Bioaccessibility of vitamin A, vitamin C and folic acid from dietary supplements, fortified food and infant formula.

In the Netherlands, vitamin intake occurs mainly via food and for some vitamins also via fortified food. In addition, some people take dietary supplements. Information on the bioavailability of vitamins is important for a good estimation of the actual exposure to vitamins. Furthermore, for a reliable intake estimation, it is important to know the accurateness of the claimed vitamin concentration on the product label. In the current study, the amount of vitamin A, vitamin C, and folic acid in different products and their maximum bioavailability (bioaccessibility) were investigated. In about half of the products, the amount of vitamins significantly deviated from the declared amounts. The vitamin bioaccessibility ranged from <1% to 100%. When assessing the dietary intake exposure of vitamins, it is important to take into account both the possible deviation from the declared level and (the variability of) the bioaccessibility of the vitamin in the products.

Carry-over of veterinary drugs from medicated to non-medicated feeds in commercial feed manufacturing plants.

Different compound feeds have to be manufactured in the same production line. As a consequence, traces of the first produced feed may remain in the production and get mixed with the next feed batches. This "carry-over" is unavoidable, and so non-medicated feed can be contaminated with veterinary drugs like antibiotics added to the previous batch of medicated feed. To monitor the carry-over of antibiotics in the Netherlands, 21 feed mills were visited and 140 samples of flushing feeds were collected and analysed for containing residues of antibiotics. Results show that 87% of all samples contain concentrations of antibiotics in the range of 0.1-154 mg/kg. It is expected that these levels - which are in the same range as previously found for the nowadays banned antimicrobial growth promoters (AMGPs) - have an effect on the occurrence of microbial resistance. Analysis of a second set of samples collected at four different feed mills directly after the production of oxytetracycline-medicated feed demonstrated that the first part of a flushing feed has much higher contamination than the last part of the batch. Furthermore, it was demonstrated that the carry-over percentage shows no correlation with the carry-over determined by one of the standard GMP+ procedures. These observations, unavoidable carry-over, inhomogeneous batches of feed with antibiotics and difficulties to predict the carry-over levels, together with the awareness of the increasing problem of microbial resistance, motivated the NEVEDI, association of Dutch Feed Producers, to announce that they will voluntarily stop the production of medicated feed in 2011. The alternatives for medicated feed are for example water or milk medication or the use of top-dressings at the farm. The consequences and possible new risks of carry-over at the farm are not completely clear yet.

Screening methods for the detection of antibiotic residues in slaughter animals: comparison of the European Union Four-Plate Test, the Nouws Antibiotic Test and the Premi®Test (applied to muscle and kidney).

Microbial growth inhibition tests are widely used as the primary screening approach for the detection of antibiotic residues in slaughter animals. In this study we evaluated and compared the performance of the European Union Four-Plate Test (EU4pt), the Nouws Antibiotic Test (NAT), and a commercial ampoule test, the PremiTest (applied to both muscle and kidney), by parallel analysis of 735 slaughter animals. The EU4pt only showed significant inhibition with two muscle samples containing 305 µg kg(-1) doxycycline and 648 µg kg(-1) tulathromycin, while an maximum residue limit (MRL) violation of 1100 µg kg(-1) sulfamethazine remained unnoticed. PremiTest-muscle only detected the sulfamethazine containing sample, all other (1.1%) suspect samples appeared false-positive results. The same test applied to kidney yielded 4.1% suspect samples, while the NAT screening (based on analysis of renal pelvis fluid) showed 4.9% suspect results. The vast majority of these samples contained tetracycline and/or aminoglycoside residues. PremiTest-kidney appeared to be more sensitive to aminoglycosides than the NAT screening, which failed to detect an MRL violation of 870 µg kg(-1) gentamicin in kidney. Detection of less than MRL levels of tetracycline residues by the NAT proved its suitability for this residue group. Whether PremiTest is sufficiently sensitive for accurate tetracycline detection in kidney remains doubtful, although changing over to kidney definitely improved the suitability of PremiTest for the detection of residues in slaughter animals.

Multiresidue analysis of beta-agonists in bovine and porcine urine, feed and hair using liquid chromatography electrospray ionisation tandem mass spectrometry.

The use of beta-agonists as growth promoters in cattle breeding is forbidden in many countries for reasons of fair trade and consumer protection. In recent years the use of liquid chromatography (LC) tandem mass spectrometry (MS/MS) has been shown to be the method of choice for the control of beta-agonists. In this study an LC-MS/MS multiresidue analysis method is presented for trace analysis of 22 beta-agonists. A truly generic concept has been designed based on mixed-mode solid-phase extraction and positive electrospray ionisation LC-MS/MS operated in the multiple reaction monitoring mode. This method allows application to a wide variety of sample matrices such as urine, feed and hair, following minor modifications to the analysis procedure only. The method features fit-for-purpose sensitivity in urine as shown by CCalpha and CCbeta values of less than 0.2 and less than 0.5 microg/l respectively, for all beta-agonists studied (terbutaline and reproterol, less than 0.3 and less than 1.0 respectively). Similar but semiquantitative application to feed and hair showed CCbeta values of less than 10.0 and less than 5.0 microg/kg, respectively. A further simplification and improvement is demonstrated using Ultra Performance LC (UPLC) and fast-switching MS/MS. The successful validation of this method following the latest EU requirements and its application to real samples demonstrate that a new versatile tool has been achieved for veterinary control of beta-agonists.

Can high-performance liquid chromatography coupled with fluorescence detection under all conditions be regarded as a sufficiently conclusive confirmatory method for B-group substances?

Commission Decision 2002/657/EC requires confirmatory analysis of B-group compounds when detected at levels above the permitted limit. In contrast to banned substances, for B-group substances, the use of mass spectrometric techniques is not obligatory and several techniques including liquid chromatography (LC)-ultraviolet light (UV) on two different LC columns and (single-column) high-performance liquid chromatography (HPLC)-fluorescence (Flu) are considered to deliver sufficient evidence for the identification of the detected substance. The analysis of sodium salicylate in animal drinking water collected at poultry farms is presented here as an example to show that even in a simple matrix such as animal drinking water, fluorescence detection in some cases may provide inadequate specificity. Of 50 samples analysed by LC-Flu, 18 tested positive for sodium salicylate. However, only in one sample was the presence of the analyte confirmed with mass spectrometric detection; the others were blank. Consequently, the LC-Flu results obtained were false-non-compliant for sodium salicylate. A second case concerning the analysis of avermectins in milk by HLPC-Flu is briefly described. For a number of samples analysed in the framework of a proficiency test, false non-compliant results for emamectin were reported due to a background interference sometimes present that practically co-eluted with the analyte. The observed retention time difference (1%) was well below the criterion (2.5%) specified in Commission Decision 2002/657/EC. Considering the impact of positive findings on individual farmers as well as on trade, product image and food safety perception by the consumer, it is concluded that also for B-group substances false-non-compliant results should be avoided whenever possible. This is especially important when the results are treated as and are expected to have the same repercussions as in the case of banned A-group substances. In these circumstances, only results obtained by mass spectrometry should be considered for confirmatory purposes.

Determination of nifursol metabolites in poultry muscle and liver tissue. Development and validation of a confirmatory method.

A method is described for the identification and quantitative determination of 3,5-dinitrosalicylic acid hydrazide (DSH), the marker residue of nifursol metabolites in poultry (turkey, broiler) muscle and liver tissue. The method is based on the acid-catalysed hydrolysis of tissue-bound metabolites to free DSH and in situ derivatisation with 2-nitrobenzaldehyde to the corresponding nitrophenyl derivative NPDSH. A structural analogue of DSH, 4-hydroxy-3,5-dinitrobenzoic acid hydrazide (HBH) was synthesised to serve as an internal standard. The analytes were isolated from the matrix by liquid-liquid extraction with ethyl acetate. Determination was performed by LC-MS/MS with negative electrospray ionisation. The [M - H](+) ions of NPDSH and NPHBH at m/z 374 were fragmented by collision induced dissociation (CID) producing transition ions at m/z 182, 183 and 226. The transition ions at m/z 182 and 226 were selected for monitoring of NPDSH while the transition ion at m/z 183 was selected for NPHBH. The method has been validated according to the EU criteria of Commission Decision 2002/657/EC at 0.5, 1.0 and 1.5 microg kg(-1) in muscle and liver tissue. A decision limit (CC(alpha)) was obtained of 0.04 and 0.025 microg kg(-1) in muscle and liver, respectively. Similarly a detection capability (CC(beta)) was obtained of 0.10 and 0.05 microg kg(-1) in muscle and liver, respectively. The introduction of HBH as an internal standard did not lead to a significant improvement of the quantitative performance of the method. In fact for liver better performance characteristics were obtained when the IS was not taken into account. Nevertheless, as a qualitative marker for recovery, HBH could still be very useful in the analysis of unknown samples.

Validation and use of the CALUX-bioassay for the determination of dioxins and PCBs in bovine milk.

There is a strong need for the development of relatively cheap and rapid bioassays for the determination of dioxins and related compounds in food. A newly developed CALUX (Chemical-Activated LUciferase gene eXpression) bioassay was tested for its possible use to determine low levels of dioxins in bovine milk. Data show that this mammalian cell-based test is very sensitive for 2,3,7,8-substituted dioxins and related PCBs, thereby reflecting the relative potencies of these compounds in comparison to TCDD (TEF-values). The limit of detection was about 50 fg of TCDD. Furthermore, the response obtained with a mixture of dioxins was additive, in accordance with the TEF-principle. Milk fat was isolated by centrifugation followed by clean-up of the fat with n-pentane, removal of the fat on a 33% H2SO4 silica column, and determination of Ah receptor agonist activity with the CALUX-bioassay. An equivalent of 67 mg fat was tested per experimental unit, resulting in a limit of quantification around 1 pg i-TEQ/g fat. To investigate the performance of the method, butter fat was cleaned and spiked with a mixture of 17 different 2,3,7,8-substituted PCDD and PCDF congeners at 1, 3, 6, 9, 12 and 15 pg TEQ/g fat, as confirmed by GC/MS. In this concentration range, the method showed a recovery of TEQs around 67% (58-87%). The reproducibility, determined in three independent series showed a CV varying between 4% and 54%, with the exception of the sample spiked at 1 pg i-TEQ (CV 97%). The repeatability determined with the sample spiked at 6 pg i-TEQ/g showed a CV of 10%. Testing of 22 bovine milk samples, taken at different sites in The Netherlands, in the CALUX-assay showed combined dioxin and dioxin-like PCB levels equivalent to 1.6 pg TCDD/g fat (range 0.2-4.6). GC/MS analysis of these samples revealed an average level of 1.7 pg i-TEQ/g fat, varying between 0.5 and 4.7 pg i-TEQ/g fat. All five samples showing a GC/MS determined dioxin content of more than 2 pg i-TEQ/g fat gave a response in the CALUX-assay corresponding to more than 2 pg TCDD/g fat. These data clearly show that the CALUX-bioassay is a promising method for the rapid and low cost screening of dioxins in bovine milk.

Experiences with the new Audiant XA-II implant and the behind-the-ear (BTE) device.

Patients with large conductive hearing loss do not always tolerate conventional air conduction or bone conduction hearing aids. They can be helped sometimes with an implantable bone conductor device. The so-called bone-anchored hearing aid consists of an implantable titanium skull screw and an external sound processor unit. In this study, we report our experiences with 10 subjects who have been implanted with the recently improved Audiant screw-type XA-II and their experience with the newly developed behind-the-ear (BTE) external processor, the at-the-ear processor and the body-level device with a new transducer with variable magnetic strength. Evaluation shows that the Audiant XA-II system is well accepted and does not show any tissue reaction. The BTE processor is an important new complement to the Audiant system. Furthermore, the system is effective in compensating the air-bone gap almost completely at the higher frequencies but only partially at the lower ones. The maximum output appears to limit its feasibility for perceptive losses beyond 20 dB (PTA). When this system is utilized for proper hearing loss, however, the system usually improves speech intelligibility both in quiet and in background noise. When the system is prescribed for hearing losses with a moderate or large perceptive component, the system is ineffective and leads to negative evaluations.

Influence of glucose absorption on ion activities in cells and submucosal space in goldfish intestine.

Mucosal glucose addition evokes in goldfish intestinal epithelium a fast depolarization of the mucosal membrane potential (delta psi mc = 12 mV) followed by a slower repolarization (delta psi mc = -7 mV). The intracellular sodium activity, aiNa+, rises from 13.2 +/- 2.4 meq/l by 6.7 +/- 0.5 meq/l within 5 min, aiCl- rises about 3 meq/l above the control value of 37.7 +/- 2.2 meq/l, while aiK is constant (97.7 +/- 7.4 meq/l). The potassium activity measured in the submucosal interstitium near the basal side of the cells (asK+) is 5.2 +/- 0.2 meq/l in non-absorbing tissue compared to 4.2 meq/l in the bathing solution and shows a transient increase due to glucose absorption (1.1 +/- 0.1 meq/l). In chloride-free media asK+ = 4.2 +/- 0.1 meq/l and psi mc hyperpolarizes by -13 mV. The depolarization due to glucose absorption increases (delta psi mc = 14.1 +/- 1.4) and the repolarization (delta psi repolmc) disappears. In addition, aiNa+ rises from 16.3 +/- 2.4 meq/l by 9.9 +/- 1.5 meq/l within 5 min, aiK+ remains constant and equal to the value in chloride containing solutions (88.5 +/- 2.8 meq/l); asK+ increases transiently (1.1 +/- 0.1 meq/l). Serosal Ba2+ (5 mM) depolarizes psi mc (+14.2 +/- 1.0 mV) and abolishes the repolarization. Increased serosal or mucosal potassium activity depolarizes psi mc and abolishes the repolarization. These effects are discussed in terms of changes of ion activities, the basolateral potassium conductance, the influence of intracellular Ca2+, the functional state of the Na/K-pump, and modulation of membrane permeabilities by extracellular potassium.