AQC柱前衍生化RP-HPLC法测定蒜氨酸及其有关物质的含量

采用6-氨基喹啉-N-(羟基琥珀酰亚胺基)氨基甲酸酯(6-aminoquinolyl -N- Hydroxysuccinimide Carbamate ,AQC)为柱前衍生化试剂,建立了AQC柱前衍生化RP-HPLC法测定蒜氨酸及其有关物质的含量。该衍生化方法反应瞬间完成,衍生化产物稳定。色谱条件为：Kromasil C18柱（250mm×4.6mm,5mm）,流动相A为0.1%乙酸铵(含0.03%乙酸),流动相B为水-乙腈（40∶60）,线性梯度洗脱,流速1.0ml/min,检测波长248nm。蒜氨酸在1.1719~1500μg /ml浓度范围内线性关系良好（r=0.9998）, 日内、日间精密度良好(RSD <1.8%,n=5), 加样回收率为99.1%（RSD1.9%,n=5）,检测限为3ng,该方法准确、方便、快速。     

Monitoring of 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine by high-performance liquid chromatography after pre-column derivatization with glyoxal reagents

A new, simple and sensitive pre-column fluorescence derivatization high-performance liquid chromatographic method for the determination of the oxidative DNA stress marker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, was developed. Solid-phase extraction using an Oasis HLB cartridge avoided troublesome sample preparation steps, interference from charged species and frequent and essential electrode maintenance in electrochemical procedures. 8-Oxo-7,8-dihydro-2'-deoxyguanosine and other guanine compounds were selectively derivatized with glyoxal reagents (phenylglyoxal, 3,4-methylenedioxyglyoxal, 2-naphtylglyoxal and 6-methoxynaphthylglyoxal) at 40-60 degrees C. Derivatization with 6-methoxynaphthylglyoxal at 40 degrees C for 30 min gave the strongest fluorescence product. The fluorescence derivatives from reaction with 6-methoxynaphthylglyoxal were separated on a Capcell Pak C18 SG 120A column (4.6 mm i.d. x 150 mm, 5 microm) with acetonitrile-5 mM phosphate buffer (pH 6.0; 3:7, v/v) as mobile phase. The detection wavelength of the fluorescence derivative of 8-oxo-7,8-dihydro-2'-deoxyguanosine was lambda(ex) 400 nm and lambda(em) 510 nm. The detection limit of 8-oxo-7,8-dihydro-2'-deoxyguanosine was 1 ng/mL using 50 mL of urine. The calibration graphs were linear up to 30 microg/mL for 8-oxo-7,8-dihydro-2'-deoxyguanosine. The relative standard deviation of 20 ng/mL of 8-oxo-7,8-dihydro-2'-deoxyguanosine was 7.0%. The proposed method was compared with the enzymatic ELISA 8-oxo-7,8-dihydro-2'-deoxyguanosine analysis method (8-OH-dG Check, JaICA, Shizuoka, Japan). The correlation coefficient was 0.79 (n = 20) and y = 0.85x + 5.34. The proposed method was applied to the monitoring of 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine from male heavy smokers.     

Determination of atractylenolide II in rat plasma by reversed-phase high-performance liquid chromatography

A method for quantitative determination of atractylenolide II in rat plasma using reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with UV spectrometry was established. From a variety of compounds and solvents tested, atractylenolide III was selected as the internal standard (IS) and ethyl acetate was found to be the best solvent for extracting atractylenolide II from plasma samples. RP-HPLC analysis of the extracts was performed on an analytical column (DIKMA ODS, 150 x 4.6 mm; i.d., 5 microm) equipped with a security guard pre-column system. There was good linearity over the range 0.05-5.0 microg/mL (r > 0.99). The recoveries were more than 90.0% in plasma, and the intra- and inter-day coefficients of variation were less than 10.0% in all cases. The limit of detection (LOD) was 0.025 microg/mL and the lower limit of quantification (LLOQ) was 0.05 microg/mL. The RP-HPLC method was applied to quantitate atractylenolide II in rat plasma within 24 h in a pharmacokinetics study where experimental rats received a single dose of atractylenolide II (60 mg/kg).     

Development of a simple and sensitive high-performance liquid chromatography method for determination of glucosamine in pharmaceutical formulations

A column high-performance liquid chromatography (HPLC) method was developed for the determination of glucosamine in dosage forms. Glucosamine was derivatized by addition of a solution containing orthophthaldialdehyde. The HPLC separation was achieved on a Spherimage 80 ODS2 column (250 x 4 mm id, 5 microm particle size) using an isocratic mobile phase containing phosphate buffer-methanol (90 + 10, v/v, pH 6.50) and methanol-tetrahydrofuran (97 + 3, v/v) in proportions of 85 + 15 at a flow rate of 1 mL/min, followed by fluorescence detection. The method was validated for specificity, linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ). The detector response for glucosamine HCI was linear over the concentration range of 0.1-20 microg/mL with a correlation coefficient of 0.9980. The accuracy was between 99.4 and 100.8%. The LOD and the LOQ were 0.009 and 0.027 microg/mL, respectively. The method was applied to determination of glucosamine in solid dosage forms.     

高效液相色谱-柱前衍生化法测定饲料中的含硫氨基酸

发展了一种饲料中含硫氨基酸的检测方法。样品经过甲酸氧化,将其中的胱氨酸和蛋氨酸分别氧化为磺基丙氨酸和蛋氨酸砜;再经酸水解后,采用2,4-二硝基氟苯进行柱前衍生化,衍生物经高效液相色谱分析。使用Elite AAK C18色谱柱(250 mm×4.6 mm, 5 μm)分离;以0.05 mol/L乙酸钠和乙腈-水(50:50, v/v)为流动相,梯度洗脱,流速为1.2 mL/min;检测波长为360 nm;柱温为31 ℃。结果表明,胱氨酸在0.4～16.0 mg/L、蛋氨酸在0.7～29.6 mg/L范围内的线性相关系数分别为0.9999、0.9998;胱氨酸和蛋氨酸的定量限(信噪比(S/N)=10)分别为2.6 μg/kg和3.1 μg/kg;3次样品平行测定的胱氨酸和蛋氨酸含量的相对标准偏差(RSD)分别为0.79%和2.56%,加标回收率分别为100.28%～102.00%和105.72%～107.89%。该方法分析成本低,测定结果准确,灵敏度高,符合饲料中含硫氨基酸的检测要求。     

半叶马尾藻粗糖中单糖的离子色谱法分析

以60～80 ℃的水从半叶马尾藻干粉中提取粗多糖,用Sevage溶剂去蛋白纯化后,将粗多糖用4.0 g/L 的三氟乙酸在 80 ℃下水解,水解液在CarboPacTM PA10离子色谱柱(2 mm i.d.×250 mm)上以14.0 mmol/L NaOH溶液为流动相进行 分离,以电化学检测器检测半叶马尾藻粗糖水解产生的单糖成分及含量。结果表明,半叶马尾藻粗糖中木糖、半乳糖、阿 拉伯糖、葡萄糖、鼠李糖和果糖的含量分别为2200,820,98,4560,358和740 mg/kg,加标回收率范围为86.0%~108.0%,检出限范围为5.6～89.6 μg/kg。该方法具有灵敏度高、精密度好、样品不需 要衍生化处理等优点,适合藻类样品中单糖的分析。     

Simultaneous determination of caffeine, theophylline and theobromine in human plasma by on-line solid-phase extraction coupled to reversed-phase chromatography

A reversed-phase liquid chromatographic column switching system was described for the determination of caffeine (CF), theophylline (TH) and theobromine (TB) in human plasma with a direct injection procedure. A short protein-coated mu Bondapak CN silica pre-column (20 x 3 mm, i.d.) was used for enrichment of the drugs and clean up from weakly retained plasma components using phosphate buffer saline pH 7.4. After washing step, the retained drugs were flushed into a reversed-phase column (5 microm TSK gel ODS-80 TM, 150 x 4.6 mm i.d.) with a mobile phase of methanol-0.01 M phosphate buffer, pH 3.5 (30:70, v/v) for the final separation. The eluent was monitored with a UV detector at 275 nm. The resulting chromatograms showed no interference from endogenous plasma components. A linear relationship between the concentration of drug and peak height was confirmed in the range of 0.5-20 microg/mL for all drugs. High extraction recoveries from plasma ranging from 96.12 to 100.32% were achieved. Validation of the method was examined performing intra- and inter-day accuracy and precision and was found to be satisfactory. The coefficients of variation of the three drugs were less than 3% for intra-day and less than 4% for inter-day run assays.     

荧光衍生化试剂2-(4-羧基苯基)-4,5-萘并咪唑的合成及在HPLC测定醇类中的应用

 合成了一种用于醇类分析的高灵敏度的荧光衍生化试 剂2-(4-羧基苯基)-4,5-萘并咪唑(CNI),将其在二氯甲烷中于80 ℃条件下与醇缩合成酯 ,并采用RP-HPLC法进行分离检测,色谱柱为Zorbax Bp C8柱(250　mm×4.6　mm i.d.) ,流动 相为V(乙腈)∶V(甲醇)=90∶10的溶液,荧光检测波长λex 345 nm, λem 485　nm。同时,测定了人血清中的胆固醇,其最低检出质量浓度为1.0 μg /L。     

Simultaneous determination of 16 estrogens,dehydroepiandrosterone and their glucuronide and sulfate conjugates in serum using sodium cholate micelle capillary electrophoresis

The simultaneous determination of 16 estrogens, dehydroepiandrosterone (DHEA) and their glucuronide and sulfate conjugates by micellar electrokinetic chromatography (MEKC) with sodium cholate micelle is reported. Sodium cholate, sodium dodecylsulfate (SDS) and alpha-, beta-, gamma-cyclodextrins were studied as micelle reagents in the pH range of 7.0-10.0. Estrogens, DHEA and their glucuronide and sulfate conjugates were separated using a 50 cm x 50 microm capillary with 10 mM borate-phosphate buffer (pH 8.0) containing 50 mM sodium cholate as carrier. The method could simultaneously determine 1.0-1000 microg/mL of steroids and metabolites in 100 microL of serum by photometric detection at 214 nm within 14 min and 80 ng/mL steroids could be determined by using 2.0 mL of serum. The relative standards deviations were 6.7-7.7% at 10 microg/mL in serum. The recoveries were 89.1-92.0% with 10 microg/mL serum samples.     

A sensitive and selective RP-HPLC method for simultaneous determination of picroside-I and picroside-II in rat plasma and its application in pharmacokinetics studies

A sensitive and selective HPLC method with UV detection for the simultaneous determination of picroside-I and picroside-II (active components of total glycoside of Picrorhiza scrophulariiflora Pennell) was developed and validated in rat plasma. After simple deproteinization using acetonitrile, analysis was performed on an RP-C18 column (250 mm x 4.6 mm id, 5 microm) with a mobile phase consisting of acetonitrile and water at a flow rate of 1.0 mL/min used in a gradient elution program. The UV detection wavelength was set at 262 and 277 nm. Linear calibration curves were obtained in the concentration range of 0.10-50 microg/mL for picroside-I and 0.25-200 microg/mL for picroside-II. The lower limits of quantification were 0.1 and 0.25 microg/mL for picroside-I and picroside-II, respectively. The recoveries from spiked control samples were up to 80% for both picroside-I and picroside-II. Accuracy and precision of the validated method were both within the acceptable limits of <15% at three quality control concentrations. The analytes were stable after three freeze-thaw cycles. The method was successfully used to determine concentrations of picroside-I and picroside-II after intravenous administration of total glycoside of Picrorhiza scrophulariiflora Pennell to rats.     

A Novel Fully Enzymatic Method for Determining Glucose and 1,5-Anhydro-<Emphasis Type="SmallCaps">d</Emphasis>-Glucitol in Serum of One Cuvette

The aim of the study was to set up a novel fully enzymatic method for screening glucose and 1,5-anhydro-D-glucitol (1,5-AG) in one cuvette. We have determined glucose and 1,5-AG, based on glucokinase (GK) converting glucose to G6P, a compound that can be catalyzed ultimately into 6-PGA by G-6PD and its coenzyme NADP(+), and then calculated glucose concentration according to absorbance variety. Furthermore, pyranose oxidase was used to oxidize 1,5-AG with the formation of 1, 5-anhydro-fructose and H(2)O(2). Measurement was done according to Trinder's reaction principle. The mean within-run and day-to-day precision (CV) of this method for glucose was 0.88% and 1.4%, and also that for 1,5-AG was 1.05% and 1.94%, respectively. The mean recovery rate of two targets was 100.2% and 101.6%, respectively. The correlation (R(2)) between the results of 1,5-AG obtained with our proposed method (y) and those obtained with LanaAG method (x) was 0.999 (y=1.002x-0.675 micromol/l; n=86), and the correlation (R(2)) of glucose between the results obtained with our GK method (y) and those obtained with recommendatory hexokinase method (x) was 0.9999 (y=1.0043x+0.1229 mmol/l; n=86). The reference range (95%) of serological glucose and 1,5-AG was 3.7 to 5.7 mmol/l (4.70+/-0.51 mmol/l) and 83.1 to 240.7 micromol/l (161.9+/-40.2 micromol/l), respectively; and there was no difference with age and sex (P>0.05). This newly developed method was dependable and steady-going, with analysis automatization, and allows quicker and easier measurement of serum glucose and 1,5-AG in one identical reaction cuvette in-phase than previously described methods.     

High-performance liquid chromatographic method for the determination and pharmacokinetic study of wogonoside in rat serum after oral administration of traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction

A high-performance liquid chromatographic method for the determination of wogonoside in plasma of rats administrated orally with the traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction was developed. Sample preparation was carried out by protein precipitation with a mixture of acetonitrile and methanol (1:1, v/v). The extracted sample was separated on a Hypersil C(18) (150 x 5 mm i.d., 5 microm) analytical column by linear gradient elution using 0.05% (v/v) phosphoric acid (containing 5 mm sodium dihydrogen phosphate) and acetonitrile as mobile phase at a flow rate of 1.5 mL/min. The eluate was detected using a UV detector at 276 nm. The assay was linear over the range 0.109-7.0 microg/mL (R(2) = 0.9999, n = 5). Mean recovery was determined as 98.39%. Intra- and inter-day precisions (RSD) were < or =7.59%. The limit of quantitation was 0.109 microg/mL. After validation, the HPLC method developed was applied to investigate the preliminary pharmacokinetics of wogonoside in rat after oral administration of Huang-Lian-Jie-Du decoction.     

高效液相色谱法同时测定化妆品中的7种磺胺及甲硝唑和氯霉素

建立了化妆品中7种磺胺(磺胺醋酰、磺胺吡啶、磺胺甲基嘧啶、磺胺二甲嘧啶、磺胺甲氧嘧啶、磺胺间甲氧嘧啶、磺胺甲基异唑)和甲硝唑及氯霉素的高效液相色谱测定方法。样品经0.1%甲酸水溶液-乙腈(体积比为8∶2)混合液超声提取后进行液相色谱分析。方法的定量检测限为3～80 μg/g,7种磺胺在20～200 μg/mL时,甲硝唑及氯霉素在40～400 μg/mL时方法的线性关系良好(r≥0.9993)。加标回收率为83.8%～105.3%(7种磺胺的添加水平为50 μg/mL和150 μg/mL,甲硝唑及氯霉素的添加水平为100 μg/mL和300 μg/mL),其相对标准偏差均小于5%。     

柱前衍生高效液相色谱测定小儿复方氨基酸注射液中氨基酸

建立了高效液相色谱(HPLC)测定小儿复方氨基酸注射液中各种氨基酸含量的方法.采用Kromasil C18(250×4.6 mm,5 μm)色谱柱,以2,4-二硝基氟苯为衍生试剂,梯度洗脱.流动相A为乙腈∶水(50∶50,V/V),流动相B为0.04 mol/L的磷酸二氢钾溶液(pH=6.8);流速为1.0 mL/min;检测波长为360 nm.所测氨基酸的线性范围为0.008～0.2213 mg/mL,平均回收率在97.85%～102.86%之间.     

Selective determination of trimethylamine in air by liquid chromatography using solid phase extraction cartridges for sampling

The selective determination of trimethylamine (TMA) in air by liquid chromatography is reported. Sampling is effected by flushing air through C18-packed solid-phase extraction (SPE) cartridges at a flow rate of 15 mL/min for 15 min. Next, TMA is desorbed from the cartridges and injected into the chromatographic system. The analyte is then selectively retained on a precolumn (20 mm x 2.1 mm i.d., packed with 30 microm, Hypersil C18 phase), and derivatized on-line by injecting 9-fluorenylmethyl chloroformate (FMOC). Finally, the TMA-FMOC derivative is transferred to the analytical column (125 mm x 4 mm i.d., LiChrospher 100 RP18, 5 microm), and monitored at 262 nm. The method was applied to the measurement of TMA in air in the 0.25-2.5 microg interval (equivalent to concentrations of TMA of 1.1-11 mg/m3), providing good linearity, reproducibility and accuracy. The mean recovery of TMA was (96 +/- 7%) (n = 12), and the limit of detection was 0.05 microg. The proposed procedure allows the selective determination of TMA in the presence of other primary and secondary short-chain aliphatic amines.     

Simultaneous determination of rifampicin and sulbactam in mouse plasma by high-performance liquid chromatography

A simple and rapid high-performance liquid chromatographic (HPLC) method with ultraviolet detection has been developed and validated for the simultaneous determination of rifampicin and sulbactam in mouse plasma. Plasma samples were deproteinized with acetonitrile and separated by HPLC on a RP-18 (125 x 4 mm, 5 microm) column and gradient elution with potassium dihydrogen phosphate solution (pH 4.5; 50 mm) and acetonitrile at a flow-rate of 1.0 mL/min. Rifampicin and sulbactam were monitored at 230 nm and confirmed by means of their UV spectra using a diode-array detector. The method was linear at plasma levels from 1 to 100 microg/mL for rifampicin and from 5 to 200 microg/mL for sulbactam. The limits of quantification were 0.6 microg/mL for rifampicin and 4.2 microg/mL for sulbactam. The intra- and inter-day precisions of the method (RSD) were lower than 5% for both compounds. Average recoveries of rifampicin and sulbactam from mice plasma were 98.2 and 89.3%, respectively. The developed method was successfully applied to the determination of the pharmacokinetic profile of both compounds in mice.     

Liquid chromatography-tandem mass spectrometric method for determination of the anti-inflammatory compound vicenin-2 in the leaves of L. ericoides Mart

This paper reports a rapid and sensitive method for determination of the anti-inflammatory compound vicenin-2 in L. ericoides Mart. using liquid chromatography-tandem mass spectrometry. Separation of the compound of interest was performed on a VP-ODS(18) (150 x 2 mm, Shimadzu, Japan) column and a pre-column packed with GPV-ODS C(18) (5 x 2 mm, Sigma-Aldrich, USA) with acetonitrile-water (15:85) mobile phase containing 2% acetic acid using isocratic flow at 0.5 mL/min for 2 min. Multiple-reaction monitoring of vicenin-2 was performed using electrospray positive ionization. The linear calibration curves were generated using a concentration range of 5-2500 ng/mL with correlation coefficients >0.99. The values of limit of detection and limit of quantitation were found to be 1 and 5 ng/mL, respectively. The method developed based on LC-ESIMS/MS is advantageous because it permits the rapid and selective detection of vicenin-2. Furthermore, the method can be easily applied to the routine analysis of vicenin-2 in plant extracts using a minimal amount of sample.     

Sensitive determination of bisphenol A and alkylphenols by high performance liquid chromatography with pre-column derivatization with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole

A new and sensitive high-performance chromatographic method for the determination of bisphenol A and 8 alkylphenols with fluorescence detection is reported. Each phenol was derivatized by reaction with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole at 40 degrees C for 60 min. The fluorescence derivatives were separated on a Wakosil 5C18 column (4.0 i.d. x 300 mm, 5 microm) with methanol:water (10:90) as mobile phase (detection wavelength: lambda(ex) 336 nm, lambda(em) 440 nm). The detection limits were in the range of 0.1-10.0 pg/mL in serum. The calibration graphs were linear to 1.0 microg/mL. The relative standard deviations were 7.2-8.9%, respectively. The proposed method was applied to the determination of bisphenol A in mother and infant rat serum.     

Simultaneous determination of palladium, platinum, rhodium and gold by on-line solid phase extraction and high performance liquid chromatography with 5-(2-hydroxy-5-nitrophenylazo)thiorhodanine as pre-column derivatization regents

In this paper, 5-(2-hydroxy-5-nitrophenylazo)thiorhodanine (HNATR) was synthesized. A new method for the simultaneous determination of palladium, platinum, rhodium and gold ions as metal-HNATR chelates was developed using a rapid analysis column high performance liquid chromatography equipped with on-line solid phase extraction technique. The samples (Water, human urine, geological samples and soil) were digested by microwave acid-digestion. The palladium, platinum, rhodium and gold ions in the digested samples were pre-column derivatized with HNATR to form colored chelates. The Pd-HNATR, Pt-HNATR, Rh-HNATR and Au-HNATR chelates can be absorbed onto the front of the enrichment column when they were injected into the injector and sent to the enrichment column [Zorbax Stable Bound, 10 mm x 4.6 mm, 1.8 microm] with a buffer solution of 0.05 mol L(-1) phosphoric acid as mobile phase. After the enrichment had finished, by switching the six ports switching valve, the retained chelates were back-flushed by mobile phase and travelling towards the analytical column. These chelates separation on the analytical column [Zorbax Stable Bound, 10 mm x 4.6 mm, 1.8 microm] was satisfactory with 72% acetonitrile (containing 0.05 mol L(-1) of phosphoric acid and 0.1% of Triton X-100) as mobile phase. The palladium, platinum, rhodium and gold chelates were separated completely within 2.5 min. Compared to the routine chromatographic method, more then 80% of separation time was shortened. By on-line solid phase extraction system, a large volume of sample (10 mL) can be injected, and the sensitivity of the method was greatly improved. The detection limits (S/N=3, the sample injection volume is 10 mL) of palladium, platinum, rhodium and gold in the original samples reaches 1.4, 1.8, 2.0 and 1.2 ng L(-1), respectively. The relative standard deviations for five replicate samples were 2.4-3.6%. The standard recoveries were 88-95%. This method was applied to the determination of palladium, platinum, rhodium and gold in human urine, water and geological samples with good results.     

Simultaneous determination of 23 amino acids and 7 biogenic amines in fermented food samples by liquid chromatography/quadrupole time-of-flight mass spectrometry

A novel liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOFMS) method was developed for the simultaneous determination of 23 amino acids and 7 biogenic amines in food samples. These analytes were pre-column derivatized with dansyl chloride and then separated in an Acquity column (1.7 μm; 2.1 mm × 100 mm). The separation of 31 compounds including an internal standard was achieved within 25 min at a flow rate of 0.2 mL/min. The method linearity for each amino acid and biogenic amine had a relatively wide range with r(2)>0.99. The intra- and inter-day precision, expressed as relative standard deviation (RSD), ranged from 1.1 to 4.6% and from 2.0 to 11.2%, respectively. The limit of detection was between 0.005 and 0.4 μg/mL. With a simple dilution, recoveries of around 80-120% were obtained for most of the compounds. No significant matrix effect was observed, and the developed method was successfully applied to the analysis of amino acids and biogenic amines in beer, cheese and sausage samples.