Determination of 3',4',5',5,7-pentamethoxyflavone in the plasma and intestinal mucosa of mice by HPLC with UV detection

In a preliminary experiment 3',4',5',5,7-pentamethoxyflavone (PMF) inhibited adenoma development in Apc(Min) mice, a model of the human heritable condition familial adenomatous polyposis. An HPLC method for tricin was modified and validated to permit measurement of PMF in mouse plasma and intestinal mucosa. HPLC analysis was carried out on a Hypersil-BDS C(18) column with detection at 324 nm and tricin as internal standard. The assay was linear in the range of 100-2000 ng/mL plasma and 1.0-40 microg/mL mucosa. PMF in plasma was efficiently extracted using solid-phase columns. In the case of mucosa organic solvent protein precipitation displayed satisfactory accuracy and precision. The assay recovery at low, medium and high concentrations was between 85 and 103% for both biomatrices, with a relative standard deviation of <15%. The lower limits of quantitation for plasma and mucosa were 100 ng/mL and 1.0 microg/mL, respectively. This method allowed measurement of PMF steady-state median concentrations in plasma (1.08 nmol/mL, n = 11; 10th and 90th percentiles: 0.633 and 2.385 nmol/mL) and mucosa (108.5 nmol/g, n = 9; 10th and 90th percentiles: 38.9 and 164.4 nmol/g) in mice which had received PMF (0.2%, w/w) with their diet.     

高效液相色谱法测定小鼠血浆和组织中的阿昔洛韦

改良HPLC用于测定小鼠血浆和组织中阿昔洛韦浓度。使用Beckinan Gold系列高效液相色谱仪,ODS柱,流动相为甲醇－水（5：95,V／V）,流速1．0mL/min,检测波长254nm,样品采用12％三氯醋酸去蛋白。血浆和组织中阿昔洛韦浓度在0．05－20mg/L范围内与峰高呈良好线性关系,最低检测浓度分别为0.02.0.05mg/L,天间精密度均优于6％。     

Mechanism for ribonucleotide reductase inactivation by the anticancer drug gemcitabine

Gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) is a very promising anticancer drug, already approved for clinical use in three therapeutic indications. It is metabolized intracellularly to 5'-diphosphate (dFdCDP), which is known to be a potent inhibitor of ribonucleotide reductase (RNR). Although several nucleotide analogs show in vitro capacity of RNR inactivation, none has shown the in vivo efficacy of dFdCDP. Accordingly, the experimental data suggests that its mechanism of inhibition is different from the other known RNR suicide inhibitors. Enzyme inhibition in the absence of reductive species leads to complete loss of the essential radical in subunit R2, and formation of a new nucleotide-based radical. Interestingly, however, the presence of the reductants does not prevent inhibition--the radical is not lost but the targeted subunit of RNR becomes R1, which is inactivated possibly by alkylation. We have conducted a theoretical study, which led us to the first proposal of a possible mechanism for RNR inhibition by dFdCDP in the absence of reductants. This mechanism turned out to be very similar to the natural substrate reduction pathway and only deviates from the natural course after the formation of the well-known disulphide bridge. This deviation is caused precisely by the F atom in the beta-face, only present in this inhibitor. The essential radical in R2 is lost, and so is the enzyme catalytic activity. The nucleotide-based radical that constitutes the end product of our mechanism has been suggested in the literature as a possible candidate for the one detected experimentally. In fact, all experimental data available has been reproduced by the theoretical calculations performed here.     

Chiral separation of 1,1'-bi-2-naphthol and its analogue on molecular imprinting monolithic columns by HPLC

Two molecular imprinting polymer (MIP) monolithic columns with (S)-(-)-1,1'-bi-2-naphthol and (R)-(+)-5,5',6,6',7,7',8,8'-octahydro-1,1'-bi-2-naphthol as the templating molecules, respectively, have been prepared by in situ polymerization using 4-vinylpyridine and ethylene dimethacrylate as functional monomer and cross-linker, respectively. The columns with good flow-through properties were obtained by changing the molar ratio of the functional monomer and the template molecule. The effects of mobile-phase composition on separation of enantiomers were systematically investigated. The results indicate that hydrophobic interaction in aqueous solution and hydrogen-bonding interaction in ACN between the enantiomers and polymers could play important roles in the retention and resolution. The effects of chromatographic conditions, such as flow rate, column temperature, sample loading, on the enantioseparation were also studied. Further, these two MIP columns show a cross-reactivity.     

Preparative isolation of guaipyridine sesquiterpene alkaloid from Artemisia rupestris L. flowers using high-speed counter-current chromatography

Although the medicinal plant Artemisia rupestris L. has been widely researched for several decades, its alkaloids have never been isolated before. To our surprise, the alkaloids in the plant were not detected in the stems but detected in the flowers. Herein, a novel and strange guaipyridine sesquiterpene alkaloid with a carboxyl group named rupestine was purified successfully from the total alkaloids extracted from the flowers by high-speed counter-current chromatography (HSCCC). The two-phase solvent system used was composed of ethyl acetate-methanol-water (8:1:7, v/v/v). Fifty six milligrams of rupestine was obtained at over 97% purity and 95% recovery from 200 mg of the total alkaloids in one-step separation. Its structure was elucidated by spectroscopic methods including high resolution ESI-MS, (1)H NMR, (13)C NMR, Heteronuclear Multiple Bond Correlation (HMBC), Heteronuclear Single Quantum Coherence (HSQC), and Nuclear Overhauser Enhancement Spectroscopy (NOESY).     

Hollow-fiber liquid-phase microextraction for the determination of pesticides and metabolites in soils and water samples using HPLC and fluorescence detection

A new and simple method has been developed for the determination of a group of four benzimidazole pesticides (carbendazim/benomyl, thiabendazole, and fuberidazole), a carbamate (carbaryl), and an organophosphate (triazophos), together with two of their main metabolites (2-aminobenzimidazole, metabolite of carbendazim/benomyl, and 1-naphthol, metabolite of carbaryl) in soils. First, an ultrasound-assisted extraction (UAE) was performed, followed by evaporation and reconstitution in water. Then, extraction and preconcentration of the analytes was accomplished by two-phase hollow-fiber liquid-phase microextraction (HF-LPME) using 1-octanol as extraction solvent. Parameters that affect the extraction efficiency in HF-LPME technique (organic solvent, pH of the sample, extraction time, stirring speed, temperature, and ionic strength) were deeply investigated. Optimum HF-LPME conditions involved the use of a 2.0 cm polypropylene fiber filled with 1-octanol to extract 10 mL of an aqueous soil extract at pH 9.0 containing 20% (v/v) of NaCl for 30 min at 1440 rpm. Separation and quantification was achieved by HPLC with fluorescence detection (FD). The proposed optimum UAE-HF-LPME-HPLC-FD methodology provided good calibration, precision, and accuracy results for two soils of different physicochemical properties. LODs were in the range 0.001-6.94 ng/g (S/N = 3). With the aim of extending the validation, the HF-LPME method was also applied to different types of waters (Milli-Q, mineral and run-off), obtaining LODs in the range 0.0002-0.57 μg/L.     

Creatinine determination in serum by capillary electrophoresis

Creatinine in human serum was separated in a fused-silica capillary with H3PO4 (75 mmol/L, pH 2.5) as BGE, followed by UV detection at 200 nm. Serum with methylimidazole added as internal standard was deproteinized with acetonitrile and the supernatant, after dilution with water was injected at pressure mode. Creatinine and methylimidazole were baseline-resolved in 6.5 min. Linearity in the 0-880 micromol/L range gave an r2 > or = 0.998, recovery was 102 +/- 2.8% (n = 6). Enzymatic breakdown with creatininase confirmed that serum does not interfere. The within-day and between-days coefficient of variation (CV) were < or = 2.16 and 2.7%, respectively. The accuracy, determined for lyophilized samples by isotope dilution gas chromatography-mass spectrometry was < or = +/- 2.0%. The results were compared with HPLC for 32 lyophilized samples and on 27 serum pools. Capillary electrophoresis, rapid and inexpensive, seems a promising alternative to high-performance liquid chromatography (HPLC) for creatinine determination in human serum.     

Determination of the active metabolites of sibutramine in rat serum using column-switching HPLC

A simple and direct analysis using column-switching HPLC method was developed and validated for the quantification of active metabolites of sibutramine, N-mono-desmethyl metabolite (metabolite 1, M1) and N-di-desmethyl metabolite (metabolite 2, M2) in the serum of rats administered sibutramine HCl (5.0 mg/kg, p.o.). Rat serum was directly injected onto the precolumn without sample prepreparation step following dilution with mobile phase A, i. e., methanol-ACN-20 mM ammonium phosphate buffer (pH 6.0 with phosphoric acid) (8.3:4.5:87.2 by volume). After the endogenous serum components were eluted to waste, the system was switched and the analytes were eluted to the trap column. Active metabolites M1 and M2 were then back-flushed to the analytical column for separation with mobile phase B, i. e., methanol-ACN-20 mM ammonium phosphate buffer (pH 6.0 with phosphoric acid) (35.8:19.2:45 by volume) and detected at 223 nm. The calibration curves of active metabolites M1 and M2 were linear in the range of 0.1-1.0 microg/mL and 0.15-1.8 microg/mL. This method was fully validated and shown to be specific, accurate (10.4-10.7% error), and precise (1.97-8.79% CV). This simple and rapid analytical method using column-switching appears to be useful for the pharmacokinetic study of active metabolites (M1 and M2) of sibutramine.     

Rapid analysis of aflatoxins B1, B2, and ochratoxin A in rice samples using dispersive liquid–liquid microextraction combined with HPLC

A novel, simple, and rapid method is presented for the analysis of aflatoxin B₁, aflatoxin B₂, and ochratoxin A in rice samples by dispersive liquid–liquid microextraction combined with LC and fluorescence detection. After extraction of the rice samples with a mixture of acetonitrile/water/acetic acid, mycotoxins were rapidly partitioned into a small volume of organic solvent (chloroform) by dispersive liquid–liquid microextraction. The three mycotoxins were simultaneously determined by LC with fluorescence detection after precolumn derivatization for aflatoxin B₁ and B₂. Parameters affecting both extraction and dispersive liquid–liquid microextraction procedures, including the extraction solvent, the type and volume of extractant, the volume of dispersive solvent, the addition of salt, the pH and the extraction time, were optimized. The optimized protocol provided an enrichment factor of approximately 1.25 and with detection of limits (0.06–0.5 μg/kg) below the maximum levels imposed by current regulations for aflatoxins and ochratoxin A. The mean recovery of three mycotoxins ranged from 82.9–112%, with a RSD less than 7.9% in all cases. The method was successfully applied to measure mycotoxins in commercial rice samples collected from local supermarkets in China.     

Rapid determination of polyprenylated xanthones in gamboge resin of Garcinia hanburyi by HPLC

A rapid ion-pair HPLC method was developed and validated for the determination of eight polyprenylated xanthones including three pairs of epimers, namely morellic acid (MA), 30-hydroxygambogic acid (HGA), 30-hydroxyepigambogic acid (HEGA), isogambogic acid (IGA), epiisogambogic acid (EIGA), gambogenic acid (GNA), gambogic acid (GA), and epigambogic acid (EGA), in gamboge resin of Garcinia hanburyi. The separation was performed on a narrow bore C8 column with isocratic elution using a mixture of methanol-ACN-40 mM KH2PO4 buffer (37.5:37.5:25 v/v/v, containing 0.1% tetradecyltrimethylammonium bromide). The newly developed method was used to determine the contents of the eight compounds present in the gamboge. Results showed that GA and EGA are the dominant components of gamboge. The content ratio of each epimer pair remained constant, indicating that the content ratio of epimers can be used as a specific characteristic for the quality control of gamboge.     

Isolation and determination of estrogens in water samples by solid‐phase extraction using molecularly imprinted polymers and HPLC

Advanced SPE with molecularly imprinted polymers (MIP) was used in this study. A noncovalent imprinting approach was applied to separate 17β‐estradiol, estriol, and estrone from water samples. Polymer material was prepared by bulk polymerization with methacrylic acid as a functional monomer, divinylbenzene and ethyleneglycol dimethacrylate as crosslinkers, and acetonitrile, acetonitrile/toluene (3:1, v/v) or isooctane/toluene (1:99, v/v) as a porogen. We also prepared an MIP film on a silica gel surface with methacrylic acid and ethyleneglycol dimethacrylate as monomers and acetonitrile as a solvent. Qualitative and quantitative hormone analyses were carried out by HPLC with various detection techniques, including UV/visible spectroscopic detection (diode array detection) and electrochemical detection (CoulArray). The results of the study indicate that MIP technology is an excellent method for the quality control of estrogens in environmental analyses with a low quantification limit for 17β‐estradiol of around 26 (diode array detection) and 0.25 ng/mL (electrochemical detection). The proposed method was found to be suitable for routine determinations of the analyzed compound in environmental laboratories.     

反相高效液相色谱法同时测定水样中辛硫磷和氯菊酯

采用AgilentSBC8(5μm,4.6mmi.d.×150mm)色谱柱,以V(乙腈)∶V(水)=70∶30为流动相进行分离,二极管阵列检测器(PDA)在210nm波长处检测,建立了反相高效液相色谱法同时测定辛硫磷和氯菊酯的分析方法。辛硫磷和氯菊酯的线性范围分别为0.1～10μg mL和0.02～10μg mL,检出限分别为0 05和0.01μg mL,实际水样的加标回收率为90.3%和95.3%,相对标准偏差为1.3%和3.2%(n=7)。     

Rapid determination of nosiheptide in feed based on dispersive SPE coupled with HPLC

A novel, simple, and reliable method based on high‐performance liquid chromatography coupled with fluorescence detection has been developed for the determination of nosiheptide in feed. The feed samples were extracted with acetonitrile 0.1% formic acid aqueous solution and then purified via a dispersive solid‐phase extraction procedure using silica gel powder as the sorbent. Using a mixture of acetonitrile and 5 mM ammonium acetate solution (containing 0.1% formic acid) as the mobile phase, good separation and peak shape were obtained for nosiheptide on a Poroshell C₈ column (250 × 4.6 mm id, 4 μm) via the isocratic elution program. The resulting calibration curve shows high levels of linearity (r² > 0.999) for nosiheptide concentrations of 50–1000 μg/L. At three spiked levels, i.e., 0.500, 2.50 and 5.00 mg/kg, the intra‐ and interday recoveries of nosiheptide in five types of feed ranged from 78.5–96.8 and 84.9–94.2%, respectively. The intra‐ and interday relative standard deviations were less than 10.8%. The limits of quantification for nosiheptide in complete feed and premixes were measured as 50 and 100 μg/kg, respectively. Compared with other common adsorbents, silica gel presents stronger recovery and purification results for feed samples during the dispersive solid‐phase extraction process.     

Rapid determination of nitrite in foods in acidic conditions by high‐performance liquid chromatography with fluorescence detection

In this study, a simple, rapid, and sensitive method for the determination of nitrite (NO₂^?) in food samples by high‐performance liquid chromatography with fluorescence detection in acidic conditions had been developed. The derivatization of the nitrite with 2,3‐diaminonaphthalene was performed in acidic conditions to yield the highly fluorescent 2,3‐naphthotriazole, which was directly analyzed by high‐performance liquid chromatography with fluorescence detection without adjusting the solution to alkaline. The analysis column was reversed‐phase C₈ column. A constant flow rate of 1.0 mL/min was employed using water/acetonitrile as the mobile phase in isocratic mode (70:30, v/v). Fluorescence was monitored with excitation at 375 nm and emission at 415 nm. The standard calibration curves were linear for nitrite in different matrixes in the concentration range of 0–100 μg/L, and the correlation coefficients ranged from 0.9978 to 0.9998. The limits of detection and quantification were in the ranges of 0.012–0.060 and 0.040–0.20 mg/kg, respectively. The recoveries of nitrite from samples spiked at three different concentrations were 74.0–113.2%, and the relative standard deviations of the recovery results (n = 6) were 1.67–10.8%. The proposed method has good repeatability and is very sensitive and simple. It has been successfully used to determine nitrite in foods.     

Validation of SPE‐HPLC determination of 1,4‐benzodiazepines and metabolites in blood plasma,urine, and saliva

A simple, sensitive, selective, and reproducible RP‐HPLC method with DAD detection at 240 nm was developed for the determination of six 1,4‐benzodiazepines: bromazepam (BRZ), clonazepam (CLZ), diazepam (DZP), flunitrazepam (FNZ), lorazepam (LRZ), alprazolam (APZ); and two metabolites: α‐hydroxyalprazolam (HALZ) and α‐hydroxytriazolam (HTZL) in human plasma, urine, and saliva, using colchicine as internal standard, after SPE using Nexus Varian cartridges. Separation was performed on a Kromasil C₈ (250 mm×5 mm, 5 μm) analytical column with a gradient mobile phase containing methanol, ACN and 0.05 M ammonium acetate. Linearity was held within the range 0.3–20.0 ng/μL, with coefficients of determination (r²) better than 0.997. The within‐ and between‐day assay RSD at 2, 4, 8 ng/μL ranged from 0.03 to 4.7% and 0.5 to 7.0%, respectively in standards, from 1.3 to 7.9% and 3.3 to 7.3%, respectively in plasma, from 2.1 to 6.0% and 2.1 to 7.8%, respectively in urine and at 0.5, 1.0, 2.0 ng/μL ranged from 2.22 to 5.8% and 2.2 to 8.1%, respectively, in saliva. The mean relative recoveries were 96.3–108.6, 96.0–108.2, 94.3–107.1, 97.0–107.0% in within‐day assay and 96.8–107.7, 94.6–107.6, 93.2–105.8, 96.0–108.6 in between‐day assay for standard, plasma, urine, and saliva, respectively. The LOD and LOQ were 0.02–0.47 and 0.07–1.57 ng/μL, respectively.     

Direct injection HPLC method for the determination of selected phenothiazines in plasma using a Hisep column

A direct plasma injection HPLC method has been developed for the determination of selected phenothiazines (promethazine, promazine, chlorpromazine) using a Hisep column. The method is easy to perform and requires 20 microL of a filtered plasma sample. The chromatographic run time is less than 11 min using a mobile phase of 15:85 v/v acetonitrile-0.18 m ammonium acetate pH 5.0 and UV detection at 254 nm. The method is linear in the concentration range 0.1-25 microg mL(-1) (r > 0.99, n = 6) for each analyte with RSD less than 6%. Interday and intraday variability were found to be < or =14%. The limits of detection and quantitation were 0.1 (S/N > 3) and 0.25 microg mL(-1) (S/N > 10), respectively, for each of the three phenothiazines. We can also apply this method to separate three other phenothiazines (ethopromazine, trifluoroperazine, prochlorperazine), although it lacks the selectivity to determine the concentration of all six drugs concurrently. The separation is feasible using these drugs in certain combinations.     

蔗果低聚糖合成液的高效液相色谱分析

建立了用高效液相色谱法测定蔗果低聚糖合成液中各糖含量的方法，该法简便易行，结果准确。采用示差折光检测器，以水为流动相，各糖相对标准偏差均小于２４％。