Separation and Quantification of Abscisic Acid and its Metabolites by High-performance Liquid Chromatography

A technique for the separation and quantitative estimation ofabscisic acid and its metabolic products, phaseic acid and dihydrophaseicacid by high performance liquid chromatography (HPLC) is described.The efficiency of two HPLC columns, LiChrosorb RP-18 and LiChrosorbNH₂ is compared. The method has been tested using extracts fromtomato plants and some of the results are presented. Abscisic acid, dihydrophaseic acid, phaseic acid, chromatography, tomato, Lycopersicon esculentum, Mill     

Solid phase extraction on sax columns as an alternative for ochratoxin A analysis in maize

A sensitive and reliable method is described for the determination of ochratoxin A (OTA) in maize. An extraction and clean-up procedure was used, with chloroform-phosphoric acid as the extractant, and liquid-liquid partition and anion-exchange chromatography (SAX columns) for the clean-up. Quantification of toxin is achieved by high performance liquid chromatography (HPLC). Recoveries were between 81-94 % at 3-90 ng/g levels. The detection limit was 0.02 ng.     

Simultaneous determination of tryptophan and its metabolites in mouse brain by high-performance liquid chromatography with fluorometric detection

A new method for the determination of tryptophan and its metabolites in a single mouse brain using high-performance liquid chromatography (HPLC) with fluorometric detection is described. Tryptophan, serotonin, 5-hydroxyindoleacetic acid, indoleacetic acid, and tryptophol were clearly separated by a C8 reverse-phase column. Tissue preparation is performed only to centrifuge homogenates of brain prior to the injection to HPLC. The sensitivity is in the range from 10 to 15 pg.     

Quantitative determination of 5-hydroxytryptophan in dissected brain regions by on-line trace enrichment HPLC with electrochemical detection

A sensitive, specific and rapid quantitative HPLC assay for 5-hydroxytryptophan (5-HTP) in samples of brain regions of widely differing size is described. The method utilizes off-line prepurification of tissue supernatants on gravity-fed strong cation exchange columns, on-line enrichment of the entire cation exchange column eluate on short reverse phase enrichment precolumns, further separation by reverse phase chromatography on an analytical column and electrochemical detection. On-line trace enrichment permits the efficient incorporation of off-line column chromatography to maximize assay specificity without compromising assay sensitivity. A reliable, working limit of detection of 200 pg 5-HTP/sample permits the estimation of in vivo tryptophan hydroxylase activity by determining the rate of 5-HTP accumulation following L-aromatic amino acid decarboxylase inhibition in small discrete brain regions or larger tissue samples only poorly innervated by 5-HT terminals.     

Microsomal lauric acid 11- and 12-hydroxylation: a new assay method utilizing high pressure liquid chromatography.

A new assay method using high pressure liquid chromatography has been developed which permits the simultaneous isolation, determination, and quantitation of lauric acid and its hydroxylated products after methylation of extracts from kidney or liver microsomal incubation mixtures. The small differences in polarity between the lauric acid, 11-hydroxy- and 12-hydroxy-lauric acid after methylation permit their separation on reverse phase columns packed with octadecyltrichlorosilane bonded to silicone polymers. The total time required for the chromatography is less than 1 hr. Using this method, the formation of hydroxylated products was shown to have a linear dependence on protein concentration and time. The K_m for lauric acid and NADPH were determined to be 8 μm and 54 μm in kidney microsomes, respectively.     

高效液相色谱法同时检测棉花根中的多种植物激素

目的：探寻高效液相色谱同时检测棉花根中多种植物激素含量的方法。方法：采用WatersC18反相色谱柱（4.6×250mm,5μm）,在柱温为35℃、流速为1mL.min-1的条件下,以乙腈和三乙胺溶液为流动相梯度洗脱,在每种物质的保留时间附近切换至最大吸收峰（GA3除外）波长作为检测波长,并与254nm同一波长检测多种植物激素含量的方法进行比较,分离和检测棉花根中的玉米素（Z）、玉米素核苷（ZR）、赤霉酸（GA3）、生长素（IAA）和脱落酸（ABA）的含量。结果：切换波长法检测5种植物激素的灵敏度和回收率均较高,检出限均较低。回收率为：Z 96.82%、ZR 94.14%、GA3 92.75%、IAA 93.38%、ABA 95.57%;检出限为：Z 0.1μg.mL-1、ZR 0.1μg.mL-1、GA3 0.5μg.mL-1、IAA 0.3μg.mL-1;ABA 0.05μg.mL-1,能准确检测出棉花根中Z、ZR、GA3、IAA和ABA的含量。结论：采用Waters C18反相色谱柱（4.6×250mm,5μm）,在柱温为35℃、流速为1mL.min-1的条件下,以乙腈和三乙胺溶液为流动相梯度洗脱,结合切换波长法能同时检测出植物组织中多种植物激素含量。     

Direct analysis of salicylic acid,salicyl acyl glucuronide,salicyluric acid and gentisic acid in human plasma and urine by high-performance liquid chromatography

A method for the simultaneous direct determination of salicylate (SA), its labile, reactive metabolite, salicyl acyl glucuronide (SAG), and two other major metabolites, salicyluric acid and gentisic acid in plasma and urine is described. Isocratic reversed-phase high performance liquid chromatography (HPLC) employed a 15-cm C₁₈ column using methanol-acetonitrile-25 mM acetic acid as the mobile phase, resulting in HPLC analysis time of less than 20 min. Ultraviolet detection at 310 nm permitted analysis of SAG in plasma, but did not provide sensitivity for measurement of salicyl phenol glucuronide. Plasma or urine samples are stabilized immediately upon collection by adjustment of pH to 3–4 to prevent degradation of the labile acyl glucuronide metabolite. Plasma is then deproteinated with acetonitrile, dried and reconstituted for injection, whereas urine samples are simply diluted prior to injection on HPLC. m-Hydroxybenzoic acid served as the internal standard. Recoveries from plasma were greater than 85% for all four compounds over a range of 0.2–20 μg/ml and linearity was observed from 0.1–200 μg/ml and 5–2000 μg/ml for SA in plasma and urine, respectively. The method was validated to 0.2 μg/ml, thus allowing accurate measurement of SA, and three major metabolites in plasma and urine of subjects and small animals administered salicylates. The method is unique by allowing quantitation of reactive SAG in plasma at levels well below 1% that of the parent compound, SA, as is observed in patients administered salicylates.     

Highly sensitive assay for the measurement of serotonin in microdialysates using capillary high-performance liquid chromatography with electrochemical detection

A highly sensitive isocratic capillary high-performance liquid chromatographic (HPLC) method with electrochemical detection (ED) for the simultaneous measurement of serotonin (5-hydroxytryptamine, 5-HT) and its metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in microdialysates has been developed using a 0.5 mm i.d. capillary column and a 11-nL detection cell. This method, validated on both pharmacological and analytical bases, can be performed using injection volumes as low as 1 microL. The limits of detection were 5.6 x 10(-11)mol/L and 3.0 x 10(-9)mol/L for 5-HT and 5-HIAA. Several applications of the present method are given on microdialysates from rodent brain and human spinal cord.     

Studies on the dynamic accumulations of Sophora alopecuroides L. Alkaloids in different harvest times and the appropriate harvest time

A sensitive and accurate method for the simultaneous determination of five alkaloids, namely 9α-hydroxymatrine (M1), matrine (M2), sophoridine (M3), oxymatrine (M4), alopecurin A (M5) in different parts (seed, legume, stem, and root) and different harvest times of Sophora alopecuroides L. was developed by high performance liquid chromatography (HPLC) with photodiode array detector (PDA) for the first time. The separation by gradient elution was achieved on Scienhome Kromasil C(18) (4.6×250 mm, 5 μm) column at 30°C with acetonitrile (A)/0.1% phosphatic acid+0.1% triethylamine (B) as the mobile phase. The detection wavelength was 205 nm. The optimized method provided a good linear relation (r≥0.9993 for all the target compounds), satisfactory precision (RSD values less than 2.3%) and good recovery (96.4-103.6%). The limits of detection ranged between 0.11×10(-3) and 4.70×10(-3) μg for the different analytes. The method was successfully applied to analysis and quality control of alkaloid extracts from the traditional Chinese herbal drugs of S. alopecuroides L.     

Polystyrene/Divinylbenzene Based Monolithic and Encapsulated Capillary Columns for the Analysis of Nucleic Acids by High‐Performance Liquid Chromatography‐Electrospray Ionisation Mass Spectrometry

Monolithic capillary columns are prepared by copolymerization of styrene and divinylbenzene, encapsulated capillary columns by immobilizing silica particles with different pore sizes inside a 200 μm i.d. fused silica capillary by encapsulation of the derivatized silica sorbent in a poly(styrene/divinylbenzene) (PS/DVB) matrix. Both allow the rapid and highly efficient separation of single‐ and double‐stranded DNA by ion‐pair reversed‐phase high‐performance liquid chromatography (IP‐RP‐HPLC). The high resolving power of monolithic and encapsulated capillary columns can be utilized for mutation screening in polymerase chain reaction (PCR) amplified polymorphic loci by denaturing HPLC (DHPLC). Recognition of mutations is based on the separation of homo‐ and heteroduplex species by IP‐RP‐HPLC under denaturing conditions, resulting in characteristic peak patterns both for homozygous and heterozygous samples. Separations can be readily hyphenated to electrospray ionization‐mass spectrometry.     

A high-pressure liquid chromatographic method for measuring 3', 5'-cyclic AMP in brain.

A method is described for the estimation of adenosine 3′,5′-monophosphate (3′,5′-cyclic AMP) in rat brain by high-pressure liquid chromatography (HPLC). The nucleotide is purified initially by being passed through two columns, alumina and AG-1X2. The peak in HPLC was identified by a number of methods. Optimum parameters for HPLC were obtained by using 1 mm KH₂PO₄ buffer, pH 4.8, at a flow rate of 57 ml/hr at room temperature. Using this technique the concentration of 3′,5′-cyclic AMP in rat brain was found to be 2.53 ± 0.40 nmol/g (mean ± SD, n = 5).     

Rapid purification of protein kinase C by high performance liquid chromatography

Protein kinase C was purified from rat brain cytosol by using a high performance liquid chromatography (HPLC), Pharmacia FPLC system. This procedure employed a column chromatography on DE-52, followed by three steps of HPLC procedures with threonine-Sepharose (prepared as described in this report), TSK gel Phenyl-5PW (Toyo Soda), and TSK gel G3000SW (Toyo Soda) columns. Starting from about 30 g of rat brain, approximately 200 micrograms of pure enzyme was obtained. The procedure was very simple and highly reproducible. The enzyme thus obtained was nearly pure by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of 10% (w/v) glycerol and 0.05% (w/v) Triton X-100, the enzyme could be stored at -80 degrees C for several months.     

Highly sensitive and rapid determination of theophylline, theobromine and caffeine in human plasma and urine by gradient capillary high-performance liquid chromatography-frit-fast atom bombardment mass spectrometry

A sensitive and reliable analytical procedure has been established for the detection of theophylline (TH), theobromine (TB) and caffeine (CA) in human plasma and urine by gradient capillary high-performance liquid chromatography (HPLC)-frit-fast atom bombardment mass spectrometry (FAB-MS) (LC-frit-FAB-MS). Two capillary columns and a column-switching valve were used in this LC system to allow all of the sample injected to be introduced into the MS system. 7-Ethyltheophylline was used as the internal standard (I.S.). The xanthines in the specimen were extracted with an Extrelut column. The lowest detected amount was ca. 5 ng/ml using this method.     

Comparison of folate quantification in foods by high-performance liquid chromatography-fluorescence detection to that by stable isotope dilution assays using high-performance liquid chromatography-tandem mass spectrometry

A comparison study on folate quantitation was carried out between the recently developed stable isotope dilution assay using liquid chromatography-tandem mass spectrometry (LC-MS-MS) and the frequently used HPLC with fluorimetric detection (LC-FD). By applying LC-MS-MS, spinach, wheat bread, beef, and blood plasma were found to contain 159.2, 19.8, 1.2, and 5.6 microg/100 g total folates, respectively, whereas the respective quantitative data obtained by LC-FD were 95.5, 16.2, 0.7, and 6.8 microg/100 g. In all samples, LC-MS-MS revealed superior selectivity and precision and circumvented the shortcomings of conventional LC techniques, i.e., ambiguous peak assignment as well as high detection limits for 5-formyltetrahydrofolate, 10-formylfolic acid, and folic acid. The affinity chromatography columns used in this study showed excellent cleanup performance and permitted detection limits as low as 0.1, 0.5, 0.1, 0.08, and 0.1 microg/100 g for tetrahydrofolate (H(4)folate), 5-methyl-H(4)folate, 5-formyl-H(4)folate, 10-formylfolate, and pteroylglutamic acid, respectively. Thus, a 10-fold higher sensitivity compared to solid-phase anion-exchange cartridges was achieved. However, affinity chromatography columns revealed a significantly higher affinity toward the natural vitamers than to the racemic isotopomeric standards, which has to be considered when applying the latter in stable isotope dilution assays.     

High-performance liquid chromatography using spherical aggregates of hydroxyapatite micro-crystals as adsorbent

High-performance liquid chromatography (HPLC) using spherical aggregates of hydroxyapatite (HA) microcrystals as adsorbent has been developed; preliminary performance tests were carried out by using several types of protein. In comparison with previously developed plate-like HA packed columns for HPLC, spherical HA packed columns show considerably high chromatographic resolutions in spite of extremely reduced column lengths of 0.5-3 cm. The pressure generated by the latter columns is much higher than that generated by the former, however.     

Quantitative high-performance liquid chromatography-based detection method for calphostin C,a naturally occurring perylenequinone with potent antileukemic activity

Calphostin C is a potent inhibitor of protein kinase C and can induce Ca²⁺-dependent apoptosis in human ALL cells. Further development of calphostin C will require detailed pharmacodynamic studies in preclinical animal models. Therefore, we established a sensitive and accurate high-performance liquid chromatography (HPLC)-based quantitative detection method for the measurement of calphostin C levels in plasma. Extraction of calphostin C from plasma was performed by precipitation of plasma protein using acetonitrile and an aliquot of extracted supernatant was injected onto a Hewlett-Packard HPLC system constituting a 250×4 mm LiChrospher 100, RP-18 (5 μm) in conjunction with a 4×4 mm LiChrospher 100, RP-18 guard column (5 μm). The eluted compounds were detected by diode array detection set at a wavelength of 479 nm. Acetonitrile–water containing 0.1% trifluoroacetic acid and 0.1% triethylamine (70:30, v/v) was used as the mobile phase. The average extraction recovery from plasma was 97.3%. Good linearity (r>0.999) was observed throughout the concentration range of 0.05–40 μM for calphostin C in 50 μl of plasma. Intra- and inter-assay variabilities were less than 6% in plasma. The lowest detection limit of calphostin C in 50 μl plasma was 0.02 μM at a signal-to-noise ratio of ∼3. The availability of this assay will now permit detailed pharmacodynamic and pharmacokinetic studies of calphostin C in vivo.     

高效液相荧光法测定大鼠脑内氨基酸类神经递质方法的改良

目的 改良测定大鼠脑组织氨基酸类神经递质的反相高效液相色谱荧光法.方法 改良使用磷酸盐-甲醇-乙腈作为流动相,反相高效液相色谱洗脱,高丝氨酸作为内标,邻苯二甲醛柱前衍生和荧光检测器,检测大鼠大脑皮质、海马、纹状体、中脑、小脑和下丘脑6个脑区中天冬氨酸(Asp)、谷氨酸(Glu)、谷氨酰胺(G1n)、甘氨酸(Gly)、γ-氨基丁酸(GABA)和牛磺酸(Tau)6种氨基酸类神经递质含量.结果 6种氨基酸在20 min内洗脱完全,分离效果良好;在6.25～ 400 μmol/L浓度范围有较好的线性关系,其相关系数不低于0.99;6种氨基酸日内试验精密度范围为1.38％ ～7.59％;日间试验精密度为2.7％～8.68％;6种氨基酸回收率不低于80％.结论 改良后的反相高效液相色谱荧光法灵敏度较高、重复性好,能有效分离检测大鼠脑组织分区中氨基酸类神经递质含量.     

Isolation of neuronal parvalbumin by high-performance liquid chromatography. Characterization and comparison with muscle parvalbumin

Neuronal parvalbumin has been isolated from rat brain and purified to homogeneity by high-performance liquid chromatography (HPLC) on reverse-phase supports. This procedure includes four consecutive chromatographic steps with an overall protein recovery of 74% and a 26 400-fold purification. The concentration of parvalbumin was found to be approximately 10 mg/kg wet weight in brain tissue, which is about 100 times lower than that in rat muscle. The physical properties of brain parvalbumin are described and compared with those of the muscle counterpart. These proteins were identical in their molecular weights (12 000), isoelectric points (4.9), retention times on C-18 reverse-phase HPLC columns, Ca2+ content (two per molecule), amino acid compositions, and immunological properties. A comparison of the tryptic peptide maps of brain and muscle parvalbumin by analytical HPLC also revealed identity and showed that the isolation method described here did not alter the chemical structure of the protein.     

魔芋中神经酰胺类物质的HPLC-ELSD分析及其含量测定

建立高效液相色谱-蒸发光散射检测器分析神经酰胺的方法并进行了含量的测定.色谱柱:ZORBZX Eclipse XDB-C18(4.6mm×250mm,5μm),洗脱方法:梯度洗脱,柱温:35℃,流动相:甲醇/水,流速:1ml/min;检测器:蒸发光散射检测器,漂移管温度:40℃,氮气流速:1.5L/min.系统探讨了梯度洗脱的起始浓度、洗脱的时间和洗脱梯度的程序设置,最佳的梯度洗脱条件为5min内,甲醇浓度从60%线性增加为90%,从5min到25min,甲醇浓度线性增加为95%,在此条件下样品和标准品的分离色谱峰对称性较好.随后测定了各种样品中神经酰胺的含量,并进行了方法学验证,结果神经酰胺在0.2～2μg之间线性关系良好,最低检测限为0.01mg/ml,R2=0.9992;平均回收率为93.3%,RSD=1.65%(n=5).本法灵敏、方便、准确,重现性好,可用于魔芋神经酰胺类物质的分离及其含量的测定.     

高效液相色谱串联质谱法同时检测粪便中三种抗抑郁药物*

目的:建立同时检测粪便中度洛西汀、氟西汀、艾斯西酞普兰三种抗抑郁药物的高效液相色谱串联质谱(HPLC-MS)方法。方法:样品经正己烷-异丙醇(95:5,v/v)预处理后,以超纯水和乙腈混合液作为流动相进行梯度洗脱,在Agilent ZORBAX SB-C18液相色谱柱(2.1 mm×100 mm,3.5 μm)上分离后用电喷雾串联质谱法检测,内标法定量。结果:粪便样品中度洛西汀、氟西汀、艾斯西酞普兰的加标回收率为61.6%～116.5%,精密度为 2.80%～12.9%(n=5),线性方程相关系数(r)均大于0.995,检出限分别为0.1 μg/g、1 μg/g、0.001 μg/g,定量限分别为0.5 μg/g、2 μg/g、0.005 μg/g,符合高效液相色谱串联质谱的分析要求。结论:该方法能够简单、准确检测出粪便中度洛西汀、氟西汀、艾斯西酞普兰这三种抗抑郁药的含量。