Vancomycin as chiral selector for enantioselective separation of selected profen nonsteroidal anti-inflammatory drugs in capillary liquid chromatography

The chiral selector vancomycin was used either as mobile phase additive or bound as a chiral stationary phase (CSP) for the stereoselective separation of seven racemic nonsteroidal anti-inflammatory drugs (NSAIDs), fenoprofen, carprofen, flurbiprofen, indoprofen, flobufen, ketoprofen, and suprofen, by capillary liquid chromatography. The effect of the type of stationary phase, the chiral column Chirobiotic V or the achiral stationary phases Nucleosil 100 C8 HD and Nucleosil 100 C18 HD, and the concentration of vancomycin in the mobile phase on separation of the drug enantiomers were evaluated. All the drugs, except flobufen, were successfully enantioseparated on Nucleosil 100 C8 HD with 4 mM vancomycin present in the mobile phase (composed of methanol and buffer) in the reversed phase mode. On the vancomycin-bonded chiral stationary phase, it was difficult to get enantioseparations of the profen NSAIDs. However, flobufen gave better enantioseparation on the vancomycin CSP. The better enantioresolution of the majority of profen derivatives on the achiral columns with vancomycin added to the mobile phase can be attributed in particular to the higher separation efficiency of this capillary chromatographic system. In addition, vancomycin dimers, formed in the mobile phase, seem to offer a better steric arrangement for stereoselective interaction to these analytes than the vancomycin bonded on the CSP. These substantial differences in the CS structure significantly influence the chiral discrimination mechanism.     

Use of a naphthylethylcarbamoylated-β-cyclodextrin chiral stationary phase for the separation of drug enantiomers and related compounds by sub- and supercritical fluid chromatography

Enantiomeric separation of a variety of drugs and related compounds was achieved on an (S)-naphthylethylcarbamoylated-β-cyclodextrin (S-NEC-CD) chiral stationary phase (CSP) using sub- and supercritical fluid chromatography (SFC). Compounds previously resolved on native or derivatized cyclodextrin CSPs in liquid chromatography (LC) using reversed phase or polar organic mobile phase modes could be resolved in SFC using a simple carbon dioxide/methanol eluent. Resolution of cromakalim, which is not possible on the S-NEC-CD column in LC, was readily accomplished in SFC. The importance of modifier, temperature, and pressure was assessed in relation to retention, selectivity, and resolution. The nature of the modifier and the modifier concentration were found to be crucial parameters. © 1996 Wiley-Liss, Inc. Contribution of the National Institute of Standards and Technology. Not subject to copyright.     

Simultaneous determination of residual veterinary drugs in bovine, porcine, and chicken muscle using liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A simple and rapid method using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of 130 veterinary drugs and their metabolites in bovine, porcine, and chicken muscle was developed. The drugs (1 to 10 ng/g, in muscle) were extracted from bovine, porcine, or chicken muscles with acetonitrile-methanol (95:5, v/v), and the extracts were delipidated with n-hexane saturated with acetonitrile. The extracts were evaporated, dissolved with methanol, analyzed by liquid chromatography with gradient elution on a C18 column, and determined by electrospray ionization tandem mass spectrometry. The detection limits ranged from 0.03 to 3 ng/g. The quantitation limits ranged from 0.1 to 10 ng/g. One hundred eleven, 122, and 123 drugs from bovine, porcine, and chicken muscle respectively showed recoveries between 70 and 110%.     

Repair replication in human cells studied by sodium iodide isopycnic centrifugation of DNA in a fixed-angle rotor

A method for purification of ethynyl steroids from biological fluids has been developed using silver-sulfoethyl cellulose column chromatography. Ethynyl steroids were applied in methanol, and the firm binding allowed the columns to be washed with methanol, ethyl acetate, or diethyl ether to remove endogenous materials. Nonethynylated steroids did not bind to silver-sulfoethyl cellulose. Release of ethynylated steroids was achieved with a saturated NaCl/methanol solution. Dehydration or de-ethynylation of ethynyl estradiol, ethynodiol diacetate, and ethynodiol was not observed. The utility of this technique for purification of ethynyl steroid metabolites from the urine of a beagle metabolizing norethynodrel was demonstrated.     

Determination of hydrophobicity of non-homologous series of anticancer drugs by reversed-phase high-performance liquid chromatography

The hydrophobiciy and specific hydrophobic surface area of 21 commercial anticancer drugs were determined by reversed-phase high-performance liquid chromatography on an octadecyl-silica column using methanol-water mixtures as eluents. Linear correlations were calculated between the log k′ values and the methanol concentration of the eluent, the intercept and slope were considered as the best estimation of the hydrophobicity and specific hydrophobic surface area. The relationship between retention characteristics and physicochemical parameters of drugs was evaluated by multivariate mathematical statistical methods, such as principal component analysis followed by two-dimensional non-linear mapping, varimax rotation and by cluster analysis. Anticancer drugs can be well separated by reversed-phase HPLC. Various multivariate mathematical statistical calculations indicate that the retention of the investigated drugs is mainly governed by hydrophobic and steric parameters. The results suggest that the use of principal component analysis followed by two-dimensional non-linear mapping is superior to cluster analysis for the evaluation of large retention data matrices.     

Chromatographic behaviour of rat-liver monophosphoinositide

1. Chromatography of rat-liver lipids on a column of silicic acid or a mixture of silicic acid and Hyflo Super-Cel, with chloroform–methanol mixtures, gave monophosphoinositide-containing fractions which were invariably contaminated by the presence of nitrogen-containing phospholipids. The behaviour of the inositide was extremely sensitive to column loading and the results with different batches of silicic acid were not reproducible. 2. However, when chromatography on an alumina column was used, the solvent system chloroform–methanol–water (23:23:4, by vol.) completely eluted the neutral lipids, choline-containing phospholipids and phosphatidylethanolamine. An increase of the water content of the solvent to 14% (by vol.) then led to the elution of the monophosphoinositide component, now free from nitrogen-containing phospholipids, but still contaminated by the presence of a phospholipid, which from its properties was taken to be polyglycerophosphatide. 3. Most of the polyglycerophosphatide could be removed from a rat-liver lipid extract by silicic acid chromatography with chloroform–methanol (19:1, v/v). The other phospholipids were then eluted and applied to an alumina column, whereby a monophosphoinositide fraction of much greater purity was obtained. 4. Further purification of the monophosphoinositide was achieved by chromatography on a mixture of silicic acid and cellulose powder. The final product was virtually pure by thin-layer chromatography and gave the expected analysis for monophosphoinositide.     

High-performance liquid chromatographic determination of hippuric acid in human urine

A method is described for the determination of urinary hippuric acid by high-performance liquid chromatography. The method used ethyl acetate extraction for partial clean-up of the urine. The separation was carried out on a reversed-phase column using 20% methanol in 0.01 M aqueous potassium phosphate containing 0.5% acetic acid as a mobile phase. The column effluent was monitored with a UV detector at 254 nm. Hippuric acid was separated from other normal urine constituents in less than 10 min. Metabolites of xylene and styrene did not interfere with the assay. Analytical recoveries from urine were excellent and peak height and concentration were linearly related.     

Enantioselective determination of FCE 28833, a new potential antiischemic agent,in gerbil plasma using column switching HPLC with UV detection

A sensitive and selective high performance liquid chromatographic method using an automated column switching technique for the determination of FCE 28833 enantiomers in gerbil plasma was developed. After solid-liquid extraction using a Supelcosil C₁₈ cartridge FCE 28833 was eluted on a clean-up column (Spherisorb CN) and the enantiomers were separated using an analytical chiral column (Crownpack CR(+)). The mobile phase (15% methanol in HClO₄ 1 mM) was directed through the columns at a flow rate of 1 ml/min and the fraction eluted between 13 and 40 min was transferred from the clean-up column into the analytical column. FCE 28833 enantiomers were monitored at 257 nm. The limit of quantitation of the method was 20 ng/ml plasma for both enantiomers and proved to be linear, precise, and accurate for the assay of both enantiomers in the 20–6,000 ng/ml concentration range. No interference from the blank gerbil plasma sample was observed. The suitability of the method was assessed using plasma samples obtained from male gerbils treated with a single oral dose (400 mg/kg) of FCE 28833. Chirality 9:133–138, 1997. © 1997 Wiley-Liss, Inc.     

Robust isocratic high performance liquid chromatographic method for simultaneous determination of seven antiepileptic drugs including lamotrigine, oxcarbazepine and zonisamide in serum after solid-phase extraction

A rapid, simple and robust method is presented for the simultaneous determination of seven antiepileptic drugs (AEDs), including primidone, phenobarbital, phenytoin, carbamazepine with its two major metabolites carbamazepine-10,11-epoxide and carbamazepine-10,11-(trans)-dihydrodiol and the new AEDs lamotrigine, hydroxycarbazepine (active metabolite of oxcarbazepine) and zonisamide in serum by high performance liquid chromatography (HPLC)-diode array detector (DAD). After solid-phase extraction, separation is achieved on an Alltima 3C18 analytical column using isocratic elution with a mixture of acetonitrile, methanol and phosphate buffer at 45 degrees C. The method is exhaustively validated, including experimental design in combination with statistical evaluation (ANOVA) to study the robustness of chromatography and sample preparation. Commonly co-administered antiepileptic drugs do not interfere with the method. Intra-day precision (RSD<1.9%), linearity, lower limit of quantitation (LOQ<0.065 mg/l) and robustness make the method suitable for daily therapeutic drug monitoring and pharmacokinetic studies.     

Evaluation of reversible and irreversible models for the determination of the enantiomerization energy barrier for N-(p-methoxybenzyl)-1,3,2-benzodithiazol-1-oxide by supercritical fluid chromatography

It has been found that the interconversion of enantiomers on a chromatographic column during the separation process can be studied by the first-order kinetic equations derived both for reversible and irreversible reactions in a stationary system if the extent of interconversion is not too high. The equation derived for irreversible reactions gives, however, results also for higher degrees of enantiomerization while that derived for reversible interconversion failed. The irreversible equation was used to determine the enantiomerization barrier of N-(p-methoxybenzyl)-l,3,2-benzodithiazol-l-oxide enantiomers by supercritical fluid chromatography. The racemate of N-(p-methoxybenzyl)-l,3,2-benzodithiazol-l-oxide was separated by supercritical fluid chromatography on the (R,R)-Whelk-Ol column with supercritical carbon dioxide containing 20% methanol as a mobile phase. Peak areas of enantiomers prior to and after the separation used for the calculation of the enantiomerization barrier were determined by computer-assisted peak deconvolution of peak clusters registered on chromatograms using commercial software.     

Simultaneous,quantitative determination of opiates,amphetamines, cocaine and benzoylecgonine in oral fluid by liquid chromatography quadrupole-time-of-flight mass spectrometry

A method using liquid chromatography coupled to tandem mass spectrometry is described for the determination of drugs of abuse in oral fluid. The method is able to simultaneously quantify amphetamines (amphetamine, methamphetamine, MDA, MDMA and MDEA), opiates (morphine and codeine), cocaine and benzoylecgonine. Only 200 micro of oral fluid is spent for analysis. The sample preparation is easy and consists of mixed mode phase solid-phase extraction. Reversed-phase chromatography is carried out on a narrow bore phenyl type column at a flow-rate of 0.2 ml/min. A gradient is applied ranging from 6 to 67.6% methanol with ammonium formate (10 mM, pH 5.0) added to the mobile phase. The column effluent was directed into a quadrupole-time-of-flight instrument by electrospray ionization, without the use of a splitter. A validation study was carried out. Recovery ranged from 52.3 to 98.8%, within-day and between-day precision expressed by relative standard deviation were less than 11.9 and 16.8%, respectively, and inaccuracy did not exceed 11.6%. The limit of quantification was 2 ng/ml (0.66 x 10(-5)-1.48 x 10(-5) M) for all compounds. Internal standards were used to generate quadratic calibration curves (r(2)>0.999). The method was applied to real samples obtained from suspected drug users. An interference was observed from the device used to sample the oral fluid, consequently this was excluded from the method which was validated on oral fluid obtained by spitting in a test-tube.     

Isolation and identification of arctiin and arctigenin in leaves of burdock (Arctium lappa L.) by polyamide column chromatography in combination with HPLC-ESI/MS

A simple method involving polyamide column chromatography in combination with HPLC-PAD and HPLC-ESI/MS for isolating and identifying two kinds of lignans, arctiin and arctigenin, in the leaves of burdock (Arctium lappa L.) has been established. After extraction of burdock leaves with 80% methanol, the aqueous phase of crude extracts was partitioned between water and chloroform and the aqueous phase was fractionated on a polyamide glass column. The fraction, eluting with 100% methanol, was concentrated and gave a white precipitate at 4 degrees C from which two main compounds were purified by semi-preparative HPLC. In comparison with the UV and ESI-MS spectra and the HPLC retention time of authentic standards, the compounds were determined to be arctiin and arctigenin. The extraction/separation technique was validated using an internal standard method.     

High-performance liquid chromatographic determination of irinotecan (CPT-11) and its active metabolite (SN-38) in human plasma

A simplified method for the simultaneous determination of irinotecan (CPT-11, I) and its active metabolite (SN-38, II) in human plasma by high-performance liquid chromatography (HPLC) with fluorescence detection has been developed. Following the addition of the internal standard (I.S.) camptothecin, the drugs were extracted from plasma using methanol. The average extraction efficiencies were 87% for I, 90% for II and 90% for the I.S. Chromatography was performed using a TSK gel ODS-80Ts column, monitored at 556 nm (excitation wavelength, 380 nm) and the mobile phase was acetonitrile-50 mM disodium hydrogen phosphate (28:72) containing 5 mM heptanesulphonate (pH 3.0). The linear quantitation ranges for I and II were 30–2000 and 1–30 ng/ml, respectively.     

Reversal of elution order for profen acid enantiomers in packed-column SFC on Chiralpak AD

Enantiomeric separations of four 2-substituted propionic acid drugs have been studied using packed-column supercritical fluid chromatography (SFC) with amylose tris(3,5-dimethylphenylcarbamate) coated on silica as support (Chiralpak AD). Under standard conditions (i.e., flow rate, 1.5 ml/min; column temperature, 30 degrees C; back-pressure, 150 bar), the order of elution could be reversed when the polar alcohol modifier methanol in carbon dioxide was replaced by 2-propanol for ibuprofen, ketoprofen, and naproxen. For flurbiprofen, with the highest selectivity factor, no reversal was observed, although selectivity was reduced significantly with higher alcohols. Naproxen and flurbiprofen were also investigated with 2-butanol and 2-pentanol. The former showed reversal of elution order but not the latter. For higher alcohol modifiers, including 2-propanol, the peak symmetry was poor but could be improved by addition of citric acid in the alcohol modifier. These results stress the importance to investigate enantiomer elution order during the development of enantioselective methods and when chromatographic conditions are optimized. Preliminary experiments with column temperatures over the range of -15 to 45 degrees C revealed that, in a few cases, reversal took place with a change in temperature only.     

Quantification of antidepressants and antipsychotics in human serum by precipitation and ultra high pressure liquid chromatography-tandem mass spectrometry

The present article describes the quantification of mirtazapine, O-desmethylvenlafaxine, quetiapine, venlafaxine, and ziprasidone (group 1), and amitriptyline, citalopram, clomipramine, clozapine, desmethylclomipramine, desipramine, imipramine, and nortriptyline (group 2) in human serum for therapeutic drug monitoring. The method was developed to replace old techniques which applied solid phase extraction and ultra-violet detection. The old methods had reached their limit of capacity regarding the number of samples and co-medicated drugs interfering with the detection. Serum samples were precipitated with zinc sulphate and methanol containing a stable isotope labelled analog for each analyte. Quantitative analysis was performed by ultra high pressure liquid chromatography combined with a tandem mass spectrometer using a Zorbax SB-C8 column (2.0 × 50 mm; 1.8 μm) with a mobile phase consisting of 0.1% formic acid in water and methanol, respectively. The total run time of the chromatography was 4 min. Precision and trueness varied from 2.6% to 14.9% and 87.6% to 103.5%, respectively. At the lower limit of quantification, precision was up to 17.9% and trueness varied from 89.5% to 111.5%. A five point standard curve covering the clinically relevant ranges with a power function fit was applied for calibration. Ion suppression from matrix effects and internal standards were thoroughly investigated and are discussed. Process efficiency rates varied from 42% to 99%. The method has shortened the response time, reduced interference from other drugs, avoided acetonitrile usage, and reduced the amount of serum needed for analysis 50-fold.     

Preparative high-performance liquid chromatography of minor products ofStreptomyces cinnamonensis

Analytical and preparative high-performance liquid chromatography of 3 phenazines and furonaphthoquinone derivative on resersed-phase column are described. The mobile phase was methanol and water. The injected amount of the mixture was about 30 mg for a preparative chromatographic run requiring 80 min. Substances were detected directly in the column effluent by UV detection.     

Preparative isolation of polyphosphoinositide fractions from ox brain

A simple preparative method for chromatographic isolation of pure fractions of di- and triphosphoinositides (1-phosphatidylinositol 4-phosphate and 1-phosphatidylinositol 4,5-bisphosphate) from ox brain is described. Polyphosphoinositide fractions have been obtained by ion-exchange chromatography of the lipid extract using gradient elution with 0-0.6 M ammonium acetate in chloroform/methanol/water (20:9:1) from a DEAE-cellulose column. Before chromatography, divalent metal ions were removed from the lipid extract by passing through a Dowex-50 (H+) column and lipids were converted to the sodium salt by neutralisation with sodium hydroxide in methanol solution. After chromatography, fractions of di- and triphosphoinositides were precipitated in methanol/water mixture (1:1) by evaporation in a vacuum to a final concentration of about 4 M ammonium acetate. Necessary salts of di- and triphosphoinositides were obtained by passing the ammonium salts of the lipids through Dowex-50 (H+) and neutralising with corresponding base in methanol solution. About 0.35 mmol of diphosphoinositide and 0.63 mmol of triphosphoinositide were obtained from 1 kg of wet ox brain tissue.     

Preparative method of isolating phosphoinositide fractions from brain tissue]

A simple technique for preparative isolation of chromatographically homogenous fractions of mono-, di- and triphosphoinositides from ox brain tissue is described. Podyphosphoinositides fractions were obtained after chromatography of lipid extract on DEAE cellulose, phosphomonoinositides fraction--after chromatography of polyphosphoinositide-free material on aluminium hydroxide column. Bivalent metal ions were eliminated from lipid extract using chromatography on Dowex-50 H+. Ammonium acetate was removed after precipitation of lipids in water: methanol (1:1) in the presence of 4 M this salt. The average yield of mono-, di- and triphosphates was 40, 22 and 58 mg respectively per 1 kg of brain tissue as callulated for lipid phosphorus.     

Pentacyclic triterpenoids and sterols from seven species of mangrove

The isolation of pentacyclic triterpenoids from seven species of fresh mangrove leaves using a simple and rapid method is described. The leaves were homogenized using chloroform—methanol and the extract was diluted with water to precipitate out triterpenoids which were separated into neutral and acidic fractions. These were analysed by gas-liquid chromatography as acetyl and trimethylsilyl ether derivatives on a 3% OV-17 column. Sterols were isolated from the chloroform layer by preparative thin layer chromatography and were analysed by gas-liquid chromatography as their trimethylsilyl ether derivatives on a 3% OV-17 column. The triterpenoids found were α-amyrin, β-amyrin, lupeol, oleanolic acid and ursolic acid in most of the samples. Sterols found in all the samples were cholesterol, campesterol, stigmasterol, sitosterol and stigmast-7-en-3β-ol. Retention indices of the triterpenoids and sterols have been determined.     

SPE-HPLC法快速检测发酵液中紫杉醇的含量

针对红豆杉内生真菌发酵液中紫杉醇的含量测定进行探讨,以建立快速高效低耗的检测方法.采用C_(18)固相萃取柱对紫杉醇进行吸附,用不同浓度的甲醇-乙酸铵和甲醇分别作为洗脱剂对其进行洗脱,比较两者的洗脱效果,洗脱液用HPLC进行检测;色谱条件为:流动相甲醇(v):水(v):乙腈(v)=20: 45: 35,流速:0.70 ml/min,检测波长:227 nm.结果表明,浓度为80%的甲醇溶液洗脱效果较好,紫杉醇的回收率为87.6%.