Curry-Odorants and Their Metabolites Transfer into Human Milk and Urine.

SCOPE: The excretion of dietary odorants into urine and milk is evaluated and the impact of possible influencing factors determined. Furthermore, the metabolic relevance of conjugates for the excretion into milk is investigated. METHODS AND RESULTS: Lactating mothers (n = 20) are given a standardized curry dish and donated one milk and urine sample each before and 1, 2, 3, 4.5, 6, and 8 h after the intervention. The concentrations of nine target odorants in these samples are determined. A significant transition is observed for linalool into milk, as well as for linalool, cuminaldehyde, cinnamaldehyde, and eugenol into urine. Maximum concentrations are reached within 1 h after the intervention in the case of milk and within 2-3 h in the case of urine. In addition, the impact of glucuronidase treatment on odorant concentrations is evaluated in a sample subset of twelve mothers. Linalool, eugenol, and vanillin concentrations increased 3-77-fold in milk samples after treatment with ß-glucuronidase. CONCLUSION: The transfer profiles of odorants into milk and urine differ qualitatively, quantitatively, and in temporal aspects. More substances are transferred into urine and the transfer needs a longer period compared with milk. Phase II metabolites are transferred into urine and milk.

Condensation of delta-1-piperideine-6-carboxylate with ortho-aminobenzaldehyde allows its simple, fast, and inexpensive quantification in the urine of patients with antiquitin deficiency.

Antiquitin (ATQ) deficiency leads to tissue, plasma, and urinary accumulation of alpha-aminoadipic semialdehyde (AASA) and its Schiff base delta-1-piperideine-6-carboxylate (P6C). Although genetic testing of ALDH7A1 is the most definitive diagnostic method, quantifications of pathognomonic metabolites are important for the diagnosis and evaluation of therapeutic and dietary interventions. Current metabolite quantification methods use laborious, technically highly complex, and expensive liquid chromatography-tandem mass spectro-metry, which is available only in selected laboratories worldwide. Incubation of ortho-aminobenzaldehyde (oABA) with P6C leads to the formation of a triple aromatic ring structure with characteristic absorption and fluorescence properties. The mean concentration of P6C in nine urine samples from seven ATQ-deficient patients under standard treatment protocols was statistically highly significantly different (P < .001) compared to the mean of 74 healthy controls aged between 2 months and 57 years. Using this limited data set the specificity and sensitivity is 100% for all tested age groups using a P6C cut-off of 2.11 µmol/mmol creatinine, which represents the 99% prediction interval of the P6C concentrations in 17 control urine samples from children below 6 years of age. Plasma P6C concentrations were only elevated in one ATQ subject, possibly because P6C is trapped by pyridoxal-5-phosphate (PLP) blocking fusing with oABA. Nevertheless, both urine and plasma samples were amenable to the quantification of exogenous P6C with high response rates. The P6C quantification method using fusion of oABA with P6C is fast, simple, and inexpensive and might be readily implemented into routine clinical diagnostic laboratories for the early diagnosis of neonatal pyridoxine-dependent epilepsy.

Offline derivatization LC-MS/MS method for simultaneous estimation of vanillin and vanillic acid in guinea pig plasma.

AIM: Vanillin used as a positive control substrate of aldehyde oxidase activity gets metabolized to vanillic acid. Low MW and low sensitivity in negative ion mode are challenges with these analytes. Our objective was to develop a simple offline derivatization LC-MS/MS method to address these challenges. METHODOLOGY/RESULTS: A simple dansyl chloride derivatization of the phenolic groups on vanillin and vanillic acid was adopted to enable easy ionization in commonly used acidic mobile phases. Calibration curves were linear over the concentrations of 4.88-1250 nM with an LLOQ of 0.64 fmoles on column for both analytes. CONCLUSION: The qualified method was successfully applied to simultaneously measure vanillin and vanillic acid in plasma and urine from a guinea pig pharmacokinetic study.

In-syringe reversed dispersive liquid-liquid microextraction for the evaluation of three important bioactive compounds of basil, tarragon and fennel in human plasma and urine samples.

In the present study, an efficient and environmental friendly method (called in-syringe reversed dispersive liquid-liquid microextraction (IS-R-DLLME)) was developed to extract three important components (i.e. para-anisaldehyde, trans-anethole and its isomer estragole) simultaneously in different plant extracts (basil, fennel and tarragon), human plasma and urine samples prior their determination using high-performance liquid chromatography. The importance of choosing these plant extracts as samples is emanating from the dual roles of their bioactive compounds (trans-anethole and estragole), which can alter positively or negatively different cellular processes, and necessity to a simple and efficient method for extraction and sensitive determination of these compounds in the mentioned samples. Under the optimum conditions (including extraction solvent: 120 µL of n-octanol; dispersive solvent: 600 µL of acetone; collecting solvent: 1000 µL of acetone, sample pH 3; with no salt), limits of detection (LODs), linear dynamic ranges (LDRs) and recoveries (R) were 79-81 ng mL(-1), 0.26-6.9 µg mL(-1) and 94.1-99.9%, respectively. The obtained results showed that the IS-R-DLLME was a simple, fast and sensitive method with low level consumption of extraction solvent which provides high recovery under the optimum conditions. The present method was applied to investigate the absorption amounts of the mentioned analytes through the determination of the analytes before (in the plant extracts) and after (in the human plasma and urine samples) the consumption which can determine the toxicity levels of the analytes (on the basis of their dosages) in the extracts.

Ultrasound-assisted temperature-controlled ionic-liquid dispersive liquid-phase microextraction method for simultaneous determination of anethole, estragole, and para-anisaldehyde in different plant extracts and human urine: a comparative study.

In this study, the performances of four ionic-liquid-based microextraction methods, ionic-liquid-based dispersive liquid-liquid microextraction (IL-DLLME), ionic-liquid-based ultrasound-assisted emulsification microextraction (IL-USA-ME), temperature-controlled ionic-liquid dispersive liquid-phase microextraction (TC-IL-DLME), and ultrasound-assisted temperature-controlled ionic-liquid dispersive liquid-phase microextraction (USA-TC-IL-DLME), were investigated for extraction of three bioactive compounds (anethole, estragole, and anisaldehyde) from different plant extracts and human urine. Anethole and estragole were chosen because they can alter cellular processes positively or negatively, and an efficient method is needed for their extraction and sensitive determination in the samples mentioned. Because there is no previous report on the separation of anethole and estragole (structural isomers), first, simultaneous gradient elution and flow programming were used. The microextraction methods were then applied and compared for analysis of these compounds in plant extracts and human urine by use of high-performance liquid chromatography (HPLC). The effect of conditions on extraction efficiency was studied and under the optimum conditions, the best enrichment factors (58-64), limits of detection (14-18 ng mL(-1)), limits of quantification (47-60 ng mL(-1)), and recovery (94.4-101.7 %) were obtained by use of USA-TC-IL-DLME. The optimized conditions were used to determine anethole, estragole, and para-anisaldehyde in fennel, anise, and tarragon extracts and in human urine.

Quantitative determination of helicid in rat biosamples by liquid chromatography electrospray ionization mass spectrometry.

A simple liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) method with highly improved sensitivities for the determination of helicid in rat bile, urine, feces and most tissues was developed. The tissues and feces were firstly homogenized mechanically using deionized water as the media. Bile, urine, tissues and feces homogenates were extracted by liquid-liquid extraction with n-butyl alcohol for sample preparation. The subsequent analysis procedures were performed on a Shimadzu LCMS2010A system (electrospray ionization single quadrupole mass analyzer). A Luna C(18) column (150 mm x 2.00 mm, 5 microm) was used as the analytical column, while a mixture of acetonitrile and ammonium chloride water solution was used as the mobile phase. The proportions of mobile phase were changed timely according to gradient programs. Chlorinated adducts of molecular ions [M+Cl](-) at m/z 319.00 and 363.05 were used to quantify helicid and bergeninum (internal standard), respectively. The method was validated to be accurate, precise and rugged with good linearity. The proposed method was successfully applied to the preclinical tissue distribution and excretion studies of helicid in rats.

Dynamic liquid phase nanoextraction coupled to GC/MS for rapid analysis of methoxyacetophenone and anisaldehyde isomers in urine.

This study introduces a novel extraction technique in the nanoscale and challenges the limits of solvent extraction in the GC/MS using electronic ionization (EI) method for quantitative determination of six methoxyacetophenone (MAP) and anisaldehye (AAH) isomers in one drop of water and urine. This technique is termed as dynamic liquid phase nanoextraction (DLPNE). The optimum parameters for the DLPNE technique were: selection of solvent, toluene; sampling volume, 0.44 microL; dwell time, 2 s; number of sampling, 15; extraction time, 1.5 min; volume of extraction solvent, 60 nL; and no salt addition. The LODs for this technique were 5-20 ng/mL. The RSDs were in the range of 9.7-12.6% (n = 6). The linear dynamic range of the calibration curve of DLPNE is from 0.02 to 0.5 microg/mL with correlation coefficient (r(2)) >0.9705. The advantages of the DLPNE technique are rapidity, ease of operation, simple device, and extremely little solvent and sample consumption. This technique was also compared with the static liquid phase nanoextraction (SLPNE) while the SLPNE failed to detect any signal for the six isomers. We believe that this technique can be very useful for the detection of volatile organic compounds in environmental science from microscale of water or it can be applied to clinical or pharmaceutical application such as diagnosis of microamount of urine or blood samples by GC/MS.

Single-drop microextraction and gas chromatography/mass spectrometric determination of anisaldehyde isomers in human urine and blood serum.

The application of single-drop microextraction (SDME) followed by gas chromatography/chemical ionization mass spectrometry (GC/CI-MS) was investigated for the determination of anisaldehyde isomers in human urine and blood serum. The effects of extraction solvent, sample agitation rate, salt addition, sampling time and temperature on the extraction efficiency were examined and optimized. Analytical parameters such as linearity, reproducibility, detection limit and relative recovery were evaluated under the optimized experimental conditions. Good reproducibilities of replicate extractions (n = 5) were obtained, with relative standard deviation (RSD) values below 6%. The limits of detection (LOD) using an extraction time of 5 min were found to be in the range 2-5 ng/mL under the selected ion monitoring (SIM) mode of GC/MS. Recoveries of 82-98% were achieved after 5 min extraction.

Urinary benzylmercapturic acid as a marker of occupational exposure to toluene.

OBJECTIVE: To examine if benzylmercapturic acid (or N-acetyl- S-benzyl cysteine) in urine can be used as a marker of occupational exposure to toluene. METHODS: A factory survey was conducted in the latter half of a working week. A group of 46 men, who volunteered for the study, was engaged in ink preparation, surface coating or printing work. Diffusive samplers were used to measure average solvent exposure in an 8-h shift. End-of-shift urine samples were analyzed for benzylmercapturic acid (BMA) by a modification of an HPLC method originally developed for phenylmercapturic acid determination. RESULTS: The workers were exposed primarily to toluene [TOL; 13 ppm as the geometric mean (GM) and 86 ppm at the maximum] together with isopropyl alcohol (<1 and 4 ppm), ethyl acetate (2 and 127 ppm) and methyl ethyl ketone (2 and 142 ppm). BMA in urine correlated closely [correlation coefficient ( r) =0.7] with TOL in air, irrespective of correction for urine density. The lowest TOL concentration at which urinary BMA increased to a measurable level was approximately 10 ppm, and urinary BMA could separate the exposed from the non-exposed when TOL exposure was 15 ppm or higher. CONCLUSIONS: BMA in end-of-shift urine samples is a good marker of occupational TOL exposure. Urinalysis for BMA is sensitive enough to detect TOL exposure at 15 ppm, and therefore BMA appears to be more sensitive than hippuric acid and possibly o-cresol as a urinary marker of TOL exposure.

Simultaneous determination of p-hydroxybenzaldehyde, p-hydroxybenzyl alcohol, 4-(beta-D-glucopyranosyloxy)-benzyl alcohol, and sugars in Gastrodia elata blume measured as their acetylated derivatives by GC-MS.

A method for the simultaneous separation and determination of the active constituents and three sugars in the roots of Gastrodia elata Blume (GE), which is used as a famous Chinese traditional herbal medicine, by gas chromatography-mass spectrometry is established. The samples are acetylated with pyridine-acetic anhydride. The contents of 4-hydroxybenzaldehyde, 4-hydroxybenzyl alcohol (HA), fructose, glucose, 4-(beta-D-glucopyranosyloxy)-benzyl alcohol (GA), and sucrose in GE are 0.004%, 0.03%, 1.36%, 1.12%, 1.97%, and 4.25%, respectively, and the detection limits are 1.5, 3.0, 11.0, 5.0, 33.0, and 35.0 pg, respectively. The contents of HA and GA in the urine and brain of a mouse are also determined. This method is simple, reliable, and quick for the simultaneous determination of the active constituents and sugars in GE.

High-performance liquid chromatographic method for determination of vanillin and vanillic acid in human plasma, red blood cells and urine.

A simple high-performance liquid chromatographic method was developed for the determination of vanillin and its vanillic acid metabolite in human plasma, red blood cells and urine. The mobile phase consisted of aqueous acetic acid (1%, v/v)-acetonitrile (85:15, v/v), pH 2.9 and was used with an octadecylsilane analytical column and ultraviolet absorbance detection. The plasma method demonstrated linearity from 2 to 100 microg/ml and the urine method was linear from 2 to 40 microg/ml. The method had a detection limit of 1 microg/ml for vanillin and vanillic acid using 5 microl of prepared plasma, red blood cells or urine. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of vanillin in patients undergoing treatment for sickle cell anemia.

Pharmacokinetic studies of benzalazine.

The pharmacokinetic properties of benzalazine ((2-hydroxy-5-[(4-carboxyphenyl)azo]benzoic acid, CAS 64896-26-0), a new agent for the treatment of ulcerative colitis and Crohn's disease of the large intestine, were investigated. From jejunal loops of rats in situ no noteworthy absorption of benzalazine was observed. All attempts to demonstrate metabolic conversion of benzalazine in mucosal homogenate of the small intestine of rats were without any success. In faecal suspensions, the half-life of the metabolic conversion of benzalazine was determined as 15 min and the formation of the metabolite 5-aminosalicylic acid (5-ASA) was demonstrated qualitatively. 72 h after single oral administration of 300 mg benzalazine/kg b.w. to rats, an average of 71.83% of the administered dose was recovered in urine and faeces. Only a small amount of unmetabolized benzalazine was excreted with urine and faeces (0.75% and 1.47% of the administered dose, respectively). The benzalazine metabolite 5-ASA and the 5-ASA metabolite acetyl-5-aminosalicylic acid (Ac-5-ASA) were excreted mainly with the faeces (29.22% and 20.66% of the administered dose, respectively) and only in small amounts with the urine (2.54% and 11.06% of the administered dose, respectively).

Specific determination of 3-methoxy-4-hydroxyphenylethylene glycol in urine by liquid chromatography with post-column reaction.

We describe a "high-performance" liquid-chromatographic method for determining 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) in human urine. MHPG is separated on a reversed-phase column with isocratic elution, oxidized with sodium metaperiodate, and its absorbance measured at 365 nm. This method shows higher specificity, less interference for MHPG than methods involving electrochemical or fluorescence detection. Post-column derivatization of MHPG with periodate yields vanillin. The detection limit (twice the signal-to-noise ratio) in urine samples was 0.08 mg/L. Mean analytical recovery was 72%. Within-assay and day-to-day CVs were 2.9% and 6.5%, respectively. Reference intervals for MHPG in 24-h urine from apparently healthy subjects were 0.85-3.24 mg/day for men and 0.63-2.20 mg/day for women. In terms of creatinine excretion, the respective reference intervals were 0.55-1.99 and 0.70-1.96 mg per gram of creatinine.

Biodistribution and excretion of [11C]benzaldehyde by the rat after two-minute inhalation exposures.

1. Based on the decay characteristics of short-lived gamma-emitting radioisotopes a new acute exposure method was developed for studying the kinetics and biodistribution of inhaled toxic agents. Such body-penetrating radiation allows direct, non-invasive determination of the radiation in the animal at any time. 2. Rats exposed to 11C-labelled benzaldehyde for 2 min accumulated an average of 2.5 micrograms of this aldehyde. 3. Inhaled benzaldehyde was rapidly absorbed and at 1.5 min after exposure only 0.8% of the administered dose was resident in the lungs. This aldehyde was quickly distributed with the peak radioactivity in the organs occurring at 1.5 min after exposure. Subsequent loss of radiolabel from tissues was rapid and paralleled the removal from the blood. The adipose tissue cleared most slowly. 4. Benzaldehyde was rapidly excreted via the renal system with the kidneys containing 17% of the total administered activity at 5 min. The excreted radiolabelled compound co-chromatographed with hippuric acid on t.l.c.