Identification of flurbiprofen and its photoproducts in methanol by gas chromatography-mass spectrometry

A sample of 10 mM flurbiprofen in methanol (or ethanol) was photoirradiated with sixteen 8 W low-pressure quartz mercury lamps irradiated at 306 nm in a Panchum PR-2000 photochemical reactor. In total, four major photoproducts derived from each sample were observed from the HPLC chromatogram. The photoproducts were separated and their structures elucidated by various spectroscopic methods. Alternatively, using GC-MS, 11 major photoproducts were observed. A reaction scheme of flurbiprofen in methanol is proposed: the photochemical reaction routes occur mainly via esterification and decarboxylation, followed by oxidation with singlet oxygen to produce a ketone, alcohols and other derivatives.     

Photolysis of NSAIDs. Part 3: Structural elucidation of photoproducts of tolmetin in methanol

A sample of 10 mM tolmetin in methanol was photo-irradiated with a Hanovia 200 W high-pressure quartz Hg lamp for four days. In total, eight photoproducts were observed from the HPLC chromatogram. Three major photoproducts were separated, and their structures were elucidated by spectroscopic methods. The structures of all photoproducts were further determined by LC-ESI-MS. A reaction scheme of tolmetin was proposed.     

Assay of naproxen by high-performance liquid chromatography and identification of its photoproducts by LC-ESI MS

A rapid, accurate and reliable reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of naproxen and its photodegradation products in methanol was developed and validated. An Inertsil 5-ODS-3V column (5 microm, C18, 250 x 4.6 mm i.d.) was used with a mobile phase of acetonitrile-methanol-1% HOAc in H2O (40:20:40, v/v/v). UV detection was set at 230 nm. The developed method satisfies system suitability criteria, peak integrity and resolution for the parent drug and its photoproducts. The intraday and interday standard deviations of five replicate determinations for five consecutive days at the working concentrations of 5.0, 10, 25, 50, and 100 microm were 0.23-0.98 with coefficients of variance (CVs) of between 0.96 and 4.56% for the former, and 0.14-1.15 with CVs of between 1.13 and 3.82% for the latter. The percentage recoveries were determined to be 98.34, 99.19, 100.18, 102.97 and 99.81%, respectively, at the five concentrations between 5.0 and 100 microm. The limit of quantitation of naproxen was determined to be 0.29 microg/mL, while the detection limit was 64 ng/mL. Four major photoproducts were observed from the HPLC chromatogram using a Panchum PR-2000 reactor which equipped with 8 W x 16 low-pressure quartz mercury lamps as the light source for irradiation of a naproxen sample in methanol. The structures of the photoproducts were confirmed by LC-ESI MS.     

Separation and structure determination of nicardipine photoproducts by LC-ESI-MS

A sample of 0.104 m nicardipine in methanol was photoirradiated with a Philips 400 W UV lamp for 3 h in a photochemical chamber. A total of four major photoproducts were found from the HPLC chromatogram. The same sample was used for taking LC-MS, while eight major photoproducts were observed and the structures elucidated by analyzing the CID patterns of their respective mass spectra. A reaction scheme of nicardipine is proposed that the photochemical reactions occur mainly via oxidation of 1,4-dihydropyridine moiety, following the stepwise photo-reduction of the m-nitro group and demethylation of the ester group at 5-position of the pyridine ring. Copyright (c) 2008 John Wiley & Sons, Ltd.     

PHOTOPRODUCTS OF BROMOURACIL-LABELED DNA AND THE STRUCTURE OF 5-BROMODEOXYURIDYLYL-(3' → 5')-THYMIDINE PHOTOPRODUCT

Abstract— –An attempt was made to identify some of the ultraviolet (u.v.) photoproducts of 5-bromouracil-labeled DNA (BrU-DNA). Two synthetic dinucleotides, 5-bromodeoxyuridylyl-(3' →5 ')-thymidine (BrdUpT) and 5-bromodeoxyuridylyl-(3' → 5')-deoxycytidine (BrdUpdC), were prepared. Each gave a single u.v. photoproduct which in turn gave a single acid hydrolysis product. 2-¹⁴C-BrU-DNA. prepared from E. coli B3, was irradiated (275–280 nm), hydrolyzed, and paper chromatographed in four systems. Comparison with the two synthetic photoproducts showed that if present at all, BrdUpT and BrdUpdC photoproducts could account for no more than 10 and 3.5 per cent respectively of the total photoproducts. At 55 per cent conversion of BrU into photoproducts, the major ¹⁴C-photoproduct was uracil (78 per cent); the remaining 22 per cent was made up of at least six products, three of which were reversed by 232 nm irradiation.
The debrominated cyclobutane structure proposed by Haug for BrdUpT photoproduct has been shown to be incorrect. It was found to contain one atom of bromine per molecule. On the basis of nuclear magnetic resonance and u.v. spectra, two possible structures are proposed for the photoproduct, each containing an eight-membered ring.     

2,3-吡啶二甲酰亚胺及其工程菌转化产物的高效液相色谱检测

建立了利用含D-海因酶的基因工程菌转化吡啶二甲酰亚胺（PDI）生成3-氨基甲酰基-α-吡啶甲酸（α-3CP）的高效液相色谱（HPLC）检测方法。将工程菌pET3a-hyd/BL21（DE3）诱导表达后收集菌体,以PDI为底物,37℃摇床反应30 min后,以13000 r/min离心,取上清液进行HPLC检测。色谱条件：Hypersil^TM GOLD C18色谱柱（250 mm×4.6 mm,5 μm）;流动相为H₂O-乙腈（体积比为90：10,含0.1%（体积分数）三氟乙酸）;检测波长为254 nm。当底物PDI达到饱和浓度时,测得工程菌pET3a-hyd/BL21（DE3）的比活力为0.61 U/（mL·10OD_{600 nm}）。该研究为今后利用生物法制备复杂半酰胺有机物提供了坚实的理论基础。     

Stereoselective determination of allethrin by two-dimensional achiral/chiral liquid chromatography with ultraviolet/circular dichroism detection

A two-dimensional achiral/chiral HPLC method with circular dichroism (CD) detection was optimized for the stereochemical resolution and determination of the elution order of the eight stereoisomers of synthetic allethrin. A monolithic silica HPLC column (Chromolith, Merck, 100 mm x 4.6 mm i.d.) was put orthogonally to an enantioselective OJ Daicel column (250 mm x 4.6 mm i.d.) by means of a switching valve. The resolution of cis and trans diastereoisomers on the silica column was obtained by using a mobile phase consisting of n-hexane:tert-butyl methyl ether (96:4) (v/v) at a flow rate of 1 ml min(-1). The cis and trans peaks were then switched to the enantioselective OJ column separately in two subsequent injections. The resolution of the four trans stereoisomers was accomplished by using n-hexane:tert-butyl methyl ether (90:10) (v/v), while the mobile phase composition for the four cis stereoisomers consisted of n-hexane:isopropanol (99.3:0.7) (v/v). The CD based detection system allowed the determination of the elution order on the basis of the CD signals of the single stereoisomers, together with the injection of pure stereoisomers. Under the final conditions, the validated method was applied to the determination of stereoisomeric composition and absolute configuration of the prevailing stereoisomers of real samples, i.e. commercial batches of different sources of d-allethrin.     

Separation and purification of four compounds from Desmodium styracifolium using off‐line two‐dimensional high‐speed counter‐current chromatography

An off‐line 2D high‐speed counter‐current chromatography technique in preparative scale has been successfully applied to separate and purify the main compounds from the ethyl acetate extract of Desmodium styracifolium. A two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water at an optimized volume ratio of 1:2:1:2 v/v/v/v was used. Conventional high‐speed counter‐current chromatography was used as the first dimension, and the upper phase of the solvent system was used as the stationary phase in the head‐to‐tail elution mode at a flow rate of 2.0 mL/min and a rotation speed of 900 rpm. Recycling high‐speed counter‐current chromatography served as the second dimension to separate an impure fraction of the first dimension. A total of four well‐separated substances including vanillic acid ( 1 ), β‐sitosterol ( 2 ), formononetin ( 3 ), and aromadendrin ( 4 ) were obtained, and their purities and structures were identified by HPLC–MS and ¹H NMR spectroscopy. The results illustrated that off‐line 2D high‐speed counter‐current chromatography is an effective way to isolate compounds in complex samples.     

Large-scale separation of hydroxyanthraquinones from Rheum palmatum L. by pH-zone-refining counter-current chromatography

pH-zone-refining counter-current chromatography was successfully applied to purify four hydroxyanthraquinones, rhein, emodin, aloe-emodin and chrysophanol, from three crude extracts of Rheum palmatum L.. After the two-phase solvent system methyl tert-butyl ether-tetrahydrofuran-water at an optimized volume ratio of 2:2:3 (v/v) was equilibrated, trifluoroacetic acid (10 mM) was added to the organic phase as a retainer and ammonia (10 mM), sodium carbonate (15 mM) and sodium hydroxide (15 mM) were added to the aqueous phase as the eluter, respectively, for three individual runs. Three separation runs of 1.25, 1.53 and 1.41 g of the three crude samples yielded four hydroxyanthraquinones: 0.70 g rhein, 0.81 g emodin, 0.41 g aloe-emodin and 0.94 g chrysophanol at a high purity of over 99.0, 98.5, 98.2 and 97.8% (determined by HPLC), respectively. The structures were identified by electrospray ionization MS-MS and (1)H NMR.     

A simple and sensitive assay for the quantitative analysis of paclitaxel and metabolites in human plasma using liquid chromatography/tandem mass spectrometry

A sensitive and specific LC-MS/MS assay for the determination of paclitaxel and its 3'p- and 6-alpha-hydroxy metabolites is presented. A 200 microL plasma aliquot was spiked with a 13C6-labeled paclitaxel internal standard and extracted with 1.0 mL tert-butylmethylether. Dried extracts were reconstituted in 0.1 M ammonium acetate-acetonitrile (1:1, v/v) and 25 microL volumes were injected onto the HPLC system. Separation was performed on a 150 x 2.1 mm C18 column using an alkaline eluent (10 mm ammonium hydroxide-methanol, 30:70, v/v). Detection was performed by positive ion electrospray followed by tandem mass spectrometry. The assay quantifies a range for paclitaxel from 0.25 to 1000 ng/mL and metabolites from 0.25 to 100 ng/mL using 200 microL human plasma samples. Validation results demonstrate that paclitaxel and metabolite concentrations can be accurately and precisely quantified in human plasma. This assay is now used to support clinical pharmacologic studies with paclitaxel.     

Preparative isolation of alkaloids from Corydalis bungeana Turcz. by high-speed counter-current chromatography using stepwise elution

High‐speed counter‐current chromatography (HSCCC) was successfully applied for the preparative separation and purification of alkaloids from Corydalis bungeana Turcz. (Kudiding in Chinese) for the first time. After the measurement of partition coefficient of seven target alkaloids in the nine two‐phase solvent systems composed of CHCl₃–MeOH–(0.1 M; 0.2 M; 0.3 M) HCl (4:1.5:2; 4:2:2; 4:3:2, v/v), CHCl₃–MeOH–0.2 M HCl (4:2:2, v/v) and CHCl₃–MeOH–0.3 M HCl (4:3:2, v/v) were finally selected for the HSCCC separation using the first upper phase as the stationary phase and the stepwise elution of the two lower mobile phases. Consequently, sanguinarine (10 mg), corynoline (25 mg), protopine (20 mg), corynoloxine (18 mg), and 12‐hydroxycorynoline (8 mg) were obtained from 200 mg of crude alkaloid extracts with purities of 94–99% as determined by HPLC. Their chemical structures were characterized on the basis of ¹H‐NMR, ¹³C‐NMR, and LC‐ESI‐Q‐TOF‐MS/MS analyses.     

Preparative separation of flavonoids in plant extract of Smilacis Glabrae Roxb. by high performance counter‐current chromatography

Four flavonoids, isoastilbin, astilbin, isoengelitin, and engelitin were isolated and purified simultaneously from Smilacis Glabrae Roxb. for the first time by high performance counter‐current chromatography using a system consisting of n‐hexane–n‐butanol–water (1:2:3, v/v/v). A total of 392.6 mg of astilbin, 71.4 mg of isoastilbin, 47.4 mg of engelitin, and 10.3 mg of isoengelitin were purified from 1.89 g of the ethyl acetate extract of Smilacis Glabrae Roxb. in six runs, each at over 94.51% purity as determined by HPLC. The structures of the four compounds were identified by their retention time, the LC‐ESI‐MSⁿ in the negative ion mode, and confirmed by ¹H‐NMR experiments. The characteristic LC‐ESI‐MS fragmentation patterns of the four compounds were discussed.     

Isolation and purification of macrocyclic components from Penicillium fermentation broth by high‐speed counter‐current chromatography

In this paper, high‐speed counter‐current chromatography (HSCCC), assisted with ESI‐MS, was first successfully applied to the preparative separation of three macrolide antibiotics, brefeldin A (12.6 mg, 99.0%), 7′‐O‐formylbrefeldin A (6.5 mg, 95.0%) and 7′‐O‐acetylbrefeldin A (5.0 mg, 92.3%) from the crude extract of the microbe Penicillium SHZK‐15. Considering the chemical nature and partition coefficient (K) values of the three target compounds, a two‐step HSCCC isolation protocol was developed in order to obtain products with high purity. In the two‐step method, the crude ethyl acetate extract was first fractionated and resulted in two peak fractions by HSCCC using solvent system n‐hexane/ethyl acetate/methanol/water (HEMWat) (3:7:5:5 v/v/v/v), then purified using solvent systems HEMWat (3:5:3:5 v/v/v/v) and HEMWat (7:3:5:5 v/v/v/v) for each fraction. The purities and structures of the isolated compounds were determined by HPLC, X‐ray crystallography, ESI‐MS and NMR. The results demonstrated that HSCCC is a fast and efficient technique for systematic isolation of bioactive compounds from the microbes.     

A sensitive and selective determination method of histamine by HPLC with intramolecular excimer-forming derivatization and fluorescence detection

A highly sensitive, selective and simple method is described for the determination of histamine by high-performance liquid chromatography (HPLC) with fluorescence detection. The method is based on an intramolecular excimer-forming fluorescence derivatization of histamine with 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase HPLC. Histamine, having two amino moieties in a molecule, was converted to the dipyrene-labeled derivative by reaction with PSE. The derivative afforded intramolecular excimer fluorescence (450-540 nm), which can clearly be discriminated from the monomer fluorescence (370-420 nm) emitted from PSE. Typically, a 10 micro L sample solution was mixed with 100 micro L of derivatization reagent solution, which was a mixture of 0.5 mm PSE in acetonitrile and 0.5 mm potassium carbonate in water (8:2, v/v). The derivatization was carried out at 100 degrees C for 90 min. The PSE derivative of histamine could be separated by reversed-phase ODS column with isocratic elution using acetonitrile:water (82:18, v/v) containing 0.03% triethylamine. The detection limit (singnal-to-noise ratio = 3) of histamine was 0.5 fmol for a 30 micro L injection. The method was successfully applied to the determination of histamine in human urine, and had enough selectivity and sensitivity for urinary histamine quantification.     

Single-run analysis of retinal isomers, retinol and photooxidation products by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) procedure was developed to allow the rapid separation, in a single run, of a mixture of the main retinal isomers (all-trans, 13-cis, 9-cis), all-trans-retinol, and of the two major photooxygenated photoproducts (5,8-peroxyretinal and 5,6-epoxyretinal). The mixture was separated by HPLC on an octadecyl (ODS) column with 16% (v/v) diethyl ether in hexane as mobile phase and anthracene as the internal standard. A commercial type cosmetic formulation containing 0.05% all-trans-retinal was analyzed successfully for this analyte.     

HPLC analysis and pharmacokinetic study of quercitrin and isoquercitrin in rat plasma after administration of Hypericum japonicum thunb. extract

A simple HPLC method was developed for determination of quercitrin and isoquercitrin in rat plasma. Reversed-phase HPLC was employed for the quantitative analysis using kaempferol-3-O-beta-D-glucopyranoside-7-O-alpha-L-rhamnoside as an internal standard. Following extraction from the plasma samples with ethyl acetate-isopropanol (95:5, v/v), these two compounds were successfully separated on a Luna C(18) column (250 x 4.6 mm, 5 microm) with isocratic elution of acetonitrile-0.5% aqueous acetic acid (17:83, v/v) as the mobile phase. The flow-rate was set at 1 mL/min and the eluent was detected at 350 nm for both quercitrin and isoquercitrin. The method was linear over the studied ranges of 50-6000 and 50-5000 ng/mL for quercitrin and isoquercitrin, respectively. The intra- and inter-day precisions of the analysis were better than 13.1 and 13.2%, respectively. The lower limits of quantitation for quercitrin and isoquercitrin in plasma were both of 50 ng/mL. The mean extraction recoveries were 73 and 61% for quercitrin and isoquercitrin, respectively. The validated method was successfully applied to pharmacokinetic studies of the two analytes in rat plasma after the oral administration of Hypericum japonicum thunb. ethanol extract.     

High-performance liquid chromatography-electrospray ionization mass spectrometry determination of mitiglinide in human plasma and its pharmacokinetics

A selective and sensitive method employing high-performance liquid chromatography-electrospray ionization mass spectrometry was developed and validated for the determination of mitiglinide in human plasma.With gliclazide as the internal standard, mitiglinide was extracted from plasma with n-hexane: = 80 : 20 (v/v). The organic layer was evaporated and the residue was redissolved in methanol: water (10 mM CH3COONH4, pH = 3.0) = 65 : 35 (v/v). An aliquot of 10 microl was chromatographically analyzed on a prepacked Shimadzu VP-ODS (5 microm, 150 x 2.0 mm i.d.) using the mobile phase comprising methanol: water (10 mM CH3COONH4) = 65 : 35 (v/v) by means of selected-ion monitoring mode mass spectrometry. Standard curves were linear (r2 = 0.9972) over the concentration range of 2.84-11 300 pmol/ml and had good accuracy and precision. The within- and between-batch precisions of the method were within 15% of standard deviation. The lower limit of detection was 1.42 pmol/ml. The validated HPLC/ESI-MS method has been successfully applied in the pharmacokinetics of mitiglinide in 12 healthy Chinese volunteers.     

Determination, purity assessment and chiral separation of levodopa methyl ester in bulk and formulation pharmaceuticals

A sensitive, reliable and reproducible HPLC method with electrochemical detection (HPLC-ECD) has been developed for the separation and quantification of levodopa methyl ester (LDME) and its impurities such as levodopa (l-DOPA), 3-methoxytyrosine (MTS) and l-tyrosine (TS) in bulk drug and pharmaceutical dosage form. The separation was performed on an LC18 column by isocratic elution with methanol-acetonitrile-50 mm potassium dihydrogen phosphate (8:2:90, v/v/v) containing 5 mm sodium 1-hexanesulfonate, 5 mm EDTA and 5 mm sodium chloride, adjusted with phosphoric acid to a pH of 3.2 as mobile phase. The correlation coefficients of linear regression for LDME, L-DOPA, MTS and TS were more than 0.999. The detection limits for L-DOPA, MTS and TS were 3.15, 2.04 and 2.88 ng/mL, respectively. The precision was checked in terms of F-test variance ratio using potentiometric titration as reference. The separation of dopa methyl ester enantiomers by chiral chromatography is also described. This method is capable of separating the two enantiomers with a selection of 1.4 and a resolution of 8.4. Both methods were found to be stable and useful in the quality control of the bulk material and formulations.     

SEPARATION OF THE MINOR FLAVONOLS FROM FLOS GOSSYPII BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY

An effective high-speed countercurrent chromatography (HSCCC) method was established for further separation and purification of four minor flavonols in addition to five major flavonols which were reported by our previous study from extracts of Flos Gossypii. HSCCC was performed with three two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (7.5:15:6:7, v/v), (2.5:15:2:7, v/v) and (0:1:0:1, v/v). The separation was repeated 3 times, and 3.8 mg of 8-methoxyl-kaempferol-7-O-β-D-rhamnoside (HPLC purity 98.27%), 6.7 mg of astragalin (HPLC purity 94.18%), 3.3 mg of 4'-methoxyl-quercetin-7-O-β-D-glucoside (HPLC purity 94.30%) and 8.2 mg of hyperoside (HPLC purity 93.48%) were separated from 150 mg of the crude sample. The chemical structures of the flavonols were confirmed by MS, (1)H NMR and (13)C NMR. Meanwhile, the results indicated that the target compound with smaller K value (<0.5) can be separated by increasing column length of HSCCC. And four separation rules of flavonols according to the present study and references were summarized, which can be used as a useful guide for separation of flavonols by HSCCC.     

An HPLC‐UV method for determining plasma dimethylacetamide concentrations in patients receiving intravenous busulfan

Dimethylacetamide (DMA) is a solvent used in the preparation of intravenous busulfan, an alkylating agent used in blood or marrow transplantation. DMA may contribute to hepatic toxicity, so it is important to monitor its clearance. The aim of this study was to develop an HPLC‐UV assay for measurement of DMA in human plasma. After precipitation of plasma proteins with acetonitrile followed by dilution (1:4) with water, the extract was injected onto the HPLC and detected at 195 nm. Separation was performed using a Cogent‐HPS 5 μm C₁₈ column (250 × 4.6 mm) preceded by a Brownlee 7 μm RP₁₈, pre‐column (1.5 cm × 3.2 mm). The mobile phase was 25 mm sodium phosphate buffer (pH 3), containing 2.5% (v /v) acetonitrile and 0.0005% (v /v) sodium‐octyl‐sulfonate. Using a flow rate of 1 mL/min, the retention times of DMA and the internal standard (IS), 2‐chloroacetamide, were 9.5 and 3.5 min, respectively. Peak area ratio (DMA:IS) was a linear function of concentration from 1 to 1000 μg/mL. There was excellent intraday precision (<5% for 5–700 μg/mL DMA), accuracy (<3% deviation from the true concentration) and recovery (74–98%). The limits of detection and quantification were 1 and 5 μg/mL, respectively. In eight children who received intravenous busulfan, DMA concentrations ranged from 110 to 438 μg/mL.