Thin layer-chromatographic separation of fat soluble and water soluble food dyes

Summary The use of fat soluble and water soluble food dyes in food technology requires reliable proofs and precise identification of these substances in foodstuffs. This problematic has been studied more detailed in our Institute. During these studies some suitable procedures for thin layer chromatographic separation of these substances have been developed.For the separation of fat soluble food dyes the chromatography on thin layers of aluminium oxide and the chromatography on thin layers of starch with reversed phases were used. In the first case good separation was achieved with a solvent system containing petroleum ether and carbon tetrachlorid and their mixtures. In the second case the starch plates were impregnated with paraffin or vegetable oil, and as mobile phases a solvent system containing methanol-water-acetic adic (16 : 3 : 1) and others solvent mixtures currently used in paper chromatography were used.Water soluble food dyes were separated on thin layers of polyamide powder, which was used also for their quantitative isolation from foodstuffs. The best results were achieved with basic solvent system, for example, with a mixture containing ammonia-methanol water (5 : 15 : 80).The developed analytical procedures are suitable for the proof and the separation of fat soluble and water soluble food dyes in all foodstuffs.

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Zusammenfassung Die Verwendung von wasser- und fettlöslichen Lebensmittelfarben erfordert verläßlichen Nachweis und genaue Identifikation dieser Stoffe in den Lebensmitteln. Wir haben diese Problematik in unserem Institut eingehend studiert und einige Verfahren der chromatographischen Trennung dieser Stoffe mittels DC erarbeitet.Zur Trennung der fettlöslichen Farbstoffe verwendeten wir einerseits DC-Platten mit Aluminiumoxid, andererseits führten wir die Trennung dieser Stoffe auf einer dünnen Stärkeschicht mit umgekehrter Phase durch. Im ersten Falle wurde eine gute Trennung mit Laufmitteln erreicht, die Petroläther und Tetrachlormethan und deren Gemische enthalten. Im zweiten Falle wurde als stationäre Phase Paraffinöl, als mobile Phase eine Gemisch von Methanol, Wasser und Essigsäure im Verhältnis 16 : 3 : 1 und andere Gemische in gleichen Verhältnissen verwendet, die sich bei der Papier-Verteilungschromatographie bewährt haben.Wasserlösliche Farbstoffe wurden auf einer dünnen Schicht von Polyamidpulver getrennt, das auch zu deren quantitativer Isolation aus den Lebensmitteln verwendet wurde. Die besten Ergebnisse wurden mit alkalischen Laufmitteln erzielt, z.B. mit dem Gemisch von Ammoniak, Methanol und Wasser im Verhältnis 5 : 15 : 80.Die erarbeiteten analytischen Verfahren für Nachweis und Trennung fett- und wasserlöslicher Lebensmittelfarben bewährten sich für alle Lebensmitteltypen.

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Résumé L'utilisation des colorants alimentaires solubles dans l'eau et dans la graisse exige des preuves sûres et l'identification exacte de ces substances dans les aliments. Nous avons étudié ces problèmes en détail à notre institut et élaboré quelques procédés de séparation chromatographique de ces substances par chromatographie sur couche mince.A la séparation des colorants solubles dans la graisse, nous avons utilisé d'une part des couches versées sur l'alumine, d'autre part la séparation de ces substances sur une couche mince de fécule à phase inverse. Au premier cas une bonne séparation fut atteinte aux systèmes comprenant l'éther de pétrole et le tétrachlore carbonique, ou leurs mélanges. A l'autre cas on applique, comme une phase ancrée, de l'huile paraffinique, comme une phase mobile, du mélange de méthanol, d'eau et d'acide acétique en proportion 16 : 3 : 1 et d'autres en mêmes proportions qui avaient fait leurs preuves à la chromatographie de séparation sur le papier.Les colorants solubles dans l'eau furent séparés sur une couche mince de poudre polyamidé qui avait été de même appliqué à leur isolement quantitatif de l'aliment. Les meilleurs résultats furent atteints avec les systèmes alcalins p.e. avec le mélange de méthanol, d'ammoniac et d'eau en proportions 5 : 15 : 80.Les procédés analytiques élaborés pour des preuves et les séparations des colorants solubles dans la graisse et dans l'eau prouvèrent leur attestation avec tous les types de matières alimentaires.

     

Quantitative PCR assays to enumerate Rhizobium leguminosarum strains in soil also target non viable cells and overestimate those detected by the plant infection method

The plant infection method is commonly used to estimate the Most Probable Number (MPN) of soil rhizobia. Here, a qPCR method was set-up and validated with newly developed ANU (strain specific) and RHIZ (more general) primers to quantify the specific Rhizobium leguminosarum bv. trifolii ANU843 strain or general R. leguminosarum strains. Detection limits of qPCR protocols in soil were 1.2 × 10⁴ (ANU) and 4.2 × 10³ (RHIZ) cells per g soil. The qPCR assay appears robust and accurate in freshly inoculated soils but overestimated MPN for indigenous soil rhizobia. An incubation experiment showed that qPCR detected added DNA or non viable cells in soils up to 5 months after addition and incubation at 20 °C in moist conditions.     

Effect of intravenous administration of ketamine on the minimum alveolar concentration of isoflurane in anesthetized dogs

OBJECTIVE: To determine the effect of 6 plasma ketamine concentrations on the minimum alveolar concentration (MAC) of isoflurane in dogs. ANIMALS: 6 dogs. PROCEDURE: In experiment 1, the MAC of isoflurane was measured in each dog and the pharmacokinetics of ketamine were determined in isoflurane-anesthetized dogs after IV administration of a bolus (3 mg/kg) of ketamine. In experiment 2, the same dogs were anesthetized with isoflurane in oxygen. A target-controlled IV infusion device was used to administer ketamine and to achieve plasma ketamine concentrations of 0.5, 1, 2, 5, 8, and 11 microg/mL by use of parameters obtained from experiment 1. The MAC of isoflurane was determined at each plasma ketamine concentration, and blood samples were collected for ketamine and norketamine concentration determination. RESULTS: Actual mean +/- SD plasma ketamine concentrations were 1.07 +/- 0.42 microg/mL, 1.62 +/- 0.98 microg/mL, 3.32 +/- 0.59 microg/mL, 4.92 +/- 2.64 microg/mL, 13.03 +/- 10.49 microg/mL, and 22.80 +/- 25.56 microg/mL for target plasma concentrations of 0.5, 1, 2, 5, 8, and 11 microg/mL, respectively. At these plasma concentrations, isoflurane MAC was reduced by 10.89% to 39.48%, 26.77% to 43.74%, 25.24% to 84.89%, 44.34% to 78.16%, 69.62% to 92.31%, and 71.97% to 95.42%, respectively. The reduction in isoflurane MAC was significant, and the response had a linear and quadratic component. Salivation, regurgitation, mydriasis, increased body temperature, and spontaneous movements were some of the adverse effects associated with the high plasma ketamine concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: Ketamine appears to have a potential role for balanced anesthesia in dogs.     

Blastomycotic osteomyelitis associated with severe lameness in a horse

A 12-year-old Quarter horse gelding was presented for evaluation of severe right forelimb lameness, 2 draining tracts over the lateral aspect of the right proximal antebrachium, and weight loss. A presumptive diagnosis of blastomycotic osteomyelitis was established based on radiographs and cytology of the exudate. This diagnosis was confirmed at necropsy.     

The influence of bedding on the time horses spend recumbent

To determine if bedding has any influence on the time horses spend recumbent, 8 horses kept on straw and 8 kept on wood shavings were observed from 10:00 to 5:30 for two successive nights. Observations were conducted using time-lapse video recordings. Lying down and rising behavior, as well as frequency and duration of bouts spent in lateral and sternal recumbency, was registered. The results showed that horses on straw were lying in lateral recumbency three times longer than horses on shavings (P < .001), whereas the time horses spent in sternal recumbency did not differ. The longest period of noninterrupted lateral recumbency was longer for horses on straw than for those on shavings. Because horses must lie down, preferably in lateral recumbency, to achieve paradoxical sleep, the reduced time spent in lateral recumbency in horses on wood shavings may affect their welfare and performance. Independent of the bedding, we further observed that, as the horses got up from recumbency, most of them made attempts to roll over before rising. This behavior appeared to be caused by some difficulty in rising, possibly due to the box size, and might have a connection with the fact that horses sometimes get stuck against the box wall.

Introduction

Many riding horses spend the majority of their life in an artificial environment. Horse owners keep their horses under certain conditions because of tradition, because they want to make the horse feel comfortable from a human point of view, or to reduce the amount of work involved in horse husbandry. Often the choice of bedding substrate is made from a subjective point of view without assessing both short-term and long-term effects of the bedding. Part of the reason is that only few studies have analyzed horses' preferences for different bedding substrates and their effect on the time horses spend recumbent. In one study comparing straw and wood shavings, no significant preference was found.[1] In another study comparing plastic, wheat straw, and wood shavings, the time horses spent standing, sleeping, or lying down was not affected significantly by the bedding substrates. [2] Mills et al [3] found that horses, given a choice between straw and wood shavings, spent significantly more time on straw. Whereas the substrates had no significant effect on behaviors such as eating, lying, and standing alert, horses spent more time performing bedding-directed behaviors on straw but more time dozing on shavings. Finally, it has been reported that the use of nonstraw bedding may increase the risk of abnormal behaviors such as weaving. [4]As far as bedding properties are concerned, Airaksinen et al[5] concluded that air quality in the stable and utilization of manure can be improved by selecting a good bedding material. According to Reed and Redhead, [6] both straw and shavings are economical and easy to obtain, and they make a bright, comfortable bed. Straw bales are convenient to store, but may be eaten by the horse, are labor intensive, and may be dusty or contain fungal spores. Wood shavings are not eaten by the horse and are good for respiratory problems but need to be kept very clean because they are porous. In addition, they are not as warm as straw because they do not trap air the way straw does.Electroencephalographic (EEG) studies in cats have demonstrated that sleep can be divided into two stages of differing electrocorticographic (EcoG) patterns, ie, slow-wave-sleep (SWS) and paradoxical sleep (PS).[7] During PS, bursts of rapid eye movements (REM) can be seen at irregular intervals. [8] In humans, dreaming occurs during this stage. [9 and 10] Horses are able to sleep while standing, [11] but in this position they only go into SWS. [14, 15 and 16] During PS there is a complete abolition of muscular tone of antigravity muscles and of neck muscles, as shown in cats. [17] In horses, there is a gradual loss of muscular tone until the middle of the recorded SWS period, whence it decreases to a negligible amount during PS. [15] Consequently, muscular tone disappears entirely at the onset of PS. [18] Horses are unable to complete a sleeping cycle without lying down to enter PS. [8, 19 and 20] They normally fall asleep while standing and, when they feel confident about their environment, lie down in sternocostal recumbency. [8] Thereafter, they proceed to lateral recumbency and enter PS. [14 and 19] Dallaire and Ruckebusch [18] demonstrated that the SWS state was infrequent in the standing animal and most often occurred during sternocostal recumbency with the head resting or not on the ground. PS occurred in both sternocostal and lateral recumbency, although the animal frequently had to readjust its position into sternocostal recumbency due to the disappearance of neck muscular tone.The sleep pattern of horses depends on many circumstances, such as age,[21, 22 and 23] diet, [16] and familiarity with the environment. When horses are put outdoors it may take some days before they lie down. If one horse that is familiar with the environment lies down, the others usually follow. [8 and 13] Dallaire and Ruckebusch [16] subjected three horses to a four-day period of perceptual (visual and auditive) deprivation. After this period total sleep time increased due to an augmentation of both SWS and PS. Finally, there is large individual variation between horses in the time they spend recumbent and sleeping. [15]Horses spend 11% to 20% of the total time in recumbency.[11 and 15] Lateral recumbency represents about 20% of total recumbency time, and uninterrupted periods of lateral recumbency vary from 1 to 13 minutes (mean, 4.6 min). [14 and 16] Steinhart [11] found that the mean length of uninterrupted lateral recumbency periods was 23 minutes, the longest period being one hour. Total sleeping time in the stabled horse averages 3 to 5 hours per day or 15% of the total time. [8, 13 and 16] Keiper and Keenan [24] found similar time budgets in feral horses that were recumbent approximately 26% of the night. PS is about 17% to 25% of total sleeping time, and the mean length of a single PS period is 4 to 4.8 minutes. [13 and 18]In stabled horses sleep is mainly nocturnal and occurs during three to seven periods during the night.[8, 13 and 16] Ruckebusch [13] observed that neither sleep nor recumbency occurred during daytime in three ponies observed for a month and, in another experiment conducted on horses, PS occurred only during nighttime. [15] A group of ponies observed for more than a month between 8:45 and 4:45 spent only 1% of the daytime recumbent.[25] The maximum concentration of sleep occurs from 12:00 to 4:00 .[8, 16, 18 and 24]The purpose of this study was to examine two groups of horses in a familiar environment, one group kept on a bedding consisting of straw, and the other kept on wood shavings, and to determine if there was any difference between the two groups in the time they spend recumbent.

Materials and methods

Housing. The study was conducted in one of the biggest riding clubs in Denmark, housing about 150 horses. The 18 horses used in the study stood in three different parts of the stable. They were all stabled in boxes measuring 3 × 3 m and subjected to the same feeding and management routine. They were unable to see their next-door neighbor because of a tall wooden board, but they were able to see the horses stabled on the opposite side of the corridor through bars. Nine horses were stabled on wheat straw (15 cm long, dry matter content 87-88%) and nine on oven-dried wood shavings (80% spruce and 20% pine, dry matter content 82%).Animals. All horses used in the study were privately owned. They had been kept in the boxes in which they were observed a minimum of three weeks. Three of the horses were mares and 15 were geldings. Most of them were Danish Warmblood used for dressage riding. Their ages ranged from 5 to 18 years (mean, 10.6 y) and their height ranged from 1.60 to 1.76 m (mean, 1.68 m). All horses wore a blanket. Age and sex distribution between the two groups is shown in Table 1.