HPLC法同时测定石蒜中加兰他敏和石蒜碱

建立了高效液相色谱同时测定石蒜中加兰他敏和石蒜碱含量的方法。 采用phenomenex-C18色谱柱,以乙腈(A)-0.1%TFA水溶液(B)为流动相梯度洗脱,检测波长289 nm。 结果表明,加兰他敏和石蒜碱均在0.5～10 mg/L(r=0.9999)呈现良好的线性关系,最低检测限(S/N=3)分别为0.09和0.15 mg/L,平均加样回收率分别为99.53%和96.82%。 该方法简单、快速、准确,适合于石蒜中加兰他敏和石蒜碱的测定。     

超高效液相色谱-串联质谱法测定尿液中7种苯系物代谢物

建立了同时测定人体尿液中7种苯系物代谢物的超高效液相色谱-串联质谱检测方法。0.5 mL尿液经盐酸水解、EVOLUTE^?EXPRESS ABN固相萃取板(10 mg)净化、洗脱、稀释后测定。使用ACQUITY UPLC HSS T3色谱柱(100 mm×2.1 mm, 1.8μm),以0.1%甲酸水溶液和甲醇作为流动相进行梯度洗脱，分离目标化合物，负离子电喷雾多反应监测模式下测定含量。7种目标化合物在各自范围内线性关系良好，相关系数(r²)>0.995;方法检出限为马尿酸(HA)0.9 mg/L,其余目标化合物0.02～4μg/L,定量限为HA 3 mg/L,其余目标化合物0.05～12μg/L;在实际尿液中低、中、高3个水平的加标回收率为84%～123%,日内精密度为1.8%～8.6%,日间精密度为1.9%～21.4%。应用该方法测定吸烟和非吸烟人群尿液样品各16份，吸烟人群中7种目标化合物检出率均为100%;非吸烟人群中反-反式黏糠酸(MU)、苄基巯基尿酸(BMA)、HA和2-甲基马尿酸(2MHA)的检出率为100%,(S)-苯巯基...     

液相色谱-串联质谱法测定尿液中的内源性类固醇激素

建立了液相色谱-串联质谱(LC-MS/MS)测定尿液中的内源性类固醇激素的方法。尿样经葡萄糖醛酸甙酶酶解后进行液-液提取,以甲醇-0.1%甲酸缓冲液(含0.02 mol/L乙酸铵)(体积比为68:32)为流动相,采用Cosmosil C18色谱柱分离,并以三重四极杆串联质谱多反应监测扫描方式对尿样中的脱氢表雄酮(DHEA)、睾酮、表睾酮、雄酮和苯胆烷醇酮等5种激素进行检测。方法的最低检出限为0.01～10 ng/mL,平均回收率为96.7%～106.5%,日内和日间相对标准偏差(RSD)分别小于7%和11%。应用所建立的方法测定了健康志愿者口服DHEA后尿液中内源性类固醇激素的变化情况,结果表明该方法样品处理简便,色谱分离完全,结果准确可靠,可替代气相色谱-质谱法用于体液中内源性类固醇激素兴奋剂的常规分析。     

超高效液相色谱-串联质谱法同时测定尿液与血清中23种双酚类化合物

建立了基于吡啶-3-磺酰氯衍生和超高效液相色谱-串联质谱(UPLC-MS/MS)检测尿液和血清中23种双酚类化合物(BPs)的方法。尿液样品采用乙酸乙酯提取;血清样品采用乙腈提取,上清液经PRiME HLB柱净化。提取液经吡啶-3-磺酰氯衍生后,以乙腈和0.1%甲酸水溶液为流动相,采用ACQUITY UPLC BEH C_(18)(2.1 mm×100 mm,1.7μm)色谱柱进行分离。质谱离子源为电喷雾电离源,正离子模式检测。23种目标物在0.005～100μg/L范围内线性关系良好,检出限为0.002～0.030μg/L,定量下限为0.005～0.100μg/L,回收率为76.6%～122%,相对标准偏差(RSD)为0.60%～14%。使用建立的方法分别对20份尿液和20份血清样品进行了检测。该方法样品用量少,灵敏度高,可以满足尿液和血清样品中23种BPs的同时测定要求。     

高效液相色谱-串联质谱法同时测定人尿液中7种邻苯二甲酸酯代谢物

建立了同时检测人尿液中7种邻苯二甲酸酯代谢物的高效液相色谱-串联三重四极杆质谱法。尿液经酶水解后,采用萃取柱净化,以2%(v/v)甲酸甲醇溶液为洗脱剂,经苯基柱分离,以0.1%(v/v)乙酸水溶液和0.1%(v/v)乙酸乙腈溶液为流动相进行梯度洗脱,采用电喷雾离子源负离子模式和多反应监测模式采集信号,用同位素内标法进行定量分析。尿液中7种邻苯二甲酸酯代谢物在0.2~200.0 μg/L范围内定量离子的相对峰面积比值与质量浓度均呈良好线性关系(r≥0.99976);检出限(LOD)为13.43~80.21 ng/L,定量限为44.77~267.37 ng/L; 3个水平的加标回收率为88.8%~108.9%,日内和日间精密度均不大于17.05%。该方法可同时准确、灵敏、简便地测定人尿液中7种邻苯二甲酸酯代谢物的暴露水平。     

在线固相萃取-高效液相色谱法同时测定人尿液中7种多环芳烃代谢物

采用双三元液相色谱系统结合荧光检测器,建立了在线固相萃取-液相色谱法同时测定人尿液中7种多环芳烃代谢物的方法。目标化合物首先在Turboflow Cyclone固相萃取柱上在线富集浓缩,然后通过六通阀转移至Hypersil Green PAH色谱柱,以乙腈-水为流动相进行梯度洗脱分离,流速1.0 mL/min,柱温30℃,荧光检测器检测,分离周期为20 min。在优化的色谱条件下,5~2000 ng/L或50~20000 ng/L范围内,7种多环芳烃代谢物均呈良好的线性关系(r≥0.999),方法检出限为0.5~15 ng/L,加标回收率为80.7％~110.7％。应用本方法对吸烟和非吸烟人群尿液中7种多环芳烃代谢物的含量进行了测定,吸烟者尿液中的2-羟基萘、1-羟基萘、2-羟基菲、2-羟基芴、4-羟基菲、6-羟基显著高于非吸烟者。     

HPLC法测定人尿中尿酸含量

建立了一种人体尿液中尿酸含量的高效液相色谱测定方法.采用ACE5 AQ亲水色谱柱,pH3.2的乙酸水溶液为流动相,检测波长280nm.尿酸含量在7.1～224.6μg/mL范围内线性关系良好,平均加样回收率为99.7%～100.5%,RSD小于1.4%.将该法用于健康人和肝硬化病人尿液样本的测定,两类样本中尿酸含量无显...     

固相萃取-液相色谱-串联质谱法同时测定尿液中9种多环芳烃代谢物

建立了固相萃取-液相色谱-串联质谱同时测定尿中2-羟基萘、1-羟基萘、2-羟基芴、3-羟基菲、1-羟基芘等9种多环芳烃代谢物的液相色谱-串联质谱测定方法。尿样中结合态的多环芳烃代谢物在β-葡萄糖苷酸酶-芳基硫酸酯酶缓冲液(pH 5.0)作用下,于37℃水浴中避光水解4 h后,以C18固相萃取小柱富集、净化,以甲醇洗脱,采用Waters Symmetry C18色谱柱,流动相为乙腈-0.2%氨水(72∶27,V/V)等度淋洗分离后进入质谱测定。在喷雾电压4 kV,毛细管温度300℃下,以3-羟基菲13C为内标,采用SRM模式负离子扫描方式测定,内标法定量。9种多环芳烃代谢物在尿中的线性范围为0.90～100μg/L;相关系数为0.9970～0.9990;回收率为79.0%～119.8%;相对标准偏差为4.3%～12.4%;检出限为0.04～0.90μg/L;结果表明,本方法可用于尿中9种多环芳烃代谢物的测定。     

在线固相萃取-超高效液相色谱-串联质谱法测定尿液中芬太尼及其代谢物的含量

建立了在线固相萃取(on-line SPE)-超高效液相色谱-串联质谱法(UHPLC-MS/MS)测定尿液中芬太尼及其代谢物去甲芬太尼的方法,并将该方法运用于大鼠尿液中芬太尼及其代谢物的检测时限研究。将尿液用pH 9.2的2 mmol·L~(-1)甲酸铵溶液稀释,离心后直接进样。采用Oasis HLB固相萃取小柱通过在线固相萃取富集净化目标物,用pH 9.2的2 mmol·L~(-1)甲酸铵溶液淋洗,以分析泵的初始流动相将目标物洗脱至Hypesil GOLD C_(18)分析柱上,以不同体积比的含有2 mmol·L~(-1)甲酸铵和0.1%(体积分数)甲酸的甲醇溶液、含有2 mmol·L~(-1)甲酸铵和0.1%(体积分数)甲酸的水溶液的混合液为流动相进行梯度洗脱。串联质谱分析中采用电喷雾正离子源(ESI~+)和选择离子监测(SRM)模式检测。将芬太尼以20μg·kg~(-1)的给药剂量对大鼠尾静脉注射后进行尿液收集,考察原体和代谢物的检测时限。结果表明:芬太尼和去甲芬太尼的质量浓度在2.0～800 ng·L~(-1)内与其峰面积呈线性关系,检出限均为0.200 ng·L~(-1),测定值的相对标准偏差(n=5)均小于15%。在大鼠尿液中芬太尼原体的检测时限为5～7 d,而其代谢物去甲芬太尼的检测时限长达28 d。     

胶束电动毛细管色谱-电喷雾质谱联用法同时测定妇宁栓中的5种有效成分

应用胶束电动毛细管色谱-电喷雾电离质谱联用法同时测定了妇宁栓中的小檗碱、巴马汀、苦参碱、儿茶素和黄芩苷5种主要有效成分的含量。在未涂层石英毛细管柱(80 cm×50 μm)中,以40 mmol/L月桂酸-100 mmol/L氨水溶液(含25%的乙腈,pH 9.5)为缓冲液,分离电压为25.0 kV,各组分在16 min内得到完全分离。电喷雾质谱检测时采用50%异丙醇水溶液(含3 mmol/L乙酸)为鞘液。结果表明,小檗碱、巴马汀、苦参碱、儿茶素、黄芩苷的线性范围分别为0.03～15、0.05～15、0.2～250、1.5～300和2.0～500 mg/L,检出限分别为0.01、0.02、0.05、0.5、0.6 mg/L。5种组分的加标回收率为94.0%～104.0%,相对标准偏差(RSD)在0.3%～3.2%之间。该法简便、快速、准确,重现性好,可用于妇宁栓中小檗碱、巴马汀、苦参碱、儿茶素、黄芩苷含量的同时测定。     

微流蒸发光散射检测器与毛细管液相色谱的联用及其在银杏叶提取物分析中的应用

将微流蒸发光散射检测器( μELSD)与毛细管液相色谱(cLC)联用,应用于中药银杏叶提取物及其分散片制剂的分离检测领域。首先对 μELSD仪器参数进行优化。通过调节漂移管温度与载气流量,提高了分析物的响应,并减小了噪声。然后,搭建了cLC-μELSD分离检测平台,其相对常规LC可大大减小实验试剂消耗。流动相A为0.05%(体积分数,下同)三氟乙酸(TFA)水溶液,流动相B为含0.05% TFA的甲醇溶液。最优的洗脱梯度条件为:0~10 min,5%B~25%B;10~25 min,25%B~38%B;25~35 min,38%B;35~40 min,38%B~42%B;40~55 min,42%B~50%B。银杏叶提取物和复杂中药制剂银杏叶提取物分散片都得到了较好的分离,并在其中鉴定到紫外波段几乎无吸收的重要内酯类活性成分白果内酯以及银杏内酯A、B和C。测定了不同厂家银杏叶提取物中萜类内酯洗脱时间的相对标准偏差,结果均不大于2.42%,表明该体系在目标物的分析上具有良好的重现性。实验证明所建立的cLC-ELSD体系在复杂中药体系的分离检测中有良好的应用性。     

Simultaneous determination of urinary hippuric acid, o-, m- and p-methylhippuric acids, mandelic acid and phenylglyoxylic acid for biomonitoring of volatile organic compounds by gas chromatography-mass spectrometry

A sensitive and precise method for the simultaneous determination of hippuric acid, o-, m- and p-methylhippuric acids, mandelic acid and phenylglyoxylic acid, which are major urinary metabolites of toluene, o-, m- and p-xylenes, styrene and ethylbenzene, respectively, was developed. These metabolites were converted into their methyl ester derivatives with methanol in hydrochloric acid, and then quantitated by gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring using a DB-1 capillary column. The injected compounds were quantitatively and reproducibly resolved within 19 min with a detection limit of 8-27 pg. The calibration curves were linear in the range of 0.05-25 μg for each compound, with correlation coefficients above 0.9999. This method was successfully used to analyze small amounts of both rat and human urine samples without any interference from coexisting substances. Overall recoveries of these compounds spiked in urine samples were 92-104%. The analytical results of the contents of these metabolites in the rat and human urine samples are presented.     

Ultra-fast gradient LC method for omeprazole analysis using a monolithic column: assay development, validation, and application to the quality control of omeprazole enteric-coated pellets

A method was optimized for the analysis of omeprazole (OMZ) by ultra-high speed LC with diode array detection using a monolithic Chromolith Fast Gradient RP 18 endcapped column (50 x 2.0 mm id). The analyses were performed at 30 degrees C using a mobile phase consisting of 0.15% (v/v) trifluoroacetic acid (TFA) in water (solvent A) and 0.15% (v/v) TFA in acetonitrile (solvent B) under a linear gradient of 5 to 90% B in 1 min at a flow rate of 1.0 mL/min and detection at 220 nm. Under these conditions, OMZ retention time was approximately 0.74 min. Validation parameters, such as selectivity, linearity, precision, accuracy, and robustness, showed results within the acceptable criteria. The method developed was successfully applied to OMZ enteric-coated pellets, showing that this assay can be used in the pharmaceutical industry for routine QC analysis. Moreover, the analytical conditions established allow for the simultaneous analysis of OMZ metabolites, 5-hydroxyomeprazole and omeprazole sulfone, in the same run, showing that this method can be extended to other matrixes with adequate procedures for sample preparation.     

Elimination of rutaecarpine and its metabolites in rat feces and urine measured by liquid chromatography

Rutaecarpine is an alkaloid isolated from the medicinal herb Evodia rutaecarpa. This study was to evaluate the elimination pathway of rutaecarpine in rat feces and urine. Rutaecarpine and its metabolites (3-, 10-, 11- and 12-hydroxyrutaecarpine) in urine were measured after incubation with beta-glucuronidase. After the rutaecarpine was administered (25 and 100 mg/kg) orally to rats, the urine and fecal samples were collected using a metabolic cage for five consecutive days. For determining rutaecarpine, the mobile phase consisted of acetontrile-10 mM NaH(2)PO(4) (60:40, v/v, pH 4.2 adjusted with orthophosphoric acid) with a flow rate of 1 mL/min. The calibration curve was linear in concentrations of 0.05-50 microg/mL in fecal and urine sample. The results indicated that more than 42% of the rutaecarpine was excreted by feces after oral administration (25 and 100 mg/kg), but only a small amount of rutaecarpine was detected in urine at a higher dose of rutaecarpine (100 mg/kg). After incubation with beta-glucuronidase, the hydroxyrutaecarpine in urine was eluted using methanol-acetonitrile-0.04% formic acid (6:30:64, v/v) with a flow rate of 1.2 mL/min. We conclude that the metabolic pathway of rutaecarpine went through phase I hydroxylation and phase II conjugation, and the major metabolite is 10-hydroxyrutaecarpine eliminated from urine of the rat.     

On-line stacking and sweeping capillary electrophoresis for detecting heroin metabolites in human urine

Heroin metabolites including morphine, codeine, and 6-acetylmorphine were determined by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI–sweep-MEKC). Liquid–liquid extraction was used for urine pretreatment. An uncoated fused silica capillary (Ld = 30 cm, 50 μm ID) was filled with phosphate buffer (50 mM, pH 2.5) containing 30% methanol, then high conductivity buffer (100 mM phosphate, 41.3 kPa for 18 s) was followed. Samples were injected electrokinetically (20 kV, 300 s). The sweeping and separation were performed at −25 kV using phosphate buffer (20 mM, pH 2.5) and 80 mM sodium dodecyl sulfate. The baseline separation was done within 10 min. During method validation, the calibration curves were linear over a range of 50–500 ng/mL (r ≧ 0.994). The RSD and RE values in intra-day and inter-day assays were all below 20%, which showed good precision and accuracy. Their detection limits were 10 ng/mL (S/N = 3). The optimized method was applied to determine real urine samples from addicts. These samples were confirmed by liquid chromatography/mass spectrometry.     

A liquid chromatography method for determination of selected amino acids, coenzymes, growth regulators, and vitamins from Cicer arietinum (L.) and Solanum lycopersicum (L.)

A bottleneck in crosstalk and QC research has been the quantification of diverse chemotypes in small amounts of tissue. An LC-UV method for estimating 28 selected metabolites of the regulatory network underlying growth, development, maintenance, vital functions, defense reactions, and food quality is reported. The method was based on binary gradient resolutions of the analytes in an RP C18 column. The mobile phase comprised solvent A [water+0.1% trifluoroacetic acid (TFA)] and B (acetonitrile + 0.085% TFA at a flow rate of 1 ml/min. Twenty-three metabolites (selected amino acids, coenzymes, growth regulators, phenolic antioxidant, and water-soluble vitamins) were detected at 254 nm, and four fat-soluble vitamins at 280 nm. Jasmonic acid was quantified at 210 nm. The RSDs of peak area and retention time for each metabolite were <5.8%. The calibration graphs were linear with R2 values ranging from 0.98 to 0.99. The LODs (microg/mL) were about 0.01-1.0 for 23 metabolites quantified at 254 nm, 0.1-0.2 for fat-soluble vitamins, and 0.1 for jasmonic acid. The recoveries ranged from 80 to 105%, with RSDs of 2.8 to 11.2%. The method has been satisfactorily applied for determination of 28 metabolites from Cicer arietinum (L.) and Solanum lycopersicum (L.). It could be an alternative and competitive method of choice that can cheaply and easily perform routine analysis for food quality and targeted metabolomics of chickpea and tomato in response to stressors.     

Simultaneous determination of MK-0767 and seven metabolites in rat urine using liquid chromatography/tandem mass spectrometry

MK-0767, 5-[2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide (I, Table 1), is a dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist previously studied for the treatment of type 2 diabetes and dyslipidemia. To support further toxicological studies in one of the animal species used in chronic testing of I, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous quantification of I and seven metabolites in rat urine was developed and validated. In this method, urine samples were diluted with acetonitrile/methanol (50:50, v/v) and injected directly onto the column of an LC system. Detection was achieved by MS/MS using a turbo ion spray probe monitoring precursor --> product ion combinations in selected reaction monitoring (SRM) mode. The linear range for I and three metabolites was 0.8-800 ng/mL, and 8-8000 ng/mL for four other metabolites found to be present in urine at higher concentrations than I. Intra-day and inter-day variation using this method were < or = 13.0%. The method exhibited good linearity, reproducibility, specificity and sufficient sensitivity when used for the analysis of rat urine samples. Concentrations of I and its major metabolites in rat urine were determined in samples collected between 0-24 h after dosing on the last day of administration of nine daily oral doses to three male (1000 mg/kg/day) and three female (300 mg/kg/day) Sprague-Dawley rats. The urinary concentrations of I and its metabolites were similar in male and female rats. The average concentrations of I were 0.51 and 0.33 microg/mL in male and female rats, respectively. Concentrations of four of the seven metabolites quantified were 6- to 45-fold higher than those of I. The most abundant metabolite, with concentrations of 24.2 and 13.3 microg/mL in male and female rat urine, respectively, was a methyl sulfoxide derivative formed by oxidative cleavage of the thiazolidinedione ring, followed by S-methylation and oxidation of the sulfide intermediate.     

Identification of key metabolites of tectorigenin in rat urine by HPLC-MS(n)

This is a report about the identification of key metabolites of tectorigenin in rat urine using high-performance liquid chromatography-electrospray ionization ion trap tandem mass spectrometric method (HPLC-ESI-MS(n)). Six healthy rats were administered a single dose (80 mg/kg) of tectorigenin by oral gavage. Urine was sampled for 0-24 h and centrifuged at 12,000 rpm for 10 min to obtain the supernatants, then the supernatants were purified by solid-phase extraction with a C(18) cartridge. The chromatographic separation was carried out on a reversed-phase C(18) column with a gradient elution program whereas acetonitrile-0.1% formic acid water was used as mobile phase. Mass spectra were acquired in negative ionization mode and a data-dependant scan was used for the identification of the key metabolites of tectorigenin in the urine samples. As a result, four phase II metabolites and the parent drug tectorigenin were found and identified in rat urine for the first time.     

Simultaneous Determination of Hydrochlorothiazide and Reserpine in Human Urine by LC with a Simple Pre-Treatment

A simple, selective and sensitive reversed-phase high performance liquid chromatography method for simultaneous analysis of hydrochlorothiazide and reserpine in human urine was developed and subjected to primary pharmacokinetic study. After a simple protein precipitation using methanol and extraction with ethyl acetate, the analytes were separated on an Elite C(18) column at a flow rate of 0.8 mL min(-1). The mobile phase was composed of acetonitrile (A) and 0.2% ammonium chloride solution (B) for a gradient elution starting at A:B at 30:70, v/v for 0~6 min, linearly raising the percent of A from 30% to 50% (6~9 min) and ending at 50:50, v/v (9~25 min). The standard curves were linear over the range of 0.05-20 μg mL(-1) for hydrochlorothiazide and 0.02-5.0 μg mL(-1) for reserpine, respectively (r > 0.999). The limit of detection (LOD) and the limit of quantification (LOQ) were 5.5 ng mL(-1) and 18.2 ng mL(-1) for hydrochlorothiazide, and 7.1 ng mL(-1) and 23.6 ng mL(-1) for reserpine, respectively. The recoveries for both analytes were above 89.0±1.35%. The intra-day and inter-day precision for hydrochlorothiazide were less than 1.91% and 1.38%, and those for reserpine were below 1.61% and 2.64%, respectively. The method indicated good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy, and it was employed successfully for the simultaneous determination of hydrochlorothiazide and reserpine in human urine samples.