QuEChERS as alternative extraction procedure in doping analyses.

QuEChERS is a dispersive solid phase extraction commonly applied in food analysis for residues, such as pesticides or mycotoxins for more than 20 years. Due to the quick and easy sample preparation procedure, a QuEChERS method based on ammonium acetate combined with formic acid in acetonitrile was tested for the preparation of urine samples for doping control purposes. Testing urine samples with different pH and specific gravity, using the combination of 10 M ammonium acetate with 3% formic acid in acetonitrile, 312 out of 342 tested compounds could be extracted at their respective minimum required performance levels according to the World Anti-Doping Agency (WADA) technical documents. For nine selected analytes representing six categories of WADA's Prohibited List, we validated the QuEChERS extraction method fulfilling WADA's requirements for a confirmation procedure of the nonthreshold substances investigated. Especially for the intact stanozolol-glucuronides analyzed by high-resolution mass spectrometry, the described extraction method might be an alternative for confirmation procedures as it is time- and cost-saving compared with the commonly applied solid phase extraction.

Identification of human in vitro metabolites of the haemoglobin S polymerization inhibitor voxelotor for doping control purposes.

Voxelotor (GBT440) is a haemoglobin S polymerization inhibitor used to treat anaemia in sickle cell disease. Due to an increase of arterial oxygen saturation as well as serum erythropoietin and haemoglobin, the World Anti-Doping Agency included voxelotor in the list of prohibited substances and methods in 2023. The objective of the present study was to identify and characterize metabolites of voxelotor to detect a potential misuse by athletes. The biotransformation was studied in vitro using the human hepatocellular cell line HepG2 and pooled human liver microsomes. The metabolites were analysed using high-performance liquid chromatography (high-resolution) mass spectrometry. In total, three phase I metabolites and six phase II metabolites (resulting from glucuro-conjugation and O-methylation) were formed by the HepG2 cells in a time-dependent manner, and two phase I metabolites were generated by the liver microsomes, among them one also found in the HepG2 incubations. A reduced metabolite and the glucuro-conjugate of a reduced metabolite were the most abundant formed by HepG2 cells. In addition, metabolites resulting from mono-hydroxylation, reduction and O-methylation in different combinations were identified. Voxelotor was also found as glucuro-conjugate with a low abundance. With the spectrometric behaviour of voxelotor and its in vitro metabolites described herein, an implementation in doping control screening and, consequently, a detection of an abuse in an athlete urine sample might be possible.

Detection of 18-methyl steroids: Case report on a forensic urine sample and corresponding dietary supplements.

The detection of a putative 18-methyl-19-nortestosterone metabolite in a forensic bodybuilder's urine sample collected as part of a criminal proceeding has triggered a follow-up investigation. Four different dietary supplements in the possession of the suspect were examined with regard to possible precursor steroids. This led to the detection of the declared ingredient methoxydienone, which was confirmed by both, GC-MSMS and LC-HRMSMS. As neither 18-methyl-testosterone, nor 18-methyl-19-nortestosterone were detectable in the supplements, the possibility that the metabolite originates from methoxydienone was investigated. For this purpose, the metabolic fate of methoxydienone was studied in vitro using human HepG2 cells and in vivo by a single oral administration. While the 18-methyl-19-nortestosterone metabolite was not generated by HepG2 cells incubated with methoxydienone, it was observed in the urine samples collected at 2, 6, 10 and 24 h after methoxydienone administration. Moreover, the potential binding of methoxydienone as ligand to the human androgen receptor was modelled in silico in comparison with 18-methylnandrolone, for which androgen receptor activation had been shown in an in vitro approach before. In conclusion, we could ascribe the presence of the 18-methyl-19-nortestosterone metabolite in a forensic urine sample to originate from methoxydienone present in dietary supplements. Methoxydienone was observed to slowly degrade by demethylation of the methoxy substituent in liquid solutions. While no compound-specific intermediates were identified that allowed differentiation from other 18-methyl steroids, the 18-methyl-19-nortestosterone metabolite proved to be a suitable marker for reliable detection in doping analysis.

Agreement of steroid profiles in Athlete Biological Passport residues and corresponding serum samples.

The steroid module of the Athlete Biological Passport (ABP) is based on the analysis of six endogenous steroids in urine samples and a Bayesian statistical approach. However, the urinary steroid concentrations may be affected by confounders like microbial degradation, possible co-administration of diuretics as masking agents, insufficient conjugate hydrolysis or UGT2B17 gene polymorphisms affecting glucuronidation. Therefore, it can be helpful to use other matrices (ABP blood and serum samples) to quantify steroids and thereby support noticeable deviations in the Athlete Biological Passport, for example, abnormally increased urinary testosterone/epitestosterone (T/E) ratios. Aim of the study was to investigate the feasibility to re-use plasma obtained from athlete ABP blood samples for measuring a steroid profile. Therefore, testosterone, androstenedione, cortisol and cortisone were quantified in 36 intra-individual matching ABP blood and serum samples. The steroid levels measured in both matrices showed a high agreement indicating a good stability uninfluenced by storage temperature and duration. Our results pointed out the possibility to expand the athlete ABP blood analysis for steroid profiling.

Emergence of the less common cannabinoid Δ8 -Tetrahydrocannabinol in a doping sample.

Δ8 -Tetrahydrocannabinol (Δ8 -THC) as isomer of the well-known Δ9 -THC has a similar mode of action, and the potency was estimated to be two thirds compared with Δ9 -THC. Content of Δ8 -THC in plant material is low, but formulations containing Δ8 -THC in high concentrations are gaining popularity. Δ8 -THC is to be regarded as prohibited substance according to the Prohibited List of the World Anti-Doping Agency (WADA). Contradictory results between initial testing procedure and confirmatory quantitation for 11-Nor-9-carboxy-Δ9 -tetrahydrocannabinol (Δ9 -THC-COOH) of a doping control sample gave rise for follow-up testing procedures. After alkaline hydrolysis and liquid-liquid extraction, the sample was analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) using isocratic elution instead of gradient elution, which is used for standard procedure. Isocratic elution resulted in two peaks instead of one using gradient elution. Both peaks showed same fragmentation. Using certified reference materials, one peak could be assigned to Δ9 -THC-COOH and the other one with higher intensity to the less common 11-Nor-9-carboxy-Δ8 -Tetrahydrocannabinol (Δ8 -THC-COOH) in a concentration of approximately 1200 ng/ml. As complementary method, gas chromatography tandem mass spectrometry (GC-MS/MS) can also be used for identification. Here Δ8 - and Δ9 -THC-COOH can be distinguished by chromatography and by fragmentation. Additional investigations of doping control samples containing Δ9 -THC-COOH revealed the simultaneous presence of Δ8 -THC-COOH in low concentrations (0.22-8.91 ng/ml) presumably due to plant origin. Percentage of Δ8 -THC-COOH varies from 0.05 to 2.83%. In vitro experiments using human liver microsomes showed that Δ8 -THC is metabolized in the same way as Δ9 -THC.

Pharmacokinetics of salmeterol and its main metabolite α-hydroxysalmeterol after acute and chronic dry powder inhalation in exercising endurance-trained men: Implications for doping control.

As of 2020, use of beta2 -agonist salmeterol is restricted by the World Anti-Doping Agency (WADA) and is only permitted by inhalation at therapeutic doses not exceeding 200 µg in 24 h. In contrast to beta2 -agonists salbutamol and formoterol, WADA has not established a urine threshold for salmeterol despite its muscle hypertrophic actions observed in animals. Herein, we investigated plasma (0-4 h) and urine (0-24 h) concentrations (by ultra-high-performance liquid chromatography-tandem mass spectrometry [UHPLC-MS/MS]) of salmeterol and α-hydroxysalmeterol after dry powder inhalation at supratherapeutic (400 µg) and high therapeutic (200 µg) doses, and after seven consecutive days of therapeutic inhalation (200 µg × day-1 ) in 11 healthy endurance-trained men. During each trial, participants inhaled salmeterol before 1½ h moderate-intensity cycling. Mean ± SD maximum urine concentrations of salmeterol unadjusted for specific gravity reached 4.0 ± 1.6, 2.1 ± 1.5, and 2.2 ± 1.1 ng × ml-1 for 400 µg, 200 µg, and seven consecutive days of 200 µg, respectively, with corresponding maximum urine concentrations of α-hydroxysalmeterol being 11.6 ± 6.1, 5.7 ± 4.6, and 6.5 ± 2.6 ng × ml-1 . Within the relevant window for doping control (first 6 h post-inhalation), the present data (119 samples), along with 64 biobank urine samples, showed that a combined salmeterol and α-hydroxysalmeterol urine threshold with equal cut-offs of 3.3 ng × ml-1 was superior to a salmeterol-only threshold to discriminate therapeutic (200 µg) from supratherapeutic use (400 µg) with a sensitivity of 24% with 0% false positives when applying the WADA technical document (TD2019DL.v2) method of specific gravity adjustment. Thus, a combination of urine salmeterol and α-hydroxysalmeterol concentrations may be suitable for discriminating between therapeutic and supratherapeutic prohibited inhalation of salmeterol.

Evaluation of uncertainty sources in the determination of testosterone in urine by calibration-based and isotope dilution quantification using ultra high performance liquid chromatography tandem mass spectrometry.

Three quantification methodologies, namely calibration with internal standard (Cal-IS, non-weighted), weighted calibration with internal standard (wCal-IS) and isotope pattern deconvolution (IPD) have been used for the determination of testosterone in urine by LC-MS/MS. Uncertainty has been calculated and compared for the three methodologies through intra- and inter-laboratory reproducibility assays. IPD showed the best performance for the intra-laboratory reproducibility, with RSD and combined uncertainty values below 4% and 9% respectively. wCal-IS showed similar performance, while Cal-IS where not constant and clearly worse at the lowest concentration assayed (2ng/mL) reaching RSD values up to 16%. The inter-laboratory assay indicated similar results although wCal-IS RSD (20%) was higher than IPD (10%) and Cal-IS get worse with RSD higher than 40% for the lowest concentration level. Uncertainty budgets calculated for the three procedures revealed that intercept and slope were the most important factors contributing to uncertainty for Cal-IS. The main factors for wCal-IS and IPD were the volumes of sample and/or standard measured.

Terbutaline Accumulates in Blood and Urine after Daily Therapeutic Inhalation.

PURPOSE: This study investigated pharmacokinetics of terbutaline after single and seven consecutive days of inhalation in exercising trained men. METHODS: Twelve healthy trained men underwent two pharmacokinetic trials comparing single dose (2 mg) and seven consecutive days (2 mg·d) of inhaled terbutaline. After inhalation of terbutaline at each trial, subjects performed 90 min of bike ergometer exercise at 55%-65% of maximal oxygen consumption after which they stayed inactive. Blood and urine samples were collected before and after inhalation of terbutaline. Samples were analyzed by high-performance liquid chromatography-tandem mass spectrometry. RESULTS: Maximum serum concentration of terbutaline (Cmax) (6.4 ± 1.2 vs 4.9 ± 1.2 ng·mL, P = 0.01) (mean ± 95% confidence interval) and area under serum concentration-time curve from 0 to 4 h after inhalation (AUC0-4) (16 ± 3 vs 13 ± 2 ng·mL·h, P ≤ 0.005) were higher after 7 d of inhalation compared with the first day. Seven days of terbutaline inhalation resulted in accumulation of terbutaline in urine, in which total urine excretion of terbutaline was higher after 7 d of inhalation compared with the first day (274 ± 43 vs 194 ± 33 µg, P ≤ 0.001). These differences were partly attributed to systemic accumulation of terbutaline after consecutive days of inhalation, in that baseline serum and urine samples revealed incomplete elimination of terbutaline. CONCLUSION: Terbutaline accumulates in serum and urine after consecutive days of inhalation. For doping control purposes, these observations are of relevance if a urine threshold and decision limit is to be introduced for terbutaline on the World Anti-Doping Agency's list of prohibited substances because asthmatic athletes may use their bronchorelievers for consecutive days.

Pharmacokinetics of Oral and Inhaled Terbutaline after Exercise in Trained Men.

AIM: The aim of the study was to investigate pharmacokinetics of terbutaline after oral and inhaled administration in healthy trained male subjects in relation to doping control. METHODS: Twelve healthy well-trained young men (27 ±2 years; mean ± SE) underwent two pharmacokinetic trials that compared 10 mg oral terbutaline with 4 mg inhaled dry powder terbutaline. During each trial, subjects performed 90 min of bike ergometer exercise at 65% of maximal oxygen consumption. Blood (0-4 h) and urine (0-24 h) samples were collected before and after administration of terbutaline. Samples were analyzed for concentrations of terbutaline by high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). RESULTS: Pharmacokinetics differed between the two routes of administration. Serum Cmax and area under the serum concentration-time curve (AUC) were lower after oral administration compared to inhalation (Cmax: 4.2 ± 0.3 vs. 8.5 ± 0.7 ng/ml, P ≤ 0.001; AUC: 422 ± 22 vs. 1308 ± 119 ng/ml × min). Urine concentrations (sum of the free drug and the glucuronide) were lower after oral administration compared to inhalation 2 h (1100 ± 204 vs. 61 ± 10 ng/ml, P ≤ 0.05) and 4 h (734 ± 110 vs. 340 ± 48 ng/ml, P ≤ 0.001) following administration, whereas concentrations were higher for oral administration than inhalation 12 h following administration (190 ± 41 vs. 399 ± 108 ng/ml, P ≤ 0.05). Urine excretion rate was lower after oral administration than inhalation the first 2 h following administration (P ≤ 0.001). Systemic bioavailability ratio between the two routes of administration was 3.8:1 (inhaled: oral; P ≤ 0.001). CONCLUSION: Given the higher systemic bioavailability of inhaled terbutaline compared to oral, our results indicate that it is difficult to differentiate allowed inhaled use of terbutaline from prohibited oral ingestion based on urine concentrations in doping control analysis. However given the potential performance enhancing effect of high dose terbutaline, it is essential to establish a limit on the WADA doping list.

Pharmacokinetics of nebulized and oral procaterol in asthmatic and non-asthmatic subjects in relation to doping analysis.

The purpose of the present study was to investigate pharmacokinetics of procaterol in asthmatics and non-asthmatics after nebulized and oral administration in relation to doping. Ten asthmatic and ten non-asthmatic subjects underwent two pharmacokinetic trials. At first trial, 4 µg procaterol was administered as nebulization. At second trial, 100 µg procaterol was administered orally. Serum and urine samples were collected before and after administration of procaterol. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Serum and urine concentrations of procaterol were markedly higher after oral administration compared to nebulized administration. After oral administration, serum procaterol concentration-time area under the curve (AUC) was higher (P ≤ 0.05) for asthmatics than non-asthmatics. Likewise, urine concentrations were higher (P ≤ 0.01) for asthmatics than non-asthmatics 4 (47 ± 12 vs. 28 ± 9 ng/mL) and 8 h (39 ± 9 vs. 15 ± 5 ng/mL) after oral administration. Detection of serum procaterol was difficult after nebulized administration with 38 samples (27%) below limit of quantification (LOQ) and only trends were observed. No differences were observed between asthmatics and non-asthmatics in the urine concentrations of procaterol after nebulized administration. In summary, our data showed that asthmatics had higher urine concentrations of procaterol than non-asthmatics after oral administration of 100 µg, whereas no difference was observed between the groups after nebulized administration. For doping control purposes, our observations indicate that it is possible to differentiate therapeutic nebulized administration of procaterol from prohibited use of oral procaterol. Copyright © 2016 John Wiley & Sons, Ltd.

The influence of exercise and dehydration on the urine concentrations of salbutamol after inhaled administration of 1600 µg salbutamol as a single dose in relation to doping analysis.

The present study investigated the influence of exercise and dehydration on the urine concentrations of salbutamol after inhalation of that maximal permitted (1600 µg) on the 2015 World Anti-Doping Agency (WADA) prohibited list. Thirteen healthy males participated in the study. Urine concentrations of salbutamol were measured during three conditions: exercise (EX), exercise+dehydration (EXD), and rest (R). Exercise consisted of 75 min cycling at 60% of VO2max and a 20-km time-trial. Fluid intake was 2300, 270, and 1100 mL during EX, EXD, and R, respectively. Urine samples of salbutamol were collected 0-24 h after drug administration. Adjustment of urine concentrations of salbutamol to a specific gravity (USG) of 1.020 g/mL was compared with no adjustment. The 2015 WADA decision limit (1200 ng/mL) for salbutamol was exceeded in 23, 31, and 10% of the urine samples during EX, EXD, and R, respectively, when unadjusted for USG. When adjusted for USG, the corresponding percentages fell to 21, 15, and 8%. During EXD, mean urine concentrations of salbutamol exceeded (1325±599 ng/mL) the decision limit 4 h after administration when unadjusted for USG. Serum salbutamol Cmax was lower (P<0.01) for R(3.0±0.7 ng/mL) than EX(3.8±0.8 ng/mL) and EXD(3.6±0.8 ng/mL). AUC was lower for R (14.1±2.8 ng/mL·âˆ™h) than EX (16.9±2.9 ng/mL·âˆ™h)(P<0.01) and EXD (16.1±3.2 ng/mL·âˆ™h)(P<0.05). In conclusion, exercise and dehydration affect urine concentrations of salbutamol and increase the risk of Adverse Analytical Findings in samples collected after inhalation of that maximal permitted (1600 µg) for salbutamol. This should be taken into account when evaluating doping cases of salbutamol. Copyright © 2015 John Wiley & Sons, Ltd.

Urine concentrations of oral salbutamol in samples collected after intense exercise in endurance athletes.

Our objective was to investigate urine concentrations of 8 mg oral salbutamol in samples collected after intense exercise in endurance athletes. Nine male endurance athletes with a VO2max of 70.2 ± 5.9 mL/min/kg (mean ± SD) took part in the study. Two hours after administration of 8 mg oral salbutamol, subjects performed submaximal exercise for 15 min followed by two, 2-min exercise bouts at an intensity corresponding to 110% of VO2max and a bout to exhaustion at same intensity. Urine samples were collected 4, 8, and 12 h following administration of salbutamol. Samples were analyzed by the Norwegian World Anti-doping Agency (WADA) laboratory. Adjustment of urine concentrations of salbutamol to a urine specific gravity (USG) of 1.020 g/mL was compared with no adjustment according to WADA's technical documents. We observed greater (P = 0.01) urine concentrations of salbutamol 4 h after administration when samples were adjusted to a USG of 1.020 g/mL compared with no adjustment (3089 ± 911 vs. 1918 ± 1081 ng/mL). With the current urine decision limit of 1200 ng/mL for salbutamol on WADA's 2013 list of prohibited substances, fewer false negative urine samples were observed when adjusted to a USG of 1.020 g/mL compared with no adjustment. In conclusion, adjustment of urine samples to a USG of 1.020 g/mL decreases risk of false negative doping tests after administration of oral salbutamol. Adjusting urine samples for USG might be useful when evaluating urine concentrations of salbutamol in doping cases.

Distribution and quantification of flavan-3-ols and procyanidins with low degree of polymerization in nuts, cereals, and legumes.

The monomeric flavan-3-ols catechin and epicatechin as well as procyanidins are of great interest due to their potential beneficial health effects observed in epidemiological studies. However, the occurrence and concentration of these compounds is not well-known due to the fact that reference compounds are not commercially available. In this study we determined the pattern and concentration of catechin, epicatechin, and different dimeric and trimeric procyanidins in 38 food samples (nuts, cereals, legumes) using a reversed phase high-performance liquid chromatography electrospray ionization tandem mass spectrometry (RP-HPLC-ESI-MS/MS) approach based on isolated authentic reference compounds. Of the analyzed food samples 21 were found to contain dimeric and trimeric procyanidins and their monomeric building units catechin and epicatechin. Mainly the monitored nut samples contained the analyzed procyanidins as well as catechin and epicatechin whereas only 3 cereals were identified as sources of the analyzed compounds. The concentration ranged from 148 µg/100 g in macadamia nut to 55 mg/100 g in pinto bean. Catechin and procyanidin B3 were found to be the most abundant analytes. The only A-type procyanidin that could be identified was procyanidin A2, which was found in peanut. The achieved data could be used for authenticity control and furthermore in combination with dietary studies to calculate the daily intake of monomeric flavan-3-ols and procyanidins. To our knowledge this is the first detailed study quantifying monomeric flavan-3-ols and dimeric and trimeric procyanidins in various nuts, cereals, and legumes.

Systematic approach for structure elucidation of polyphenolic compounds using a bottom-up approach combining ion trap experiments and accurate mass measurements.

Polyphenols are a group of plant secondary metabolites with a wide range of structural differences. In many cases, in vitro and in vivo studies of polyphenols revealed beneficial health effects. The mass spectrometric characterization of polyphenols can be the key to understanding the metabolism and resorption of this group of substances. For structure elucidation of polyphenolic compounds nuclear magnectic resonance spectroscopy is the method of choice. Due to the broad structure variability and the sometimes relatively low concentrations of polyphenols and/or their metabolites in foods as well as physiological samples, mass spectrometry could be an alternative for structure elucidation. Especially high-resolution mass spectrometry, for example, Fourier transformation mass spectrometry (FTMS), is a valuable tool. Using a FTMS system, a systematic approach to the fragmentation behavior of phenolic and polyphenolic compounds was chosen to verify the influence of the structure on the fragmentation pattern of the different substances. Depending on the structure, specific fragment ions could be detected. Therefore, it is possible to gain reliable information about the structure of the pseudomolecular ion from its fragmentation spectrum, which is of great aid in the structure elucidation of unknown polyphenols and/or their metabolites.

Urinary excretion and metabolism of procyanidins in pigs.

SCOPE: Aim of this study was to investigate urinary excretion and metabolism of procyanidins a group of secondary plant metabolites with many beneficial health effects described in literature. METHODS AND RESULTS: To investigate the metabolism of procyanidins in the absence of flavan-3-ols, centrifugal partition chromatography was used for their reduction in a grape seed extract to a level of almost zero. After administration of the monomer reduced grape seed extract (mredGSE) containing procyanidins B1, B2, B3, B4, C1 to pigs flavan-3-ols, their methyl derivatives, dimeric and trimeric procyanidins were determined in urine by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Maximal concentrations of procyanidins 6 h after administration vary from 5 to 30 ng/mg creatinine. Total excretion of flavan-3-ols and their methyl derivatives indicates an increasing trend for pigs given mredGSE in comparison to pigs of the control group. Flavan-3-ols were conjugated and methylated to a great extent in comparison to dimeric and trimeric procyanidins. In the case of low molecular weight metabolites, an increasing trend was observed for hippuric acid, not for phenolic acids. CONCLUSIONS: Ratios of total excretion of procyanidins to administrated amounts between 0.004% (C1) and 0.019% (B4) suggest a poor urinary excretion by pigs. A transfer of these results to humans is possible due to their similar gastrointestinal tract.

Intestinal metabolism of two A-type procyanidins using the pig cecum model: detailed structure elucidation of unknown catabolites with Fourier transform mass spectrometry (FTMS).

Procyanidins, as important secondary plant metabolites in fruits, berries, and beverages such as cacao and tea, are supposed to have positive health impacts, although their bioavailability is yet not clear. One important aspect for bioavailability is intestinal metabolism. The investigation of the microbial catabolism of A-type procyanidins is of great importance due to their more complex structure in comparison to B-type procyanidins. A-type procyanidins exhibit an additional ether linkage between the flavan-3-ol monomers. In this study two A-type procyanidins, procyanidin A2 and cinnamtannin B1, were incubated in the pig cecum model to mimic the degradation caused by the microbiota. Both A-type procyanidins were degraded by the microbiota. Procyanidin A2 as a dimer was degraded by about 80% and cinnamtannin B1 as a trimer by about 40% within 8 h of incubation. Hydroxylated phenolic compounds were quantified as degradation products. In addition, two yet unknown catabolites were identified, and the structures were elucidated by Fourier transform mass spectrometry.

Isolation and structure elucidation of two new cytotoxic metabolites from red yeast rice.

In this study, 10 already described secondary metabolites and 2 unknown metabolites were identified in an extract of Monascus purpureus by high-performance liquid chromatography-diode array detection. The unknown metabolites were isolated and their chemical structures were elucidated. The new metabolites possess the molecular formulas C(21)H(27)NO(4) and C(23)H(31)NO(4). They were named monascopyridines E and F due to their pyridine backbone. The cytotoxicity of the new compounds was studied using immortalised human kidney epithelial cells displaying IC(50) values in the micromolar range.

Analysis of Flavan-3-ols and procyanidins in food samples by reversed phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (RP-HPLC-ESI-MS/MS).

Concentrations of the main dimeric and trimeric procyanidins (PC) and their monomeric constitutive units catechin (CT) and epicatechin (EC) were determined in food samples by using reversed phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (RP-HPLC-ESI-MS/MS). In a first step, 12 PCs (PC B1, B2, B3, B4, B5, B6, B7, B8, C1, C2, and A2 and cinnamtannin B1), of which most are not commercially available, were isolated from plant materials or synthesized and purified by a combination of column chromatographic separation techniques with different stationary phases. These PCs in combination with CT and EC were used as standard substances for identification and quantification during the following screening of food samples by RP-HPLC-ESI-MS/MS analysis. The main focus of the newly developed RP-HPLC-ESI-MS/MS method is the compensation of matrix effects by using the echo-peak technique simulating internal standard injection. The suitability of this new method was demonstrated by the determination of recovery rates being 90% or higher. Use of this method allowed the determination of patterns and concentrations of PCs in 55 food samples.

Complex flavonoids in cocoa: synthesis and degradation by intestinal microbiota.

Rarely occurring flavan-3-ol derivatives such as C-glycosides can be generated during food processing, for example, by cocoa production. These astringent taste compounds may also exert interesting behavior toward microbial metabolism, as other C-glycosides have been shown to be quite stable. Oligomeric flavan-3-ols, the procyanidins, bear also a C-C bond between the main moieties and are suspected to resist microbial metabolism for a prolonged time compared to other flavonoids. This paper describes a semisynthetic approach for the generation of flavan-3-ol C-glycosides. Results of incubation experiments studying five flavan-3-ol C-glycosides bearing different sugars, linkage positions, and stereochemistries are presented as well as the behavior of di- and trimeric B-type procyanidins toward intestinal microbiota. Low molecular weight degradation products are considered as well as concentration-time courses of degraded and liberated compounds. All metabolic studies were performed with the well-proven pig cecum model.