Profiling and imaging of tissues by imaging ion mobility-mass spectrometry

Molecular profiling and imaging mass spectrometry (IMS) of tissues can often result in complex spectra that are difficult to interpret without additional information about specific signals. This report describes increasing data dimensionality in IMS by combining two-dimensional separations at each spatial location on the basis of imaging ion mobility-mass spectrometry (IM-MS). Analyte ions are separated on the basis of both ion-neutral collision cross section and m/z, which provides rapid separation of isobaric, but structurally distinct ions. The advantages of imaging using ion mobility prior to MS analysis are demonstrated for profiling of human glioma and selective lipid imaging from rat brain.     

DETECTING LOW-LEVEL SYNTHESIS IMPURITIES IN MODIFIED PHOSPHOROTHIOATE OLIGONUCLEOTIDES USING LIQUID CHROMATOGRAPHY - HIGH RESOLUTION MASS SPECTROMETRY

An LC-MS method based on the use of high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS) for profiling oligonucleotides synthesis impurities is described.Oligonucleotide phosphorothioatediesters (phosphorothioate oligonucleotides), in which one of the non-bridging oxygen atoms at each phosphorus center is replaced by a sulfur atom, are now one of the most popular oligonucleotide modifications due to their ease of chemical synthesis and advantageous pharmacokinetic properties. Despite significant progress in the solid-phase oligomerization chemistry used in the manufacturing of these oligonucleotides, multiple classes of low-level impurities always accompany synthetic oligonucleotides. Liquid chromatography-mass spectrometry has emerged as a powerful technique for the identification of these synthesis impurities. However, impurity profiling, where the entire complement of low-level synthetic impurities is identified in a single analysis, is more challenging. Here we present an LC-MS method based the use of high resolution-mass spectrometry, specifically Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS or FTMS). The optimal LC-FTMS conditions, including the stationary phase and mobile phases for the separation and identification of phosphorothioate oligonucleotides, were found. The characteristics of FTMS enable charge state determination from single m/z values of low-level impurities. Charge state information then enables more accurate modeling of the detected isotopic distribution for identification of the chemical composition of the detected impurity. Using this approach, a number of phosphorothioate impurities can be detected by LC-FTMS including failure sequences carrying 3'-terminal phosphate monoester and 3'-terminal phosphorothioate monoester, incomplete backbone sulfurization and desulfurization products, high molecular weight impurities, and chloral, isobutyryl, and N(3) (2-cyanoethyl) adducts of the full length product. When compared with low resolution LC-MS, ~60% more impurities can be identified when charge state and isotopic distribution information is available and used for impurity profiling.     

离子淌度质谱技术在中药化学成分分析中的研究进展

离子淌度质谱(IM-MS)是一种将离子淌度分离与质谱分析相结合的新型分析技术。IM-MS的主要优势不仅是在质谱检测前提供了基于气相离子形状、大小、电荷数等因素的多一维分离,而且能够提供碰撞截面积、漂移时间等质谱信息进而辅助化合物鉴定。近年来,随着IM-MS技术的不断发展,该技术在中药化学成分分析中受到越来越多的关注。首先,IM-MS已成功应用于改善中药复杂成分尤其是同分异构体或等量异位素等成分的分离;其次,IM-MS可通过多重碎裂模式辅助高质量中药小分子质谱信息的获取;此外,IM-MS提供的高维质谱数据信息还可促进中药复杂体系多成分的整合分析。该文在对IM-MS分类和基本原理进行概述的基础上,从分离能力及分离策略、多重碎裂模式、多维质谱数据处理策略3个方面,重点综述了IM-MS在中药化学成分分析中的应用,以期为IM-MS在中药化学成分研究提供参考。     

Metabolic profiling of Escherichia coli by ion mobility-mass spectrometry with MALDI ion source

Comprehensive metabolome analysis using mass spectrometry (MS) often results in a complex mass spectrum and difficult data analysis resulting from the signals of numerous small molecules in the metabolome. In addition, MS alone has difficulty measuring isobars and chiral, conformational and structural isomers. When a matrix-assisted laser desorption ionization (MALDI) source is added, the difficulty and complexity are further increased. Signal interference between analyte signals and matrix ion signals produced by MALDI in the low mass region (<1500 Da) cause detection and/or identification of metabolites difficult by MS alone. However, ion mobility spectrometry (IMS) coupled with MS (IM-MS) provides a rapid analytical tool for measuring subtle structural differences in chemicals. IMS separates gas-phase ions based on their size-to-charge ratio. This study, for the first time, reports the application of MALDI to the measurement of small molecules in a biological matrix by ion mobility-time of flight mass spectrometry (IM-TOFMS) and demonstrates the advantage of ion-signal dispersion in the second dimension. Qualitative comparisons between metabolic profiling of the Escherichia coli metabolome by MALDI-TOFMS, MALDI-IM-TOFMS and electrospray ionization (ESI)-IM-TOFMS are reported. Results demonstrate that mobility separation prior to mass analysis increases peak-capacity through added dimensionality in measurement. Mobility separation also allows detection of metabolites in the matrix-ion dominated low-mass range (m/z < 1500 Da) by separating matrix signals from non-matrix signals in mobility space.     

Real-time reaction monitoring using ion mobility-mass spectrometry

The potential of ion mobility (IM) spectrometry in combination with mass spectrometry (MS) for real-time reaction monitoring is reported. The combined IM-MS approach using electrospray ionization affords gas-phase analyte characterization based on both mass-to-charge (m/z) ratio and gas-phase ion mobility (drift time). The use of IM-MS analysis is demonstrated for the monitoring of the reaction products formed when 7-fluoro-6-hydroxy-2-methylindole is deprotonated by aqueous sodium hydroxide. Real-time reaction monitoring was carried out over a period of several hours, with the reaction mixture sampled and analysed at intervals of several minutes. Product ion relative intensity is enhanced selectively in the ion mobility-selected mass spectrum, compared to mass spectrometry alone. The combined IM-MS approach has potential as a rapid and selective technique to aid pharmaceutical process control and for the elucidation of reaction mechanism.     

Characterisation of nicotine and related compounds using electrospray ionisation with ion trap mass spectrometry and with quadrupole time-of-flight mass spectrometry and their detection by liquid chromatography/electrospray ionisation mass spectrometry

Electrospray ionisation ion trap mass spectrometry (ESI-MS(n)) has been used to study the fragmentation patterns of nicotine and nine of its related compounds. From this study certain characteristic fragmentations are apparent with generally the pyrrolidine or piperidine ring being subject to chemical modifications. The structures of the product ions proposed for the ESI-MS(n) study have been supported by results from electrospray ionisation quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS). Compounds with pyrrolidine and piperidine rings that possess an unsubstituted N atom have been shown to lose NH(3) at the MS(2) stage. Those compounds with N-methyl groups lose CH(3)NH(2) at the MS(2) stage. The loss of NH(3) or CH(3)NH(2) leaves the corresponding rings opened and this is followed by ring closure at the pyridine-2 carbon atom. Mono-N-oxides fragment in a similar way but the di-N-oxide can also fragment by cleavage of the bond between the pyridine and pyrrolidine rings. Cotinine also can undergo cleavage of this bond between the rings.This data therefore provides useful information on how substituents and the nature of the non-pyridine ring can affect the fragmentation patterns of nicotine and its related compounds. This information can be used in the characterisation of these compounds by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) which results in the separation of nicotine and its related compounds with limits of detection (LODs) ranging from 15 to 105 ng/mL. The use of LC/ESI-MS to study nicotine-containing samples resulted in the simultaneous and unambiguous identification of seven of the compounds discussed in this paper: cotinine identified at retention time 12.5 min (with its [M+H](+) ion at m/z 177), nornicotine 16.0 min (m/z 149), anatabine 18.0 min (m/z 161), myosmine 18.5 min (m/z 147), anabasine 20.4 min (m/z 163), nicotine 22.2 min (m/z 163), and nicotyrine 31.4 min (m/z 159). For quality control of nicotine replacement therapy products, these nicotine impurities can be readily identified and determined at levels up to 0.3% for single impurities and up to 1.0% for total impurities.     

Analysis of ceramides in cosmetics by reversed-phase liquid chromatography/electrospray ionization mass spectrometry with collision-induced dissociation

A rapid, sensitive and selective method involving reversed-phase liquid chromatography (LC) with electrospray ionization (ESI) mass spectrometry (MS) was employed for determination of commercial ceramides in cosmetics for quality control of the product formulation. Using this LC/ESI-MS technique, simultaneous separation and characterization of ceramides and an impurity substance were possible. Informative fragmentation patterns were obtained by employing LC/ESI-MS in both positive and negative ionization modes to identify the structures of both sphingoid base and N-acyl chains of ceramides, and also of an impurity. The combination of positive and negative mass spectra can be used for unambiguous confirmation of ceramides and for characterization of unknown species. In-source collision-induced fragmentation resulted in characteristic product anions for the ceramides containing a phytosphingosine moiety at m/z 267, 255 and 225, and for those with a sphingosine moiety at m/z 263 and 237, regardless of the length of the fatty acyl chains. The detection limit was about 0.5 pmol in selected-ion monitoring mode. Quantification using internal standards showed good linearity and a relative standard deviation of 4%. These ceramides were more sensitively detected in positive than in negative ion mode.     

Qualitative determination of urinary morphine by capillary zone electrophoresis and ion trap mass spectrometry

A rapid, sensitive method for the determination of morphine and amphetamine was developed using capillary zone electrophoresis coupled with electrospray interface (ESI), ion-trap tandem mass spectrometry (CE-ESI-MS2). Morphine and amphetamine were separated in 20 mM ammonium acetate buffer (pH 6.6) and detected by ion-trap mass detector in different analytical time segments (0-6.25 min for amphetamine and 6.25-12.0 min for morphine) in which the tune file for each compound was used separately. Molecular ions of morphine (m/z 286) and amphetamine (m/z 136) were detected at 5.77 and 6.83 min, respectively, while product ions of MS2 for each compound (m/z 229, 201 for morphine and m/z 119 for amphetamine) were detected almost exactly at the same time with their parent compounds. The limits of detection (LOD) for MS2 determination were 30 and 50 ng/mL for amphetamine and morphine, respectively, with an S/N ratio of 3. For more sensitive detection of morphine, the sample was injected for a longer time (i.e., 80 s) and hydrodynamically transported into a CE capillary for MS detection. Morphine and its product ion appear at 0.36 and 0.39 min on the ion chromatogram, respectively, with a five-fold increase of detection sensitivity (LOD, 10 ng/mL). The CE-MS system thus established was further applied for forensic urine samples screened as morphine-positive by fluorescence polarization immunoassay (FPIA). These results indicated the feasibility of CE-ESI-MS2 for confirmative testing of morphine in urine sample.     

Rapid and sensitive liquid chromatography tandem mass spectrometry method for the quantification of ambroxol in human plasma

A sensitive, specific and rapid high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was described and validated for the quantification of ambroxol in human plasma using enalaprilat as the internal standard (IS). Chromatographic separation was performed on a Lichrospher CN column with a mobile phase of methanol and water (containing 0.1% formic acid) (70:30, v/v). The total run time was 5.0 min for each sample. The analytes was detected by mass spectrometry with electrospray ionization source in positive selected reaction monitoring mode. The precursor-fragment ion reaction for ambroxol was m/z 378.9 --> 263.8, and for IS was m/z 349.0 --> 205.9. The linearity was established over the concentration range of 1.56-400.00 ng/mL. The inter-day and the intra-day precisions were all within 10%. A simple protein precipitation with methanol was adopted for sample preparation. The extraction recoveries of ambroxol and IS were higher than 90.80%. The validated method was successfully applied in pharmacokinetic study after oral administration of 90 mg ambroxol to 24 healthy volunteers.     

Drug impurity profiling by capillary electrophoresis/mass spectrometry using various ionization techniques

Capillary electrophoresis/mass spectrometry (CE/MS) is predominantly carried out using electrospray ionization (ESI). Recently, atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) have become available for CE/MS. With the VUV lamp turned off, the APPI source may also be used for CE/MS by thermospray ionization (TSI). In the present study the suitability of ESI, APCI, APPI and TSI for drug impurity profiling by CE/MS in the positive ion mode is evaluated. The drugs carbachol, lidocaine and proguanil and their potential impurities were used as test compounds, representing different molecular polarities. A background electrolyte of 100 mM acetic acid (pH 4.5) provided baseline separation of nearly all impurities from the respective drugs. APPI yielded both even‐ and odd‐electron ions, whereas the other ionization techniques produced even‐electron ions only. In‐source fragmentation was more pronounced with APCI and APPI than with ESI and TSI, which was most obvious for proguanil and its impurities. In general, ESI and TSI appeared the most efficient ionization techniques for impurities that are charged in solution achieving detection limits of 100 ng/mL (full‐scan mode). APPI and APCI showed a lower efficiency, but allowed ionization of low and high polarity analytes, although quaternary ammonium compounds (e.g. carbachol) could not be detected. Largely neutral compounds, such as the lidocaine impurity 2,6‐dimethylaniline, could not be detected by TSI, and yielded similar detection limits (500 ng/mL) for ESI, APPI and APCI. In many cases, impurity detection at the 0.1% (w/w) level was possible when 1 mg/mL of parent drug was injected with at least one of the CE/MS systems. Overall, the tested CE/MS systems provide complementary information as illustrated by the detection and identification of an unknown impurity in carbachol. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantification of zolpidem in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A simple and robust method for quantification of zolpidem in human plasma has been established using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). Es-citalopram was used as an internal standard. Zolpidem and internal standard in plasma sample were extracted using solid-phase extraction cartridges (Oasis HLB, 1 cm3/30 mg). The samples were injected into a C8 reversed-phase column and the mobile phase used was acetonitrile-ammonium acetate (pH 4.6; 10 mm) (80:20, v/v) at a flow rate of 0.7 mL/min. Using MS/MS in the selected reaction-monitoring (SRM) mode, zolpidem and Es-citalopram were detected without any interference from human plasma matrix. Zolpidem produced a protonated precursor ion ([M+H]+) at m/z 308.1 and a corresponding product ion at m/z 235.1. The internal standard produced a protonated precursor ion ([M+H]+) at m/z 325.1 and a corresponding product ion at m/z 262.1. Detection of zolpidem in human plasma by the LC-ESI MS/MS method was accurate and precise with a quantification limit of 2.5 ng/mL. The proposed method was validated in the linear range 2.5-300 ng/mL. Reproducibility, recovery and stability of the method were evaluated. The method has been successfully applied to bioequivalence studies of zolpidem.     

Determination of glycoalkaloids and relative aglycones by nonaqueous capillary electrophoresis coupled with electrospray ionization-ion trap mass spectrometry

Glycoalkaloids are naturally occurring nitrogen-containing compounds present in many species of the family Solanaceae, including cultivated and wild potatoes (Solanum spp.), tomatoes (Lycopersicon spp.), etc. These compounds have pharmacological and toxicological effects on humans due to their significant anticholinesterase activity and disruption of cell membranes. Herein is reported the development of a capillary electrophoresis (CE) method using nonaqueous (NA) separation solutions in combination with ion trap mass spectrometry (MS and MS/MS) detection for the identification and quantification of glycoalkaloids and their relative aglycones. A mixture 90:10 v/v of MeCN-MeOH containing 50 mM ammonium acetate and 1.2 M acetic acid (applied voltage of 25.5 kV) was selected as a good compromise for the separation and detection of these compounds. The electrospray MS measurements were carried out in the positive ionization mode using a coaxial sheath liquid, methanol-water (1:1) with 1% of acetic acid at a flow rate of 2.5 microL/min. Under optimized experimental conditions, the predominant ion was the protonated molecular ion ([M+H](+)) of solanidine (m/z = 398), tomatidine (m/z = 416), chaconine (m/z = 852), solanine (m/z = 868), and tomatine (m/z = 1034). MS/MS experiments were carried out systematically by changing the relative collisional energy and monitoring the intensities of the fragment ions that were not high enough to allow better quantification than with the mother ions. The method was used for analyzing glycoalkaloids in potato extracts.     

Determination of glycyrrhizin in dog plasma by liquid chromatography-mass spectrometry and its application in pharmacokinetic studies

A sensitive liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method was established and validated for the determination of glycyrrhizin in dog plasma. After treatment with methanol to precipitate proteins, plasma samples were analyzed on a reversed-phase C18 (ODS) column with a mobile phase of methanol:1% formic acid solution (75:25, v/v). MS determination was performed using negative electrospray ionization (negative ESI) in the selected ion monitoring mode. Glycyrrhizin was monitored at the m/z 821 channel and internal standard (gliquidone) at the m/z 526 channel. The calibration curve was linear over the range from 0.05 μg mL(-1) to 10 μg mL(-1) with a correlation coefficient above 0.99. This method was successfully applied to the pharmacokinetic studies in beagle dogs. The absolute bioavailability of glycyrrhizin in beagle dogs was 3.24%.     

衍生化-离子阱气相色谱质谱联用检测乳粉中三聚氰胺时不同质谱检测方法研究

利用N,O-双三甲基硅基三氟乙酰胺(BSTFA)和三甲基氯硅烷(TMCS)衍生化试剂对乳粉中三聚氰胺进行衍生化处理,利用离子阱气相色谱质谱联用仪,建立了全扫描、选择离子监测、二级质谱3种测定三聚氰胺的质谱方法.选择离子监测以三聚氰胺衍生物的特征离子m/z342,327,171,99为定性离子,以m/z327为定量离子;全扫描法二级质谱特征峰为定性依据,以特征离子m/z327为定量离子;二级质谱法以衍生物二级质谱m/z285,171,213为定性离子,以m/z 285为定量离子.3种方法的线性范围为0.05～2.0 mg/L,线性相关系数分别为0.9986、0.9990、0.9988;检测限分别为0.005、0.002、0.003 mg/kg,RSD分别为6.3%、5.7%、6.1%(n=6),方法的回收率为84%～105%.3种不同质谱检测方法应用到乳粉的检测中效果良好,均能够满足乳粉中三聚氰胺的检测要求.     

高效液相色谱-离子阱质谱法测定人血浆中的头孢拉定和青霉素G

目前,β-内酰胺类抗生素在临床抗感染药物中占有十分突出的地位,但在近年来的药品不良反应报告中,抗生素类药物引起的不良反应也占据了很高的比例,其中有我国生活环境影响、感染性疾病多的客观因素,但病人用药盲目性大、医生用药随意性多的问题也普遍存在。因此,进一步加强对抗生素类药物的监测,开发快速有效的分析测试方法显得十分重要。本文基于实际全血未知样品,开发了基于固相萃取及液质联用技术,快速准确地对血液中青霉素G及头孢拉定进行定性及质谱定量分析的检测方法。     

Rapid and sensitive liquid chromatography-electrospray ionization-mass spectrometry method for the determination of eperisone in human plasma: method and clinical applications

A sensitive liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method for determination of eperisone in human plasma using buflomedil as the internal standard (IS) is established. After being made alkaline with saturated sodium bicarbonate solution, plasma samples are extracted with a mixture of diethyl ether-cyclohexane (1:1, v/v) and separated by high-performance liquid chromatography on a reversed-phase C(18) column with a mobile phase of 10mM ammonium acetate buffer (adjusted the pH to 3.9 with acetic acid)-methanol (20:80, v/v). Eperisone is determined using ESI in a single-quadrupole MS. LC-ESI-MS is performed in the selected ion monitoring mode using target ions at m/z 260 for eperisone and m/z 308 for the IS. Calibration curves are linear over the ranges 0.02-20 ng/mL for eperisone. The intra- and interassay variability values are less than 9.0% and 11.5%, respectively. The mean plasma extraction recovery of eperisone is 91.7 +/- 6.6%. The method has been successfully applied to study the pharmacokinetics of eperisone in healthy, male, Chinese volunteers. Pharmacokinetic parameters of the reference and test tablets have been compared.     

高效液相色谱-质谱法同时测定清热解毒口服液中的5种有效成分

建立了同时测定清热解毒口服液中的绿原酸、栀子苷、黄芩苷、连翘苷和靛玉红5种有效成分的高效液相色谱-电喷雾电离质谱（HPLC-ESI/MS）分析方法。采用Zorbax SB C18色谱柱,以含0.2%甲酸的0.4 mmol/L醋酸钠(A相)、乙腈(B相)为流动相进行梯度洗脱,在ESI正离子模式下,采用选择离子监测方法进行测定,用峰面积进行定量。结果表明,绿原酸、栀子苷、黄芩苷、连翘苷和靛玉红的线性范围分别为0.050～50 mg/L,0.020～20 mg/L,0.005～30 mg/L,0.010～15 mg/L和0.010～10 mg/L;检出限分别为0.010,0.005,0.001,0.002和0.003 mg/L。5种成分的加样回收率为97.0%～101.7%,相对标准偏差小于2.2%。该法快捷、准确、重复性好,可用于清热解毒口服液中的5种有效成分含量的同时测定。     

Specific detection of Lewis x-carbohydrates in biological samples using liquid chromatography/multiple-stage tandem mass spectrometry

The Lewis x structure [Lex, Galbeta1-4(Fucalpha1-3)GlcNAc] motif is one of the tumor antigens and plays an important role in oncogenesis, development, cellular differentiation and adhesion. The detection of Lex-carbohydrates and their structural analysis are necessary to clarify the role of Lex in several biological events. Mass spectrometry has been preferably used for the structural analysis of carbohydrates. Especially, collision-induced dissociation (CID) tandem mass spectrometry (MS/MS), which causes a glycosidic bond cleavage, is used for carbohydrate sequencing. However, Lex cannot be identified by MS/MS due to the existence of the positional isomers, such as Lewis a [Galbeta1-3(alpha1-4Fuc)GlcNAc]. In the present study, we demonstrate the specific detection of Lex-carbohydrates in a biological sample by using multiple-stage MS/MS (MSn). Using pyridylaminated oligosaccharides bearing Lex, we found that the Lex-motif yields a cross-ring fragment by the cleavage of a bond between C-3 and C-4 of GlcNAc in Gal(Fuc)GlcNAc. The Lex-specific cross-ring fragment ion at m/z 259 was effectively detected by sequential scans, consisting of a full MS1 scan, data-dependent CID MS2 scan, MS3 of [Gal(Fuc)GlcNAc+Na]+ at m/z 534, and MS4 of [GalGlcNAc+Na]+ at m/z 388. The sequential scan was applied to N-linked oligosaccharide profiling using a LC/ESI-MSn system equipped with a graphitized carbon column. We successfully detected the Lex-motif and elucidated the structures of several Lex and Lewis y [(Fucalpha1-2)Galbeta1-4(Fucalpha1-3)GlcNAc] oligosaccharides in the murine kidney used as a model tissue. Our method is expected to be a powerful tool for the specific detection of the Lex-motif, and structural elucidation of Lex-carbohydrates in biological samples.     

The detection of phenotypic differences in the metabolic plasma profile of three strains of Zucker rats at 20 weeks of age using ultra-performance liquid chromatography/orthogonal acceleration time-of-flight mass spectrometry

Analysis of biological fluids using ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) (metabonomics) can allow new insights to be gained into disease processes, with advances in chromatographic techniques enabling the detection of thousands of metabolites. In this work metabonomics has been used to investigate the metabolic processes involved in type II diabetes in the Zucker obese rat. Plasma was analyzed from three different strains, the Zucker (fa/fa) obese, Zucker lean and the lean/(fa) obese cross. Using UPLC/MS, ca. 10,000 ions were detected due to the narrow peak widths and excellent peak shapes achieved with this technology. Confidence in the chromatographic performance was demonstrated by the use of quality control standards. The positive and negative ion total ion chromatograms obtained from the three strains were readily distinguishable using multivariate statistical analysis. The greatest difference was observed between the Zucker lean and Zucker lean/(fa) rats compared to the Zucker (fa/fa) obese rats. Positive ions m/z 220 (4.36 min), 282(3.78 min), 359 (5.33 min) and 405 (7.77 min) were elevated in the plasma derived from Zucker lean rats whilst ions m/z 385 (6.80 min) and 646 (4.36 min) were at a lower concentration compared to the plasma from the Zucker (fa/fa) obese animals. Negative ions elevated in the Zucker lean rats included m/z 212 (2.30 min), 514 (2.85 min), 295 (4.39 min), 329 (3.11 min), 343 (2.86 min) and 512 (2.86 min) with ions m/z 538 (4.18 min), 568 (4.18 min), 568 (5.09 min) and 612 (4.30 min) being raised in the samples derived from Zucker (fa/fa) obese animals. The ion m/z 514 (3.85 min) was found to correspond to taurocholate, providing further support for an involvement of taurine metabolism in diabetes.     

Online solid-phase extraction liquid chromatography-electrospray-tandem mass spectrometry analysis of buprenorphine and three metabolites in human urine

An online solid-phase extraction (SPE) liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) method for the determination of buprenorphine (Bup), norbuprenorphine (nBup), buprenorphie-3-beta-d-glucuronide (Bup-3-G) and norbuprenorphie-3-beta-d-glucuronide (nBup-3-G) in human urine was developed and validated. A mixed mode SPE column with both hydrophilic and lipophilic functions was used for online extraction. A C18 column was employed for LC separation and ESI-MS/MS was utilized for detection. Buprenorphine-D(4) (Bup-D4) and norbuprenophine-D3 (nBup-D3) were used as internal standards for quantitative determination. The extraction, clean-up and analysis procedures were controlled by a fully automated six-port switch valve. Identification and quantification were based on the following transitions: m/z 468-->414 for Bup, m/z 414-->364 for nBup, m/z 644-->468 for Bup-3-G and m/z 590-->414 for nBup-3-G, respectively. Good recoveries from 93.6% to 102.2% were measured and satisfactory linear ranges for these analytical compounds were determined. Minimal ion suppression effect (approximately 7% response decrease) was determined. Intra-day and inter-day precision showed coefficients of variance, CV, ranged from 3.3% to 10.1% and 4.4% to 9.8%, respectively. Accuracy ranging from 97.0% to 104.0% was determined. The applicability of this newly developed method was demonstrated by analyzing human urine samples from the patients in Bup treatment program for therapeutic monitoring purpose.