Separation and quantification of alkylphosphocholines by reversed phase high performance liquid chromatography

Alkylphosphocholines represent a new class of drugs with remarkable antineoplastic and antiprotozoal activity. For instance, hexadecylphosphocholine has been approved for the topical treatment of skin metastasis. In addition, it was successfully studied in India for the treatment of leishmaniasis. Different phase-I and phase-II-trials resulted in cure rates of more than 97%. To optimize antitumor or antiprotozoal activity, we have prepared alkylphosphocholines differing in chain length and unsaturation. For the qualitative and quantitative analysis of these longer chain analogues, we have used isocratic high performance liquid chromatography. The separation of the alkylphosphocholines with different chain lengths in this reversed phase HPLC system was achieved on a YMC-TMS column with a mobile phase consisting of methanol-water (85:15; v/v) at a flow rate of 1.0 ml/min. Furthermore the cis-/trans-isomers such as oleylphosphocholine and elaidylphosphocholine were clearly separated on a YMC-C8 column with a methanol-water mixture (80:20; v/v) as mobile phase. In the described reversed phase HPLC systems simple refractive index detection and UV detection allow the sensitive and quantitative determination of alkylphosphocholines. These methods are very important for reproducible identification and quantitative determination of saturated and mono-unsaturated alkylphosphocholines with alkyl residues containing up to 25 carbon atoms.     

大孔树脂吸附与反相色谱纯化恩拉霉素

恩拉霉素作为多肽类抗生素,是一种新型、安全的饲料添加剂。本文建立了一条基于大孔树脂初纯和反相色谱精制的分离纯化工艺。该工艺路线首先使用AB-8大孔树脂在0.012 mol/L盐酸溶液-甲醇(50:50,V/V)缓冲液条件下洗脱实现恩拉霉素初步纯化,再使用制备型C18反相色谱柱在0.05 mol/L磷酸二氢钠-乙腈(70:30,V/V)(p H 4.5)缓冲液洗脱下实现恩拉霉素a和b的有效分离,a、b两个组分纯度分别达到98.5%和98.0%,a和b两种有效成分的总收率为29.2%。本研究为恩拉霉素a和b两种纯品的制备以及高纯度恩拉霉素产品的生产提供了参考。     

Determination of methylputrescine oxidase by high performance liquid chromatography

N-Methyl-Δ¹-pyrrolinium chloride, the product of the title enzyme, was synthesized by methylation of aminobutyraldehyde diethylacetal followed by acidic cleavage. After purification to homogeneity, it was characterized by NMR and UV spectroscopy. The compound had an absorption maximum at 210 nm; previous data indicating a maximum at 267 nm were shown to arise from an impurity. An HPLC method for the assay of N-methylputrescine oxidase from plant material was developed based on the separation of N-methyl-Δ¹-pyrrolinium chloride on a cation exchange column and direct detection at 210 nm. The enzyme activity was measured in the protein fraction extracted from plant roots and treated by gel filtration on disposable PD 10 columns. A K_m value of 1.9 mM was determined for methylputrescine and the enzyme from tobacco roots. The enzyme activities from N. tabacum and Datura stramonium were compared.     

Determination of stereo- and positional isomers of oxygenated triterpenoids by reversed phase high performance liquid chromatography

Twenty four oxygenated triterpenoids, including eight pairs of stereoisomers and five pairs of positional isomers, could be separated by reversed phase HPLC. The capacity factors obtained in methanol-water and acetonitrile-water solvent systems made it possible to correlate the molecular polarities due to the presence of multiple oxygenated functional groups in these compounds. It was found that the number and position of functional groups as well as the stereochemistry of these functional groups played important roles in governing the polarity of these lanostanoid acids. The polarity weighting factors were in the following order: 3 beta-OH greater than 3 alpha-OH greater than 3 alpha-OAc greater than 3 beta-OAc. The contribution to polarity due to 15 alpha-OAc and 22 beta-OAc was probably very similar. The unique stereochemical character and eluting sequences of the lanostanoid acids provide information to generate empirical rules for predicting the role of individual polar functional groups in the chromatographic behavior in reversed phase HPLC.     

HPLC法测定牙膏中柚皮苷的含量

建立了高效液相色谱测定牙膏中柚皮苷含量的方法。采用的色谱条件:Hypersil BDS C18色谱柱(250 mm×4.6 mm,5μm);柱温为40℃;以水(A相)和乙腈(B相),梯度洗脱程序为:0～15 min,10%～100%B;流速为1.0 mL/min;检测波长为283 nm;进样量为20μL。结果表明,柚皮苷的质量浓度在14.55～116.40μg/mL范围内与峰面积呈良好的线性关系(r=0.9999),平均加标回收率为97.56%。该方法稳定、准确,重现性好,可作为牙膏中柚皮苷的含量测定和质量控制方法。     

Determination of putrescine N-methyltransferase by high performance liquid chromatography

A novel procedure is described for the chemical synthesis of N-methylputrescine, the product of the title enzyme. This is obtained from putrescine by formylation followed by the reduction of the monoformylputrescine intermediate with LiA1H₄. An assay method for putrescine N-methyltransferase was developed which depends on the determination of N-methylputrescine in the presence of an excess of putrescine. This method, which makes use of a radiolabeled substrate unnecessary, is based on dansylation of the product followed by HPLC separation on a reversed-phase column. The enzyme activity of the protein peak extracted from plant material was measured after treatment by gel filtration on prepacked disposable PD 10 columns. The specific enzyme activities determined in the extract from the roots of Nicotiana tabacum and Datura stramonium plants, and from a root culture of D. stramonium, are reported. With an enzyme preparation from the last root culture, K_m values for putrescine and S-adenosylmethionine (SAM) were determined as 0.88 mM and 0.15 mM, respectively.     

Mobile phase compositions for ceramide III by normal phase high performance liquid chromatography

Ceramide III was prepared by the cultivation ofSaccharomyces cerevisiae. Ceramide III was partitioned from the cell extracts by solvent extraction and analyzed by Normal Phase High Performance Liquid Chromatography (NP-HPLC) using Evaporative Light Scattering Detector (ELSD). We experimentally determined the mobile phase composition to separate ceramide III with NP-HPLC. Three binary mobile phases of n-hexane/ethanol,n-hexane/Isoprophyl Alcohol (IPA) andn-hexane/n-butanol and one ternary mobile phase ofn-hexane/IPA/methanol were demonstrated. For the binary mobile phase ofn-hexane/ethanol, the first mobile phase composition, 95/5 (v/v), was step-increased to 72/23 (v/v) at 3 min. In the binary mobile phase, the retention time of ceramide III was 7.87 min, while it was 4.11 min respectively in the ternary system, where the mobile phase composition ofn-hexane/IPA/methanol, 85/7/8 (v/v/v), was step-increased to 75/10/15 (v/v/v) at 3 min. However, in the ternary mobile phase, the more peak area of ceramide III was observed.     

Analysis of protein-glutathione mixed disulfides by high performance liquid chromatography

After precipitation of proteins; serum, hepatocytes, or glutathione-derivatized bovine serum albumin, by perchloric acid, dithiotheritol was used to reduce glutathione-protein mixed disulfides in the ether-washed, resuspended pellet. Following neutralization and S-carboxymethylation of free sulfhydral groups in the acid soluble fraction by iodoacetic acid, 2,4-dinitrophenyl derivatives of released compounds were produced by addition of ethanolic fluorodinitrobenzene. The 2,4-dinitrophenyl derivative of S-carboxymethylglutathione was measured by high-performance liquid chromatography. The method was found to be reproducible and limited only by the sensitivity of the glutathione analysis (about 10 pmol/sample). Quantitation of protein-bound glutathione was shown to be indepedent of the ratio of bound to soluble glutathione as well as the protein concentration in the sample. This method was found to produce glutathione values identical to those measured after borohydride reduction without the problems of foaming, sample loss, and the need of continuous pH adjustment during reduction.     

Metallothionein separation and analysis by reversed phase high performance liquid chromatography coupled with graphite furnace atomic absorption spectrometry

Abstract

The feasibility of using reversed-phase high performance liquid chromatography (RP-HPLC) for the separation of metallothioneins (MTs) and subsequent determination of cadmium in MTs by graphite furnace atomic absorption spectrometry (GFAAS) in rabbit kidney and liver has been studied. RP-HPLC was used to isolate, characterise and quantitate liver and kidney MT isoforms. The MTs were eluted from a radially compressed C18 column with a neutral sodium phosphate buffer and detected by UV absorbance at 254 nm. Rabbit liver MTs was found to be comprised of seven distinct isoforms with five of which were found to be subspecies of the MT-I isoform. Rabbit kidney MTs exhibited only two predominant isoforms. A standard calibration curve was constructed using purified rabbit kidney MT-I and MT-II which demonstrated excellent linear correlation between peak height and the quantity of MT injected into the column. Recovery of MT from RP-HPLC was found to exceed 90%. Kidney and liver tissues from rabbit by feeding low levels of cadmium in diets was assayed using the RP-HPLC analysis of cytosol samples. Feeding stable cadmium in the diet resulted in the deposition of MT in the kidney rather than in the liver. The cadmium content in MT isoforms was determined by GFAAS. Less than 10% of the total cadmium in kidney was associated with MTs.     

III. Reversed phase high pressure liquid chromatography for the identification and purification of neuropeptides

Reversed phase HPLC has wide applications in studies on neuropeptides. It provides a fast and effective technique for assessing the purity of synthetic peptides and for purifying mg amounts of synthetic peptides (examples: angiotensins II and III and analogues; neurohypophysial hormones). Due to the very small quantities of peptides which can usually be safely recovered after HPLC, the method is also useful in the isolation, purification and sequencing of peptides from biological sources (examples: urotensins I and II), and in the identification of neuropeptides in tissues when coupled with radioligand-binding displacement assays (example: [arginine⁸]vasotocin in the anterior ganglia of $\text{Aplysia}$$\text{california}$).     

反相高效液相色谱法测定食用菌中7种有机酸的研究

利用反相高效液相色谱法同时分析食用菌中7种有机酸,优化后的色谱条件为:Green ODS-AQ柱(4.6mm×250mm,5μm),流动相为10mmol/L的磷酸二氢钾(pH2.8),流速为1.0mL/min,柱温30℃,检测波长为210nm,进样量10μL。该方法精密度、重复性、稳定性实验中7种有机酸的RSD值均小于5%,加样回收率在94.6%–99.3%之间;该方法简便,可应用于食用菌中7种有机酸的检测。运用建立的方法测定了8种食用菌中7种有机酸成分,结果发现不同食用菌中有机酸的种类和含量均差异显著。     

Comparison of plasma sample purification by manual liquid–liquid extraction, automated 96-well liquid–liquid extraction and automated 96-well solid-phase extraction for analysis by high-performance liquid chromatography with tandem mass spectrometry

Three extraction procedures were developed for the quantitative determination of a carboxylic acid containing analyte (I) in human plasma by high-performance liquid chromatography (HPLC) with negative ion electrospray tandem mass spectrometry (MS–MS). The first procedure was based on the manual liquid–liquid extraction (LLE) of the acidified plasma samples with methyl tert.-butyl ether. The second procedure was based on the automation of the manual LLE procedure using 96-well collection plates and a robotic liquid handling system. The third approach was based on automated solid-phase extraction (SPE) using 96-well SPE plates and a robotic liquid handling system. A lower limit of quantitation of 50 pg/ml was achieved using all three extraction procedures. The total time required to prepare calibration curve standards, aliquot the standards and plasma samples, and process a total of 96 standards and samples by manual LLE was three-times longer than the time required for 96-well SPE or 96-well LLE (4 h, 50 min vs. 1 h, 43 min). Even more importantly, the time the bioanalyst physically spent on the 96-well LLE or 96-well SPE procedure was only a small fraction of the time spent on the manual LLE procedure (<10 min vs. 4 h, 10 min). It should be noted that the 96-well SPE procedure incorporated the two steps of evaporation of the eluates to dryness and subsequent reconstitution of the dried extract. The total time required for the 96-well SPE could be reduced by 50% if the eluates were injected directly, eliminating the drying and reconstitution steps, which is achievable when sensitivity is less of an issue.     

Analysis of the nucleotide pool during growth of suspension cultured cells of Nicotiana tabacum by high performance liquid chromatography

In suspension cultured cells of Nicotiana tabacum L. cv. Wisconsin 38 the .concentration of 18 nucleotides and 3 nucleosides have been determined using a new procedure developed for the extraction, purification and HPLC separation of these compounds from plant tissue. The different nucleotide pools increase in size immediately at the onset of the batch culture and reach distinct maxima at the very beginning of the proliferative phase. The main component are the uracil nucleotides with UDP-sugars as the predominant fraction followed by the adenine nucleotides; the energy charge is maintained at a high and constant value throughout the whole culture time. During the growth interval the increases in the nucleotide pools reveal that the cell proliferation phase is followed by an extensive phase of cell elongation. Whereas the concentration of the total nucleotides varies by a factor of about 3 along the growth curve, the ratio of uracil to adenine nucleotides is kept fairly constant indicating regulatory mechanisms for correlation of the individual nucleotide pools.     

The simple and sensitive measurement of malondialdehyde in selected specimens of biological origin and some feed by reversed phase high performance liquid chromatography

A method for the determination of malondialdehyde (MDA) concentrations in specimens of animal tissues and feed has been developed using high performance liquid chromatography. The MDA concentration in acidified urine samples was determined after its conversion with 2,4-dinitrophenylhydrazine (DNPH) to a hydrazone (MDA-DNPH). Samples of blood plasma, muscle, liver and feed were prepared by saponification followed by derivatisation with DNPH to MDA-DNPH. The MDA concentration in chicken and hen feed samples was analysed after saponification and derivatisation followed by extractions with hexane. The free MDA in plasma samples was determined after deproteinization followed by derivatisation of MDA with DNPH. The chromatographic separation of MDA-DNPH samples was conducted using Phenomenex C(18)-columns (Synergi 2.5 μm, Hydro-RP, 100 ?, the length of 100mm) with an inner diameter of 2 or 3mm. MDA in processed biological samples was analysed using a linear gradient of acetonitrile in water, and the photodiode detector was set to 307 or 303 nm for detection. The current method that was utilised was based on the high-efficient derivatisation of MDA and was more sensitive compared to previously used methods. The selective and sensitive photodetection of the column effluent was found to be suitable for the routine analysis of MDA in urine, plasma, muscles and liver of animals and some feed samples. Because urine or blood plasma samples can be derivatised in a simple manner, the proposed method can also be suitable for the routine, non-invasive evaluation of oxidative stress in animals and humans.     

Principles and methods for the analysis and purification of synthetic deoxyribonucleotides by high-performance liquid chromatography

The need for high-purity oligodeoxyribonucleotides for various applications has resulted in the development of novel synthesis, purification, and analytical techniques, A diversity of methods, including polyacrylamide slab gel electrophoresis, capillary gel electrophoresis, as well as high-performance liquid chromatography (HPLC), have been successfully used to aid in the characterization and isolation of these synthetic compounds. The information contained in this review article primarily details both the theoretical and practical aspects related to the use of HPLC for the analysis and purfication of synthetic DNA. In addition, a variety of postsynthesis sample preparation protocols, commonly employed prior to and after HPLC, are described.     

Development of a reversed phase high performance liquid chromatography method based on the use of cyclodextrins as mobile phase additives to determine pterostilbene in blueberries

In this work, a reversed phase high performance liquid chromatography (RP-HPLC) method was developed for the determination of pterostilbene in food samples. The novel method is based on the addition of cyclodextrins (CDs) to the mobile phase where the complexation of pterostilbene by CDs is carried out. In order to select the most suitable conditions for the RP-HPLC method, the effect of several physico-chemical parameters on the complexation of pterostilbene by CDs was studied. Our results show that the addition of 12 mM HP-β-CD to a 50:50 (v/v) methanol:water mobile phase at 25°C and pH 7.0 significantly improves the main analytical parameters. In addition, it was seen that pterostilbene forms a 1:1 complex with HP-β-CD, showing an apparent complexation constant of 251±13 M(-1). Finally, in order to study the validity of the proposed method, blueberries were analyzed and the concentration of pterostilbene has been determined.     

15种獐牙菜属植物中主要药用成分的高效液相色谱测定

对青藏高原和云贵高原的15种獐牙菜属植物进行了3种苦味苷,即獐牙菜苦苷(swertiamarin)、龙胆苦苷(gentiopicroside)、苦龙苷(amarogentin)、一种黄酮苷-当药黄素(swertisin)、及5种口山酮苷-芒果苷(mangiferin)、当药醇苷(swertianolin)、7-O-[a-L-吡喃鼠李糖-(1→2)-β-D-吡喃木糖]-1,8-二羟基-3-甲氧基口山酮(7-O-[a-L-rhamnopyranosyl-(1-2)-β-D-xylopyranosyl]-1,8-dihydroxy-3-methoxyxanthone)、7-O-β-D-吡喃木糖-1,8-二羟基-3-甲氧基口山酮(7-O-β-D-xylopyranosyl-1,8-dihydroxy-3-methoxyxanthone)、3-O-β-D-吡喃葡萄糖-1,8-二羟基-5-甲氧基口山酮(3-O-β-D-glucopyranosyl-1,8-dihydroxy-5-methoxyxanth-one)等9种主要药效成分同时进行了高效液相色谱的含量测定(Kromasil C18柱,甲醇一水梯度洗脱,二级管阵列检测);并对其主要药效成分的分布进行了比较。     

双歧杆菌发酵果蔬汁中低聚果糖的高效液相色谱法分析

目的采用高效液相色谱法（HPLC）对双歧杆菌发酵果蔬汁中的低聚果糖进行分析。方法 HPLC分析条件为：ZORBAX NH2 Analytical色谱柱,乙腈∶水（75∶25）为流动相,柱温35℃,流速为1.0ml/min,示差折光检测器（RID）。结果果蔬汁中的低聚果糖及几种主要糖类均得到有效分离。此分离方法的加标回收率和精密度（RSD）均较高。结论分析结果表明双歧杆菌发酵果蔬汁中含有较丰富的功能性低聚果糖,尤其是蔗果三糖含量很高。     

Evaluation of Daphne genkwa diterpenes: fingerprint and quantitative analysis by high performance liquid chromatography

Daphne genkwa contains a novel class of anticancer diterpene esters that inhibit DNA topoisomerase I. Fingerprint and quantitative analysis by HPLC were performed in order to characterise and evaluate D. genkwa. A standard fingerprint of Daphne diterpene esters from the root extract was first established by HPLC-UV, and the major peaks in the fingerprint profile were preliminarily determined using HPLC-MS. The principal Daphne diterpene esters, yuanhuacine (1), yuanhuadine (2), yuanhuajine (3) and yuanhuagine (4), were isolated and identified using a combination of UV, IR, MS, 1H-NMR and 13C-NMR spectral data. Quantitative analysis indicated that 1 was the principal component in the root, and that 2 was the major component in the buds. The average extraction rates of 1 and 2 were 0.0151 and 0.0033% (n=10) from the root, respectively, and 0.0020 and 0.0078% (n=3) from the buds, respectively.     

Determination of epitestosterone in human urine by off-line immunoaffinity solid-phase extraction coupled with high performance liquid chromatography

Epitestosterone (ET) has been used as a masking agent and prohibited by the World Anti-Doping Agency (WADA) because its administration will decrease the urinary T/ET ratio, a marker of testosterone (T) administration. In this study, an off-line immunoaffinity extraction coupled with high performance liquid chromatography (HPLC) was developed to quantify the endogenous steroid ET in human urine. The immunoaffinity column (IAC) was prepared by immobilizing the anti-ET monoclonal antibodies on CNBr-activated Sepharose 4B, which can remove the contaminations and non-target compounds from matrix to enrich the target analyte ET. The mobile phase was ammonium acetate (10 mM, pH 4.0)/acetonitrile (45/55, v/v) at an isocratic flow of 1.0 mL/min and the UV absorbance detection wavelength was 244 nm for the detection of ET. The IAC showed good reliability and durability since it had been used for more than 100 runs in a year. The limit of quantification (LOQ) was 1 ng/mL. Satisfied repeatability and precision of the day-to-day and within-day were obtained with the RSD values less than 10%. Results of the recovery of the urine samples were ranged from 98% to 102% with repeatability less than 9%, indicating that the method developed can be used for the real urine sample analysis.