Nanoconfinement effect based in-fiber extraction and derivatization method for ultrafast analysis of twenty amines in human urine by GC-MS: Application to cancer diagnosis biomarkers’ screening

Selective liquid-chromatographic determination of native fluorescent biogenic amines in human urine based on fluorous derivatization

A number of amines have been shown to result from metabolism of various pesticides. From an epidemiological standpoint, it may be possible to monitor human exposure to these pesticides through the excretion of their corresponding amines in urine. An investigation has been initiated to develop and apply methods of analysis of amines in human urine. The results of a survey of derivatization techniques involving several substituted anilines are presented. These include conditions for derivatization, utilizing a number of halo- and nitro- substituted reagents; electron capture and gas chromatographic properties of the derivatives; and stability of the derivatives to extraction and column chromatography for purposes of separation and cleanup. The recoveries of anilines from spiked water and urine samples at the 1.0 ppm and 0.1 ppm levels were between 85 and 90%. The advantages and disadvantages of the various derivatives and techniques are discussed and a rationale is presented for the preliminary selection of a particular derivative for application of the analysis of aniline metabolites in urine.

5- and 6-Hydroxydopamine, which we had earlier identified as naturally occurring amines in human urine, were quantified in Parkinson's patients treated with L-DOPA, Parkinson's patients whose treatment did not include L-DOPA and in age matched controls. Analysis was carried out by GC-MS of the ditrifluoromethylbenzoyl-trimethylsilyl (DTFMB-TMS) derivatives of the compounds. The concentrations of 5- and 6-hydroxydopamine in the urine of DOPA treated Parkinson's patients were significantly higher than the concentrations from patients not treated and from normal controls. Urinary dopamine levels were greatly elevated in DOPA treated Parkinson's patients while p-tyramine levels were suppressed. No marked differences were seen between the three groups in terms of the urinary concentrations of any of the other amines measured.

Aromatic amines found in tobacco smoke are carcinogenic to humans. There are no doubts that they cause of cancers in the lungs, bladder, kidneys, pancreas, esophagus, larynx, pharynx, and oral cavities associated with public health. Therefore, monitoring, control, and awareness toward the smoking effects are important subjects to be conveyed. Thus, rapid, sensitive, simple and accurate analytical methods for the identification of aromatic amines and their metabolites are required to provide a clear and complete visualization for the occurrence of aromatic amines by investigating the urine of smokers and nonsmokers. This comprehensive review serves to give an overview of the previous and recent studies about the analytical trends for the determination of urinary aromatic amines. This review covers the sampling methodologies and sample preparation techniques such as solvent extraction, solid phase extraction, magnetic solid phase extraction, and solid phase microextraction couple with different separation methods including gas chromatography and liquid chromatography. To add more value to this review paper, the advantages, disadvantages, challenges, and the future prospects of these methods are discussed as well. This review is hopefully beneficial for researchers to access and monitor the level of potentially carcinogenic aromatic amines in human urine.

Nanoconfined liquid phase nanoextraction combined with in-fiber derivatization for simultaneous quantification of seventy amino-containing metabolites in plasma by LC-MS/MS: Exploration of lung cancer screening model

Aromatic amines (arylamines) such as o-toluidine, 2-aminonaphthalene, and 4-aminobiphenyl occur in the environment and are constituents of tobacco smoke. Human exposure to these aromatic amines has long been associated with an elevated risk of bladder cancer. A validated, specific, and sensitive method for measuring o-toluidine, 2-aminonaphthalene, and 4-aminobiphenyl in cigarette smokers and nonsmokers was developed. The method uses acid hydrolysis of the arylamine conjugates in urine, extraction with n-hexane, derivatization with pentafluoropropionic anhydride, and subsequent analysis with gas chromatography combined with mass spectrometry using negative ion chemical ionization. The limits of detection were 4 ng/L for o-toluidine and 1 ng/L for 2-aminonaphthalene and 4-aminobiphenyl. Smokers (N = 10) excreted significantly higher amounts of o-toluidine (204 versus 104 ng/24 h), 2-aminonaphthalene (20.8 versus 10.7 ng/24 h), and 4-aminobiphenyl (15.3 versus 9.6 ng/24 h) than nonsmokers (N = 10). Urinary arylamine excretion in smokers was associated with the extent of smoking as assessed by daily cigarette consumption, urinary excretion of nicotine equivalents (nicotine plus its five major metabolites), cotinine in saliva, and carbon monoxide in exhaled breath. All nonsmokers investigated had quantifiable amounts of o-toluidine, 2-aminonaphthalene, and 4-aminobiphenyl in their urine, confirming that other environmental sources of exposure to these compounds also occur. In conclusion, the analytical method is suitable for measuring short-term exposure to arylamines in urine of non-occupationally exposed smokers and nonsmokers.

Recent progress on media for biological sample preparation

Determination of amines in biological samples as markers of exposure to the amines or the corresponding isocyanates is an important tool for industrial exposure assessment. In this study, a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method for determination of amines in biological samples as perfluorofatty amides derivatives is presented. The method enables determination of diamines such as methylene diamine (MDA), toluene diamine (TDA), naphthalene diamine (NDA), hexamethylene diamine (HDA), isophorone diamine (IPDA), methylenedi(cyclohexylamine)(HMDA) and 4,4'-methylene-(2-chloroaniline)(MOCA) in human urine and plasma. The work-up procedure included hydrolysis of the biological samples with 3 M H(2)SO(4) at 100 degrees C for 16 h and extraction of the amines into toluene, where derivatisation of the amines with perfluorofatty acid anhydride was performed. Following removal of excess reagent and the acid formed and an exchange of solvent, the derivatives were analysed using gradient elution with an acetonitrile/water mobile phase composition and electrospray ionisation (ESI) with multiple reaction monitoring (MRM) of [M - H](-)-->[M - H - 120](-) or [119](-). Several perfluorofatty acid anhydrides were evaluated as derivatisation reagents, but the LC chromatographic properties of the pentafluoropropionic acid anhydride (PFPA) derivatives were favourable. Quantification of amine-PFPA derivatives was performed using deuterium labelled amine-PFPA derivatives as internals standards with good precision and linearity in the investigated range of 0-20 ng ml(-1) urine. The instrumental detection limits for the amine-PFPA derivatives were 0.2-3 fmol for MRM of [M - H](-)-->[119](-) and 0.3-8 fmol for [M - H](-)-->[M - H - 120](-). In 10 urine and 6 plasma samples from workers exposed to isocyanates, determination of TDA and MDA as PFPA derivatives was performed using LC-MS/MS and a reference GC-MS method. No significant difference between the two methods was observed.

Meeting report

We observed pollen in the late development stages of fresh wild Arabidopsis thaliana by scanning electron microscope, using direct spraying of gold and the conventional biological electron microscope sample preparation method, and made comparative analyses. Arabidopsis pollen prepared according to the conventional biological electron microscope sample preparation method can maintain intact and clear outer wall structure under scanning electron microscopy. However, after direct spraying of gold, only mature pollen could be observed relatively clear outer wall structure, and no clear observation results could be obtained for pollen in other stages. Our results showed that the outer wall morphology of the mature pollen can be observed by direct gold spraying with fresh materials. This method is easy to operate and saves time. For the immature pollen, especially the pollen in the later stage of development, direct gold spraying will affect the pollen morphology, so we recommend using the conventional biological electron microscope sample preparation method to obtain the best shooting effect. The results of this study might provide some useful reference for researchers to observe the pollen outer wall structure in the late development stages of Arabidopsis thaliana by scanning electron microscope.

This paper provides a review of novel strategies for sample preparation in forensic toxicology. The review initially outlines the principle of each technique, followed by sections addressing each class of abused drugs separately. The novel strategies currently reviewed focus on the preparation of various biological samples for the subsequent determination of opiates, benzodiazepines, amphetamines, cocaine, hallucinogens, tricyclic antidepressants, antipsychotics and cannabinoids. According to our experience, these analytes are the most frequently responsible for intoxications in Greece. The applications of techniques such as disposable pipette extraction, microextraction by packed sorbent, matrix solid-phase dispersion, solid-phase microextraction, polymer monolith microextraction, stir bar sorptive extraction and others, which are rapidly gaining acceptance in the field of toxicology, are currently reviewed.

It is difficult to detect biogenic amines (BAs) in biological fluids because of their very low concentrations. In this paper, we reported an on-line sample preconcentration method in CE-amperometric detection (CE-AD) based on a dynamic pH junction, and a concentration enhancement of approximately 100-fold was achieved compared with the classical CE-AD methods in the simultaneous analysis of six BAs in urine (dopamine, epinephrine, norepinephrine, tyramine, tryptamine, and serotonin). The technique is proposed based on the sharp pH change generated at the boundary between an acidic sample and the basic BGE zone. Under optimized conditions, all analytes were successfully focused and well separated within 20 min with high efficiency and sensitivity (LODs at S/N = 3 ranging from 5.34 to 68.3 nM). For the analysis of urine samples by this method, satisfactory recoveries were obtained without a complicated pretreatment step or derivatization process. Therefore, it is self-evident that this approach for the analysis of real biological samples has great potential in the future.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the reference technique for bioanalysis, due to its high sensitivity and selectivity. In bioanalysis, a sample preparation step is usually necessary to eliminate most endogenous compounds and to concentrate the target analytes that often exist at trace levels in biological matrixes. Recent trends in biological sample preparation have focused on miniaturized systems, the development of selective new sorbent materials, and high throughput performance with online coupling to analytical instruments. A miniaturized system requires a smaller amount of sample and organic solvents, and an online system reduces the sample preparation steps, analysis time, and costs and improves accuracy. In this context, online capillary solid-phase microextraction (in-tube SPME) coupled to LC systems is worth mentioning. This technique uses a capillary column as the extraction device and enables continuous extraction, concentration, and desorption online with LC systems. This chapter describes the principle of in-tube SPME-LC, configurations, optimization of parameters, new stationary phases, bioanalysis application, and the present state of this technique and summarizes current trends and future perspectives.

Analytical derivatization is an important for the vast majority of substances an indispensable sample preparation step for their quantitative GC-MS and GC-MS/MS analysis in biological samples. Pentafluorobenzyl bromide (PFB-Br), pentafluorobenzoyl chloride (PFB-COCl), pentafluorobenzyl hydroxylamine (PFB-NHNH2), pentafluorophenyl hydrazine (PFPh-ONH2), pentafluoropropionic anhydride (PFPA), and heptafluorobutyric anhydride (HFBA) are versatile derivatization reagents in analytical chemistry. In the present work, the utility of the above mentioned derivatization reagents for the GC-MS analysis of carboxylic, aldehydic, hydroxylic and amine groups containing analytes including amino acids is reviewed and discussed. Derivatization requires different conditions for solvents, reaction temperature and time, and possibly for catalysts. The perfluorinated derivatives are electrically neutral and best soluble in water-immiscible organic solvents such as toluene. Under negative-ion chemical ionization (NICI) conditions, the perfluorinated derivatives readily and abundantly ionize that allows for sensitive analysis. In addition, the perfluorinated analyte derivatives emerge earlier from GC columns than protiated, thus enabling shorter analysis times. Externally added 2H-, 13C-, 15N and 18O-isotopologs for use as internal standards undergo similar changes during derivatization, extraction by organic solvents, ionization in the ion-source of GC-MS apparatus and have almost identical retention times with the analytes. Due to selective analytical derivatization, almost all classes of endogenous and exogenous low-molecular-mass analytes, including drugs and inorganic anions such as nitrite, nitrate, carbonate, and (pseudo)halogenides, become accessible to quantitative GC-MS and GC-MS/MS analysis. Thanks the high sensitivity of quantitative analytical methods based on GC-MS and GC-MS/MS, very low amounts of perfluorinated derivatization reagents are consumed. In consideration of the enormously high global warming potential (GWP) of F-containing derivatization reagents, this article discussed a potential abandonment of the use of perfluorinated reagents and their replacement by F-free reagents in GC-MS and GC-MS/MS.

Electrokinetic supercharging (EKS) is known as one of the most effective online electrophoretic preconcentration techniques, though pairing with it with mass spectrometry has presented challenges. Here, EKS is successfully paired with ESI-MS/MS to provide a sensitive and robust method for analysis of biogenic amines in biological samples. Injection parameters including electric field strength and the buffer compositions used for the separation and focusing were investigated to achieve suitable resolution, high sensitivity, and compatibility with ESI-MS. Using EKS, the sensitivity of the method was improved 5000-fold compared to a conventional hydrodynamic injection with CZE. The separation allowed for baseline resolution of several neurotransmitters within 16 min with LODs down to 10pM. This method was applied to targeted analysis of seven biogenic amines from rat brain stem and whole Drosophila tissue. This is the first method to use EKS with CE-ESI-MS/MS to analyze biological samples.