SWATH data independent acquisition mass spectrometry for screening of xenobiotics in biological fluids: Opportunities and challenges for data processing.

SWATH data independent acquisition (DIA) mass spectrometry (MS) has become an established technique in MS-based 'omics' research and is increasingly used for the screening of xenobiotics (e.g. drugs, drug metabolites, pesticides, toxicants). Such xenobiotic screening methods are mostly applied for tentative compound identification purposes based on spectral library searching, while additional data processing techniques are scarcely used thereby leaving the full potential of these methods often unused. Here we present an analytical workflow for screening xenobiotics in human samples using SWATH/MS based on which we highlight opportunities for unlocking unused potential of these methods. The workflow was applied to urine samples from subjects who tested positive for THC and/or cocaine during roadside drug testing with the goal of confirming the positive roadside drug tests and identifying compounds that relate to illicit drug use (e.g. cutting agents, tobacco components) or associate with corresponding lifestyle choices (e.g. nasal decongestants, painkillers). These goals could only be reached by complementing spectral library search procedures with additional multivariate data analyses due to inherent incompleteness of the spectral library that was employed. Such incompleteness represents a common challenge for applications where limited or no metadata is available for study samples, for example in toxicology, doping control in sports, and workplace or roadside drug testing. It furthermore sets the stage for employing additional data processing techniques as is outlined in the presented work.

Hydrolytic Cleavage Products of Globin Adducts in Urine as Possible Biomarkers of Cumulative Dose: Proof of Concept Using Styrene Oxide as a Model Adduct-Forming Compound.

A new experimental model was designed to study the fate of globin adducts with styrene 7,8-oxide (SO), a metabolic intermediate of styrene and a model electrophilic compound. Rat erythrocytes were incubated with SO at 7 or 22 °C. Levels of specific amino acid adducts in globin were determined by LC/MS analysis of the globin hydrolysate, and erythrocytes with known adduct content were administered intravenously to recipient rats. The course of adduct elimination from the rat blood was measured over the following 50 days. In the erythrocytes incubated at 22 °C, a rapid decline in the adduct levels on the first day post-transfusion followed by a slow phase of elimination was observed. In contrast, the adduct elimination in erythrocytes incubated at 7 °C was nearly linear, copying elimination of intact erythrocytes. In the urine of recipient rats, regioisomeric SO adducts at cysteine, valine, lysine, and histidine in the form of amino acid adducts and/or their acetylated metabolites as well as SO-dipeptide adducts were identified by LC/MS supported by synthesized reference standards. S-(2-Hydroxy-1-phenylethyl)cysteine and S-(2-hydroxy-2-phenylethyl)cysteine, the most abundant globin adducts, were excreted predominantly in the form of the corresponding urinary mercapturic acids (HPEMAs). Massive elimination of HPEMAs via urine occurred within the first day from the erythrocytes incubated at both 7 and 22 °C. However, erythrocytes incubated at 7 °C also showed a slow second phase of elimination such that HPEMAs were detected in urine up to 50 days post-transfusion. These results indicate for the first time that globin adducts can be cleaved in vivo to modified amino acids and dipeptides. The cleavage products and/or their predictable metabolites are excreted in urine over the whole life span of erythrocytes. Some of the urinary adducts may represent a new type of noninvasive biomarker for exposure to adduct-forming chemicals.

Is serum gamma-glutamyltransferase an exposure marker of xenobiotics? Empirical evidence with polycylic aromatic hydrocarbon.

BACKGROUND: We recently hypothesized that serum gamma-glutamyltransferase (GGT), within its reference range, predicts many diseases as a biomarker for background exposure to various xenobiotics. Even though normal serum GGT was associated with xenobiotics having very long half-lives (heavy metals, dioxin, or organochlorine pesticides), it was unknown whether GGT was associated with xenobiotics with short half-lives, including polycyclic aromatic hydrocarbons (PAHs), well known carcinogens. METHODS: Among 1256 adult participants in the National Health and Nutrition Examination Survey (NHANES) 2003-2004, urinary metabolites of PAH (monohydroxy-PAH), and serum GGT were measured. We selected the 10 monohydroxy-PAHs (OH-PAHs) for which at least 90% of participants had concentrations greater than the limit of detection. RESULTS: Among the 10 OH-PAHs, eight had significant positive correlations with serum GGT. These correlations were similarly observed in men and women, and in individuals under 60 years of age. Unlike serum GGT, alanine aminotransferase, another liver enzyme, was not associated with OH-PAHs. CONCLUSIONS: Taken together with the previous epidemiological evidence, the associations of serum GGT with OH-PAHs reinforce the concept that serum GGT is a marker for various environmental pollutants encountered at background levels in the general population.

Nanostructure initiator mass spectrometry: tissue imaging and direct biofluid analysis.

Nanostructure initiator mass spectrometry (NIMS) is a recently introduced matrix-free desorption/ionization platform that requires minimal sample preparation. Its application to xenobiotics and endogenous metabolites in tissues is demonstrated, where clozapine and N-desmethylclozapine were observed from mouse and rat brain sections. It has also been applied to direct biofluid analysis where ketamine and norketamine were observed from plasma and urine. Detection of xenobiotics from biofluids was made even more effective using a novel NIMS on-surface extraction method taking advantage of the hydrophobic nature of the initiator. Linear response and limit of detection were also evaluated for xenobiotics such as methamphetamine, codeine, alprazolam, and morphine, revealing that NIMS can be used for quantitative analysis. Overall, our results demonstrate the capacity of NIMS to perform sensitive, simple, and rapid analyses from highly complex biological tissues and fluids.

Application of fast gas chromatography/mass spectrometry for the rapid screening of synthetic anabolic steroids and other drugs in anti-doping analysis.

This paper describes a fast gas chromatographic/mass spectrometric (GC/MS) screening method for the detection, in urine, of 36 xenobiotics (30 synthetic anabolic steroids, four narcotics, one diuretic and one stimulant) excreted free or as glucuro-conjugates in urine and detectable as trimethylsilyl (TMS) derivatives. These drugs (and/or their urinary metabolites) can be simultaneously extracted by a single liquid/liquid separation step, at alkaline pH, after enzymatic hydrolysis with beta-glucuronidase and then assayed as TMS derivatives by GC/MS using electron ionisation (EI) and single ion monitoring (SIM) acquisition mode. The total time needed for the GC run is less than 8 min. Good reproducibility of the retention times (CV% <1) and the relative abundances of the diagnostic fragment ions (CV% <10) was observed for all target analytes. The sensitivity of the method is sufficient to match the requirements of the World Anti-Doping Agency (WADA) for the accredited laboratories, with limits of detection (LODs) that are lower than the corresponding WADA minimum required performance limits (MRPLs) for all target compounds.

Effect of creatinine and specific gravity normalization techniques on xenobiotic biomarkers in smokers' spot and 24-h urines.

Renal excretion mechanisms are xenobiotic-specific; therefore, accurate exposure assessment requires an understanding of relationships of xenobiotic biomarker concentration and excretion rate to urine flow, specific gravity and creatinine concentration. Twenty-four-hour urine collection for xenobiotic exposure assessment is considered the "gold standard" procedure. Random spot-urine collection is convenient and minimizes subject compliance concerns but requires that normalization techniques be employed to account for diuresis and diurnal variation in xenobiotic biomarker excretion. This paper examines and makes recommendations concerning normalization techniques and conditions under which spot-urine results most accurately reflect 24-h urine results. Specific gravity, creatinine, and xenobiotic biomarkers were determined in smokers' spot and 24-h urines. Normalization techniques were applied, variance-component analyses were performed to estimate variability, spot urines were pooled mathematically to simulate 24-h urines and analyses of variance were performed to evaluate spot urines' ability to reflect 24-h urine concentrations. For each xenobiotic biomarker concentration, log-linear relationships were observed with urine flow, specific gravity, and creatinine. For most xenobiotic biomarker excretion rates, log-linear relationships were observed with urine flow; creatinine, however, was unaffected by urine flow. The conventional creatinine ratio-normalization technique demonstrated greater variability (within-day, between-day and between-subject) than other normalization techniques. Comparisons of simulated 24-h urines to spot urines suggest that spot-urine collection be performed only between 2 p.m. and 2 a.m. and that the modified specific-gravity-adjusted-creatinine ratio-normalization technique and the creatinine-regression normalization technique yield the best agreement between spot- and simulated 24-h urine results.

Rapid detection and identification of N-acetyl-L-cysteine thioethers using constant neutral loss and theoretical multiple reaction monitoring combined with enhanced product-ion scans on a linear ion trap mass spectrometer.

A sensitive and specific liquid chromatography-mass spectrometry (LC-MS) method based on the combination of constant neutral loss scans (CNL) with product ion scans was developed on a linear ion trap. The method is applicable for the detection and identification of analytes with identical chemical substructures (such as conjugates of xenobiotics formed in biological systems) which give common CNLs. A specific CNL was observed for thioethers of N-acetyl-L-cysteine (mercapturic acids, MA) by LC-MS/MS. MS and HPLC parameters were optimized with 16 MAs available as reference compounds. All of these provided a CNL of 129 Da in the negative-ion mode. To assess sensitivity, a multiple reaction monitoring (MRM) mode with 251 theoretical transitions using the CNL of 129 Da combined with a product ion scan (IDA thMRM) was compared with CNL combined with a product ion scan (IDA CNL). An information-dependent acquisition (IDA) uses a survey scan such as MRM (multiple reaction monitoring) to generate "informations" and starting a second acquisition experiment such as a product ion scan using these "informations." Th-MRM means calculated transitions and not transitions generated from an available standard in the tuning mode. The product ion spectra provide additional information on the chemical structure of the unknown analytes. All MA standards were spiked in low concentrations to rat urines and were detected with both methods with LODs ranging from 60 pmol/mL to 1.63 nmol/mL with IDA thMRM. The expected product ion spectra were observed in urine. Application of this screening method to biological samples indicated the presence of a number of MAs in urine of unexposed rats, and resulted in the identification of 1,4-dihydroxynonene mercapturic acid as one of these MAs by negative and positive product ion spectra. These results show that the developed methods have a high potential to serve as both a prescreen to detect unknown MAs and to identify these analytes in complex matrix.

Stir bar sorptive extraction with in situ derivatization and thermal desorption-gas chromatography--mass spectrometry for measurement of phenolic xenoestrogens in human urine samples.

A high-sensitivity analytical method that uses stir bar sorptive extraction (SBSE) with in situ derivatization and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS) for the simultaneous measurement of trace amounts of phenolic xenoestrogens (PXs), such as 2,4-dichlorophenol (DCP), 4-tert-butylphenol (BP), 4-tert-octylphenol (OP), 4-nonylphenol technical isomers (NP), pentachlorophenol (PCP) and bisphenol A (BPA), in human urine samples was developed. The urine sample (1 ml) was de-conjugated by adding beta-glucuronidase and sulfatase. Then, protein precipitation was performed by the addition of acetonitrile. After centrifugation, the supernatant was diluted with purified water and subjected to SBSE with in situ derivatization and TD-GC-MS. The detection limits of DCP, BP, OP, NP, PCP and BPA in the urine samples were 20, 10, 10, 50, 20 and 20 pg ml-1 (ppt), respectively. The calibration curves for PXs were linear and had correlation coefficients higher than 0.99. The average recoveries of those analytes in the urine samples were higher than 95% (RSD: <10%, n=6) with correction using the added surrogate standards. This simple, accurate, sensitive and selective method can be used in the determination of PXs in human urine samples.

In vivo metabolic fate of the xeno-estrogen 4-n-nonylphenol in Wistar rats.

The distribution and the metabolic fate of 4-n-nonylphenol were investigated in male and female Wistar rats dosed orally with 1 microg/kg ("low-dose") or 10 mg/kg ("high-dose") labeled 4-n-nonylphenol. Following a 4-day metabolic balance study, neither the distribution pattern nor the residual levels of 4-n-nonylphenol were found to be different between groups, and no unexpected tissue-specific accumulation of 4-n-nonylphenol was detected. Most of the radioactivity was eliminated in urine, and consisted of hydrophilic metabolites very likely resulting from extensive beta-oxidation of the nonyl side chain and from the conjugation of the phenol to sulfate or to glucuronic acid. Traces of ring-hydroxylated nonylphenol were also characterized. Fecal excretion was mainly associated with unchanged 4-n-nonylphenol and with side chain hydroxylated 4-n-nonylphenol. Experiments carried out in pregnant rats exposed to a low-dose of 4-n-nonylphenol from day 3 to day 19 of gestation demonstrated similar metabolic pathways for this xeno-estrogen. Very limited amounts, if any, of non metabolized 4-n-nonylphenol did reach fetuses. The oxidative metabolism of 4-n-nonylphenol leads to the formation of both ring-hydroxylated and side chain hydroxylated metabolites. The latter metabolic pathway may be a major metabolic pathway for branched 4-nonyl-phenols and may be a clue to understand their biological activity.

[Selected psychoactive drugs--clinical problems and medical-legal aspects]. / Wybrane srodki odurzajace--problemy kliniczne i medyczno-sadowe.

The multifarious aspects of psychoactive drug use present a significant challenge to the contemporary analyst. During the first stage of the present experiment, the recovery from human serum and urine of some psychoactive drugs with acidic or basic properties was studied. The efficiency of this process was determined using solutions of drug standards added to serum or urine. Classic liquid-liquid extraction, as well as solid phase extraction methods were compared. The efficiency of recovery was checked using high-performance liquid chromatography (HPLC). The results of this study confirm the usefulness of RP-18 sorbent from Merck and the importance in terms of quantitative analysis of the technique selected for isolation of the xenobiotic from the biological material. The second stage of the experiment was aimed at qualitative determination of some narcotics using thin-layer chromatography (TLC). By stepwise comparison and elimination it was possible to develop an optimal system of chromatographic separation using laminar staining. The proposed system and the conditions for separation ofxenobiotics with six selected elution systems and laminar visualization confirm the feasibility of separating 22 psychoactive drugs. The practical use of the system is limited mainly to screening. Conditions for quantitative analysis of diazepam, tramadol, and pethidine in biological material (serum, urine) using high-performance liquid chromatography, as well as morphine in serum using an immunoenzyme assay have been developed. The procedures have been applied to analysis of narcotics and psychoactive drugs administered prior to anesthesia (morphine, diazepam, pethidine) or for suppression of post-operative pain (morphine, tramadol) in 31 patients of an intensive care unit. 10 ml of blood was drawn at fixed times: 30 minutes prior to surgery (S1), at start of surgery (S2), 60 minutes later (S3), 30 minutes after administration of analgesic (S4), and 60 minutes after administration of analgesic (S5). Urine samples were also collected: immediately after surgery (M1) and 90 minutes after administration of analgesic (M2).

Valores de referência do ácido hipúrico urinário na regiäo metropolitana de Belo Horizonte / Urinary hippuric acid reference values in Belo Horizonte metropolitan area, Brazil

Objetivando estabelecer a faixa de valor referência para o ácido hipúrico na regiäo metropolitana de Belo Horizonte, urinas de indivíduos näo expostos ocupacionalmente ao tolueno foram analisadas por cromatografia em fase gasosa, utilizando-se detector de ionizaçäo de chama. Os valores encontrados experimentalmente (n=281) variaram de <0,1 a 2,79 g/L, mas a aplicaçäo de estudo estatístico delimita como faixa de referência,a nível de significância de 95 porcento, os valores de 0,361 a 0,481 g/L. Säo descritas, também, as condiçöes analíticas padronizadas para a determinaçäo cromatográfica do ácido hipúrico urinário

Analytical development for low molecular weight xenobiotic compounds.

Specific and sensitive detection or precise quantification of xenobiotics in biosamples (e.g. blood, urine, saliva, sweat, hair) are great challenges in analytical toxicology. GC-MS is the most sensitive, specific and universal analytical method for low mass xenobiotics. Precise quantification can be performed using the selected ion mode (SIM) and stable isotopes as internal standards. Negative chemical ionization (NCI) can improve severalfold the sensitivity for the determination of compounds with electronegative sites (e.g. halogens). For screening and identification of most of the basic and neutral drugs (e.g. drugs of abuse, psychotropics, hypnotics, analgesics, cardiacs) in urine, a systematic toxicological analysis procedure (STA) was developed using GC-MS after acid hydrolysis, extraction and acetylation. for detection of acidic xenobiotics (e.g. anticoagulants, ACE inhibitors, diuretics, antirheumatics) in urine, a further GC-MS procedure was developed using extractive alkylation. For the detection of non-volatile xenobiotics (e.g. toxic peptides like alpha- and beta-amanitin or phase II metabolites) electrospray LC-MS procedures were developed. The procedures and examples show that in analytical toxicology GC-MS is the method of choice for low mass xenobiotics while LC-MS is that for non-volatiles.

Manifestations of chemically induced liver damage.

Possible liver damage induced by chemicals or drugs must be detected early during drug development or industrial exposure, although damage is still difficult to predict, especially when immunotoxicity is involved. Liver toxicity may result from cytolysis, steatosis, cholestasis, phospholipidosis, or vascular lesions, most the outcome of a disadvantageous balance between chemicals or metabolites vs protective mechanisms, resulting from chemical dosage, genetic factors, or the immunoallergic status of the patient. Drug metabolism, lipid peroxidation, and thiol oxidation are frequently involved in liver toxicities. Classical guidelines in toxicology propose many methods for liver toxicity assessment: histology; chemical changes in hepatic tissue (lipids, glutathione, enzymes); physiological changes in biosynthesis (proteins, glycoproteins); excretion function (fructose); drug metabolism; and concentrations of related enzymes (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and gamma-glutamyltransferase) in blood. In vitro studies in human or animal hepatocytes or tumor-derived cell lines are useful in detecting hepatocellular lesions by cell viability, glutathione concentration, amount of lactate dehydrogenase released, cellular ATP, morphology (blebs), and drug metabolism.

A urine collection system for studying the excretion of xenobiotics from Dungeness crabs.

A technique is described for collecting urine from the Dungeness crab, Cancer magister (Dana), a commercially important seafood and an indicator species of aquatic pollution. The technique has been modified from previously published methods to study the role of urinary excretion in the elimination of lipophilic pollutants by the Dungeness crab. The improved urine collection system uses chemically resistant and nonreactive materials that are better suited to pharmacological and toxicological studies than those used in previous methods. The improved method was tested by injecting a vital dye into catheterized Dungeness crabs and by measuring urine flows for 10 days. This technique can be used to determine the urinary excretion of xenobiotics and facilitate the characterization of chemicals and their metabolites accumulated by crabs.

[Reference values for xenobiotics in biological matrixes: the state of the art]. / Valori di riferimento di xenobiotici in matrici biologiche: stato dell'arte.

The basic concepts of reference values of xenobiotics in biological matrixes have been extensively discussed over the last 3-4 years by transferring the principles from clinical chemistry. In this paper three topics of current interest, i.e. the control of variability factors, metanalysis procedure for production of reference values, the reference values as a part of the system of guide values, are dealt with. The control of variability factors is identified as the most important procedure to guarantee the quality and consequently the usefulness of the reference values. Metanalysis is not considered as a correct method to produce reference values, but rather a method to compare the investigation published and to compel the investigators to standardize their working methods. Lastly reference values should be considered as the first step of an integrated system of values (action levels, limit values) which enable us to correctly interpret the significance of biological monitoring.

Determination of mercapturic acid excretions in exposure control to toxicants.

Toxicants can be converted in vivo by a variety of biotransformation reactions into substances that are more, equally, or less noxious than the parent compound. Although conjugation with glutathione is a process that usually results in less harmful products, these products might subsequently form new metabolites that exert more toxicity than the parent compound. These conjugation reactions are catalyzed by several classes of glutathione-S-transferase isoenzymes and thus result in the urinary or biliary excretion of N-acetyl-L-cysteine-S-conjugates (mercapturic acids). Inasmuch as GSH-S-transferase activity varies among different tissues, urinary excretion of mercapturic acids might reflect tissue-specific toxicity. Urinary mercapturic acids are biomarkers of internal and, in some cases, effective dose. The utility of these markers is, however, limited to times shortly after exposure. Studies on possible human deficiencies in some GSH-S-transferases might help us better understand interindividual variations in susceptibility to different toxicants and thus the differences in the pathway of mercapturic acid excretion pattern.