反相高效液相色谱法测定人血浆中异丙酚浓度

用柱前衍生化反相高效液相色谱法测定人血浆中异丙酚浓度,为临床药理学研究提供了依据。提取的异丙酚及内标麝香草酚同时以Ｇｉｂｂｓ试剂衍生化,衍生物经色谱柱分离和紫外检测,异丙酚在５０～１５００μｇ／Ｌ浓度范围内呈线性关系（ｒ＝０．９９９１）。方法平均变异系数为６．１％,最低检出浓度为２４．８μｇ／Ｌ,已满意地用于临床药理学研究。     

高效液相色谱-质谱法鉴定总合草苔虫中的草苔虫内酯成分

采用高效液相色谱分离与质谱检测的联用技术,对总合草苔虫中的草苔虫内酯成分进行定性分析。在以体积比为80∶20的甲醇-水溶液为流动相、采用ODS柱进行分离的色谱条件下,10种草苔虫内酯成分均可获得良好的分离。分析结果表明：深圳大亚湾产地的总合草苔虫中共含有9种已知和1种未知的草苔虫内酯成分。已知成分分别是bryostatin 4,5,6(9),7,8,10,16,17,18,其中bryostatin 7和17是首次从中国海域总合草苔虫中发现的2种痕量草苔虫内酯。该方法简便、准确、灵敏度高,可以很好的对总合草     

高效液相色谱法分离测定人参中的6种人参皂甙

 采用梯度法,以乙腈-水溶液为流动相,用高效液相色谱法分离人参中的6种主要人参皂甙,并采用紫外检测器检测,在203 nm处测定4种人参样品。该方法在25 mg/L～300 mg/L的范围内有良好的线性关系,回收率高于80%。     

Comparative performance of ion exchange and ion-paired reversed phase high performance liquid chromatography for the determination of nucleotides in biological samples.

We have compared anion exchange chromatography on APS-Hypersil (4.6 x 100 mm) eluted with a phosphate gradient with reversed phase chromatography on ODS-Hypersil (4.6 x 100 mm) in the presence of either tetrabutylammonium (TBA) or triethylammonium (TEA) ions with a methanol gradient. The systems have been compared both for ease of operation and for their resolving power with standard mixtures and acid extracts of both normal red cells (RBC) and ischaemic tissues. The two chromatographic modes exhibited similar separating efficiencies for standard mixtures of nucleotides but retention times were most stable using reversed phase liquid chromatography (RPLC) with TEA. Anion exchange columns slowly lost ion exchange capacity but selectivity was unchanged. RPLC in the presence of TBA gave reproducibile capacity factors only when operated isocratically due to irreversible changes to the silica surface. For RBCs the RPLC with TEA and anion exchange systems resolved 17 and 15 peaks, respectively, and for the ischaemic samples 22 and 14 peaks, respectively. However, nucleosides and bases were also resolved by the ODS column causing chromatographic crowding and uncertain peak identification.     

反相高效液相色谱法测定杜仲中的松脂醇二葡萄糖甙

建立一种测定杜仲中松脂醇二葡萄糖甙的反相高效液相色谱法。杜仲粉末的甲醇提取液经大孔树脂柱处理后,在ＹＷＧ－Ｃ１８柱上进行ＨＰＬＣ分析。流动相为２８％（Ｖ／Ｖ）的甲醇水溶液;流速１．０ｍＬ／ｍｉｎ;紫外检测波长２３２ｎｍ。方法平均回收率为９９．２２％（ｎ＝３）,变异系数０．５０％～０．７４％（ｎ＝５）。进样量在０．０６８μｇ～０．６８μｇ范围内线性关系良好,ｒ＝０．９９９９。所建立的方法可作为评定杜仲及杜仲制品降压效果的一种简便、快速和准确的测定方法。     

Validation of the RP–HPLC method for analysis of hydrochlorothiazide and captopril in tablets

A rapid and sensitive reverse-phase high performance liquid chromatography (RP–HPLC) method with ultra-violet (UV) detection for a routine control of hydrochlorothiazide and captopril in tablets was developed. The chromatographic system Hewlet Packard 1100 consisted of a HP 1100 pump, HP 1100 UV–VIS detector and HP ChemStation integrator. The samples were introduced through a Rheodyne injector valve with a 20-L sample loop. The isocratic system consisted of a Beckman Ultrasphere ODS 4.6 mm x 15 cm, 5-m-particle column and a mobile phase containing methanol/water (45:55 v/v). The pH of the mobile phase was adjusted to 3.8 with 85% ortophosphoric acid. Quantitation was accomplished using the internal standard method. At the selected conditions, the other excipients of the tablets did not interfere in the assay of active substances. The developed RP–HPLC method was validated, so linearity, precision, accuracy, robustness, limit of quantitation and limit of detection were investigated. For the robustness test, three factors were considered: the composition of the mobile phase , the pH of the mobile phase, and temperature. With the aid of response surface metodology (RSM), it was possible to precisely define the robustness of the method.     

Considerations on the possibilities and limitations of comprehensive normal phase-reversed phase liquid chromatography (NPLC x RPLC)

A comprehensive normal phase system LC-reversed phase LC (NPLC x RPLC) was evaluated for the separation of a pharmaceutical mixture and citrus oil extracts. NPLC was performed on a 25 cm x 1 mm ID x 5 microm dp diol phase. In the second dimension, an RP 18 monolithic column (10 cm L x 4.6 mm ID x 2 microm macropore size) and an octadecyl silicagel-packed column (5 cm L x 4.6 mm ID x 3.5 microm dp) were applied for the analyses of the pharmaceutical sample and the citrus oil extracts, respectively. A two-position/ten-port switching valve was used as interface. Under optimised LC conditions, the high degree of orthogonality between NP and RP resulted in peak capacities of 300 for the pharmaceutical sample and of 450 for the citrus oil extract composed of lemon and orange oil. Despite the features of NPLC x RPLC, several shortcomings related with the solvent incompatibility between the two LC modes were identified and the practical consequences were discussed.     

On-line preconcentration of niobium(V) and tantalum(V) as 4-(2-pyridylazo) resorcinol-citrate ternary complexes in geological samples by ion interaction high-performance liquid chromatography

4-(2-Pyridylazo) resorcinol (PAR) and citrate were used as pre-column complexing agents for the determination of Nb(V) and Ta(V) as ternary complexes in geological samples. Aliquots of 2 ml of the standard and sample solutions containing the Nb(V) and Ta(V) complexes were loaded onto a concentrator column (C18, 0.4 cm x 4.6 mm) with a carrier mobile phase comprising 20% (v/v) methanol and containing 5 mM acetic acid, 5 mM citric acid and 10 mM tetrabutylammonium bromide (TBABr), pH 6.5 at 2 ml/min for 2 min, with the effluent being directed to waste. An automatic switching valve was then switched to flush both complexes from the concentrator column onto a C18 analytical column using a mobile phase comprising 32% (v/v) methanol and containing 5 mM acetic acid, 5 mM citric acid and 3 mM TBABr, pH 6.5 for 2.5 min. The switching valve was then switched back to the original position, and cleaned with methanol for 7 min to eliminate unwanted species still adsorbed to the concentrator column. This procedure prevented later eluting compounds from reaching the analytical column, which reduced the overall run time. The detection limits of Nb(V) and Ta(V) (determined at a signal-to-noise ratio of 3, detection wavelength of 540 nm and a 2-ml sample volume) were 0.012 and 0.039 ppb for Nb(V) and Ta(V), respectively. Recoveries of Nb(V) and Ta(V) were 99.4 and 96.2%, respectively. The HPLC results obtained from the reference granite and basalt samples agreed well with inductively coupled plasma MS and certified values, but the HPLC method yielded slightly low values of the Nb/Ta ratio.     

Two-dimensional reversed-phase liquid chromatography using two monolithic silica C18 columns and different mobile phase modifiers in the two dimensions

Comprehensive two-dimensional (2D) HPLC in the reversed-phase liquid chromatography (RPLC) mode using C18 silica monolith columns at first dimension (1st-D) (10 cm x 4.6mm I.D.) and second dimension (2nd-D) (5 cm x 4.6mm I.D.) was carried out successfully. A mixture of water and tetrahydrofuran (THF) was used as a mobile phase in the 1st-D separation, and a mixture of water and methanol (CH3OH) in the 2nd-D separation. Sample fractions from 1st-D column were directly loaded into an injection loop of the 2nd-D HPLC equipped with two injector valves for one column. The fractionation time at the 1st-D that was equal to the separation time at the 2nd-D was 45 or 60s. Total peak capacity up to 900 was obtained in about 60 min for the isocratic mode separation of aromatic compounds in this system. Gradient elution mode applied to both 1st-D and 2nd-D separations resulted in shorter separation time and better separation efficiencies than the isocratic mode. It was demonstrated that 2D-HPLC systems employing popular C18 stationary phases with different organic modifiers in mobile phases for each dimension could produce large peak capacity. The different selectivities were provided by the difference in polar interactions between a solute and the organic modifier existing in the stationary phase.     

Comparison of Reversed Phase High Performance Liquid Chromatography and Competitive Protein Binding Assay in the Quantification of Cortisol in Bovine Plasma

Abstract

A reversed phase high performance liquid (HPL) chromato-graphic method was developed for quantification of cortisol in bovine plasma. The reliability of this method was compared to that of a competitive protein binding assay (CPBA). Samples were adjusted to > pH 12 with NaOH, extracted with methylene chloride, evaporated to dryness and reconstituted in methanol/water (55%/45%) for HPLC determination. Satisfactory separation and sensitivity were achieved using an Altex Ultrasphere C-8 column (15 cm × 4.6 mm) with isocratic elution of methanol/water (57%/43%) and UV detection (242 nm). The detection limit of the CPBA (0.16 ng) was lower than for the HPL chromatographic method (0.54 ng); however, precision was significantly better for the HPL chromatographic method than for the CPBA. Values for the CPBA were significantly higher than those obtained using HPL chromatography, probably due to the positive interference of non-specific binding of plasma components in the CPBA. The specificity of HPL chromato-graphy for cortisol was validated by mass spectrometry.     

Determination of dehydrodiisoeugenol in rat tissues using HPLC method

Dehydrodiisoeugenol (DDIE) is a bioactive neolignan from the seeds of Myristica fragrans Houtt., which exhibits good anti-inflammatory activity. A rapid and simple high-performance liquid chromatographic (HPLC) method has been developed for the determination of DDIE in rat tissues after intravenous administration. The tissue samples were processed by liquid-liquid extraction. The analyses were successfully carried out on a Diamonsiltrade mark ODS C18 column (250 x 4.6 mm i.d., 5 microm) equipped with a C18 guard column (8 x 4.6 mm i.d., 5 microm). The mobile phase was the system of methanol-water (4:1, v/v). The UV detection was set at 270 nm. The calibration curves were linear from 0.4 to 200.0 microg/g with the correlation coefficients (r(2)) greater than 0.998. The intra- and inter-day precisions in quality control samples were less than 10% and the accuracies were in the range 85.4-110.3%. The average recoveries from all the tissues were between 84.4 and 106.0%. This assay method has been successfully used to study the tissues distribution of DDIE in rats after intravenous administration. The result suggests that DDIE is distributed to rat tissues rapidly with possibly greater initial concentrations in liver and brain than in other tissues.     

Analysis of selected stilbenes in Polygonum cuspidatum by HPLC coupled with CoulArray detection

The roots of three varieties of Polygonum cuspidatum were analyzed for resveratrol and its analogs. The powder of the dried roots was extracted with aqueous ethanol (60% v/v) and the extracts obtained were analyzed using RP HPLC with coulometric detection. A simple HPLC method with a multichannel CoulArray detector was developed for the determination of four stilbenes: resveratrol, its glucoside piceid, piceatannol, and its glucoside astringin. Analyses were carried out on a LiChrospher C18 (125 x 4.6 mm id, particle size 5 microm) column with a mobile phase of ammonium acetate (pH 3) and ACN in gradient mode. Four compounds were monitored by a CoulArray electrochemical detector. Potentials of eight electrochemical cells in series were set in the range of 200-900 mV. Optimization of the mobile phase pH was performed. Calibration curves showed good linearity with correlation coefficients (r(2))--more than 0.9975.     

三尖杉悬浮培养细胞中hinokiol的定量分析

建立了高效液相色谱分析三尖杉悬浮培养细胞中次生代谢产物的方法,实现了次生代谢产物的分离以及hinokiol的定量分析。样品经甲醇提取后,再用氨水-氯仿萃取。采用Apollo C18色谱柱(250 mm×4.6 mm, 5 μm)进行分离,流动相为甲醇-水,梯度洗脱,柱温为30 ℃,检测波长为290 nm,流速为1 mL/min。在0.0125~0.2 g/L范围内,hinokiol的色谱峰面积与质量浓度之间具有良好的线性关系。采用该方法测定了实际样品中hinokiol的含量,并进行了3个水平的加标回收试验,其回收率为87.2%～94.7%,相对标准偏差(n=3)为0.9%~4.2%。该方法可靠、重现性好,适合对植物培养细胞中的hinokiol进行分析。     

Comparison of Reverse-Phase High-Performance Liquid Chromatographic Methods for Precolumn-Derivatized Amino Acids

Abstract

A comparison was made among five precolumn derivatization techniques for amino acid analysis using reverse-phase high-performance liquid chromatography (HPLC). All chromatographic analyses were conducted using the same instrumentation and a C₁₈ Ultrasphere ODS column (5 μm, 250 × 4.6 mm). The precolumn derivatization methodologies studied included the formation of OPA (o-phthaldialdehyde), DANSYL (dimethylaminonaphthalenesulphonyl), DABSYL (dimethylaminoazobenzenesulphonyl), PTH (phenylthiohydantoin), and PTC (phenylthiocarbamyl) derivatives. The derivatization procedures were evaluated for simplicity, time required, and derivative stability. HPLC analyses of the amino acid derivatives were compared in terms of resolution, sensitivity, reproducibility, and time of analysis.     

替考拉宁键合手性固定相高效液相色谱法直接拆分泮托拉唑钠对映体

以替考拉宁为手性选择剂制备了大环抗生素类手性固定相替考拉宁键合手性固定相(T-CSP),建立了T-CSP反相液相色谱直接拆分泮托拉唑钠对映体的方法。考察了流动相中有机改性剂的种类和比例、柱温以及流动相流速对拆分泮托拉唑钠对映体的影响。研究发现,用甲醇作有机改性剂比乙腈更有利于对映体的分离;在研究的温度范围内,随着柱温的升高,对映体的保留时间缩短,同时分离因子和分离度降低;在一定范围内降低流速有利于对映体的分离。采用T-CSP色谱柱(150 mm×4.6 mm i.d.,5 μm),以甲醇-水(体积比为35∶65)为流动相,在流速0.6 mL/min、检测波长290 nm、柱温20 ℃的条件下,泮托拉唑钠对映体获得了近于基线的分离,所建立的方法具有简便快速及重复性好等优点。     

朝鲜淫羊藿的色谱指纹图谱及液相色谱/电喷雾串联质谱分析

建立了长白山区朝鲜淫羊藿药材的高效液相色谱指纹图谱的分析方法. 确定了18批朝鲜淫羊藿药材的13个共有峰, 该指纹图谱的精密度、稳定性和重现性的相对标准偏差均低于3.0%. 结合液相色谱/电喷雾串联质谱对特征峰进行了结构确认, 并根据电喷雾串联质谱数据推测了13个特征化合物的结构. 结果表明采用高效液相色谱与质谱联用技术对朝鲜淫羊藿色谱指纹图谱中的特征峰进行结构确认, 使其色谱指纹图谱的特征性更强, 更适合于药材质量的鉴别与评价.     

Packed column supercritical fluid chromatographic separation and estimation of acetaminophen, diclofenac sodium and methocarbamol in pharmaceutical dosage forms

A reproducible and fast method has been developed for the assay of acetaminophen, methocarbamol, and diclofenac sodium in bulk and drug forms using packed column supercritical fluid chromatography employing internal standard method. The analytes were resolved by elution with supercritical fluid carbon dioxide doped with 11.1% (v/v) methanol on a Shendon-Phenyl (250x4.6 mm) 5 mum column with detection monitored spectrophotometrically at 225 nm. The densities and polarities of the mobile phase were optimised from the effects of pressure, temperature and modifier concentration on chromatograhic figures like retention time (t(R), min), retention factor (k(')) etc. Modifier concentration proved to be the most effective means for changing both retention and selectivity. Calibration data and recovery of the drug from spiked concentrations were determined to assess the viability of the method. The supercritical fluid chromatography (SFC) method was directly compared to an HPLC assay, developed in the laboratory, of the same analytes. With respect to speed and use of organic solvents SFC was found to be superior, while in all other aspects the results were similar to HPLC. The method has been successfully used for the assay of two formulations containing a combination of (A) acetaminophen and methocarbamol and (B) acetaminophen and diclofenac sodium. There was no interference from excipients. The present work validates the recent proposition that supercritical fluid chromatography using CO(2) and modifiers is a viable, faster alternative to reverse phase HPLC.     

Optimization of HPLC-APCI-MS conditions for the qualitative and quantitative determination of total solanesol in tobacco leaves

HPLC coupled online with atmospheric pressure chemical ionization MS (APCI-MS) technique was evaluated for the qualitative and quantitative determination of solanesol in extracts of tobacco leaves. The solanesol and other compounds in the extract were separated on an Alltima C(8) (4.6 mm x 250 mm) column with methanol and water (98:2 v/v) as mobile phase, with flow rate of 0.8 mL/min and UV detection wavelength of 211 nm. In the APCI(+) mode, abundant stable [M-H(2)O + H](+) ion (m/z at 613.5) was observed, with low abundance of other fragmentation ions. A comparison of APCI-MS and ESI-MS techniques showed that APCI mode is more sensitive than ESI mode, and thus better suited for solanesol analysis. When comparing UV 211 nm and APCI-MS in SIM for solanesol quantification, the former offered better precision and reproducibility, but the latter was more than 200 times sensitive in detection. The developed method has been successfully applied to the analysis and comparison of solanesol concentration in different tobacco leaf samples.     

反相高效液相法测定穿心莲药材及其制剂中的穿心莲内酯和脱水穿心莲内酯

 发展了穿心莲药材及其中成药中两种主要成分穿心莲内酯和脱水穿心莲内酯的反相高效液相测定方法。采用甲醇振荡提取法进行样品前处理 ,在以乙腈 水为流动相作梯度洗脱、ODS柱、检测波长为 2 2 5nm的条件下 ,穿心莲内酯和脱水穿心莲内酯在 1 5min内可达到基线分离。两种内酯在 1 0mg/L～ 1 0 0mg/L时其浓度与峰面积成良好的线性关系 ,加标回收率为 96 %～ 1 0 4 %。     

高效液相色谱法测定血浆中莲心碱的浓度

采用高效液相色谱法（甲基莲心碱为内标）测定了血浆中莲心碱的浓度。以ＵｌｔｒａｓｐｈｅｒｅＳｉ为固定相、二氯甲烷－异丙醇－二乙胺（７５１５０．２,Ｖ／Ｖ）为流动相,检测波长为２８２ｎｍ。血样用氨－氯化铵缓冲液调ｐＨ１０后用乙醚提取。平均回收率９３．６％,ＲＳＤ为１．９％,最低检测浓度０．０２５ｍｇ／Ｌ。