Determination of vitamin B5 in human urine by high-performance liquid chromatography coupled with mass spectrometry

In the present work, we have developed a simple and rapid liquid chromatography/mass spectrometry (LC/MS) method for the identification and quantification of vitamin B5 in human urine. Urine was spiked with vitamin B5 internal standard, hopantenic acid (HOPA), and then diluted with the LC mobile phase prior to its analysis by LC/MS. The quantification was performed in single ion monitoring mode. The calibration curve was linear (r2 = 0.999) between 0.25 to 10 microg/mL. With a limit of detection of 0.1 microg/mL the method was sensitive enough to determine low levels of vitamin B5 in urine. The overall quantitative efficiency of the method was evaluated by spiking urine samples with four different concentrations of vitamin B5; the intra-assay coefficient of variation was below 5% and the recoveries were between 96 to 108%. The results of the present study show that the proposed method is selective and sensitive enough for the quantification of vitamin B5 in urine.     

Determination of polyamines in human urine by precolumn derivatization with benzoyl chloride and high-performance liquid chromatography coupled with Q-time-of-flight mass spectrometry

A simple and sensitive HPLC/Q-TOF MS method for simultaneous determination of 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine and acetyl-spermine in human urine was developed in electrospray-ionization source by positive ion mode. The samples were firstly pretreated by 10% HClO₄ and then derivatized by benzoyl chloride with 1,6-diaminohexane as internal standard. The derived polyamines were separated on a C₁₈ column by a gradient elution with methanol-water, and then sensitively detected with Q-TOF MS. The limits of detection for polyamines ranged from 0.02 to 1.0 ng ml⁻¹ with excellent linearity within the range from 1 to 1000 ng ml⁻¹ except acetyl-spermine from 5 to 1000 ng ml⁻¹. The intra- and inter-day R.S.D. for all polyamines were 2.0-14.7% and 3.9-12.9%, respectively. The method was applied to determine the polyamines in human urine from 10 cancer patients and 15 healthy volunteers. Results showed that the mean levels of polyamines in urine of patients were all higher than those in healthy volunteers. The cluster analysis was used to establish the distinction mode between cancer sufferers and healthy individuals.     

Simultaneous determination of triptolide, tripdiolide and tripterine in human urine by high-performance liquid chromatography coupled with ion trap atmospheric-pressure chemical ionization mass spectrometry

An accurate and selective method for the simultaneous determination of triptolide, tripdiolide and tripterine in human urine using hydrocortisone as an internal standard (IS) by high-performance liquid chromatography coupled with atmospheric-pressure chemical ionization mass spectrometry in negative ion mode has been developed. After triptolide, tripdiolide and tripterine in human urine were extracted with ethyl acetate and cleaned by solid-phase extraction with C(18) cartridges, a satisfactory separation was achieved on an XDB C(18) short column (30 x 2.1 mm i.d., 3 microm) using the mobile phase of acetic acid-ammonium acetate (5 mmol/L, pH = 4.5)-acetonitrile-methanol in gradient elution. Detection was operated by APCI in selected ion monitoring mode. The target ions m/z 359, m/z 375, m/z 449 and m/z 419 were selected for the quantification of triptolide, tripdiolide, tripterine and IS, respectively. The linear range was 1.0-100.0 ng mL(-1), and the limits of quantification in human urine were found to be 0.1-0.5 ng mL(-1) for the three compounds. The precisions (CV%) and accuracies were 6.6-12.9 and 85.1-97.0%, respectively. The developed method could be applied to the determination of triptolide, tripdiolide and tripterine in human urine for diagnosis of the intoxication and for forensic purposes.     

Development of HPLC-MS/MS method for the simultaneous determination of environmental phenols in human urine

We present a sensitive method for simultaneous determination of bisphenol A (BPA), benzophenone-3 (BP-3), 4-tert-octylphenol (t-OP), ortho-phenylphenol (OPP), four parabens (methyl, ethyl, propyl, butyl parabens) and five chlorophenols (2,4-dichlorophenol (2,4-DCP), 2,5-dichlorophenol (2,5-DCP), 2,4,5-trichlorophenol (2,4,5-triclorophenol), 2,4,6-trichlorophenol (2,4,6-TCP), and triclosan (TCS)), in human urine by high-pressure liquid chromatography (HPLC) mass spectrometry (MS). Samples were processed using enzymatic deconjugation of glucuronides followed by solid phase extraction (SPE) on a C18 cartridge and the eluate was concentrated. Analytes were separated by reversed-phase HPLC and then detected by atmospheric pressure chemical ionisation (APCI) MS and quantified by isotope dilution method. We describe details for optimisation of each step of the procedure. The sample treatment steps are straightforward and not labour-intensive and, therefore, permit a high sample throughput with excellent prospects for automation. This method shows low inter-day variation, and detection limits for most of the compounds are below 1 ng/mL in 1 mL of urine. The method accuracy was also verified by the analysis of proficiency testing urine samples.     

Determination of macrolide antibiotics in porcine and bovine urine by high-performance liquid chromatography coupled to coulometric detection

A high-performance liquid chromatography method coupled to coulometric detection has been applied for the determination, in a single run, of up to eight macrolide antibiotics (erythromycin [ERY], tylosin [TYL], tilmicosin [TILM], spiramycin 2 [SPI 2], spiramycin 3 [SPI 3], josamycin [JOS], kitasamycin [KIT], and rosamicin [ROS]) in spiked porcine and bovine urine. Quantification was performed using matrix-matched calibration with roxithromycin (ROX) as the internal standard. The detection limits for each drug were below 3.5 ng injected (equivalent to an initial concentration below 0.07 mg L^–1) for porcine urine and below 5 ng injected (equivalent to an initial concentration below 0.10 mg L^–1) for bovine urine. Recoveries from urine samples spiked at three different concentrations within the linear range were not significantly dependent on concentration. The entire procedure provides average macrolide recoveries ranging from 69.7 to 96.6% for bovine urine and from 75.5 and 96.1% for porcine urine.     

Development and validation of a specific and sensitive liquid chromatography tandem mass spectrometry method for determination of tetrodotoxin in human urine and its pharmacokinetic study

Tetrodotoxin (TTX) exhibits the therapeutic potential in blocking pain and in low doses can safely relieve severe pain. The urinary excretion profiles of TTX in humans have not been reported due to the extremely low lethal dose. In this study, a rapid and specific method based on protein precipitation coupled to liquid chromatography tandem mass spectrometry was developed to determine the level of TTX in human urine samples. 11-Deoxytetrodotoxin was used as an internal standard (IS). Multiple reaction monitoring mode was used for quantification using target fragment ions m/z 320.0 → 162.1 for TTX and m/z 304.0 → 176.0 for 11-deoxyTTX. The separation of analytes was achieved on a hydrophilic interaction liquid chromatography column (250 × 4.6 mm, 5.0 μm). The mobile phase consisted of 5 mM ammonium formate in water (pH = 4.50) and 5 mM ammonium formate in acetonitrile (pH = 4.50). The flow rate was set at 0.80 mL/min in a gradient condition. Calibration plots were linear throughout the range 0.986–98.6 ng/mL of TTX in human urine. The intra-assay accuracies and precisions were within the acceptable range. The method was successfully applied to a urinary excretion study after intravenous administration of TTX to healthy volunteers. The developed method will be helpful for future pharmacological studies of TTX.     

Quantitative determination of BMS-378806 in human plasma and urine by high-performance liquid chromatography/tandem mass spectrometry

BMS-378806 is a human immunodeficiency virus (HIV) entry inhibitor that is being developed for the oral treatment of HIV infection. Human plasma and urine LC/MS/ MS methods have been developed and validated for the quantitation of BMS-378806. For human plasma method, methyl t-butyl ether was used to extract BMS-378806 from plasma in a 96-well format, and the organic layers were dried down and then reconstituted for the injection, while a dilute-and-shoot approach was used for human urine method in a 96-well format. Chromatographic separation was achieved isocratically on a Phenomenex C18 (2) Luna column (2 x 50 mm2, 5 microm). The mobile phase contained 60:40 v/v of 0.1% formic acid in water and ACN. Detection was by positive ion electrospray MS/MS. The standard curves ranged from 1.25 to 1000 ng/mL for the plasma assay and from 10 to 5000 ng/mL for the urine assay. The curves were fitted to a 1/x2 weighted quadratic regression model for both methods. The validation results demonstrated that both methods had satisfactory precision and accuracy across the calibration ranges. The methods were applied to the analysis of human plasma and urine samples from a single ascending dose clinical study to assess the pharmacokinetics of the drug. The pharmacokinetic analysis results indicated the absorption and disposition of the drug was rapid. The systemic exposure of BMS-378806 was generally dose proportional among the doses from 100 to 1200 mg, but not dose proportional to 1600 mg. There were modest increases in the systemic exposure when the drug was given with food or given as a solution formulation. Renal excretion was not a substantial elimination pathway of the drug. BMS378806 was safe and well tolerated over a dose range of 100-1600 mg administered as a single oral dose.     

Identification and analysis of alkaloids in cortex Phellodendron amurense by high‐performance liquid chromatography with electrospray ionization mass spectrometry coupled with photodiode array detection

Alkaloids from Cortex Phellodendron amurense Rupr. were identified to determine the material basis for the bioactivity of this herb. HPLC–ESI‐MS with photodiode array detection coupled to XCharge C₁₈ column was applied to analyze the alkaloids qualitatively and quantitatively. A total of 37 alkaloids were identified and tentatively characterized from the ethanol extract by online ESI‐MSⁿ fragmentation and UV spectral analysis. A total of ten alkaloids, including four novel natural products, were tentatively identified for the first time in P. amurense. The fragmentation pathways for certain compounds were analyzed. The contents of a pair of isomers (columbamine and jatrorrhizine) and four main alkaloids (phellodendrine, magnoflorine, berberine, and palmatine) were simultaneously quantified using the aforementioned method. Results showed that the newly discovered and known components of P. amurense were helpful in determining the material basis for the bioactivity of the herb. The application of the XCharge C₁₈ column is a suitable and practical method for the isolation of alkaloids in plants.     

超高效液相色谱-三重四极杆质谱法同时检测人尿中12种单羟基多环芳烃代谢物

建立了高效、准确的同时测定人体尿样中多种多环芳烃代谢物的方法。取10.0 mL尿液,酶解,经固相萃取净化浓缩、0.2 μm滤膜过滤后,以Acquity UPLC^®HSS T3（100 mm×2.1 mm,1.8 μm）为分析柱,甲醇和水为流动相,采用负离子电喷雾多反应监测模式检测样品中多环芳烃代谢物的含量。12种多环芳烃代谢物在0.04~20.0 μg/L范围内线性关系良好,相关系数>0.99,方法检出限为0.01~0.41 μg/L,平均回收率为80.0%~105%,批内RSD为1.21%~9.12%,批间RSD为4.43%~19.7%。应用该方法对淮河流域某区域的100份人体尿样进行了检测,多环芳烃代谢物的检出率为98%。该方法操作简单,灵敏度高,选择性好,可同时检测尿中12种羟基多环芳烃代谢物。     

Rapid method for simultaneous determination of flavonoid,saponins and polyacetylenes in Folium Ginseng and Radix Ginseng by pressurized liquid extraction and high-performance liquid chromatography coupled with diode array detection and mass spectrometry

A rapid pressurized liquid extraction (PLE) and high-performance liquid chromatography coupled with diode array detection and mass spectrometry (HPLC-DAD–MS) method for the simultaneous determination of one flavonoid (panasenoside), nine saponins (ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3 and Rd) and two polyacetylenes (panaxydol and panaxynol) in Folium Ginseng and Radix Ginseng was developed. A Prevail C₁₈ rocket column (33 mm × 7 mm, 3.0 μm) and gradient elution were used during the analysis. Flavonoid was quantified at 355 nm, and saponins and polyacetylenes were determined at 203 nm. The chromatographic peaks of 12 investigated compounds in samples were unambiguously identified by compared their UV spectra and/or MS data with the related reference compounds. All calibration curves showed good linearity (r > 0.999) within the test ranges. The intra- and inter-day variations for 12 analytes were less than 1.17% and 2.17%, respectively. The developed method was successfully applied to determine the investigated compounds in 10 samples of Radix Ginseng and Folium Ginseng, respectively. The result showed that PLE combined with rocket column HPLC analysis could provide a rapid method for analysis of compounds in traditional Chinese medicines (TCMs), which is helpful to comprehensive evaluation of quality of Radix Ginseng and Folium Ginseng.     

A rapid and sensitive method for quantitation of nucleosides in human urine using liquid chromatography/mass spectrometry with direct urine injection

Oxidized nucleosides are biochemical markers for tumors, aging, and neurodegenerative diseases. However, during the last decade, the analytical methods for nucleosides by gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography (HPLC) with single-parameter detectors like electron-capture detection (ECD) have not been sufficiently rapid or reliable to detect nucleosides in urine and to analyze clinical samples. It has been reported (Dudley et al., Rapid Commun Mass Spectrom. 2000; 14: 1200) that liquid chromatography with electrospray mass spectrometry (LC/ESI-MS) is more specific and sensitive for analysis of nucleosides than HPLC with conventional detectors; however, this method required complex extraction steps. In the present work a direct LC/ESI-MS method for nucleosides without extraction of urine samples has been developed. Analysis of nucleosides using positive-ion mode with selected reaction monitoring effectively eliminated potential interferences from endogenous constituents of the urine. This highly selective and sensitive method made it possible to analyze urinary nucleosides with a lower limit of quantitation of 0.2 nmol/mL. The method has been validated, with both excellent linearity and reproducibility, in the calibration range from 0.2-400 nmol/mL. The correlation coefficients of the calibration curves were higher than 0.987. The coefficients of variation were in the range 0.03-14.92% (inter-day) and 0.54-14.39% (intra-day), respectively.     

Quantitative analysis of docetaxel in human plasma using liquid chromatography coupled with tandem mass spectrometry

An assay for the quantitative determination of docetaxel in human plasma is described. Docetaxel was extracted from the matrix using liquid-liquid extraction with ter-butylmethylether, followed by high-performance liquid chromatographic analysis using an alkaline eluent. Paclitaxel was used as internal standard. Positive ionization electrospray tandem mass spectrometry was performed for selective and sensitive detection. The method was validated according to the FDA guidelines on bioanalytical method validation. The validated range for docetaxel was from 0.25--1000 ng/mL using 200 microL plasma aliquots. The method requires only a limited volume (200 microL) of human plasma and the method can be applied in studies requiring a low lower limit of quantitation of 0.25 ng/mL. The assay was applied successfully in several clinical and pharmacological studies with docetaxel.     

High-throughput determination of atrasentan in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Atrasentan (A-147627) is an endothelin antagonist receptor being developed at Abbott Laboratories for the treatment of prostate cancer. A quick and sensitive method for the determination of atrasentan in human plasma has been developed and validated using high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. A dual-column, single mass spectrometer system is used to provide a reliable and routine means to increase sample throughput. The analytical method involves liquid-liquid extraction and internal standard (A-166790). The plasma samples and internal standard are acidified with 0.3 M hydrochloric acid prior to being extracted into 1:1 (v/v) hexanes--methyl t-butyl ether. The organic extract was evaporated to dryness using heated nitrogen stream and reconstituted with mobile phase. Atrasentan and internal standard were separated with no interference in a Zorbax SB-C(18) analytical column with 2.1 x 50 mm, 5 microm, and a Zorbax C(8) guard column using a mobile phase consisting of 50:50 (v:v) acetonitrile--0.05 M ammonium acetate, pH 4.5, at a flow rate of 0.30 mL/min to provide 4 min chromatograms. For a 250 microL plasma sample volume, the limit of quantitation was approximately 0.3 ng/mL. The calibration was linear from 0.30 to 98.0 ng/mL (r(2) > 0.995). A significant advantage of the method is the ability to employ parallel HPLC separations with detection by a single MS/MS system to provide sensitivity and selectivity sufficient to achieve robust analytical results with a lower limit of quantitation of 0.30 ng/mL and high throughput.     

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。     

Rapid bioanalysis of vancomycin in serum and urine by high-performance liquid chromatography tandem mass spectrometry using on-line sample extraction and parallel analytical columns

A novel high-performance liquid chromatography tandem mass spectrometry (LC/MS/MS) method is described for the determination of vancomycin in serum and urine. After the addition of internal standard (teicoplanin), serum and urine samples were directly injected onto an HPLC system consisting of an extraction column and dual analytical columns. The columns are plumbed through two switching valves. A six-port valve directs extraction column effluent either to waste or to an analytical column. A ten-port valve simultaneously permits equilibration of one analytical column while the other is used for sample analysis. Thus, off-line analytical column equilibration time does not require mass spectrometer time, freeing the detector for increased sample throughput. The on-line sample extraction step takes 15 seconds followed by gradient chromatography taking another 90 seconds. Having minimal sample pretreatment the method is both simple and fast. This system has been used to successfully develop a validated positive-ion electrospray bioanalytical method for the quantitation of vancomycin. Detection of vancomycin was accurate and precise, with a limit of detection of 1 ng/mL in serum and urine. The calibration curves for vancomycin in rat, dog and primate were linear in a concentration range of 0.001-10 microg/mL for serum and urine. This method has been successfully applied to determine the concentration of vancomycin in rat, dog and primate serum and urine samples from pharmacokinetic and urinary excretion studies.     

Measurement of caffeine and five of the major metabolites in urine by high-performance liquid chromatography/tandem mass spectrometry

Analysis of caffeine and its metabolites is of interest with respect to caffeine exposure, for kinetic and metabolism studies and for opportunistic in vivo estimation of drug metabolizing enzyme activity in humans and animals. For the latter, analysis is usually done by high-performance liquid chromatography (HPLC) with UV detection. However, this method is close to the detection limit for certain of the metabolites and requires very long chromatography, 30-60 min. We have developed a fast method for the quantification of caffeine and its metabolites 1-methylxanthine, 1-methyluric acid, 1,7-dimethyluric acid, 5-acetylamino-6-amino-3-methyluracil (AAMU) and 5-acetylamino-6-formylamino-3-methyluracil (AFMU) by HPLC tandem mass spectrometry (MS/MS) in urine that requires only its dilution with buffer and centrifugation before injection into the HPLC/MS/MS system. The chromatography lasts 7 min and is followed by 4.5 min for re-equilibration of the HPLC column, giving a total analysis time of 11.5 min. The method provides a great sensitivity improvement with detection limits for all analytes 相似文献   

Extraction and analysis of pharmaceuticals in polluted sediment using liquid chromatography mass spectrometry

A multi-residue method for the extraction and clean-up of sediment samples was developed for the analysis of pharmaceutical residues. Sediment samples were collected in the proximity of sewage water plant in Stockholm, Sweden. Target analytes were the basic β-blocker propranolol, the neutral neuroleptic carbamazepine and the acidic anticoagulant warfarin, the painkiller diclofenac and the lipid regulator gemfibrozil. The extraction solvent was optimised with regard to pH and organic modifer. Extraction and clean up were performed with liquid-liquid extraction and ultra-sonication followed by solid-phase extraction. One extraction solvent, containing acetone/McIlvaine buffer pH4, provided satisfactory extraction for all substances. LC/MSMS in the MRM mode was used for determination. The recoveries of the extraction and clean-up steps were 60–75% (±2–8%) and LOQs were in the range 0.4–8?ng/g sediment (dry weight). The pharmaceuticals found in the sediment samples were propranolol and carbamazepine, representing substances with basic and neutral properties. Additionally, the samples were analysed with LC/QTOF for verification with the use of accurate mass measurement in the full-scan mode. Pharmaceuticals not represented in the original method were looked for. Non-target pharmaceuticals found using the LC/QTOF system were the basic β-blocker metoprolol and the acidic painkiller naproxen.     

A general screening method for doping agents in human urine by solid phase extraction and liquid chromatography/time-of-flight mass spectrometry

A general screening method based on solid phase extraction (SPE) and liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) was developed and investigated with 124 different doping agents, including stimulants, -blockers, narcotics, -adrenergic agonists, agents with anti-estrogenic activity, diuretics and cannabinoids. Mixed mode cation exchange/C8 cartridges were applied to SPE, and chromatography was based on gradient elution on a C18 column. Ionization of the analytes was achieved with electrospray ionization in the positive mode. Identification by LC/TOFMS was based on retention time, accurate mass and isotopic pattern. Validation of the method consisted of analysis of specificity, analytical recovery, limit of detection and repeatability. The minimum required performance limit (MRPL), established by World Anti-Doping Agency (WADA), was attained to 97 doping agents. The extraction recoveries varied between 33 and 98% and the median was 58%. Mass accuracy was always better than 5 ppm, corresponding to a maximum mass error of 0.7 mDa. The repeatability of the method for spiked urine samples, expressed as median of relative standard deviations (RSD%) at concentrations of MRPL and 10 times MRPL, were 14% and 9%, respectively. The suitability of the LC/TOFMS method for doping control was demonstrated with authentic urine samples.     

Solid-phase microextraction coupled with microcolumn liquid chromatography for the analysis of amitriptyline in human urine

Summary Solid-phase microextraction (SPME) is a solvent-free sample-preparation technique that enables isolation and pre-concentration of analytes from a sample on a thin film coating a fused-silica fiber. In this study SPME coupled with microcolumn liquid chromatography (micro LC) has been used for the determination of four tricyclic antidepressants (amitriptyline, imipramine, nortriptyline, and desipramine) in human urine. SPME conditions which affect extraction efficiency were optimized, and under the optimum conditions the system was a few hundred times more sensitive than direct LC analysis without SPME. For amitriptyline the detection limit was 3 ng mL⁻¹ and the calibration curve was linear in the range of 5–500 ng mL⁻¹. The SPME-micro LC method has been applied to the analysis of amitriptyline in patient’s urine.     

Quantitative determination of uridine in rabbit plasma and urine by liquid chromatography coupled to a tandem mass spectrometry

Recently a pyrimidine nucleoside, uridine, has been show to have a protective effect on cultured human corneal epithelial cells, and on dry eye animal model and patients. In this study, we introduce a sensitive liquid chromatography/tandem mass spectrometry method for the determination of uridine in rabbit plasma and urine. After protein precipitation with methanol including methaqualone (internal standard), the analyte was chromatographed on a reversed-phase column with a mobile phase of 0.1% formic acid aqueous solution and methanol (1:4, v/v). The accuracy and precision of the assay were in accordance with Food and Drug Administration regulations for the validation of bioanalytical methods. This method was used to measure the concentrations of uridine in plasma and urine after a single oral administration of 450 mg/kg uridine in rabbits.