Dispersive liquid-liquid microextraction of 11-nor-Δ9-tetrahydrocannabinol-carboxylic acid applied to urine testing.

Aim: THC-COOH is the major metabolite of Δ9-tetrahydrocannabinol commonly tested in urine to determine cannabis intake. In this study, a method based on dispersive liquid-liquid microextraction was developed for testing THC-COOH in urine. Materials & methods: Hydrolyzed urine specimens were extracted via dispersive liquid-liquid microextraction with acetonitrile (disperser solvent) and chloroform (extraction solvent). Derivatization was performed with N,O-Bis(trimethylsilyl)trifluoroacetamide with 1% trichloro(chloromethyl)silane. Analysis was performed by GC-MS/MS. Results: The method showed acceptable linearity (5-500 ng/ml), imprecision (<10.5%) and bias (<4.9%). Limits of detection and quantitation were 1 and 5 ng/ml, respectively. Twenty-four authentic samples were analyzed, with 22 samples being positive for THC-COOH. Conclusion: The proposed method is more environmentally friendly and provided good sensitivity, selectivity and reproducibility.

Tweetable abstract Green analytical toxicology: Dispersive liquid­liquid microextraction applied to the analysis of THC-COOH in urine by GC­MS/MS.

Metabolic profiling of organic acids in urine samples of Cri Du Chat syndrome individuals by gas chromatography-mass spectrometry.

Cri Du Chat (CDC) syndrome is a rare genetic condition caused by the deletion of genetic material on the small arm (the p arm) of chromosome 5. A high-pitched cry that sounds like that of a cat, dysmorphic characteristics, and cytogenetic methods are often used for diagnosing the syndrome. In this study, we applied GC-MS analysis for determining organic acids in urine from 17 control volunteers without CDC syndrome, and from 16 individuals with the CDC syndrome in order to determine the profile of organic acids and biochemical pathways alterations resulting from this genetic condition. First, performing multivariate data analysis selected the best method for extracting organic acids with greater signal intensities and good reproducibility. After selection, multivariate (PLS-DA) and univariate (Mann-Whitney test) data analysis discriminated the metabolites responsible for separation between groups. Nine organic acid metabolites had values of VIP ≥ 1.0 and p-values ≤ 0.05, with highest intensities in the samples from CDC individuals, indicating the strongest discriminative power (tricarballylic acid, indoleacetic acid, anthranilic acid, 4-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, 4-hydroxyhippuric acid, pantothenic acid, homovanillic acid, and vanillylmandelic acid). These metabolites are involved in several biochemical pathways like in the tyrosine and phenylalanine metabolism, as well as the tryptophan metabolism, which could be associated (i) to some neuropsychiatric alterations commonly observed in CDC individuals, (ii) to exogenous compounds related to transformation products by intestinal microbial, and (iii) to a possible deficiency in enzyme activity due to the syndrome.

Characterizing and Mitigating Bladder Radioactivity on 18F-Fluciclovine PET/CT.

18F-fluciclovine PET is approved for prostate cancer recurrence imaging. According to the radiopharmaceutical package insert, only 3% of the tracer is expected to be excreted in the urine over the first 4 h. Yet, in clinical practice we noticed a higher percentage of bladder excretion. We sought to evaluate and quantify early 18F-fluciclovine bladder radioactivity and determine whether refraining from voiding before 18F-fluciclovine injection would mitigate it. Methods: In total, 159 patients underwent 18F-fluciclovine PET/CT imaging as part of their clinical workup. The first 36 patients were instructed to void just before 18F-fluciclovine injection; the subsequent 123 patients were not asked to void. The SUVmax and SUVmean of the bladder, aorta, marrow, liver, and bladder volumes were determined. Comparing SUVmean of bladder to background, we characterized bladder radioactivity as insignificant (bladder < aorta), mild (bladder > aorta < marrow), moderate (bladder > marrow < liver), or intense (bladder > liver). Differences between the protocols were investigated. Results: Overall, 22% (35/159) of patients had moderate bladder activity and 8.8% (14/159) had intense bladder activity. A negative association was found between bladder volume and SUVmean A significant difference was found between the voiding and nonvoiding groups, with 38.9% (14/36) versus 17.1% (21/123) of patients, respectively, having moderate bladder activity and 22.2% (8/36) versus 4.9% (6/123) of patients, respectively, having intense bladder activity. Conclusion: Refraining from voiding before 18F-fluciclovine injection results in significantly lower urinary bladder radioactivity than does purposeful voiding before injection. We have modified our practice accordingly, particularly as moderate and intense bladder activity may mask or mimic local prostate cancer recurrence. Mechanisms underlying this phenomenon should be further investigated.

GC-MS analysis of organic acids in rat urine: A protocol of direct ultrasound-assisted derivatization.

The aim of the present study was to develop a novel ultrasound-assisted derivatization method for analysis of urine that can be used for preliminary screening and monitoring of metabolic disorders. Here we describe an ultrasound-assisted derivatization method followed by GC-MS analysis to quantify 26 organic acids in urine. The optimum levels of the variables affecting the yield of derivatization were investigated, including urease doses, derivatization reagents and derivatization conditions (duration time, reaction temperature and sonic power). The method exhibited the best results with 80 µl urease. The optimal reaction conditions were 100 µl BSTFA, 80% ultrasound power, 70°C and 40 min. This method showed satisfactory linearity, good reproducibility and an acceptable limit of detection and accuracy. Therefore, it could potentially be used to as a standard method to enable comparisons between laboratories. Finally, we applied our method to urine samples from pregnant rats administered 2 or 10 mg/kg folic acid supplementation.

Urinary Organic Acids Increase After Clinical Stabilization of Hospitalized Children With Severe Acute Malnutrition.

BACKGROUND: Despite a reduction of child mortality in low-income countries, acutely ill undernourished children still have an elevated risk of death. Those at highest risk are children with severe acute malnutrition (SAM) who often show metabolic dysregulation that remains poorly understood. OBJECTIVE: We performed a pilot study to examine changes in urinary organic acids during nutritional rehabilitation of children with SAM, and to identify metabolites associated with the presence of edema or with mortality. METHODS: This study included 76 children aged between 6 and 60 months, hospitalized for SAM at the Moyo Nutritional Rehabilitation and Research Unit in Blantyre, Malawi. Urine was collected at admission and 3 days after clinical stabilization and metabolomics were performed using gas chromatography-mass spectrometry. Metabolite concentrations were evaluated with both uni- and multivariate approaches. RESULTS: Most metabolites increased 3 days after clinical stabilization, and total urinary concentration changed from 1.2 mM (interquartile range [IQR], 0.78-1.7) at admission to 3.8 mM (IQR, 2.1-6.6) after stabilization (P < .0001). In particular, 6 metabolites showed increases: 3-hydroxybutyric, 4-hydroxyhippuric, p-hydroxyphenylacetic, oxoglutaric, succinic, and lactic acids. Urinary creatinine was low at both time points, but levels did increase from 0.63 mM (IQR, 0.2-1.2) to 2.6 mM (IQR,1.6-4.4; P < .0001). No differences in urinary profiles were found between children who died versus those who survived, nor between children with severe wasting or edematous SAM. CONCLUSIONS: Total urinary metabolites and creatinine increase after stabilization and may reflect partial recovery of overall metabolism linked to refeeding. The use of urinary metabolites for risk assessment should be furthered explored. TRIAL REGISTRATION: TranSAM study (ISRCTN13916953).

Extractive Ratio Analysis NMR Spectroscopy for Metabolite Identification in Complex Biological Mixtures.

The complexity of biological mixtures continues to challenge efforts aimed at unknown metabolite identification in the metabolomics field. To address this challenge, we provide a new method to identify related peaks from individual metabolites in complex NMR spectra. Extractive ratio analysis NMR spectroscopy (E-RANSY) builds on our previously described ratio analysis method [ Wei et al. Anal. Chem. 2011 , 83 , 7616 - 7623 ] and exploits the simplified NMR spectra provided by the extraction of metabolites under varied pH conditions. Under such conditions, metabolites from the same biological specimen are extracted differentially, and the resulting NMR spectra exhibit characteristics favorable for unraveling unknown metabolite peaks using ratio analysis. We demonstrate the utility of the E-RANSY method by extracting carboxylic acid containing metabolites from human urine, one of the highly complex biological mixtures encountered in the metabolomics field. E-RANSY performs better than STOCSY and the original RANSY method and offers new avenues to identify unknown metabolites in complex biological mixtures.

Development of a new quantification method for organic acids in urine as potential biomarkers for respiratory illness.

Asthma and chronic obstructive pulmonary disease (COPD) are common respiratory disorders that have similar clinical presentation and misdiagnosis may lead to improper treatment. There is a need for a better, non-invasive test for the differentiation of asthma and COPD. In this study, we developed a new validated LC-MS/MS method for 17 urinary organic acids that could serve as potential biomarkers. Human urine samples were collected from adults with asthma or COPD. LC-MS/MS was performed using the differential isotope labeling approach. 4-(Dimethylamino) phenacyl bromide (DmPA) was used for derivatization using two different carbon isotopes, allowing for the formation of internal standard for each metabolite. Gradient elution was employed on a C18 column while the LC-MS/MS operated in the multiple reaction monitoring mode (MRM). Regulatory guidelines were used for method validation. Partial Least Squares Discriminative Analysis (PLS-DA) was applied to the log-transformed values of metabolites in each group of asthma and COPD subjects. Full validation in targeted metabolomics is scarce with usually limited number of metabolites, unlike fit-for-purpose approach. Due to the endogenous nature of the metabolites, numerous challenges were encountered during method development and validation, such as the lactic acid interference from the surrounding environment. The required specificity, accuracy and precision was successfully achieved. The method was fully validated, ensuring robustness and reproducibility when analyzing patient samples. The method was applied to analyze human urine samples and PLS-DA analysis showed differentiation of asthma and COPD subjects (R2 0.89, Q2 0.68). As targeted metabolomics is expanding to the clinical sphere, more validated methods/strategies are needed. Our work will expand the current knowledge-base regarding targeted metabolomics.

Alkylpyrazines from Coffee are Extensively Metabolized to Pyrazine Carboxylic Acids in the Human Body.

SCOPE: Coffee is a complex mixture of over 1000 compounds, including diverse heteroaromatic compounds such as alkylpyrazines. Little is known about the intake, metabolism, and bodily distribution of these compounds. Therefore, a human intervention study is conducted to investigate the excretion of alkylpyrazine metabolites in urine after the ingestion of brewed coffee containing alkylpyrazines. METHODS AND RESULTS: After consuming a diet without heat-processed food, ten volunteers consumed 500 mL of freshly brewed coffee prepared from coffee pads, providing intakes of 2-methylpyrazine (2-MeP), 2,5-dimethylpyrazine (2,5-DMeP), and 2,6-dimethylpyrazine (2,6-DMeP) amounting to 17.2, 4.4, and 4.9 µmol, respectively. These alkylpyrazines are metabolized into the corresponding pyrazine carboxylic acids, namely pyrazine-2-carboxylic acid (PA), 5-hydroxypyrazine-2-carboxylic acid (5-OHPA), 5-methylpyrazine-2-carboxylic acid (5-MePA), and 6-methylpyrazine-2-carboxylic acid (6-MePA). In total, 64% of the ingested 2-MeP is excreted as PA, as well as 26% as 5-OHPA, while 91% and 97% of the ingested 2,5-DMeP and 2,6-DMeP are recovered as 5-MePA and 6-MePA, respectively, in urine samples collected after coffee consumption. CONCLUSION: This study provides evidence that alkylpyrazines are rapidly metabolized into the corresponding carboxylic acids and excreted via urine by humans, which is consistent with earlier rodent studies.

Human urine metabolomic signature after ingestion of polyphenol-rich juice of purple grumixama (Eugenia brasiliensis Lam.).

Polyphenol intake has been associated with health promotion because of its interaction with several metabolic pathways. This study investigates changes in the urine metabolome following acute intake of polyphenol-rich juice, purple grumixama juice. Grumixama (Eugenia brasiliensis Lam.) is a cherry native to Brazil that is known to be a rich source of anthocyanins and ellagitannins. In this research 15 healthy subjects consumed a single dose of grumixama juice. Urine samples were collected before grumixama juice intake, 0-1, 1-2, 2-4 h, with fasting at 24 h after intake. Plasma samples were also collected before intake, 30' and at 1 h, 2 h and 4 h, with fasting at 24 h after juice intake. The urine primary metabolites were analysed by a metabolomic approach using gas chromatography mass spectrometry with methyl chloroformate derivatisation for amino acids and organic acids. Also, an oxygen radical absorbance capacity method was carried out to evaluate the plasma samples antioxidant capacity changes. Subjects showed increase in plasma antioxidant capacity after juice intake (p-values < .05). A total of 114 metabolites were assessed in urine (1-2 h and 2-4 h), including 17 amino acids, 47 organic acids and several other metabolites. Among the 114 metabolites, 25 were significantly changed during the first 4 h following juice intake, as shown by the Orthogonal Partial Least Squares Discriminant Analysis (0.5 > p(corr) > 0.3) and univariate analysis (p-values < .05). Some metabolites were related to mitochondrial metabolism, such as glyoxylic acid and oxalic acid. Metabolites related to amino acid metabolism were also changed, such as beta-alanine, l-phenylalanine and l-tyrosine. In conclusion, results suggest that acute intake of grumixama juice could affect amino acid metabolism and mitochondrial metabolism, but the related health implications should be explored in further studies using additional approaches.

Two-phase micro-electromembrane extraction across free liquid membrane for determination of acidic drugs in complex samples.

A dynamic two-phase micro-electromembrane extraction (µ-EME) using electrically induced transfer of charged analytes directly into free liquid membrane (FLM) is proposed as a novel technique for improving enrichment capabilities of µ-EME. The presented set-up employs aqueous sample as donor solution and water immiscible organic solvent (1-octanol) as FLM, which form the two-phase µ-EME system for efficient extraction of model acidic drugs (ibuprofen, ketoprofen, naproxen and diclofenac) from standard solutions, human urine, human serum and wastewater samples. The FLM eliminates migration of matrix components from the complex samples and simultaneously it acts as an acceptor solution for selective trapping and enrichment of the analyte ions. Electrodes are immersed directly into the sample and the FLM and replenishment of analyte ions at the sample/FLM phase interface is accomplished by stirring the sample solution using a conventional laboratory stirrer. At optimized two-phase µ-EME conditions (100 V, 15 min, 1000 rpm) and optimized volume ratio of sample to FLM (480:16 µL), extraction recoveries of 60-97% and enrichment factors up to 29.1 were achieved. Determination of the acidic drugs in resulting FLMs was achieved by capillary electrophoresis with ultraviolet detection with good linearity (r2 ≥ 0.9998) and low limits of detection (4-20 ng/mL).

Improving liquid chromatography-tandem mass spectrometry determination of polycarboxylic acids in human urine by chemical derivatization. Comparison of o-benzyl hydroxylamine and 2-picolyl amine.

Due to its high sensitivity and specificity, liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) could be considered as the gold-standard in targeted metabolomics. Although LC-MS/MS allows for the direct detection of a large number of molecules, the proper quantification of highly polar compounds such as poly-carboxylic acids in complex matrices like urine is still a challenge. Chemical derivatization offers a suitable way to improve chromatographic behavior and sensitivity for these compounds. Several derivatizing agents have been proposed for the LC-MS/MS determination of carboxylic acids but studies dealing with their comparison in challenging scenarios are scarce. Here we present the evaluation of two different derivatization agents; o-benzylhydroxyl amine (oBHA) and 2-picolyl amine (2-PA); for the quantification of the (poly)-carboxylic acids belonging to the tricarboxylic acid cycle in urine. The suitability of both derivatizating agents was compared by validation of the two approaches. Derivatization with oBHA showed important advantages against 2-PA derivatization such as (i) providing better sensitivity, (ii) more stable derivatives and (iii) allowing for the proper validation of a larger number of analytes. Moreover, while 2-PA derivatization failed in the determination of the target analytes in some stored urine samples, oBHA derivatization successfully allowed for their appropriate determination in the same samples. A comparison between the concentrations obtained using oBHA derivatization and those provided by an external laboratory using UV and GC-MS detection revealed a satisfactory agreement between both results. Additionally, the concentrations obtained by the oBHA method for a set of 38 urines are in agreement with those previously reported in the literature. As a conclusion, our results show that the use of oBHA is preferred against 2-PA for the detection and quantification of (poly)-carboxylic acids in urine.

A novel diagnostic in situ derivatization kit for the simultaneous determination of 14 biomarkers of exposure to benzene, toluene, ethyl benzene and xylenes in human urine by isotope dilution liquid chromatography tandem mass spectrometry and kit optimization using response surface methodology.

Metabolite profiling can be used as a diagnostic measure for both short and long term co-exposure by individuals to benzene, toluene, ethylbenzene and xylenes (BTEX). A novel one pot derivatization in situ kit (OPDISK) was developed and optimized using a multivariate approach based on central composite design. The OPDISK was designed to simultaneously derivatize, in a urine sample matrix, a series of fourteen carboxylic acid and phenol-bearing urinary metabolites of BTEX to enhance their chromatographic analysis and sensitivity for detection by liquid chromatography - electrospray ionization - tandem mass spectrometry (LC-ESI-MS/MS). Using the reagent kit, the less responsive functional units on the molecules were converted to permanently positively-charged functional units. The kit was composed of three components, 2-fluoro-1-methylpyridinium p-toluenesulfonate (FMP), 3-carbinol-1-methylpyridinium iodide (CMP) and triethylamine (TEA) as a basic catalyst and, only after diluting a urine sample 20 fold with acetonitrile, was applied under mild conditions of room temperature and short reaction time of 20 min. The derivatized biomarkers were then directly analyzed using isotope dilution LC-ESI-MS/MS. The method was sensitive (limit of detection on column ranged from 1.4 pg to 3.1 ng), accurate (mean accuracy from 85% to 114%), and precise (mean coefficient of variation from 1% to 14%). The method results indicated a good linearity (R2 ≥ 0.990) for all metabolites. ClinChek® urine control samples were used successfully to demonstrate the accuracy of the method.

GC-MS-based urinary organic acid profiling reveals multiple dysregulated metabolic pathways following experimental acute alcohol consumption.

Metabolomics studies of diseases associated with chronic alcohol consumption provide compelling evidence of several perturbed metabolic pathways. Moreover, the holistic approach of such studies gives insights into the pathophysiological risk factors associated with chronic alcohol-induced disability, morbidity and mortality. Here, we report on a GC-MS-based organic acid profiling study on acute alcohol consumption. Our investigation - involving 12 healthy, moderate-drinking young men - simulated a single binge drinking event, and indicated its metabolic consequences. We generated time-dependent data that predicted the metabolic pathophysiology of the alcohol intervention. Multivariate statistical modelling was applied to the longitudinal data of 120 biologically relevant organic acids, of which 13 provided statistical evidence of the alcohol effect. The known alcohol-induced increased NADH:NAD+ ratio in the cytosol of hepatocytes contributed to the global dysregulation of several metabolic reactions of glycolysis, ketogenesis, the Krebs cycle and gluconeogenesis. The significant presence of 2-hydroxyisobutyric acid supports the emerging paradigm that this compound is an important endogenous metabolite. Its metabolic origin remains elusive, but recent evidence indicated 2-hydroxyisobutyrylation as a novel regulatory modifier of histones. Metabolomics has thus opened an avenue for further research on the reprogramming of metabolic pathways and epigenetic networks in relation to the severe effects of alcohol consumption.

Metabonomics Approach To Comparing the Antistress Effects of Four Panax ginseng Components in Rats.

Different components of Panax ginseng have different properties and medicinal effects. Metabonomics was a prospective approach to analyze the global response of endogenous metabolites to physiological and pathological processes. In this study, an untargeted metabonomics method using GC/TOFMS combined with multivariate statistical techniques was applied to compare entire metabolite differences and the antistress variations among four components of P. ginseng, namely, total ginsenosides (TG), panaxadiol (PD), panaxatriol (PT), and ginseng polysaccharide (PS), in Wistar rats. The results of metabolite analysis showed that numerous urine metabolites involving neurotransmitters, amino acids, organic acids, and gut microbiota metabolites were changed after administration of the four components of P. ginseng, with TG having the least impact on urinary metabolites. The urinary metabolite profiling of these rats exposed to acute combined stress (forced swimming and behavior restriction) demonstrated that the four ginseng components attenuated urine metabolite changes involving gut microbiota metabolites, tricarboxylic acid (TCA) cycle and energy metabolites, and organic acids to different degrees, with TG improving most of the metabolites altered by stress.

Medium-chain acyl-CoA dehydrogenase deficiency: Two novel ACADM mutations identified in a retrospective screening.

Objective The aim of this study was to determine whether an expanded newborn screening programme, which is not yet available in Slovenia, would have detected the first two patients with medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in the country. Two novel ACADM mutations are also described. Methods Both patients were diagnosed clinically; follow-up involved analysis of organic acids in urine, acylcarnitines in dried blood spots, and genetic analysis of ACADM. Cut-off values of acylcarnitines in newborns were established using analysis of 10,000 newborns in a pilot screening study. Results In both patients, analysis of the organic acids in urine showed a possible ß-oxidation defect, while the specific elevation of acylcarnitines confirmed MCAD deficiency. Subsequent genetic analysis confirmed the diagnosis; both patients were compound heterozygotes, each with one novel mutation (c.861 + 2T > C and c.527_533del). The results from a retrospective analysis of newborn screening cards clearly showed major elevations of MCAD-specific acylcarnitines in the patients. Conclusions An expanded newborn screening programme would be beneficial because it would have detected MCAD deficiency in both patients before the development of clinical signs. Our study also provides one of the first descriptions of ACADM mutations in Southeast Europe.

Gas chromatography-mass spectrometry profiles of urinary organic acids in healthy captive cheetahs (Acinonyx jubatus).

In captivity, cheetahs (Acinonyx jubatus) frequently suffer from several unusual chronic diseases that rarely occur in their free-ranging counterparts. In order to develop a better understanding of their metabolism and health we documented the urine organic acids of 41 apparently healthy captive cheetahs, in an untargeted metabolomic study, using gas chromatography-mass spectrometry. A total of 339 organic acids were detected and annotated. Phenolic compounds, thought to be produced by the anaerobic fermentation of aromatic amino acids in the distal colon, as well as their corresponding glycine conjugates, were present in high concentrations. The most abundant organic acids in the cheetahs' urine were an as yet unidentified compound and a novel cadaverine metabolite, tentatively identified as N1,N5-dimethylpentane-1,5-diamine. Pantothenic acid and citramalic acid concentrations correlated negatively with age, while glutaric acid concentrations correlated positively with age, suggesting possible dysregulation of coenzyme A metabolism in older cheetahs. This study provides a baseline of urine organic acid reference values in captive cheetahs and suggests important avenues for future research in this species.

"Classical organic acidurias": diagnosis and pathogenesis.

Organic acidurias are inherited metabolic diseases due to the deficiency of an enzyme or a transport protein involved in one of the several cellular metabolic pathways devoted to the catabolism of amino acids, carbohydrates or lipids. These deficiencies result in abnormal accumulation of organic acids in the body and their abnormal excretion in urine. More than 65 organic acidurias have been described; the incidence varies, individually, from 1 out of 10,000 to >1 out of 1000,000 live births. Collectively, their incidence approximates 1 out of 3000 live births. Among these disorders, methyl malonic aciduria, propionic aciduria, maple syrup urine disease and isovaleric aciduria are sometimes referred to as classical organic acidurias. In this review, we focused on the basic GC-MS-based methodologies employed in the diagnosis of classical organic acidurias and provided updated reference values for the most common involved organic acids. We also attempted to provide the most recent updates on the pathogenetic bases of these diseases.

Robust and High-Throughput Method for Anionic Metabolite Profiling: Preventing Polyimide Aminolysis and Capillary Breakages under Alkaline Conditions in Capillary Electrophoresis-Mass Spectrometry.

Capillary electrophoresis-mass spectrometry (CE-MS) represents a high efficiency microscale separation platform for untargeted profiling of polar/ionic metabolites that is ideal for volume-restricted biological specimens with minimal sample workup. Despite these advantages, the long-term stability of CE-MS remains a major obstacle hampering its widespread application in metabolomics notably for routine analysis of anionic metabolites under negative ion mode conditions. Herein, we report for the first time that commonly used ammonia containing buffers compatible with electrospray ionization (ESI)-MS can compromise the integrity of fused-silica capillaries via aminolysis of their outer polyimide coating. Unlike organic solvent swelling effects, this chemical process occurs under aqueous conditions that is dependent on ammonia concentration, buffer pH, and exposure time resulting in a higher incidence of capillary fractures and current errors during extended operation. Prevention of polyimide aminolysis is achieved by using weakly alkaline ammonia containing buffers (pH < 9) in order to preserve the tensile strength of the polyimide coated fused-silica capillary. Alternatively, less nucleophilic primary/secondary amines can be used as electrolytes without polyimide degradation, whereas chemically resistant polytetrafluoroethylene coating materials offer higher pH tolerance in ammonia. In this work, multisegment injection (MSI)-CE-MS was used as multiplexed separation platform for high throughput profiling of anionic metabolites when using optimized buffer conditions to prevent polyimide degradation. A diverse range of acidic metabolites in human urine were reliably measured by MSI-CE-MS via serial injection of seven urine samples within a single run, including organic acids, food-specific markers, microbial-derived compounds and over-the-counter drugs as their sulfate and glucuronide conjugates. This approach offers excellent throughput (<5 min/sample) and acceptable intermediate precision (average CV ≈ 16%) with high separation efficiency as reflected analysis of 30 anionic metabolites following 238 repeated sample injections of human urine over 3 days while using a single nonisotope internal standard for data normalization. Careful optimization and rigorous validation of CE-MS protocols are crucial for developing a rapid, low cost, and robust screening platform for metabolomics that is amenable to large-scale clinical and epidemiological studies.

Quetiapine Carboxylic Acid and Quetiapine Sulfoxide Prevalence in Patient Urine.

Treatment adherence is often an issue with mental health patients. For those prescribed quetiapine (Seroquel®), the low levels of parent drug and plasma metabolite(s) (e.g., 7-hydroxyquetiapine) typically used in urine drug monitoring can result in false negatives with concomitant unfavorable impacts on patient care. Literature review coupled with liquid chromatography/time-of-flight mass spectrometry analysis of patient positive urine samples indicated the presence of quetiapine carboxylic acid and quetiapine sulfoxide as significant urinary metabolites of quetiapine. Analysis of these two metabolites determined that they are abundant in the urine of quetiapine patients and can result in apparent adherence rates that are improved relative to those determined using only quetiapine and 7-hydroxyquetiapine. For example, analysis of a random set of 114 patients who were prescribed quetiapine exhibited an apparent adherence rate of 47% using the quetiapine carboxylic acid and quetiapine sulfoxide metabolites. Traditional metabolite testing with quetiapine and 7-hydroxyquetiapine yielded apparent adherence rates of ~31% while all four analytes resulted in apparent adherence of 48%. The prevalence of these metabolites suggests that quetiapine urine drug testing would be more consistent with prescriptions when they are included in the analysis.