errors (bias) were (-5.5) to 0.9% and (-0.6) to 6.9%, respectively. Stability of irbesartan in plasma was >89%, with no evidence of degradation during sample processing and 60 days storage in a deep freezer at -70 degrees C. This validated method is sensitive and simple with between-batch precision of <3% and can be used for pharmacokinetic studies.     

Liquid chromatographic determination of amodiaquine in human plasma

A normal-phase high-performance liquid chromatographic method using dichloromethane- methanol-1M perchloric acid (100:10:0.9, v/v/v) at a flow rate of 1.0 ml min(-1) on a LiChrospher Si column with UV (254 nm) detection has been developed for the determination of amodiaquine and its metabolites desethyl amodiaquine and bisdesethyl amodiaquine in plasma. The limit of quantification was 5 ng ml(-1). Mean within-day and day-to-day coefficients of variation (CV) were 4.10 and 6.27% for amodiaquine, 3.43 and 4.80% for desethyl amodiaquine and 3.53 and 5.23% for bisdesethyl amodiaquine, respectively. Mean extraction recovery of amodiaquine, desethyl amodiaquine and bisdesethyl amodiaquine from plasma were 82.48, 74.50 and 69.65%, respectively. Chloroquine and its metabolite desethyl chloroquine, quinine, sulfadoxine and primaquine do not interfere in the detection of amodiaquine, desethyl amodiaquine and bisdesethyl amodiaquine in plasma.