Stability of Cocaine,Opiates, and Metabolites in Dried Saliva Spots

Drug abuse still represents a global problem, and it is associated with an increased risk of diseases, injuries, and deaths. Cocaine (COC) and opiates are the most abused drugs and account for a significant number of fatalities. Therefore, it is important to develop methods capable of effectively identifying and quantifying these substances. The present study aims to evaluate the long-term stability of COC, ecgonine methylester (EME), benzoylecgonine (BEG), cocaethylene (COET), norcocaine (NCOC), morphine (MOR), codeine (COD) and 6-monoacetylmorphine (6-MAM) in oral fluid samples. The analytes of interest were isolated from the matrix (50 µL) using the dried saliva spots (DSS) sampling approach and were subsequently analyzed by gas chromatography coupled with tandem mass spectrometry (GC–MS/MS). The parameters that could influence the stability of the target compounds were studied, and these were storage temperature, light, use of preservatives (and respective concentrations), and time. The effects of each parameter were evaluated using the design of experiments (DOE) approach. The stability of the target analytes was improved when the DSS were stored at room temperature, in the presence of light and using 1% sodium fluoride. The best conditions were then adopted for the DSS storage and long-term stability was assessed. COD was only stable for 1 day, EME was stable for 3 days, COC, COET, NCOC and 6-MAM were stable for 7 days, MOR for 14 days and BEG remained stable throughout the study (136 days). This is the first study that evaluates the stability of these compounds in oral fluid samples after application in DSS cards, and optimizes the conditions in order to improve their stability.     

Quantification of Zonisamide in Dried Blood Spots and Dried Plasma Spots by UPLC–MS/MS: Application to Clinical Practice

In this research, a UHPLC–MS/MS method was developed and validated for the determination of zonisamide in dried plasma spots (DPS) and dried blood spots (DBS). Detection of zonisamide and internal standard, 1-(2,3-dichlorphenyl)piperazine, was carried out in ESI+ mode by monitoring two MRM transitions per analyte. Total run time, less than 2.5 min, was achieved using Acquity UPLC BEH Amide (2.1 × 100 mm, 1.7 µm particle size) column with mobile phase comprising acetonitrile–water (85:15%, v/v) with 0.075% formic acid. The flow rate was 0.225 mL/min, the column temperature was 30 °C and the injection volume was 3 µL. Desolvation temperature, desolvation gas flow rate, ion source temperature and cone gas flow rate were set by the IntelliStart software tool in combination with tuning. All of the Guthrie cards were scanned, and DPS/DBS areas were determined by the image processing tool. The influence of hematocrit values (20–60%) on accuracy and precision was evaluated to determine the range within which method for DBSs is free from Hct or dependency is within acceptable limits. The validated method was applied to the determination of zonisamide levels in DPS and DBS samples obtained from patients confirming its suitability for clinical application. Finally, the distribution of zonisamide into the red blood cells was estimated by correlating its DPS and DBS levels.     

LC-MS/MS法快速高效检测尿液中尼古丁及其9种代谢物

以氘代尼古丁、氘代可的宁、3-OH-氘代可的宁、氘代尼古丁葡萄糖醛酸复合物、氘代可的宁葡萄糖醛酸复合物、3-OH-氘代可的宁葡萄糖醛酸复合物作内标, 在LC-MS/MS的大气压化学电离(APCI)离子化模式下, 建立了快速检测吸烟者尿液中尼古丁及其9种代谢物的方法, 并对19个实际样品进行了测试和判别分析(DA). 实验前处理简单, 色谱运行时间仅为3.8 min. 结果表明, 尼古丁及其代谢物的精密度在0.5%～5.5%之间, 回收率在94%～109%之间, 线性相关系数均大于0.995. DA分析充分区别开了不同量的吸烟者, 进一步的相关性分析表明吸烟者24 h尿液中的尼古丁及其9种代谢物含量与卷烟抽吸量的分析因子之间均有较强正相关性.     

Dried blood spot on‐card derivatization: an alternative form of sample handling to overcome the instability of thiorphan in biological matrix

Thiorphan, the active metabolite of racecadotril, can undergo oxidation in biological matrices such as blood and plasma. In bioanalysis, a general approach for the stabilization of such a molecule is to derivatize the thiol group to a more stable thioether, often requiring complex handling procedures at the clinical site. In this research, the concept of dried blood spot (DBS) on‐card derivatization was evaluated to stabilize thiorphan. DBS cards were in‐house pre‐treated with 2‐bromo‐3′‐methoxyacetophenone and left to dry prior to blood spotting. Thiorphan was shown to be effectively derivatized to thiorphan–methoxyacetophenone once applied on the in‐house pre‐treated cards. Thiorphan–methoxyacetophenone was extracted by soaking a 6 mm DBS punch in methanol containing the internal standard (thiorphan–methoxyacetophenone‐D₅). Chromatographic separation was achieved on a Waters XBridge C₁₈ column with a gradient elution of 5 m m NH₄HCO₃ and methanol in 2.5 min and detection by ESI(+)/MS/MS. A linear (weighted 1/x²) relationship was obtained over a concentration range of 5.00–600.00 ng/mL. The assay met regulatory guidelines acceptance criteria for sensitivity, selectivity, precision and accuracy, matrix effect, recovery, dilution integrity and multiple stability evaluations. The DBS on‐card derivatization has shown to be an easy and reliable alternative form of sample collection for the quantification of thiorphan. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantification of Gut Microbiota Dysbiosis-Related Organic Acids in Human Urine Using LC-MS/MS

Urine organic acid contains water-soluble metabolites and/or metabolites—derived from sugars, amino acids, lipids, vitamins, and drugs—which can reveal a human’s physiological condition. These urine organic acids—hippuric acid, benzoic acid, phenylacetic acid, phenylpropionic acid, 4-hydroxybenzoic acid, 4-hydroxyphenyl acetic acid, 3-hydroxyphenylpropionic acid, 3,4-dihydroxyphenyl propionic acid, and 3-indoleacetic acid—were the eligible candidates for the dysbiosis of gut microbiota. The aim of this proposal was to develop and to validate a liquid chromatography–tandem mass spectrometry (LC-MS/MS) bioanalysis method for the nine organic acids in human urine. Stable-labeled isotope standard (creatinine-d₃) and acetonitrile were added to the urine sample. The supernatant was diluted with deionized water and injected into LC-MS/MS. This method was validated with high selectivity for the urine sample, a low limit of quantification (10–40 ng/mL), good linearity (r > 0.995), high accuracy (85.8–109.7%), and high precision (1.4–13.3%). This method simultaneously analyzed creatinine in urine, which calibrates metabolic rate between different individuals. Validation has been completed for this method; as such, it could possibly be applied to the study of gut microbiota clinically.     

Development of LC‐MS/MS method for the determination of dapiprazole on dried blood spots and urine: application to pharmacokinetics

A rapid and highly sensitive liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method for determination of dapiprazole on rat dried blood spots and urine was developed and validated. The chromatographic separation was achieved on a reverse‐phase C₁₈ column (250 × 4.6 mm i.d., 5 µm), using 20 mm ammonium acetate (pH adjusted to 4.0 with acetic acid) and acetonitrile (80:20, v/v) as a mobile phase at 25 °C. LC‐MS detection was performed with selective ion monitoring using target ions at m/z 326 and m/z 306 for dapiprazole and mepiprazole used as internal standard, respectively. The calibration curve showed a good linearity in the concentration range of 1–3000 ng/mL. The effect of hematocrit on extraction of dapiprazole from DBS was evaluated. The mean recoveries of dapiprazole from DBS and urine were 93.88 and 90.29% respectively. The intra‐ and inter‐day precisions were <4.19% in DBS as well as urine. The limits of detection and quantification were 0.30 and 1.10 ng/mL in DBS and 0.45 and 1.50 ng/mL in urine samples, respectively. The method was validated as per US Food and Drug Administration guidelines and successfully applied to a pharmacokinetic study of dapiprazole in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

人体尿液中双酚A与壬基酚的同位素稀释的LC-MS/MS分析

采用固相萃取、高效液相色谱-质谱/质谱仪同位素稀释技术,建立了尿中双酚A(BPA)和壬基酚(NP)的检测方法.液相色谱分离用C18柱,流动相为甲醇和水,负离子电喷雾模式电离,多反应离子监测方式检测,BPA和NP的定量离子对分别为m/z 226.9/132.9、219.3/118.8, 13C和氘代同位素化合物分别做回收率控制和内标定量.BPA和NP的基质加标回收率分别为86% ～106%、90% ～114%,定量下限分别为0.97、2.3 μg/L.实际尿样的测定验证了该方法的可行性.     

环境中的医药品与个人护理品

医药品与个人护理品(PPCPs)作为一类大量使用和具有潜在生理效应的"新型"化学物质,近年来受到科学界和社会的普遍关注.环境中的PPCPs主要来源于污水处理厂不完全处理导致的排放和人类农业或畜牧业活动的直接排放.PPCPs连续性输入使得它们在环境中呈现出一种"持久"存在的状态,进而可能对生态系统和人类健康产生危害.本文综述了PPCPs的环境分析方法研究进展、在水处理过程中的行为变化和目前在各种不同环境中的浓度水平,介绍了当前PPCPs的毒性研究结果及初步的风险评价工作,并对PPCPs类物质未来的研究方向进行了展望.     

New Metabolites of Aconitine in Rabbit Urine

A sensitive analytical method to identify and determine aconitine and its metabolites in rabbit urine was developed by liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS^n). In this method,aconitine and its four metabolites in rabbit urine were isolated and deduced as 16-O-demethylaconine (M1), benzoylaconine (M2),16-O-demethylbenzoylaconine (M3) and aconine(M4).M1 and M3 are new metabolites of aconitine and M2 and M4 are first identified in rabbit urine.     

氯丙嗪在大鼠体内的代谢研究

采用气相色谱-质谱法(GC-MS)检测大鼠尿液中的氯丙嗪及其代谢产物,并对其进行质谱解析,推测氯丙嗪在大鼠体内的代谢途径.通过对Wistar大鼠进行灌胃服用药物后收集24 h内尿样,经固相萃取(SPE)净化提取后,采用GC-MS(EI,PCI)检测.对比空白尿液与阳性尿液萃取液的质谱图,并根据质谱裂解规律鉴定其结构.实...     

大鼠尿液中异丙嗪及其主要代谢物的GC-MS分析

采用气相色谱-质谱(GC-MS)分析了大鼠尿液中的异丙嗪及其代谢产物。尿液经β-葡萄糖醛酸酶酶解,Oasis(HLB 3 mL固相萃取柱提取后,以Restek R tx-5MS(30 m×0.25 mm×0.25μm)石英毛细管色谱柱为分析柱,经GC-MS分离检测。根据质谱图对代谢物的结构进行鉴定。结果表明:在大鼠尿液中检出了吩噻嗪、去甲基异丙嗪、异丙嗪亚砜、2-羟基去甲基异丙嗪、二羟基异丙嗪、顺-N-(1-丙烯基)吩噻嗪、反-N-(1-丙烯基)吩噻嗪、顺-N-(1-丙烯基)吩噻嗪砜、反-N-(1-丙烯基)吩噻嗪砜、顺-N-(1-丙烯基)吩噻嗪亚砜及反-N-(1-丙烯基)吩噻嗪亚砜11种代谢物。对异丙嗪在大鼠体内的氧化、还原等主要代谢途径进行了讨论。     

The Determination of Cannabinoids in Urine Samples Using Microextraction by Packed Sorbent and Gas Chromatography-Mass Spectrometry

Cannabis is the most consumed illicit drug worldwide, and its legal status is a source of concern. This study proposes a rapid procedure for the simultaneous quantification of Δ⁹-tetrahydrocannabinol (THC), 11-hydroxy-Δ⁹-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-Δ⁹-tetrahydrocannabinol (THC-COOH), cannabidiol (CBD), and cannabinol (CBN) in urine samples. Microextraction by packed sorbent (MEPS) was used to pre-concentrate the analytes, which were detected by gas chromatography–mass spectrometry. The procedure was previously optimized, and the final conditions were: conditioning with 50 µL methanol and 50 µL of water, sample load with two draw–eject cycles, and washing with 310 µL of 0.1% formic acid in water with 5% isopropanol; the elution was made with 35 µL of 0.1% ammonium hydroxide in methanol. This fast extraction procedure allowed quantification in the ranges of 1–400 ng/mL for THC and CBD, 5–400 ng/mL for CBN and 11-OH-THC, and 10–400 ng/mL for THC-COOH with coefficients of determination higher than 0.99. The limits of quantification and detection were between 1 and 10 ng/mL using 0.25 mL of sample. The extraction efficiencies varied between 26 and 85%. This analytical method is the first allowing the for determination of cannabinoids in urine samples using MEPS, a fast, simple, and low-cost alternative to conventional techniques.     

Development and validation of a sensitive LC‐MS/MS method for determination of tacrolimus on dried blood spots

A bioanalytical method for the quantification of tacrolimus (TAC) on dried blood spots (DBS) using liquid chromatography, electrospray ionization coupled with tandem mass spectrometry (LC‐ESI‐MS/MS) was developed and validated. It involves solvent extraction of a punch disk of DBS followed by liquid–liquid extraction. The analyte and the internal standard (IS, ascomycin) were separated on a phenyl column using an isocratic mobile phase elution at a flow rate of 0.3 mL/min. The assay was linear from 1 to 80 ng/mL. The mean recovery of TAC was 76.6%. Intra‐assay, inter‐assay imprecision and biases were all less than 15%. TAC on DBS was stable for at least 10 days at room temperature, and at least 24 h at 50°C. A chromatographic effect of the filter paper (Whatman 903) was not detected. The volume of blood (15–50 μL) and hematocrit of blood (ranging from 23.2 to 48.6%) did not show a significant influence on detection of TAC concentration by DBS‐LC‐MS/MS. Fifty samples from patients were detected by both DBS‐LC‐MS/MS and microparticle enzyme‐linked immunoassay (MEIA). TAC concentrations measured by DBS‐LC‐MS/MS method tended to be lower than those by MEIA. Copyright © 2012 John Wiley & Sons, Ltd.     

高效液相色谱-串联质谱法测定猪尿中秋水仙碱

提出了猪尿中秋水仙碱的液相色谱-串联质谱检测方法。采用pH8.0磷酸盐缓冲溶液碱化5mL尿样,用乙酸乙酯提取两次,合并提取液,于40℃吹氮蒸干,用2 mL流动相溶解残渣。在色谱分离中选用Sunfire C_(18)色谱柱,流动相为甲醇-2mmol·L~(-1)乙酸铵溶液(含0.1%甲酸)混合溶液(40+60),流量为0.2mL·min,进样量为10μL。质谱测定中采用电喷雾正离子模式离子化、多反应监测模式测定秋水仙碱,外标法定量。猪尿中秋水仙碱在1～50μg·L~(-1)内呈线性,测定下限(10S/N)为0.2μg·L~(-1),方法的回收率为90%～100%,相对标准偏差(n=6)小于9.0%。     

Development and Validation of the LC–MS/MS Method for Determination of 130 Natural and Synthetic Cannabinoids in Cannabis Oil

Dietary supplements are widely available products used by millions of people around the world. Unfortunately, the procedure of adding pharmaceutical and psychoactive substances has recently been observed, in order to increase the effectiveness of supplements in the form of hemp oils. For this reason, it is extremely important to develop analytical methods for the detection of substances prohibited in dietary supplements and food products. In the present study, using the LC–MS/MS technique, an innovative method for the detection and quantification of 117 synthetic cannabinoids and 13 natural cannabinoids in dietary supplements and food products in the form of oils during one 13-min chromatographic run was developed. Each method was fully validated by characterization of the following parameters: The limit of detection was set to 0.1 ng/mL (100 µg/g, 0.01%). The limit of quantification ranged from 0.05 ng/mL to 50 ng/mL. The criteria assumed for systematic error caused by methodological bias (±20%) resulting from the recovery of analytes after the extraction process, as well as the coefficient of variation (CV) (≤20%), were met for all 130 tested compounds. The positive results of the validation confirmed that the developed methods met the requirements related to the adequacy of their application in a given scope. Additionally, methods developed using the LC–MS/MS technique were verified via proficiency tests. The developed analytical procedure was successfully used in the analysis of hemp oils and capsules containing them in the studied dietary supplements.     

Analysis of Dried Blood Spots for Polychlorinated Biphenyls and Organochlorine Pesticides by Gas Chromatography Coupled with Tandem Mass Spectrometry

Polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCs) are anthropogenic pollutants highly resistant to chemical degradation and readily absorbed by organic tissue. Their persistence in the environment and toxicological threat to mammals prompts swift, reliable methods of analysis. This study outlines a rapid, efficient and sensitive, validated methodology utilizing a simple liquid extraction technique, and subsequent analysis by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) for the determination of PCBs and OCs from dried blood spots. The PCBs and OCs were quantified in whole marine mammal blood spotted on Whatman Protein Saver cards (PSCs) by extracting the analytes into acetonitrile acidified with formic acid, followed by GC-MS/MS analysis. The samples were analyzed in positive electron impact (EI+) ionization mode with the electron energy set to 40?eV to preserve analyte integrity. Fortified blood with the analytes of interest was used for method validation and subsequent sample screening. The recoveries of targeted analytes ranged from 62.5% to 107.8%, with relative standard deviations ranging from 0.09% to 4.6% at a 100?ng·mL^?1 concentration level. The method detection limits were from 40.4?ng·g^?1 to 179.2?ng·g^?1 for the PCBs and 37.6?ng·g^?1 to 145.1?ng·g^?1 for the OCs. The use of dried blood spots provided for numerous advantages compared to whole blood samples while demonstrating reduced matrix effects and enhanced sample lifespan while retaining analyte sensitivity.     

尿样中神经性毒剂代谢产物的HPLC/Q-TOFMS/MS检测方法研究

建立了尿样中甲基膦酸单乙酯(EMPA)、甲基膦酸单异丙酯(IMPA)、甲基膦酸频哪基酯(PMPA)3种神经性毒剂代谢产物的HPLC/Q-TOFMS/MS检测方法。以StrataSi-1型固相萃取小柱对尿样中的3种神经性毒剂代谢产物进行分离,HPLC/Q-TOFESIMS/MS进行测定,内标法定量。该方法对EMPA、IMPA、PMPA的线性范围均为5～320μg/L,相关系数均不低于0.9974;EMPA、IMPA、PMPA的加标回收率分别为57%、98%、81%;检出限(S/N≥3)均为0.1μg/L,定量下限(S/N≥10)均为1μg/L。并将该方法应用于禁化武组织(OPCW)首次生物医学样品分析演练未知尿样的检测,结果满意。     

人尿中诺龙和炔诺酮代谢物的气相色谱-质谱分析

本文对合成的19-去甲雄烷醇酮的四个差向异构体混合物进行气相色谱分离,对诺龙和炔诺酮用药后,留取的人尿样通过化学预处理,MSTFA/TMSI衍生化反应,GC/MS分析,确证了尿样中诺龙的两个主要代谢产物为19-去甲雄酮和19-去甲原胆烷醇酮,炔诺酮的两个主要代谢物是四氢炔诺酮(THINE)的两个形式,可能是3α-OH-5α-THINE和3α-OH-5β-THINE,在炔诺酮代谢前期,还发现有诺龙的上述两个代谢物存在。     

Unravelling the Helianthus tuberosus L. (Jerusalem Artichoke,Kiku-Imo) Tuber Proteome by Label-Free Quantitative Proteomics

The present research investigates the tuber proteome of the ‘medicinal’ plant Jerusalem artichoke (abbreviated as JA) (Helianthus tuberosus L.) using a high-throughput proteomics technique. Although JA has been historically known to the Native Americans, it was introduced to Europe in the late 19th century and later spread to Japan (referred to as ‘kiku-imo’) as a folk remedy for diabetes. Genboku Takahashi research group has been working on the cultivation and utilization of kiku-imo tuber as a traditional/alternative medicine in daily life and researched on the lowering of blood sugar level, HbA1c, etc., in human subjects (unpublished data). Understanding the protein components of the tuber may shed light on its healing properties, especially related to diabetes. Using three commercially processed JA tuber products (dried powder and dried chips) we performed total protein extraction on the powdered samples using a label-free quantitate proteomic approach (mass spectrometry) and catalogued for the first time a comprehensive protein list for the JA tuber. A total of 2967 protein groups were identified, statistically analyzed, and further categorized into different protein classes using bioinformatics techniques. We discussed the association of these proteins to health and disease regulatory metabolism. Data are available via ProteomeXchange with identifier PXD030744.     

Routine Analysis of Catecholamines and Metabolites in Urine by a Liquid Chromatographic Column Switching System

Abstract

A reversed-phase liquid chromatographic technique including a column switching system has been adapted for the routine measurement of catecholamines and their metabolites (14 compounds) in urine. From 1 ml of urine all the compounds and the internal standards were obtained according to combined extraction procedures involving organic solvent, anionic and weakly cationic resins. Finally four extracts (catecholamines, methoxamines, acidic and neutral derivatives) had to be chromatographed throughout a wholly automated apparatus. For each run, the column switching system determined the analytical columns to be used to obtain the separation of the compounds from interferences due to other co-extracted endogenous substances, while the analysis times remained between 20 and 40 min. Such a system allowed the rapid clean-up of columns (in direct- and back-flush mode) carried out between two consecutive injections. By coupling on-line fluorimetric and electro-chemical detections the specificity of the technique could be checked, since the ratio of the responses of both detectors was an index of the purity of the peaks. Finally the advanced automation of the equipment allowed weekly the evaluation of catecholamines and the whole range of their known metabolites in 36 urine samples.