Rapid and sensitive determination of amprolium in chicken plasma by high-performance liquid chromatography with post-column reaction

A rapid, sensitive and reproducible reversed-phase HPLC assay was developed for the determination of amprolium (APL) in chicken plasma. Protein in plasma sample was precipitated with 0.33 M perchloric acid and supernatant solution was injected into the HPLC system. Following the chromatographic separation of APL and the beclotiamine (I.S.) on a C₁₈ column, the derivatives of APL and I.S. were formed by post-column reaction and detected by fluorescence detection (excitation at 400 nm, emission at 460 nm). The method showed excellent precision, accuracy and speed with a detection limit of 2 ng/ml. The intra- and inter-assay variance of this method were less than 11.2%. This method has been successfully applied to plasma determinations after oral administration of APL to chicken.     

Direct separation of albuterol enantiomers in biological fluids and pharmaceutical formulations using α1-acid glycoprotein and pirkle urea type columns

A direct, isocratic, and simple chromatographic method is described for the resolution of racemic albuterol using the α₁-acid glycoprotein chiral stationary phase (AGP-CSP) under reverse phase conditions. The effect of various organic modifiers, temperature, and phosphate buffer ionic strength on the separation factor (α) and stereochemical resolution factor (R_s) has been studied. The enantiomeric separation of albuterol was also achieved using a urea-type CSP of (S)-indoline-2-carboxylic acid and (R)-1-(α-naphthyl)ethylamine, known as Chirex 3022, running in the normal phase mode. The effect of different organic acids added to the mobile phase was examined and the chiral recognition mechanism(s) is discussed. Solid phase extraction with C₁₈ Sep-Pak cartridges was applied as a clean-up step to determine the enantiomeric ratio between (?)-R and (+)-S-albuterol in pharmaceutical formulations and in human plasma. © 1995 Wiley-Liss, Inc.     

Copper(II) complexes of N2-alkyl-(S)-amino acid amides as chiral selectors for dynamically coated chiral stationary phases in RP-HPLC

Copper(II) complexes of N²-octyl-(S)-phenylalaninamide (Noc-Phe-NH₂), N²-dodecyl-(S)-phenylalaninamide (Ndo-Phe-NH₂), and N²-octyl-(S)-norleucinamide (Noc-NLeu-NH₂), dynamically adsorbed on a reversed-phase C₁₈ column, were able to perform the direct enantiomeric separation of unmodified amino acids, amino acid amides and esters, hydroxy acids, and dipeptides by elution with aqueous or mixed aqueous-organic solutions containing copper(II) sulphate or acetate. The role played by several parameters in the separation procedure was examined with the copper(II) complex of Noc-Phe-NH₂ [concentration of the copper(II) ion in the eluent, pH and eluent polarity, amount of adsorbed selector]. The separation was shown to occur entirely on the stationary phase. The mechanism of chiral discrimination is discussed in terms of the chromatographic parameters and of the structure of the copper(II) complexes in solution and in the solid state. The chiral stationary phase maintained its separation ability for about 3 months. However, the column could be easily restored by recovering the selector with methanol and repeating the loading procedure. © 1996 Wiley-Liss, Inc.     

Epidermal structures and composition of epicuticular waxes of Sedum album sensu lato (Crassulaceae) in Balkan Peninsula

Sedum album is a succulent plant, with phenotypic variability resulting in delimitation of a number of taxa with diverse taxonomic recognition. We analyzed the taxa from Sedum album in broad sense (S. album, S. micranthum, S. athoum and S. serpentini) from Balkan Peninsula, to check whether the variability of their epidermal structures and epicuticular wax composition corresponds to their taxonomic reliability. Epidermal structures and epicuticular waxes were analyzed using light and scanning electron microscopy, gas chromatography-mass spectrometry and gas chromatography-flame ionization detector chromatography and multivariate statistical techniques. Analyses of studied taxa have shown that significant systematic characters include adaxial epidermal and guard cell length, stomatal frequency and subsidiary cells area on abaxial epidermis. The content of n-alkanes C₂₉, C₃₀, C₂₇, C₃₂ and C₃₃ was shown to be valuable criterion for their phytochemical delimitation. The comparison of epicuticular wax microstructures has shown no significant differences. Results indicate presence of two (micromorphology) or three (phytochemistry) well-defined groups of populations, with S. serpentini manifesting a considerable level of separation toward the others. Sedum athoum have shown weak to moderate degree of differentiation, while S. micranthum has shown general resemblance to S. album.     

Enantiomeric NMR signal separation behavior and mechanism of samarium(III) and neodymium(III) complexes with (S,S)‐ethylenediamine‐N,N'‐disuccinate

Enantiomeric ¹H and ¹³C NMR signal separation behaviors of various α‐amino acids and DL‐tartarate were investigated by using the samarium(III) and neodymium(III) complexes with (S ,S )‐ethylenediamine‐N ,N' ‐disuccinate as chiral shift reagents. A relatively smaller concentration ratio of the lanthanide(III) complex to substrates was suitable for the neodymium(III) complex compared with the samarium(III) one, striking a balance between relatively greater signal separation and broadening. To clarify the difference in the signal separation behavior, the chemical shifts of β‐protons for fully bound D‐ and L‐alanine (δ_b(D) and δ_b(L)) and their adduct formation constants (K s) were obtained for both metal complexes. Preference for D‐alanine was similarly observed for both complexes, while it was revealed that the difference between the δ_b(D) and δ_b(L) values is the significant factor to determine the enantiomeric signal separation. The neodymium(III) and samarium(III) complexes can be used complementarily for higher and smaller concentration ranges of substrates, respectively, because the neodymium(III) complex gives the larger difference between the δ_b(D) and δ_b(L) values with greater signal broadening compared to the samarium(III) complex.     

A simple and rapid harvesting method for microalgae by in situ magnetic separation

A simple and rapid harvesting method by in situ magnetic separation with naked Fe₃O₄ nanoparticles has been developed for the microalgal recovery of Botryococcus braunii and Chlorella ellipsoidea. After adding the magnetic particles to the microalgal culture broth, the microalgal cells were adsorbed and then separated by an external magnetic field. The maximal recovery efficiency reached more than 98% for both microalgae at a stirring speed of 120 r/min within 1 min, and the maximal adsorption capacity of these Fe₃O₄ nanoparticles reached 55.9 mg-dry biomass/mg-particles for B. braunii and 5.83 mg-dry biomass/mg-particles for C. ellipsoidea. Appropriate pH value and high nanoparticle dose were favorable to the microalgae recovery, and the adsorption mechanism between the naked Fe₃O₄ nanoparticles and the microalgal cells was mainly due to the electrostatic attraction. The developed in situ magnetic separation technology provides a great potential for saving time and energy associated with improving microalgal harvesting.     

Estimation of amniotic fluid phospholipids by high-performance liquid chromatography

We have developed a high-performance liquid chromatographic (HPLC) method for the analyses of surface-active amniotic fluid phospholipids, lecithin (L), sphingomyelin (S), phosphatidyl glycerol (PG), phosphatidyl inositol (PI), phosphatidyl ethanolamine (PE), and phosphatidyl serine (PS), which are important in the prediction of fetal lung maturity. The method incorporates an internal standard in the amniotic fluid extract, and utilizes a 10-μl aliquot of a 2:1 chloroform—methanol extract of amniotic fluid injected onto a 5-μm DIOL or CN HPLC column, and a variable-wavelength detector set at 203 nm.Amniotic fluid phospholipid estimations were determined on 40 amniotic fluid samples by the HPLC method and by the routine thin-layer chromatographic (TLC) method. Good agreement was observed between the two methods for the L/S ratio, PG, and PI (r_PG 0.94, r_PI 0.95, r_L/S 0.97).The advantages of the HPLC procedure include: (i) Selective separation for PG, PI, PS, and PE, as well as L and S at the same time. (ii) The internal standard allows individual concentration of phospholipids to be estimated. (iii) The procedure is rapid: 16 min for a single assay compared with 50 min for the standard TLC procedure.     

On-line monitoring of the purification of GST-(His)6 from an unclarified Escherichia coli homogenate within an immobilised metal affinity expanded bed

The use of a rapid chromatographic assay to monitor the level of a specific protein during its downstream processing by expanded bed adsorption is described. An expanded bed column (5 cm diameter) has been modified to allow the abstraction of liquid samples at various heights along the bed, in an automated, semi-continuous manner throughout the separation. The withdrawn samples were filtered in-line and the level of the target protein assayed by a rapid on-line chromatographic method. Using this technique it was possible to monitor the development of adsorbate profiles during the loading, washing and elution phases of the application of an unclarified feedstock. The potential of the technique is demonstrated using the separation of histidine tagged glutathione s-transferase (GST-(His)₆) from an unclarified Escherichia coli homogenate using an expanded bed of Ni²⁺ loaded STREAMLINE Chelating^TM. The level of GST-(His)₆ in the abstracted homogenate samples was measured using Zn²⁺ loaded NTA-silica as an affinity chromatographic sensor. The approach described demonstrates potential for the on-line monitoring and control of expanded bed separations and for providing a greater understanding of adsorption/desorption and hydrodynamic processes occurring within the bed.     

Enantiomeric resolution of some nonsteroidal antiinflammatory and anticoagulant drugs using β‐cyclodextrins by capillary electrophoresis

β‐cyclodextrin (CD) and its derivatives HP‐β‐CD, DM‐β‐CD, and TM‐β‐CD have been employed as chiral selectors for the separation of three nonsteroidal antiinflammatory drugs (NSAIDs) and anticoagulant at relatively low concentration (8–15 mM) by capillary zone electrophoresis (CZE). In this study, baseline separation was achieved for ibuprofen, ketoprofen, naproxen, and warfarin. It was found that the addition of 0.1% hydroxypropyl methyl cellulose (HPMC) was effective for separation. Under these conditions, the S‐(+) enantiomer eluted before R‐(−) in terms of ibuprofen; the calculated energy values obtained from the molecular modeling correlated well with the elution order. An equation for calculating the pK_a values by capillary electrophoresis was introduced, and the pK_a values of the four chiral drugs at 25°C were obtained based on the equation. The value pK_a + 0.5 is proposed to be the suitable pH of the background electrolyte for the separation of chiral compounds containing a carboxylic group. Chirality 11:56–62, 1999. © 1999 Wiley‐Liss, Inc.     

速生槐叶蘋入侵风险评估及管理措施分析

[目的]速生槐叶蘋是一种原产于巴西东南部的多年生漂浮型水生植物,广泛引种至世界各地后逃逸,侵入湖泊、河流和稻田等各种水生生境。通过开展速生槐叶蘋入侵风险评估,进而提出其管理措施,可为其检疫决策及防治提供科学依据。[方法]通过查阅梳理文献,分别对速生槐叶蘋的国内外分布、传播方式及入侵扩散历史、防治方法、生态适应性和抗逆性、生物学和遗传特性、危害性及利用价值等方面进行定性分析。并基于外来植物风险指标体系和判断标准,对4个指标层的17个指标和32个问题进行量化赋值,完成速生槐叶蘋的定量分析。[结果]速生槐叶蘋通过自然和人为传播方式已经入侵全世界57个国家地区,可通过无性繁殖快速分裂生长建立种群,其环境适应性和抗逆性强、竞争能力强、检疫鉴定和根除难度大,对经济、生态和社会造成巨大负面影响。速生槐叶蘋的入侵性R₁为72,适应性R₂为53,扩散性R₃为82,危害性R₄为81,风险值R为73.4,应归为严格禁止引入的植物。[结论]尽管速生槐叶蘋在中国的分布范围有限,但属于高入侵风险物种,其潜在的逃逸扩散风险和危害性不可忽视,应严格禁止引入。考虑到该种已经传入我国且有作为观赏植物的市场需求,应制定相应的风险管理制度,定期开展检疫、监测和灭除,防止其逃逸至野外定殖于其他还未发生的地区。     

泥炭地苔藓植物孢子生活力的快速检测

适量的烟气能够促进有性繁殖体萌发,但迄今尚无辅助烟气处理探究孢子生活力快速检测方法的研究报道。该文选择毛缘泥炭藓（Sphagnum fimbriatum）、中位泥炭藓（S.magellanicum）和粗叶泥炭藓（S.squarrosum）作为材料,分别使用亚甲基蓝染色法、四唑（TTC）染色法、碘-碘化钾（I₂-KI）染色法和红墨水染色法对泥炭藓孢子进行染色,并比照营养液、烟溶液+营养液培养的孢子萌发试验,对比研究泥炭地苔藓植物孢子生活力快速检测的最佳方法。结果表明：亚甲基蓝染色法的染色效果最为明显,TTC和I₂-KI均未能使泥炭藓孢子着色,孢子对红墨水虽有着色反应但不清晰;与营养液培养相比,添加烟溶液使毛缘泥炭藓、中位泥炭藓和粗叶泥炭藓孢子萌发率分别提高5%、5%和18%;使用亚甲基蓝染色的孢子染色率与经烟溶液处理过的孢子萌发率最为接近。综上认为,亚甲基蓝染色法能快速检测泥炭藓孢子的生活力。     

A Single Nucleotide Polymorphism and Sequence Analysis of CSN1S1 Gene Promoter Region in Chinese BOS Grunniens (YAK)

The aim of this study was to investigate the polymorphism of the CSN1S1 gene promoter region in 4 Chinese yak breeds, and compare the yak CSN1S1 gene promoter region sequences with other ruminants. A Polymerase Chain Reaction-Single Strand Conformation Polymorphism protocol was developed for rapid genotyping of the yak CSN1S1 gene. One hundred fifty-eight animals from 4 Chinese yak breeds were genotyped at the CSN1S1 locus using the protocol developed. A single nucleotide polymorphism of the CSN1S1 gene promoter region has been identified in all yak breeds investigated. The polymorphism consists of a single nucleotide substitution G→A at position 386 of the CSN1S1 gene promoter region, resulting in two alleles named, respectively, G₃₈₆ and A₃₈₆, based on the nucleotide at position 386. The allele G₃₈₆ was found to be more common in the animals investigated. The corresponding nucleotide sequences in GenBank of yak (having the same nucleotides as allele G₃₈₆ in this study), bovine, water buffalo, sheep, and goat had similarity of 99.68%, 99.35%, 97.42%, 95.14%, and 94.19%, respectively, with the yak allele A_386.     

Determination of flunarizine in rat brain by liquid chromatography–electrospray mass spectrometry

A rapid liquid chromatography–electrospray mass spectrometry (LC–ES-MS) assay for the determination of flunarizine (FZ) in rat brain has been developed. A C₁₈ column and an isocratic elution were employed for the separation. Using post-column split, 64% of the eluent was introduced into the ES-MS system for detection. The [M+H]⁺ (m/z 406) and a fragmented ion (m/z 203) were detected using selected ion monitoring. The linear range of this assay was good, ranging from 0.05 to 5 μM (r²=0.99). The intra- and inter-day precisions showed relative standard deviations ranging from 1.4% to 2.0% and 1.3% to 2.9%, respectively. The application of this newly developed method was demonstrated by examining the pharmacokinetics of FZ in rat brain.     

The direct determination of the enantiomers of ketorolac and parahydroxyketorolac in plasma and urine using enantioselective liquid chromatography on a human serum albumin-based chiral stationary phase

An enantioselective assay has been developed for the determination of the enantiomers of ketorolac and its metabolite p-hydroxyketorolac in plasma and urine. The analytical method utilizes a coupled achiral–chiral HPLC system where the initial separation of ketorolac from p-hydroxyketorolac and matrix interferences was achieved on a C₁₈-stationary phase and the enantioselective separations of the two target solutes were accomplished on a human serum albumin-based chiral stationary phase. The two columns were attached in sequence and the assay was carried out without the necessity of column-switching techniques. The method has been validated for use in pharmacokinetic and metabolic studies and represents the initial report of the determination of ketorolac and p-hydroxyketorolac enantiomers in urine. The results of the study indicate that after the administration of racemic ketorolac there was an enantioselective distribution of ketorolac enantiomers in plasma [(R)-ketorolac: (S)-ketorolac = 3.89 ± 0.93 (n = 6) and urine (R)-ketorolac: (S)-ketorolac = 1.26 ± 0.09 (n = 7)]. The mean ratio of the p-hydroxyketorolac enantiomers was 1.77 ± 0.46 (n = 7). Both ketorolac and p-hydroxyketorolac are glucuronized in the acyl carboxyl moiety and the results of this study indicate that this process is not enantiospecific. © 1994 Wiley-Liss, Inc.     

Synthesis and pKa determination of new enantiopure dimethyl‐substituted acridino‐crown ethers containing a carboxyl group: Useful candidates for enantiomeric recognition studies

New enantiopure dimethyl‐substituted acridino‐18‐crown‐6 and acridino‐21‐crown‐7 ethers containing a carboxyl group at position 9 of the acridine ring [(S,S )‐ 8 , (S,S )‐ 9 , (R,R )‐ 10 ] were synthesized. The pK _a values of the new crown ethers [(S,S )‐ 8 , (S,S )‐ 9 , (R,R )‐ 10 ] and of an earlier reported macrocycle [(R,R )‐ 2 ] were determined by UV‐pH titrations. Crown ether (S,S )‐ 8 was attached to silica gel by covalent bonds and the enantiomeric separation ability of the newly prepared chiral stationary phase [(S,S )‐CSP‐ 12 ] was studied by high‐performance liquid chromatography (HPLC). Homochiral preference was observed and the best separation was achieved for the enantiomers of 1‐NEA. Ligands (S,S )‐ 9 and (R,R )‐ 10 are precursors of enantioselective sensor and selector molecules for the enantiomers of protonated primary amines, amino acids, and their derivatives.     

Characterization of heterochromatin in different species of the Scilla siberica group (Liliaceae) by in situ hybridization of satellite DNAs and fluorochrome banding

Satellite DNAs have been isolated from the monocotyledonous plants Scilla siberica, S. amoena, S. ingridae (all are highly GC-rich), and S. mischtschenkoana by using the Ag⁺ –Cs₂SO₄ density centrifugation technique. Hybridization in situ has been performed with ₃H-cRNA to these satellite DNAs in all four species. In each species, the endogenous satellite DNA is located mainly in intercalary and major heterochromatin bands associated with terminal regions and nucleolar organizer regions (NORs) but not in centromeric regions. Patterns observed after cross-species hybridization show a high degree of satellite DNA homology between S. siberica, S. amoena, and S. ingridae. By contrast, satellite DNA of S. mischtschenkoana consists largely of different, non homologous DNA sequences, with two exceptions: (i) the NORs of all four species contain similar satellite sequences, and (ii) a strong homology exists between the satellite DNA of S. mischtschenkoana and centromeric DNA of S. siberica but not with those of S. amoena and S. ingridae. — Heterochromatin has also been characterized by the AT-specific fluorochromes quinacrine (Q) and DAPI and the GC-specific agent chromomycin A₃ (CMA₃), in combination with two counterstaining techniques. While CMA₃-fluorescence is largely in agreement with data on base composition and location of the specific satellite DNAs, the results with Q and DAPI are conflicting. Prolonged fixation has been found to change the fluorescence character in certain instances, indicating that other factors than the base sequence of the DNA also play a role in fluorochrome staining of chromosomes. The results are discussed in relation to the taxonomy and phylogeny of the four species.     

Chromatographic profiles of tryptophan and kynurenine enantiomers derivatized with (S)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole using LC–MS/MS on a triazole‐bonded column

d ‐ and l ‐Tryptophan (Trp) and d ‐ and l ‐kynurenine (KYN) were derivatized with a chiral reagent, (S )‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole (DBD‐PyNCS), and were separated enantiomerically by high‐performance liquid chromatography (HPLC) equipped with a triazole‐bonded column (Cosmosil HILIC) using tandem mass spectrometric (MS/MS) detection. Effects of column temperature, salt (HCO₂NH₄) concentration, and pH of the mobile phase in the enantiomeric separation, followed by MS detection of (S )‐DBD‐PyNCS‐d ,l ‐Trp and ‐d ,l ‐KYN, were investigated. The mobile phase consisting of CH₃CN/10 mM ammonium formate in H₂O (pH 5.0) (90/10) with a column temperature of 50–60 °C gave satisfactory resolution (R s) and mass‐spectrometric detection. The enantiomeric separation of d ,l ‐Trp and d ,l ‐KYN produced R s values of 2.22 and 2.13, and separation factors (α) of 1.08 and 1.08, for the Trp and KYN enantiomers, respectively. The proposed LC–MS/MS method provided excellent detection sensitivity of both enantiomers of Trp and KYN (5.1–19 nM).     

A karyotypic study on Silene sect. Sclerocalycinae in Turkey

The karyology of fifteen taxa of Silene sect. Sclerocalycinae from Turkey has been investigated. Karyotype analyses has been carried out and the chromosome number of Silene caramanica, S. peduncularis, S. armena, S. laxa, S. swertiifolia, S. caesarea, S. sclerophylla, S. haradjianii and S. lycaonica have been determined for the first time. For all the taxa studied, the diploid chromosome number and the basic chromosome number were found to be 2n=24 and x=12, respectively. Except for S. chlorifolia and S. doganii, the karyomorphology of the taxa studied is here described for the first time. S. laxa was found to have the smallest chromosomes whereas the largest ones were observed in S. haradjianii. Silene chlorifolia had the highest A₁ index and S. bupleuroides subsp. bupleuroides had the highest interchromosomal asymmetry coefficient (A₂).     

Abscisic acid-triggered guard cell l-cysteine desulfhydrase function and in situ hydrogen sulfide production contributes to heme oxygenase-modulated stomatal closure

Recent studies have demonstrated that hydrogen sulfide (H₂S) produced through the activity of l -cysteine desulfhydrase (DES1) is an important gaseous signaling molecule in plants that could participate in abscisic acid (ABA)-induced stomatal closure. However, the coupling of the DES1/H₂S signaling pathways to guard cell movement has not been thoroughly elucidated. The results presented here provide genetic evidence for a physiologically relevant signaling pathway that governs guard cell in situ DES1/H₂S function in stomatal closure. We discovered that ABA-activated DES1 produces H₂S in guard cells. The impaired guard cell ABA phenotype of the des1 mutant can be fully complemented when DES1/H₂S function has been specifically rescued in guard cells and epidermal cells, but not mesophyll cells. This research further characterized DES1/H₂S function in the regulation of LONG HYPOCOTYL1 (HY1, a member of the heme oxygenase family) signaling. ABA-induced DES1 expression and H₂S production are hyper-activated in the hy1 mutant, both of which can be fully abolished by the addition of H₂S scavenger. Impaired guard cell ABA phenotype of des1/hy1 can be restored by H₂S donors. Taken together, this research indicated that guard cell in situ DES1 function is involved in ABA-induced stomatal closure, which also acts as a pivotal hub in regulating HY1 signaling.     

Chiral discrimination by ligand-exchange chromatography: A comparison between phenylalaninamide-based stationary and mobile phases

The copper(II) complexes of two new diastereomeric ligands, N²-(R)- and N²-(S)-2′-hydroxypropyl-(S)-phenylalaninamide [(R, S)-1 and (S, S)-1], have been used as additives to the eluent in high-performance liquid chromatography (HPLC) reversed phase for the chiral separation of DNS-amino acids. The aim was that of comparing the separation process obtained by the chiral eluent with that obtained by an analogous bonded stationary phase containing (S)-phenylalaninamide, previously studied [CSP-(S)-Phe-NH₂]. The affinity of the ternary complexes for the C₁₈ column was determined by adsorption experiments in HPLC. It was shown that the two systems (chiral eluent, chiral stationary phase) work according to different mechanisms. Ternary complex formation in solution was studied by fluorescence spectroscopy. It was shown that chiral separation with the Cu(II) complexes added to the eluent was determined by the relative affinities of the ternary complexes for the column-stationary phase rather than by their stabilities in solution. With CSP-(S)-Phe-NH₂ the separation is accounted for by the relative stabilities of the ternary complexes, which depends mainly on the “allowed” geometry of the complex and on the steric repulsion of the amino acid side chain with the spacer. © 1996 Wiley-Liss, Inc.