Development of quantitative imaging mass spectrometry (q-IMS) for drug visualization using animal tissues

Quantitative imaging mass spectrometry (q-IMS) is a frontier topic of research in drug analysis. Although many q-IMS methodologies have been reported, validation of the method is insufficient. We have investigated the feasibility of coupling q-IMS with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to develop a verifiable method. The approach combines quantitative LC-MS/MS information with the spatial distribution information obtained by IMS. This paper compares measured drug quantities with those estimated using IMS. The target drug, erlotinib, is a tyrosine kinase inhibitor of non-small-cell lung cancer. The quantitative accuracy of our q-IMS method in an animal model study is approximately 17%. Measurements were conducted on mouse liver and brain tissues before and after erlotinib administration. Erlotinib is delivered to the brain, although the concentration is 10⁴ times smaller than that found in the liver.     

反相高效液相色谱-串联质谱法测定尿液中儿茶酚胺和肾上腺素(英文)

The measurement of urine catecholamine and metanephrine concentrations is important for biochemical screening and diagnosis of pheochromocytoma.The goal of this work was to develop a simple liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for determining catecholamines and metanephrines in urine to replace an existing liquid chromatographic method using electrochemical detection.Urine samples were prepared using Oasis weak-cation-exchange cartridges.The eluate was analyzed on an Agilent ZORBAX Eclipse Plus Phenyl-Hexyl column in 3 min.Adrenaline,noradrenaline,dopamine,metanephrine,normetanephrine,and their deuterated internal standards were monitored in positive electrospray ionization mode by multiple reaction monitoring(MRM).No evidence of ion suppression was observed.The assay was linear up to 5μmol/L for adrenaline,5μmol/L for noradrenaline,6.1μmol/L for dopamine,5.6μmol/L for metanephrine,and 34.6μmol/L for normetanephrine,with lower limits of quantification of 5,5,12,6 and 7nmol/L,respectively.The intra-day and inter-day precisions for all analytes ranged from 0.59%to 4.64%and1.98%to 4.80%,respectively.External quality assurance samples were assayed and showed excellent agreement with the target values.This simple method provides an improved assay for determining urine catecholamines and metanephrines.     

Quantitative metabolic profiling of 21 endogenous corticosteroids in urine by liquid chromatography-triple quadrupole-mass spectrometry

Since corticosteroid metabolism may be affected by disease states, the accurate and precise measurement of endogenous corticosteroids in urine is necessary to understand their biochemical roles. An efficient quantitative profiling of 21 endogenous corticosteroids in urine has been validated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After enzymatic hydrolysis with β-glucuronidase, samples were purified using a solid-phase extraction cartridge and then separated through a sub-2 μm particle C18 column (2.1 mm × 50 mm, 1.9 μm) and quantified within 12.1 min using a triple quadrupole MS with electrospray ionization in positive ion mode. All corticosteroids resulted in the base-line separation, which is even achieved for stereo-isomers, such as α-/β-cortol, α-/β-cortolone, and allo-tetrahydrocortisol/tetrahydrocortisol. Overall recoveries ranged from 85% to 106% with limit of quantification ranged from 0.5 to 2.0 ng mL⁻¹ for the corticosteroids examined. The precision (% CV) and accuracy (% bias) of the assay were 1.7-7.8% and 95.1-105.4%, respectively, in 0.5-200 ng mL⁻¹ calibration ranges (r² > 0.9903), for quality-control samples containing 21 endogenous corticosteroids at three different urinary concentrations. Clinical application included quantitative analysis from patients with both prostate cancer and benign prostatic hyperplasia with altered cortisol concentrations. The described LC-MS/MS method eliminates interference from other urine components, has excellent chromatographic resolution achieved by a small particle LC column with a sufficient sensitivity to allow the profiling of both gluco- and mineralo-corticosteroids at a time.     

Trace iodine quantitation in biological samples by mass spectrometric methods: The optimum internal standard

Accurate quantitation of iodine in biological samples is essential for studies of nutrition and medicine, as well as for epidemiological studies for monitoring intake of this essential nutrient. Despite the importance of accurate measurement, a standardized method for iodine analysis of biological samples is yet to be established. We have evaluated the effectiveness of ⁷²Ge, ¹¹⁵In, and ¹²⁹I as internal standards for measurement of iodine in milk and urine samples by induction coupled plasma mass spectrometry (ICP-MS) and of ³⁵Cl¹⁸O₄⁻, ¹²⁹I⁻, and 2-chlorobenzenesulfonate (2-CBS) as internal standards for ion chromatography-tandem mass spectrometry (IC-MS/MS). We found recovery of iodine to be markedly low when IC-MS/MS was used without an internal standard. Percent recovery was similarly low using ³⁵Cl¹⁸O₄ as an internal standard for milk and unpredictable when used for urine. 2-Chlorobenzebenzenesulfonate provided accurate recovery of iodine from milk, but overestimated iodine in urine samples by as much as a factor of 2. Percent recovery of iodine from milk and urine using ICP-MS without an internal standard was ∼120%. Use of ¹¹⁵In predicted approximately 60% of known values for both milk and urine samples. ⁷²Ge provided reasonable and consistent percent recovery for iodine in milk samples (∼108%) but resulted in ∼80% recovery of iodine from urine. Use of ¹²⁹I as an internal standard resulted in excellent recovery of iodine from both milk and urine samples using either IC-MS/MS and ICP-MS.     

高效液相色谱-离子阱/飞行时间质谱鉴定违禁药品邻氯苯基环戊酮中杂质及其标准物质的制备

建立了高效液相色谱-离子阱/飞行时间质谱（HPLC-IT/TOF MS）分析违禁药品邻氯苯基环戊酮样品中杂质成分的方法。对邻氯苯基环戊酮标准品进行多级质谱分析,根据各碎片离子的精确质量数推测邻氯苯基环戊酮的裂解路径,并利用该方法检测出邻氯苯基环戊酮样品中的2种杂质成分：2-氯苯甲酸酸酐和1,2-二邻氯苯甲酰基环戊烯,推断出该违禁药品的合成方法,为追溯其来源提供了重要依据。同时建立了制备邻氯苯基环戊酮标准品的方法,制备高效液相色谱条件是流动相甲醇-水（85∶15,v/v）,流速8 mL/min,进样量1 mL。制备得到的邻氯苯基环戊酮标准物质纯度为99.53%。该方法简单、高效,可拓展应用于其他违禁药物标准物质的制备。     

Modifier-assisted differential mobility–tandem mass spectrometry method for detection and quantification of amphetamine-type stimulants in urine

An advantage of differential mobility spectrometry (DMS) is it provides an orthogonal mechanism to mass spectrometry (MS). The DMS-MS/MS detects analytes in the gas phase on the basis of differences in ion mobility in low and high electric fields, which makes DMS-MS/MS an alternative to chromatographic separation-MS. One drawback of DMS is its limited resolution and sensitivity, especially for detecting small molecules when using a nonpolar inert gas as the carrier gas. The present work has evaluated the effects on peak capacity of adding chemical modifiers to inert carrier gases (nitrogen, helium, argon and carbon dioxide). Use of a methanol-helium mixture gave improvements in both separation and sensitivity. Nine structurally similar amphetamine-type stimulants were determined in urine without pretreatment of the samples before analysis. After optimization of carrier gas, nature and concentration of chemical modifier, and DMS temperature, limits of detection ranging from 1.1 to 2.7 ng mL⁻¹, with a linear range of three orders of magnitude (5–5000 ng mL⁻¹) were achieved. Precision was <15% and the accuracy of the quality control samples was 87.6–113.7%. For the quantitation of urine samples from drug abusers, data obtained using DMS-MS/MS showed reasonable agreement (within ±19.5%) with that obtained using LC-MS/MS. The analysis time for DMS-MS/MS was only 1.1 min and a paired sample t-test between the two methods gave a p-value of 0.0894, which indicates that DMS-MS/MS is a reliable method, with comparable precision and sensitivity to LC-MS/MS.     

Confirmatory analysis of ethylglucuronide in urine by liquid-chromatography/electrospray ionization/tandem mass spectrometry according to forensic guidelines

beta-D-ethylglucuronide (EtG) is a stable Phase II metabolite of ethanol which can be detected in urine samples several days after elimination of ethanol. It is a useful diagnostic parameter for monitoring abstinence of alcoholics in alcohol withdrawal treatment. For this purpose, determination in urine is mainly performed by LC-MS, LC-MS/MS, or by GC-MS. For the mass spectrometric identification and detection of controlled substances in more sensitive fields such as forensic toxicology, workplace drug testing, doping analysis, and veterinary organic residue control, official guidelines have been released requiring a chromatographic separation and a minimum of two mass spectrometric transitions of the analyte. However, for detection of EtG none of the published LC-MS/MS methods could fulfill the minimum requirements of any of these guidelines. Therefore, an existing LC-MS/MS method has been modified by monitoring further MS/MS transitions instead of only one (deprotonated molecule [M - H](-)/product ions: m/z 75, 85, 113, and 159 optional) with the aim of withstanding administrative or court scrutiny in forensic or workplace drug testing cases. Full method validation has been performed in accordance to guidelines of the German Society of Toxicology and Forensic Chemistry (GTFCh) and requirements of ISO 17025. One application field in the United States is a workplace monitoring program to detect surreptitious alcohol use among recovering health professionals, who by contract had agreed on total abstinence after drug and alcohol withdrawal therapy.     

A new liquid chromatography-tandem mass spectrometry method for determination of parabens in human placental tissue samples

Endocrine disruptors are a group of organic compounds widely used, which are ubiquitous in the environment and in biological samples. The main effect of these compounds is associated with their ability to mimic or block the action of natural hormones in living organisms, including humans. Parabens (esters of p-hydroxybenzoic acid) belong to this group of compounds. In this work, we propose a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to asses the presence of parabens most commonly used in industrial applications (methyl-, ethyl-, propyl- and butyl-paraben) in samples of human placental tissue. The method involves the extraction of the analytes from the samples using ethyl acetate, followed by a clean-up step using centrifugation prior to their quantification by LC-MS/MS using an atmospheric pressure chemical ionization (APCI) interface in the negative mode. Deuterated bisphenol A (BPA-d₁₆) was used as surrogate. Found detection limits (LOD) ranged from 0.03 to 0.06 ng g⁻¹ and quantification limits (LOQ) from 0.1 to 0.2 ng g⁻¹, while inter- and intra-day variability was under 13.8%. The method was validated using standard addition calibration and a spike recovery assay. Recovery rates for spiked samples ranged from 82% to 108%. This method was satisfactorily applied for the determination of parabens in 50 placental tissue samples collected from women who live in the province of Granada (Spain).     

慢性心功能衰竭患者尿液的代谢组学研究

采用基于液相色谱-质谱联用的方法对慢性心力衰竭(Chronic heart failure, CHF)患者和正常对照(Control)人群的尿液进行分析, 筛选慢性心力衰竭患者尿液中的差异代谢物, 研究其发病机制, 并为临床治疗提供科学依据.选择15个慢性心力衰竭患者(年龄(62.27±3.14)岁)及15个正常人(年龄(65.41±4.63)岁), 采用高分辨度快速液相色谱-四极杆-飞行时间串联质谱(RRLC-QTOF/MS)技术对尿液代谢物进行分析, 采用主成分分析(PCA)对两组代谢物进行分类, 并筛选潜在生物标记物;运用偏最小二乘判别分析法(PLS-DA)建模, 考察生物标记物对疾病筛选的预测能力.研究结果表明, CHF组和Control组尿液代谢物谱能得到很好的区分, 发现并鉴定了2种潜在生物标记物尿苷及丙氨酰色氨酸, 提示嘧啶代谢和色氨酸代谢可能在心力衰竭发生发展中有重要作用.     

Identification and structural characterization of in vivo metabolites of ketorolac using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS)

In vivo metabolites of ketorolac (KTC) have been identified and characterized by using liquid chromatography positive ion electrospray ionization high resolution tandem mass spectrometry (LC/ESI‐HR‐MS/MS) in combination with online hydrogen/deuterium exchange (HDX) experiments. To identify in vivo metabolites, blood urine and feces samples were collected after oral administration of KTC to Sprague–Dawley rats. The samples were prepared using an optimized sample preparation approach involving protein precipitation and freeze liquid separation followed by solid‐phase extraction and then subjected to LC/HR‐MS/MS analysis. A total of 12 metabolites have been identified in urine samples including hydroxy and glucuronide metabolites, which are also observed in plasma samples. In feces, only O‐sulfate metabolite and unchanged KTC are observed. The structures of metabolites were elucidated using LC‐MS/MS and MSⁿ experiments combined with accurate mass measurements. Online HDX experiments have been used to support the structural characterization of drug metabolites. The main phase I metabolites of KTC are hydroxylated and decarbonylated metabolites, which undergo subsequent phase II glucuronidation pathways. Copyright © 2012 John Wiley & Sons, Ltd.     

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。     

A hybrid liquid chromatography–mass spectrometry strategy in a forensic laboratory for opioid,cocaine and amphetamine classes in human urine using a hybrid linear ion trap-triple quadrupole mass spectrometer

A rapid method has been developed to analyse morphine, codeine, morphine-3-glucuronide, 6-monoacetylmorphine, cocaine, benzoylegonine, buprenorphine, dihydrocodeine, cocaethylene, 3,4-methylenedioxyamphetamine, ketamine, 3,4-methylenedioxymethamphetamine, pseudoephedrine, lignocaine, benzylpiperazine, methamphetamine, amphetamine, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine and methadone in human urine. Urine samples were diluted with methanol:water (1:1, v/v) and sample aliquots were analysed by hybrid linear ion trap-triple quadrupole mass spectrometry with a runtime of 12.5 min. Multiple reaction monitoring (MRM) as survey scan and an enhanced product ion (EPI) scan as dependent scan were performed in an information-dependent acquisition (IDA) experiment. Finally, drug identification and confirmation was carried out by library search with a developed in-house MS/MS library based on EPI spectra at a collision energy spread of 35 ± 15 in positive mode and MRM ratios. The method was validated in urine, according to the criteria defined in Commission Decision 2002/657/EC. At least two MRM transitions for each substance were monitored in addition to EPI spectra and deuterated analytes were used as internal standards for quantitation. The reporting level was 0.05 μg mL⁻¹ for the range of analytes tested. The regression coefficients (r²) for the calibration curves (0–4 μg mL⁻¹) in the study were ≥0.98. The method proved to be simple and time efficient and was implemented as an analytical strategy for the illicit drug monitoring of opioids, cocaines and amphetamines in criminal samples from crime offenders, abusers or victims in the Republic of Ireland. To the best of our knowledge there are no hybrid LC–MS applications using MRM mode and product ion spectra in the linear ion trap mode for opioids, cocaines or amphetamines with validation data in urine.     

Imatinib metabolite profiling in parallel to imatinib quantification in plasma of treated patients using liquid chromatography-mass spectrometry

Besides affecting the systemic bioavailability of the parent drug, drug metabolizing enzymes (DMEs) may produce bioactive and/or toxic metabolites of clinical interest. We have investigated the capability to analyze simultaneously the parent drug and newly identified metabolites in patients' plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The anticancer drug, imatinib, was chosen as a model drug because it has opened a new area in cancer therapy and is given orally and chronically. In addition, resistance and rare but sometimes severe side effects have been reported with this therapy. The quantification of imatinib and the profiling of its metabolites in plasma were established following three steps: (1) set-up of a generic sample extraction and LC-MS/MS conditions, (2) metabolite identification by LC-MS/MS using either in vitro incubations performed with human liver microsomes (HLMs) or patient plasma samples, (3) the simultaneous determination of plasma levels of imatinib and 14 metabolites in the plasma samples of 38 patients. Partial or cross method validation has been done and revealed that precise determinations of metabolite levels can be performed whereas pure standards are not available. Preliminary results indicate that the disposition of imatinib and its metabolites is related to interindividual variables and that outlier metabolite profiles can be revealed. This article underscores that, in addition to usual therapeutic drug monitoring (TDM), LC-MS/MS methods can simultaneously record a complete drug metabolic profile enabling various correlation studies of clinical interest.     

高效液相色谱-串联质谱在兽药残留分析中的应用

高效液相色谱-串联质谱以其特有的快速高效分离和定性、定量准确等优势,在兽药残留检测领域应用广泛。本文系统地介地绍了高效液相色谱-串联质谱(HPLC-MS/MS)在兽药残留分析中的研究进展,并对该领域今后的发展趋势进行了展望。引用文献66篇。     

基于快速高分辨液相色谱串联质谱技术的代谢组学尿液分析方法的建立

采用快速高分辨液相色谱(RRLC)分离系统与QTRAP型及QTOF型MS/MS仪联用技术,通过考察尿液样本前处理方法,优化液相色谱条件和质谱检测参数,建立了用于尿液中代谢物分析的RRLC-MS方法.采用本方法对尿液浓度下的20种代表性代谢物进行了检测,考察了方法的灵敏度和精密度,证明本方法适用于尿液代谢组学的研究.对穿...     

液相色谱串联质谱法快速测定猪尿中10种镇静剂类药物残留量

建立了快速测定猪尿中10种镇静剂类药物(噻拉嗪、阿扎哌隆、氟哌啶、氟哌啶醇、艾司唑仑、硝西泮、地西泮、奥沙西泮、氯丙嗪和奋乃静)残留量的液相色谱串联质谱方法.猪尿样品离心后过C18固相萃取小柱,用甲醇-乙酸乙酯(1∶ 4, V/V)混合溶剂洗脱,氮气吹干后用0.1%甲酸溶液溶解进行仪器分析.采用Eclipse XDB-C18色谱柱分离,以乙腈和0.1%甲酸溶液为流动相进行梯度洗脱,电喷雾正电子(ESI+)模式电离,多反应监测(MRM)模式检测,基质校准进行定量分析.10种镇静剂类药物在5～200 μg/L范围内呈良好的线性,线性相关系数均大于0.99.方法检出限为0.11～0.52 μg/L; 定量限为0.20～0.91 μg/L.添加浓度为1.0, 2.0和10.0 μg/L时,平均回收率在74.6%～115 %之间,批内和批间相对标准偏差均小于15%.     

液相色谱-串联质谱结合谱库检索测定猪组织中的9种β-兴奋剂残留量

建立了猪组织中9种β-兴奋剂(克伦特罗、莱克多巴胺、沙丁胺醇、利托君、特布他林、班布特罗、妥布特罗、西马特罗和异舒普林)多残留的液相色谱-四极杆/线性离子阱串联质谱(QTrap LC-MS/MS)检测方法。样品经β-葡萄糖醛苷酶/芳基硫酸酯酶酶解提取,液液萃取净化,以乙腈-甲酸水溶液为流动相,经C18柱分离后用QTrap LC-MS/MS进行多反应监测(MRM)、信息相关采集(IDA)、增强子离子扫描(EPI)和谱库检索分析。9种β-兴奋剂的线性范围为0.1～50.0 μg/L,线性关系良好(r＞0.99);样品中9种目标物在0.5、1.0和5.0 μg/kg添加水平下的回收率为72.0%～95.1%,相对标准偏差为3.1%～12.1%;方法检出限为0.1～0.2 μg/kg。实际样品检测结果表明,本方法可实现猪组织样本中β-兴奋剂残留量的灵敏、准确的定性和定量分析。     

Direct quantification of creatinine in human urine by using isotope dilution extractive electrospray ionization tandem mass spectrometry

Urinary creatinine (CRE) is an important biomarker of renal function. Fast and accurate quantification of CRE in human urine is required by clinical research. By using isotope dilution extractive electrospray ionization tandem mass spectrometry (EESI–MS/MS) a high throughput method for direct and accurate quantification of urinary CRE was developed in this study. Under optimized conditions, the method detection limit was lower than 50 μg L⁻¹. Over the concentration range investigated (0.05–10 mg L⁻¹), the calibration curve was obtained with satisfactory linearity (R² = 0.9861), and the relative standard deviation (RSD) values for CRE and isotope-labeled CRE (CRE-d3) were 7.1–11.8% (n = 6) and 4.1–11.3% (n = 6), respectively. The isotope dilution EESI–MS/MS method was validated by analyzing six human urine samples, and the results were comparable with the conventional spectrophotometric method (based on the Jaffe reaction). Recoveries for individual urine samples were 85–111% and less than 0.3 min was taken for each measurement, indicating that the present isotope dilution EESI–MS/MS method is a promising strategy for the fast and accurate quantification of urinary CRE in clinical laboratories.     

Dansylation isotope labeling liquid chromatography mass spectrometry for parallel profiling of human urinary and fecal submetabolomes

Human urine and feces can be non-invasively collected for metabolomics-based disease biomarker discovery research. Because urinary and fecal metabolomes are thought to be different, analysis of both biospecimens may generate a more comprehensive metabolomic profile that can be better related to the health state of an individual. Herein we describe a method of using differential chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) for parallel metabolomic profiling of urine and feces. Dansylation labeling was used to quantify the amine/phenol submetabolome changes among different samples based on ¹²C-labeling of individual samples and ¹³C-labeling of a pooled urine or pooled feces and subsequent analysis of the ¹³C-/¹²C-labeled mixture by LC-MS. The pooled urine and pooled feces are further differentially labeled, mixed and then analyzed by LC-MS in order to relate the metabolite concentrations of the common metabolites found in both biospecimens. This method offers a means of direct comparison of urinary and fecal submetabolomes. We evaluated the analytical performance and demonstrated the utility of this method in the analysis of urine and feces collected daily from three healthy individuals for 7 days. On average, 2534 ± 113 (n = 126) peak pairs or metabolites could be detected from a urine sample, while 2507 ± 77 (n = 63) peak pairs were detected from a fecal sample. In total, 5372 unique peak pairs were detected from all the samples combined; 3089 and 3012 pairs were found in urine and feces, respectively. These results reveal that the urine and fecal metabolomes are very different, thereby justifying the consideration of using both biospecimens to increase the probability of finding specific biomarkers of diseases. Furthermore, the CIL LC-MS method described can be used to perform parallel quantitative analysis of urine and feces, resulting in more complete coverage of the human metabolome.     

Simultaneous determination of anti‐diabetes/anti‐obesity drugs by LC/PDA,and targeted analysis of sibutramine analog in dietary supplements by LC/MS/MS

The safety of dietary supplements is questionable as there have been occasional reports of products contaminated with illegal adulterants. The present study was carried out to develop trustworthy methodologies to screen for six anti‐diabetic drugs (phenformin, rosiglitazone, glipizide, glimepiride, glybenclamide and gliclazide) and six anti‐obesity drugs (ephedrine, fenfluramine, T3, T4, fluoxetine and sibutramine) in dietary supplements. A simultaneous determination method of the 12 drugs by liquid chromatography coupled with a photodiode array (LC/PDA) was established and was validated for linearity (r² > 0.99), precision (RSD <13.3%), recoveries (88.8–115.9%) and reproducibility. Sibutramine and its analogs, N‐desmethylsibutramine, were subject to further investigation by LC/MS/MS because they were one of the major illegal adulterants. Our proposed method to monitor illegal drug adulterations in dietary supplements using LC/PDA is a simple and reliable, and therefore applicable to routine drug‐adulteration screening. Copyright © 2009 John Wiley & Sons, Ltd.